The dormant blood microbiome in chronic, inflammatory diseases

Abstract

Blood in healthy organisms is seen as a 'sterile' environment: it lacks proliferating microbes. Dormant or not-immediately-culturable forms are not absent, however, as intracellular dormancy is well established. We highlight here that a great many pathogens can survive in blood and inside erythrocytes. 'Non-culturability', reflected by discrepancies between plate counts and total counts, is commonplace in environmental microbiology. It is overcome by improved culturing methods, and we asked how common this would be in blood. A number of recent, sequence-based and ultramicroscopic studies have uncovered an authentic blood microbiome in a number of non-communicable diseases. The chief origin of these microbes is the gut microbiome (especially when it shifts composition to a pathogenic state, known as 'dysbiosis'). Another source is microbes translocated from the oral cavity. 'Dysbiosis' is also used to describe translocation of cells into blood or other tissues. To avoid ambiguity, we here use the term 'atopobiosis' for microbes that appear in places other than their normal location. Atopobiosis may contribute to the dynamics of a variety of inflammatory diseases. Overall, it seems that many more chronic, non-communicable, inflammatory diseases may have a microbial component than are presently considered, and may be treatable using bactericidal antibiotics or vaccines.
© FEMS 2015.

Full text

FEMS Microbiology Reviews, fuv013
doi: 10.1093/femsre/fuv013
Review Article
REVIEW ARTICLE
The dormant blood microbiome in chronic,
inammatory diseases
Marnie Potgieter1, Janette Bester1,DouglasB.Kell
2,∗and Etheresia Pretorius1
1Department of Physiology, Faculty of Health Sciences, University of Pretoria, Arcadia 0007,
South Africa and 2School of Chemistry and The Manchester Institute of Biotechnology, The University
of Manchester, 131, Princess St, Manchester M1 7DN, Lancs, UK
∗Corresponding author: School of Chemistry and The Manchester Institute of Biotechnology, The University of Manchester, 131, Princess St,
Manchester M1 7DN, Lancs, UK. Tel: (+44)161 306 4492; E-mail: dbk@manchester.ac.uk
One sentence summary: Atopobiosis of microbes (the term describing microbes that appear in places other than where they should be), as well as the
products of their metabolism, seems to correlate with, and may contribute to, the dynamics of a variety of inammatory diseases.
Editor: Prof. Antoine Danchin
ABSTRACT
Blood in healthy organisms is seen as a ‘sterile’ environment: it lacks proliferating microbes. Dormant or
not-immediately-culturable forms are not absent, however, as intracellular dormancy is well established. We highlight here
that a great many pathogens can survive in blood and inside erythrocytes. ‘Non-culturability’, reected by discrepancies
between plate counts and total counts, is commonplace in environmental microbiology. It is overcome by improved
culturing methods, and we asked how common this would be in blood. A number of recent, sequence-based and
ultramicroscopic studies have uncovered an authentic blood microbiome in a number of non-communicable diseases. The
chief origin of these microbes is the gut microbiome (especially when it shifts composition to a pathogenic state, known as
‘dysbiosis’). Another source is microbes translocated from the oral cavity. ‘Dysbiosis’ is also used to describe translocation
of cells into blood or other tissues. To avoid ambiguity, we here use the term ‘atopobiosis’ for microbes that appear in places
other than their normal location. Atopobiosis may contribute to the dynamics of a variety of inammatory diseases.
Overall, it seems that many more chronic, non-communicable, inammatory diseases may have a microbial component
than are presently considered, and may be treatable using bactericidal antibiotics or vaccines.
Keywords: ‘sterile’ blood microbiome; culturability; dormancy; dysbiosis; atopobiosis; Parkinson’s disease; Alzheimer
disease
INTRODUCTION
‘Overall, it seems inevitable that the availability of these meth-
ods will cause the catalog of disease states recognized as having
a microbial contribution to their etiology to expand enormously
in the short term, particularly as improved methods for resusci-
tation of small cell numbers are found’ (Davey and Kell 1996).
Over the years, a variety of diseases that were previously con-
sidered non-communicable have been found to have a micro-
bial component, the role of Helicobacter pylori in ulcerogenesis
(Marshall and Warren 1984) being a particularly well-known ex-
ample. There have also been hints for a microbial component to
many other non-communicable diseases, but culturing the rele-
vant organisms has rarely been successful. However, there is in-
creasing recognition that microbes may be present in forms that
are not easily culturable, and a number of recent articles have
brought these possibilities more sharply into focus. Our aim is to
review these developments. The manuscript structure is shown
in Fig. 1.
Received: 26 January 2015; Accepted: 2 March 2015
C
FEMS 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium,
provided the original work is properly cited.
1
FEMS Microbiology Reviews Advance Access published May 3, 2015

2FEMS Microbiology Reviews
Figure 1. An overview gure summarizing the contents of this manuscript.
A note on terminology: viable, culturable, dormant and
sterile
In this eld, much confusion has arisen historically because of
a failure to recognize that most microbes reproduce by binary
ssion and that this reproduction must be a minimal property
or hallmark of a microbial cell that possesses ‘life’ or is ‘alive’
(Proal, Albert and Marshall 2011). Thus, as with Schr ¨
odinger’s cat
(e.g. Primas 1981; Gribbin 1985), we cannot say that an individ-
ual microbial cell ‘is’ alive, only (if true) that it ‘was’ alive, since
it will by then have become two cells. This implies that being
alive is not best treated as though it were an innate property of a
cell, but the denition must be operational, and include both the
cell and the ‘environment’ (experiment) used to detect the status
a posteriori (Kell et al. 1998).
Thus, as with Postgate (e.g. Postgate 1967,1969,1976), we
equate viability with culturability, and stress that culturability—
the ability to reproduce—is to be determined operationally.
Other methods that do not determine culturability are not tests
of viability per se, but merely measure what they measure (e.g.
the content of a chemical such as ATP, membrane permeability
to a dye, enzymatic activity, macromolecular sequences and so
on). In addition, it is impossible in principle to (cor)relate macro-
scopic measurements of a culture with the ability of individual
cells to divide (Kell et al. 1991; Davey and Kell 1996). In other
words, if the macroscopic ATP content of say a starving culture
were to decrease by 50%, we would not know if all of the cells
had lost half their ATP or half of the cells had lost all of their ATP
(or anything in between). The culturability of the former would
likely be 50% and of the latter 100%, despite the same macro-
scopic ATP content.
A lack of culturability may mean that a cell is non-viable
under the circumstances tested, but viability or non-viability
are not the only two possible states here. An apparent non-
culturability of a surviving cell also admits another possibility,
for which the natural term is ‘dormant’ (Kaprelyants, Gottschal
and Kell 1993;Epstein2013). This is that the cell is not presently
culturable (viable), but it is not ‘dead’ (in the sense of an oper-
ationally irreversible loss of viability) in that it may be induced
to return to a state of culturability (by a process or processes
typically referred to as ‘resuscitation’). This also means that the
term ‘viable-but-non-culturable’, while quite common in use, is
in fact an oxymoron that is to be discouraged (Kell et al. 1998).
The eminent microbial physiologist Howard Gest is similarly
Tab le 1. Operational denitions of viable, non-viable and dormant
microbes. These are the three terms we consider best suited to de-
scribe the macroscopic physiological states of microbes as regards
their ability to replicate. We note that the terms ‘not immediately
culturable’ (NIC) and ‘active but not culturable’ (ABNC) can also have
some utility (Kell et al. 1998), while dormant cells are sometimes
referred to as ‘persisters’. Other variants of ‘dormancy’ that have
been used include ‘anabiosis’ (Keilin 1959) and ‘cryptobiosis’ (e.g.
Clegg 2001;Neuman2006); all these terms imply a reversible state
between the appearance of being living and non-living in differ-
ent circumstances. This denition of dormancy also likely includes
cells that may operationally be ‘injured’, and possibly wall-less L-
forms (Domingue and Woody 1997; Mattman 2001; Allan, Hoischen
and Gumpert 2009;Dom
´
ınguez-Cuevas et al. 2012; Errington 2013;
Mercier, Kawai and Errington 2013,2014) provided they are or may
become culturable. ‘Sterile’ refers to an absence of operationally vi-
able organisms as dened in this table.
Term Properties
Viable Capable of observable replication, i.e.
culturable, by any stated means.
Non-viable Incapable of observable replication by any
stated means normally capable of effecting
replication in the relevant organism.
Dormant Not viable in the sense of not being more or
less immediately culturable, but may be
returned to a state of viability or culturability
by preincubation under suitable conditions.
scathing about the term ‘unculturable’ (Gest 2008), noting that
one just needs to try harder to culture organisms. Table 1shows
the three terms best suited to discuss these issues, while Fig. 2
shows a diagrammatic representation of the macroscopic phys-
iological microbial states we mostly consider.
The assessment of replication potential (culturability) of in-
dividual cells may be done microscopically (e.g. by microscopic
counts) or macroscopically (e.g. via colony formation on an agar
plate or through the ‘most probable number’ technique). The
latter has the advantage of potentially assessing dormancy in
the absence of any contaminating culturable cells that might
proliferate during the assay (Kaprelyants, Mukamolova and Kell
1994; Votyakova, Kaprelyants and Kell 1994;Kellet al. 1998). For
assessing culturability (=viability), we do not therefore include

Potgieter et al.3
Figure 2. A diagrammatic representation of the major macroscopic physiological states of microbes and their interrelationships.
other strategies in which cells do not actually divide, such as the
so-called direct viable count of Kogure, Simidu and Taga (1979).
Thus, we here highlight the point that the possibility of micro-
bial dormancy means that a system that appears to be devoid
of culturable microbes may still contain dormant cells or forms
that may become culturable.
The ‘sterile’ blood microbiome brought into question
The circulation is a closed system and the blood in healthy or-
ganisms was rst believed to be a sterile environment (Drennan
1942; Proal, Albert and Marshall 2014). This denition is used
in the most usual sense of an absence of culturable microbes,
since blood can of course provide a suitable growth medium
for microbes (as in blood culture; Wilson and Weinstein 1994;
Weinstein 1996; Schroeter et al. 2012; cf. Valencia-Shelton and
Loeffelholz 2014), and any bacteraemia or sepsis, even at 1–
10 cells mL−1(Murray 2015), is potentially life-threatening (e.g.
Vincent et al. 2009; Eleftheriadis et al. 2011; Havey, Fowler and
Daneman 2011; Montassier et al. 2013). However, the principle
of the presence of truly sterile blood in healthy humans has
been challenged, as operationally it does not mean that dormant
or non-culturable forms of organisms are absent (Kaprelyants,
Gottschal and Kell 1993;Kellet al. 1998; McLaughlin et al. 2002)
(see Table 1). Nearly 50 years ago, the existence of a novel bacte-
riological system was noted in 71% of blood samples taken from
diseased humans and from 7% of supposedly healthy humans,
when RBCs were lysed (Domingue and Schlegel 1977). A year
later, corynebacteria-like microorganisms developing in hemo-
cultures were shown within RBCs (Tedeschi et al. 1978), and in
2001 it was found that even ‘healthy’ blood specimens can con-
tain bacterial 16S ribosomal DNA (Nikkari et al. 2001). Domingue
and Woody (1997) and Domingue (2010) summarizes much of
this earlier literature. L-forms are bacterial variants that lack
some or all of a cell wall. Nonetheless they can divide, especially
in osmotically stabilized media, by processes that variously in-
volve membrane blebbing, tubulation, vesiculation and ssion
(Allan, Hoischen and Gumpert 2009; Errington 2013; Mercier,
Kawai and Errington 2014). While it remains unclear whether
what was seen in these earlier studies (Domingue and Woody
1997; Domingue 2010) may have been L-forms (Mattman 2001),
that could in time revert to normal bacteria under the correct
conditions (Casades ´
us 2007), L-forms are becoming a topic of
considerable current research (Devine 2012;Dom
´
ınguez-Cuevas
et al. 2012; Mercier, Kawai and Errington 2013,2014).
The presence of a blood bacterial microbiome has also been
associated with a variety of infectious, as well as non-infectious
disease states (Huang et al. 2006; Thwaites and Gant 2011;
Nielsen et al. 2012; Prajsnar et al. 2012;Wanget al. 2012a; Ki-
bru et al. 2014;Satoet al. 2014). It is, for example, known that
H. pylori can exist not only in the gastric mucosa but also in
peripheral blood, where it could cause bacteremia (Huang et al.

4FEMS Microbiology Reviews
2006), and could contribute to Parkinson’s disease (PD) or related
pathologies that precede motor symptoms (Nielsen et al. 2012).
Helicobacter pylori was also previously implicated in the develop-
ment of anemia (Wang et al. 2012b; Kibru et al. 2014). Staphylo-
coccus aureus can also use neutrophils as ‘Trojan horses’ to dis-
seminate infection (Thwaites and Gant 2011; Prajsnar et al. 2012),
while many other pathogens, such as Listeria monocytogenes (Xa-
yarath and Freitag 2012), Salmonella typhimurium (Eisenreich et al.
2010; Claudi et al. 2014; Holden 2015)andYersinia pestis (Isberg
1991), are well known to persist intracellularly; Gest (2008)gives
other historical examples. The same is true for viruses, which
are not discussed here.
The presence of an aberrant blood microbiota (as assessed by
sequencing) has been implicated in type II diabetes and cardio-
vascular disease (Amar et al. 2011,2013;Satoet al. 2014). There
is also growing evidence that periodontal disease and gingivi-
tis are closely linked to cardiovascular disease (Yang et al. 2013;
Ram´
ırez et al. 2014). Oral bacterial translocation into the blood
has been implicated in the development of periodontal disease-
induced endocarditis and myocardial and/or cerebral infarction,
especially in patients with heart valve dysfunction (Koren et al.
2011; Amar and Engelke 2014;Seringecet al. 2014).
We will arguein the next sections that the existence of poten-
tially viable (but possibly non-proliferating) pleomorphic bac-
teria in the blood of healthy humans (McLaughlin et al. 2002)
may therefore be of some signicance in pathology. If such a
microbiome can disrupt homeostasis, it can ultimately play a
fundamental role in disease development and progression. It
has therefore been proposed that the blood microbiota might
therefore represent or contribute to the rst step in the kinetics
of atherosclerosis (Sato et al. 2014), cardiovascular disease and
type II diabetes (Amar et al. 2011), and therefore ultimately serve
as biomarkers for cardiovascular disease risk (Amar et al. 2013).
However, in the quest to use the blood microbiota as biomarkers,
the question of detectability and cultivability are key concepts.
In particular, the existence of a blood microbiome is only
really meaningful and of scientic interest if it represents an
undisturbed state, and is not, for instance, an artefact caused
by the external introduction of microbes through human in-
tervention, reagent contamination (Schroeter et al. 2012;Salter
et al. 2014) and so forth. We therefore rehearse the evidence
that while such artefacts are certainly possible, and must be
excluded rigorously, the phenomenon of a human blood micro-
biome cannot be dismissed as such an artefact in toto.
Evidence that these observations are not due to
contamination
While contamination from reagents (e.g. Schroeter et al. 2012;
Salter et al. 2014), or simply poor sterile technique with nee-
dles and so on, can lead to an artefactual appearance of a blood
microbiome, we consider that the following arguments, taken
together, exclude the thought that the entire (and consider-
able) literature on a blood microbiome can be explained via
contamination.
(I) The rst argument is that there are signicant differences
between the blood microbiomes of individuals harboring
disease states and nominally healthy controls, despite the
fact that samples are treated identically (see later). Some
similar arguments apply to the assessment of drug trans-
porters under different conditions (Kell and Oliver 2014).
(II) A second argument is that the morphological type of organ-
ism (e.g. coccus versus bacillus) seems to be characteristic
of particular diseases.
(III) A third argument is that in many cases (see below) relevant
organisms lurk intracellularly, which is hard to explain by
contamination.
(IV) A fourth argument is that there are just too many diseases
where bacteria have been found to play a role in the patho-
genesis, that all of them may be caused by contamination.
(V) Finally, the actual numbers of cells involved seem far too
great to be explicable by contamination; given that blood
contains more than 109erythrocytes mL−1, if there was
just one bacterial cell per 100 000 erythrocytes (see below
and Amar et al. 2011), this will equate to 104bacteria mL−1.
These are not small numbers.
It is important to point out that molecular methods have been
used frequently to detect active sepsis. These selfsame meth-
ods are also used in environmental biology (as we pointed
out in this review), without undue concern about the poten-
tial for contamination. Contamination will always be a concern,
of course, as noted by Nikkari et al. (2001), but many papers
since 2001 have documented strategies for detecting prokaryotic
DNA in blood and serum using appropriate and careful controls
(Anthony et al. 2000; Mylotte and Tayara 2000;Jianget al. 2009;
Var ani et al. 2009;Manciniet al. 2010; Chang et al. 2011; Grif et al.
2012a;Fern
´
andez-Cruz et al. 2013;Gaibaniet al. 2013). Also, de-
tecting bacteria in blood cultures during sepsis is considered the
standard diagnostic tool for blood stream infections (Mu ˜
noz et al.
2008; Varani et al. 2009), and some laboratories consider that e.g.
PCR testing should always be a complement for the traditional
blood culture test (Grif et al. 2012b).
Theroleofdormancy
Dormancy in microbiology is of course well known, even for non-
sporulating bacteria, and has been dened as a stable but re-
versible nonreplicating state (Mariotti et al. 2013; see also Table 1
and Kaprelyants, Gottschal and Kell 1993;Kellet al. 1998,2003).
The importance of dormant or non-cultured (as opposed to ‘non-
culturable’) organisms has long been recognized in environmen-
tal microbiology (e.g. Mason, Hamer and Bryers 1986; Amann,
Ludwig and Schleifer 1995; Eilers et al. 2000; Hugenholtz 2002;
Keller and Zengler 2004; Pham and Kim 2012;Epstein2013), be-
cause of the 100-fold or greater difference between microscopi-
cally observable cells and those capable of forming a colony on
an agar plate (‘the great plate count anomaly’, see below).
Of the four main possibilities, what we do not know in gen-
eral is whether the ‘missing’ cells
(i) are incapable of growth on the enrichment/isolation media,
(ii) are killed by the enrichment/isolation media (e.g. Tanaka
et al. 2014),
(iii) have lost viability irreversibly (i.e. are operationally dead) or
(iv) are in a dormant or not-immediately-culturable state from
which we might resuscitate them (to effect culturability) if
only we knew how.
The fact that typical isolation media and incubation conditions
do not admit the measurable growth of all strains is certainly
well known (indeed it is the basis for selective isolation media!),
and it took a good while to learn how to culture pathogens such
as H. pylori (Marshall and Warren 1984; Marshall 2006), Legionella
pneumophila (Feeley et al. 1978;Saitoet al. 1981;Meyer1983),
Tropheryma whipplei (Maiwald and Relman 2001;Maiwaldet al.

Potgieter et al.5
2003; Renesto et al. 2003) and so on (Singh et al. 2013). The major-
ity of bacteria that persist in a ‘non-culturable’ form in wounds
(e.g. Dowd et al. 2008;Percivalet al. 2012), or in diseases such
as cystic brosis (Lewis 2010) or tuberculosis (Young, Stark and
Kirschner 2008; Zhang, Yew and Barer 2012), and even simply
in conventional cultures of Escherichia coli (e.g. Koch 1987;Bal-
aban et al. 2004; Keren et al. 2004a,b; Gerdes and Maisonneuve
2012; Amato, Orman and Brynildsen 2013; Germain et al. 2013;
Maisonneuve, Castro-Camargo and Gerdes 2013; Maisonneuve
and Gerdes 2014; Holden 2015), where phenotypic culture dif-
ferentiation is well established (Koch 1971), are also ‘normally
culturable’ by established means. Thus, the existence of oper-
ationally ‘non-culturable’ forms of only moderately fastidious
bacteria is very well established, and more and more bacteria
previously thought ‘unculturable’ are being brought into culture
(e.g. Zengler et al. 2002; Keller and Zengler 2004; Stevenson et al.
2004; Gich et al. 2005; Kamagata and Tamaki 2005; D’Onofrio et al.
2010; Nichols et al. 2010; Vartoukian, Palmer and Wade 2010;
Dedysh 2011; Pham and Kim 2012; Puspita et al. 2012,2013; Stew-
art 2012; Allen-Vercoe 2013; Narihiro and Kamagata 2013;Singh
et al. 2013;Walkeret al. 2014;Lagieret al. 2015a,b;Linget al. 2015).
In environmental microbiology, some bacteria pass through
the usual 0.2 μm lters, and have been referred to as ‘ultrami-
crobacteria’ (Macdonell and Hood 1982; Morita 1997). It was pro-
posed (Kaprelyants, Gottschal and Kell 1993) that rather than
being small (starved) forms of normal bacteria they were more
likely to be normal forms of small bacteria, and this seems to
have been accepted (Lysak et al. 2010;Sahinet al. 2010; Duda et al.
2012;Soinaet al. 2012).
The ability to culture certain kinds of soil bacteria by prein-
cubation in weak broth is also well established (e.g. Bakken and
Olsen 1987; Kaprelyants, Gottschal and Kell 1993), and our own
experiments showed very high levels of resuscitability of dor-
mant cells of Micrococcus luteus (e.g. Kaprelyants and Kell 1993;
Kaprelyants, Mukamolova and Kell 1994; Kaprelyants et al. 1996,
1999;Kellet al. 1998,2003; Mukamolova et al. 1998a,b,1999,
2002a,b). In a similar way, substrate-accelerated death of non-
or slowly growing microorganisms has been known for decades
(Postgate 1967; Calcott and Postgate 1972; Calcott and Calvert
1981).
Thus, any of several well-established mechanisms may con-
tribute to the (often) large differences observable between mi-
croscopic counts and the number of operationally culturable
microbes, with the greatest likelihood being that we simply
have to develop more and better methods to bring these strains
back into culture, i.e. to resuscitate them. In particular, however,
this ‘great plate count anomaly’ has, of course, been brought
into much sharper focus because of the advent of culture-
independent, sequence-based means for detecting and (to a cer-
tain extent) enumerating microbes (though not, of course, of as-
sessing their culturability).
Sequence-based methods for detecting
non-proliferating microbes
The vast majority of microbial species remain uncultivated and,
until recently, about half of all known bacterial phyla were iden-
tied only from their 16S ribosomal RNA gene sequence (Lasken
and McLean 2014). Also, single-cell genomics is a powerful tool
for accessing genetic information from uncultivated microor-
ganisms (Lasken 2012;Rinkeet al. 2013;Cavanaghet al. 2014;
Clingenpeel et al. 2014). Bacterial single-cell genome sequenc-
ing and bioinformatics are, however, challenging (Pallen, Loman
and Penn 2010;Didelotet al. 2012; Loman et al. 2012;Frickeand
Rasko 2014).
The development of sequence-based methods for microbes
(and especially non-eukaryotes) owes much to the pioneering
work of Carl Woese and colleagues, who recognized the util-
ity of small subunit ribosomal RNA (based on both its essen-
tiality and the small but signicant sequence variations) and
applied it with great effect in molecular phylogenetics (Woese
and Fox 1977; Woese, Kandler and Wheelis 1990). Notwithstand-
ing modern reinterpretations of the taxonomic details derived
therefrom (e.g. Williams et al. 2013), there can be little doubt
that this work drew the attention of microbiologists to the po-
tential of sequence-based methods for detecting microbes that
were then invisible to methods based solely on culture, e.g. in
clinical microbiology (Didelot et al. 2012; Loman et al. 2012;Proal
et al. 2013; Fricke and Rasko 2014). rRNA remains a widely used
strategy for detecting specic microbes. This has of course led to
metagenomics, the large-scale sequencing of macromolecules
and indeed (statistically) entire genomes from complex (non-
axenic) environments, increasing the requirement for a full set
of complete reference sequences (Kyrpides et al. 2014) and not
just those of 16S rRNA (Yarza et al. 2013). Even the coupling of
sequences to activities has now become possible (e.g. Radajew-
ski et al. 2000;Wanget al. 2012c).
Microbiome analyses: latest technologies employed
More recently, gut metagenomics has been systematized with
NIH’s Human Microbiome project (HMP) and the European
MetaHIT project aiming to deciphering the structure and func-
tion of the human gut microbiota (Fredricks 2013; Robles-Alonso
and Guarner 2014). The HMP has developed a reference collec-
tion of 16S ribosomal RNA gene sequences collected from sites
across the human body (Koren et al. 2013; Ding and Schloss 2014).
This information can be used to associate changes in the micro-
biome with changes in health, and particularly also the blood
microbiome. The Integrative Human Microbiome Project (iHMP,
http://hmp2.org), the second phase of the NIH HMP, aims to
study the interactions by analyzing microbiome and host ac-
tivities in longitudinal studies of disease-specic cohorts and
by creating integrated data sets of microbiome and host func-
tional properties (The Integrative HMP (iHMP) Research Network
Consortium 2014), ultimately allowing us to analyze host and
microbial DNA (genome) and RNA (transcriptome) sequences
(Morgan and Huttenhower 2014). However, in the HMP study,
the main anatomic sites where samples are collected are skin,
mouth, nose, colon and vagina (ElRakaiby et al. 2014). So far as we
are aware, these projects do not focus on the blood microbiome
(which is probably unsurprising when most commentators as-
sume that it does not exist).
The gut microbiome is by far the largest numerically, and our
purpose here is not to review it in any detail, since this has been
done very well in terms of
(i) its constitution (Lozupone et al. 2012;Weinstock2012),
(ii) temporal variation (Caporaso et al. 2011;Floreset al. 2014;
Thaiss et al. 2014),
(iii) changes associated with diet (Muegge et al. 2011),
(iv) obesity (Turnbaugh et al. 2006,2009),
(v) age and geography (Delzenne and Cani 2011; Delzenne
et al. 2011; Yatsunenko et al. 2012),
(vi) inammation (Cani et al. 2008,2012),
(vii) the immune system (Kau et al. 2011; McDermott and Huff-
nagle 2014)
(viii) and various pathologies (Pughoeft and Versalovic 2012;
Schulz et al. 2014).

6FEMS Microbiology Reviews
It was implied that a better understanding of microbiome-
encoded pathways for xenobiotic metabolism might also have
implications for improving the efcacy of pharmacologic inter-
ventions with neuromodulatory agents (Gonzalez et al. 2011),
and that the exploration of microbiome and metagenome might
give us insightful new perspectives regarding human genet-
ics and how the microbiota contribute to immunity, as well as
to metabolic and inammatory diseases (Cho and Blaser 2012;
Blaser et al. 2013; Blaser 2014; Leslie and Young 2015). This
is because it is assumed in such studies that it is the small-
molecule products of the gut microbiome that can appear in
the human serum metabolome, and thus inuence the rest of
the human body (e.g. Wikoff et al. 2009; Holmes et al. 2011;
Le Chatelier et al. 2013, and see Table 2). Here we also need
to mention lipopolysaccharide (LPS), a main constituent of the
Gram-negative outer membrane that induces the production of
cytokines and/or chemokines, which in turn regulate inamma-
tory and innate and subsequent adaptive immune responses
(Glaros et al. 2013;Rhee2014; Ronco 2014). The release of LPS
may therefore change gut homeostasis, may play a role in e.g.
inammatory bowel disease and necrotizing enterocolitis (Rhee
2014), and may certainly act as an acute phase protein in sepsis
(Ding and Jin 2014).
By contrast, our theme here is that it is additionally the mi-
crobes themselves that can pass from the gut (and other ‘exter-
nal’ surfaces) into the human body, a phenomenon sometimes
known as ‘dysbiosis’, albeit this term is more commonly used
with another meaning. We here need to discriminate a changed
(pathologic) microbiota in the place of origin from the results of a
translocation of microbiota to other areas of the body. In the fol-
lowing sections, we use the term dysbiosis to describe changes
in a microbiome in its main origin (typically the gut), and we
coin the term ‘atopobiosis’ to describe microbes that appear in
places other than where they should be.
The origin of detectable but non-proliferating microbes
appears to be mainly via ‘atopobiosis’ of the gut
microbiome
Dysbiosis, also known as dysbacteriosis, particularly referring to
microbial imbalance in the digestive tract, has been widely dis-
cussed (e.g. Scher and Abramson 2011;Scanlanet al. 2012; Amar
et al. 2013; Bested, Logan and Selhub 2013; Duytschaever et al.
2013; Vaarala 2013). Core to this literature is the idea that factors
that lead to signicant changes in the gut microbiota composi-
tion (dysbiosis) ultimately result in pathology (Larsen et al. 2010;
Amar et al. 2011,2013; Bested, Logan and Selhub 2013; Burcelin
et al. 2013; De Angelis et al. 2013; Fremont et al. 2013; Lanter, Sauer
and Davies 2014; Petriz et al. 2014;Poweret al. 2014; Tojo et al.
2014). Table 3gives a list of diseases, largely inammatory dis-
eases, which have been associated with gut dysbiosis.
In addition, we argue here that as well as gut dysbiosis, a
derangement of the gut microbiome, what we are seeing here,
often called ‘translocation’ in the context of surgery (Swank
and Deitch 1996;MacFie2004) and various diseases (Berg 1995)
(see Table 4that lists diseases and conditions where bacterial
translocation is specically implicated), is what might better be
called atopobiosis (Greek ¨ατoπoςor atopos, in the wrong place),
i.e. an appearance of members of the gut (or other) microbiome
in the wrong place. Bacterial translocation is therefore discussed
in the context of the movement of gut origin microbes [that
changed from normal (dysbiosis)] that moved across the ‘intact’
gastrointestinal tract into normally sterile tissues, including
blood, where the organisms may then directly cause infection or
inammation leading to tissue injury, organ failure, etc. (Stein-
berg 2003; Wiest and Rath 2003;Balzanet al. 2007). We stress
that they may be found in both infectious and non-infectious
diseases as well as being translocated during surgery, and
that atopobiosis of bacteria originating in the oral cavity, e.g.
in periodontal disease, may also be signicant in rheumatoid
arthritis, for instance (see below). Fig. 3provides a schematic
representation of dysbiosis, bacterial translocation and
atopobiosis.
How do gut bacteria escape into blood?
If the gut microbiome is seen as the main source of the blood
microbiome, it is necessary to establish which kinds of condi-
tions might permit this in the absence of real physical damage
(as may, for instance, be caused by surgery) leading to micro-
bial translocation. Wiest, Lawson and Geuking (2014) mention
three possible points of entrance for bacteria into the surround-
ing (sterile) tissue:
(i) by dendritic cells via processes between epithelial cells, not
affecting tight junction function,
(ii) via injured/inamed epithelium with dysfunctional epithe-
lial barrier,
(iii) and via M cells overlying Peyer’s patches as specialized
cells providing access of microbial products to antigen-
presenting cells.
We discuss bacterial translocation in this context in the fol-
lowing sections.
The role of M cells and Peyer’s patches in gut microbial
translocation and atopobiosis
While the gut epithelium represents the largest mucosal tissue,
the mechanisms underlying the interaction between the micro-
biome and the epithelial cells remain poorly understood (Math-
ias et al. 2014). Although this is a vast and complex eld that
warrants a review of its own, we briey argue that gut dysbio-
sis results in an atypical interaction of both the microbiota, as
well as their secretory products, with the gut epithelial layer.
This results in an altered barrier function, which may also lead
to changed mucosal immunity and ultimately to atopobiosis.
The gut epithelium is necessarily normally quite impermeable
to microbes, but there is increasing evidence that direct chem-
ical communication between the microbiota and the epithelial
cells regulates mucosal integrity (Venkatesh et al. 2014). A pos-
sible point of entry is by direct cellular uptake, and there is
one type of cell that can take up microbes, and these are the
M cells overlaying the Peyer’s patches (Kern ´
eis et al. 1997;Jep-
son and Clark 1998; Clark and Jepson 2003; Corr, Gahan and
Hill 2008; Lelouard et al. 2010; Fukuda, Hase and Ohno 2011).
Peyer’s patches are seen as the ‘immune sensors’ of the gut ep-
ithelium. Considerable evidence exists that they provide a pri-
mary route for the limited translocation of microbes between
the gut epithelium and the blood system (Jung, Hugot and Bar-
reau 2010). These interactions with the cells of the gut may
suggest that changes in the intestinal microbiota also inu-
ence mucosal immunity (Sato, Kiyono and Fujihashi et al. 2014).
This is indeed the case, and gut dysbiosis has been shown to
play a signicant role in the development of autoimmune dis-
eases, in particular inammatory bowel diseases (Clemente et al.
2012; Morgan et al. 2012; Hold et al. 2014; Kostic, Xavier and
Gevers 2014;OwyangandWu2014;Maet al. 2015). It was also

Potgieter et al.7
Tab le 2. Some examples of small molecule gut metabolites whose secretion has been implicated in various disease states.
Metabolite Intermediates/products Synthesis
Role in health and
disease References
Amino acids The gut microbiota is not itself an important source
of amino acids during periods of adequate protein
intake. Some commensal members produce biolog-
ically active components from amino acids. Amino
acid supplementation in a mouse model of ulcerative
colitis has been shown to promote overall growth of
commensal microbiota. The effect was considered to
be mediated via the stimulatory effect on mucin pro-
duction by amino acid supplementation.
Faure et al. (2006); Devaraj,
Hemarajata and Versalovic
(2013); Bergen (2014)
Benzoates Benzoic acid, hippurate,
2-hydroxyhippurate
Gut microbiota in mice with active colitis displayed
enrichment for genes involved in benzoate degrada-
tion. Hippurate derives from plant food polyphenols
and is a conjugate of benzoic acid with glycine. In
humans a large portion of hippurate is believed to
be derived from precursors absorbed in the small in-
testines. It is reliably decreased in IBD.
Rechner et al. (2002);
Aronov et al. (2011); De
Preter and Verbeke (2013);
Rooks et al. (2014)
Bile acids Bile acids are synthesized from cholesterol in the
liver and further metabolized into secondary bile
acids by the gut microbiota. The amino acid sides
chain of glyco- and tauro-conjugated bile acids are
cleaved by bacterial bile salt hydrolase (BSH) enzyme
to yield unconjugated bile acids (cholic and chen-
odeoxycholic acids). These products will then be fur-
ther modied by gut bacteria to produce secondary
bile acids. A decrease in this conversion is positively
correlated with liver cirrhosis. Bile acids can mod-
ulate the composition of the microbiota in the gut,
where they function as signaling molecules and may
constitute a mechanism of quorum sensing. In turn,
the microbiota strongly affect bile acid metabolism
by promoting deconjugation, dehydrogenation and
dehydroxylation. It can also inhibit bile acid synthe-
sis in the liver by alleviation of farnesoid X receptor
inhibition in the ileum. Bile acids can induce FMO3
expression by an FXR-dependent mechanism.
Martin et al. (2007); Bennett
et al. (2013); G´
erard (2013);
Kakiyama et al. (2013);
Mart´
ınez et al. (2013); Sayin
et al. (2013); Joyce et al.
(2014)
Lipids Cholesterol The gut microbiota impact on the host systemic lipid
metabolism. When administered as probiotics Bi-
dobacteria and Lactobacillus can enhance dyslipidemia
and insulin resistance. Microbiota have an inuence
on cholesterol metabolism and weight gain in the
host via the bacterial BSH mechanism.
Martin et al. (2007);
Mart´
ınez et al. (2009,2013);
Yu et al. (2013); Joyce et al.
(2014)
Methylamines and
products of choline
metabolism
Methylamine,
dimethylamine,
dimethylglycine,
trimethylamine (TMA) and
trimethylamine N-oxide
(TMAO)
Cleavage of choline and phosphatidylcholine (PC) by
the gut microbiota via the enzyme choline TMA-lyase
produces TMA. Oxidation of TMA by hepatic avin-
containing monooxygenase 3 (FMO3) forms TMAO.
Microbial metabolism of L-carnitine also produces
TMA via a novel Rieske-type protein. Risk for major
adverse cardiovascular events coincides with higher
levels of TMAO.
Wan g et al. (2011); Craciun
and Balskus (2012); Koeth
et al. (2013); Tang et al.
(2013); Zhu et al. (2014)
Neurotransmitters Serotonin, melatonin,
glutamate, GABA,
noradrenaline, dopamine
and acetylcholine
It was recently discovered that gut microbiota pro-
duce tryptophan decarboxylase, the enzyme respon-
sible for decarboxylasing tryptophan to tryptamine.
Tryptamine promotes the release of serotonin by en-
terochromafncells.Inaratmodelitwasshownthat
Bidobacteria treatment resulted in increased tryp-
tophan and kynurenic acid levels. Another study in
mice showed the potential of Lactobacillus rhamnosus
to modulate the GABAergic system. Decreased levels
of dopamine were measured in fecal samples from
active colitis mice.
Desbonnet et al. (2008);
Bravo et al. (2011); Rooks
et al. (2014); Williams et al.
(2014); O’Mahony et al.
(2015)

8FEMS Microbiology Reviews
Tab le 2. (Continued.)
Metabolite Intermediates/products Synthesis
Role in health and
disease References
Phytochemicals,
particularly
polyphenolic
compounds
Chlorogenic acids,
hydrolysable tannins and
avonoids
A signicant amount of polyphenols reaches the
colon and is believed to contribute to gut health
by promoting the growth of some commen-
sals. Polyphenolic bioconversion by microbiota
is paramount in the production of a large range
of bioactive molecules. The exact roles of these
molecules in health and disease are yet to be fully
understood. Nonetheless epidemiological stud-
ies have tied polyphenols to health benets such
as antioxidative, anticarinogenic, antiadipogenic,
antidiabetic and neuroprotective properties. Gut
microbiota can also convert dietary polyphenols to
benzoate.
Tomas-Barberan et al.
(2014); Kahle et al. (2006);
Aronov et al. (2011); van
Duynhoven et al. (2011);
Cardona et al. (2013); Mar´
ın
et al. (2015)
Polyunsaturated
fatty acids (PUFA)
Omega3and6 L. plantarum has genes encoding for the enzyme in-
volved in saturation metabolism of PUFA.
Kishino et al. (2013)
Short-chain fatty
acids (SCFAs)
Most abundant acetate,
propionate, butyrate; to a
lesser extent—formate,
fumarate, malonate,
succinate, caproate and
valerate
The SCFAs are produced from bacterial fermentation
of non-digestible polysaccharides. They play a role in
metabolic syndrome prevention and treatment. Evi-
dence point to their potential to promote metabolic
control in type 2 diabetes. SCFAs are a major source
of energy for colonocytes and also contribute up to
10% of the host’s daily caloric requirements. They
are further involved in the control of energy utiliza-
tion and maintenance of metabolic homeostasis via
the G Protein coupled Receptor 43 (GPR43) receptor.
SCFA products also dampen inammatory response
through this receptor. SCFAs have also been shown
to affect cell proliferation and apoptosis (in cancer
cells), and in epigenetic machinery such as histone
acetylation by butyrate.
Bergman (1990); Maslowski
et al. (2009); den Besten et al.
(2013); Kimura et al. (2013);
Natarajan and Pluznick
(2014); Puddu et al. (2014)
Vitamins B-group vitamins, vitamin
B12; vitamin C, biotin,
vitamin K
It is well established that the gut microbiota synthe-
size a large number of vitamins de novo.Thisisim-
portant since humans lack biosynthetic pathways for
vitamins. The deleterious effects of vitamin decien-
cies are well known. It has only recently been sug-
gested that vitamin B12 may also contribute to shap-
ing the structure and function of microbial commu-
nities in the human gut.
Hill (1997); Cooke, Behan
and Costello (2006);
Arumugam et al. (2011);
LeBlanc et al. (2013);
Degnan, Taga and
Goodman (2014)
Other noteworthy bioactives
Conjugated linoleic acid (CLA), bacteriocin CLA is associated with a diverse array of biological
activities, and predominantly associated with acti-
vation of peroxisome proliferator activated receptors
(PPARs) and the associated switching on and off of
genes. Some Bidobacteria and Lactobacillus species
have been shown to produce CLA. Bacteriocins are
peptides synthesized by bacteria and have narrow
(same species) or broad (across genera) spectrum ac-
tivity against other bacteria. A large number of ar-
chaea and bacteria are believed to produce at least
one bacteriocin.
Bowdish, Davidson and
Hancock (2005); Ross et al.
(2010)
Tetrathionate and nitric oxide Tetrathionate and nitric oxide are produced in an in-
ammatory environment and are central to the t-
ness of several Enterobacteriaceae.Tetrathionate uti-
lization positively correlated with active colitis in a
mouse model. Bacterial growth depends on the pres-
ence of nitrogen. Synthesis of amino acids by the mi-
crobiome depends on the recycling of nitrogen back
into gastrointestinal organs.
Winter et al. (2010); Bergen
(2014); Rooks et al. (2014)

Potgieter et al.9
Tab le 3. Various pathologies that have been associated with dysbiosis of the gut.
Condition References
Asthma Abrahamsson et al. (2014)
AD Karri, Martinez and Coimbatore (2010); Alam et al. (2014)
Atherosclerosis Koren et al. (2011)
Autism spectrum disorders Parracho et al. (2005); Finegold et al. (2010); Adams et al. (2011); Williams et al. (2011,2012); De
Angelis et al. (2013); Kang et al. (2013)
β-Cell autoimmunity de Goffau et al. (2014)
Cardiovascular disease Amar et al. (2011)
Crohn’s disease Seksik et al. (2003)
Chronic fatigue syndrome Sheedy et al. (2009); Proal et al. (2013)
Cystic brosis Scanlan et al. (2012); Bruzzese et al. (2014);S
´
anchez-Calvo et al. (2008); Duytschaever et al. (2011,
2013); Madan et al. (2012)
HIV/AIDS Lozupone et al. (2013); McHardy et al. (2013); Vujkovic-Cvijin et al. (2013)
IgE-associated eczema Abrahamsson et al. (2012)
Inammation Cani et al. (2008,2012); Delzenne and Cani (2011); Delzenne et al. (2011)
Inammatory bowel disease Conte et al. (2006); Clemente et al. (2012); Manichanh et al. (2012); Morgan et al. (2012);
Nagalingam and Lynch (2012); Bakhtiar et al. (2013)
Iron deciency Balamurugan et al. (2010); Zimmermann et al. (2010); Dostal et al. (2012,2014)
Liver disease Schnabl and Brenner (2014)
Multiple sclerosis Berer et al. (2011)
Obesity Delzenne and Cani (2011); Geurts et al. (2014)
Rheumatoid arthritis Detert et al. (2010); Berer et al. (2011); Scher and Abramson (2011); Bingham and Moni (2013);
Brusca, Abramson and Scher (2014); Catrina, Deane and Scher (2014); C´
enit et al. (2014);
Demoruelle, Deane and Holers (2014); Taneja (2014)
Parkinson’s Disease Scheperjans et al. (2015); Vizcarra et al. (2015)
Sarcoidosis Almenoff et al. (1996)
Systemic lupus erythematosus Hevia et al. (2014); Zhang et al. (2014a)
Symptomatic atherosclerosis/stroke Karlsson et al. (2012)
Type 1 diabetes Brown et al. (2012); Owen and Mohamadzadeh (2013); Petersen and Round (2014)
Type 2 diabetes Larsen et al. (2010); Brown et al. (2012); Qin et al. (2012); Karlsson et al. (2013); Everard et al. (2014)
Tab le 4. Diseases and conditions where bacterial translocation (of gut or oral origin) and consequent chronic infection are specically implicated
Diseases and conditions where
translocation of bacteria are present References
Communicable diseases
Fibrosis stage in HIV/HCV coinfection Balagopal et al. (2008); Montes-de-Oca et al. (2011); Page, Nelson and Kelleher (2011); Lin,
Weinberg and Chung (2013); Sacchi et al. (2015)
Hepatitis C virus (HCV) infection French et al. (2013); Munteanu et al. (2014)
HIV/AIDS infection Sandler and Douek (2012); Klatt, Funderburg and Brenchley (2013);
V´
azquez-Castellanos et al. (2014)
Pneumonia in immunocompromised
patients
Sawa (2014)
Diseases usually seen as non-communicable
Abdominal compartment syndrome Mifkovic et al. (2013)
Alcoholic liver disease Chen and Schnabl (2014); Malaguarnera et al. (2014)
Allergic disease: bacterial translocation
during pregnancy
Abrahamsson Wu and Jenmalm (2015)
Atherosclerosis Epstein, Zhou and Zhu (1999); Kozarov et al. (2006); Erridge (2008); Renko et al. (2008); Epstein
et al. (2009); Nagata, de Toledo and Oho (2011); Rosenfeld and Campbell (2011); Hopkins (2013);
Dinakaran et al. (2014); Rogler and Rosano (2014); Trøseid et al. (2014)
Burn wounds Macintire and Bellhorn (2002); Sharma (2007); Aboelatta et al. (2013)
Cirrhosis Wiest and Garcia-Tsao (2005); Jun et al. (2010); Giannelli et al. (2014); Wiest, Lawson and
Geuking (2014)
Chronic kidney disease Anders, Andersen and Stecher (2013); Sabatino et al. (2014)
Metabolic syndrome Festi et al. (2014)
Non-alcoholic fatty liver disease Bieghs and Trautwein (2014)

10 FEMS Microbiology Reviews
Tab le 4. (Continued.)
Diseases and conditions where
translocation of bacteria are present References
Obesity Vajro, Paolella and Fasano (2013); Sanz and Moya-P´
erez (2014)
Pancreatitis Mifkovic et al. (2009); Guo et al. (2014); Ol´
ah and Romics (2014)
Rheumatoid arthritis Ogrendik (2009b,2013b); Ebringer and Rashid (2014); Koziel, Mydel and Potempa (2014)
Schizophrenia Severance et al. (2013); Severance, Yolken and Eaton (2014)
Sepsis and Septic shock∗Tsujimoto, Ono and Mochizuki (2009); Wallet et al. (2011); Deitch (2012); Leli et al. (2014)
Stroke Syrj¨
anen et al. (1988); Emsley and Tyrrell (2002); Emsley et al. (2003); Emsley and Hopkins (2008);
McColl, Allan and Rothwell (2009); Emsley and Chamorro (2010); Grau, Urbanek and Palm
(2010); Wang et al. (2012a); Chien et al. (2013); Dalager-Pedersen et al. (2014); Fugate et al. (2014)
Surgical procedures
Bariatric surgery Festi et al. (2014)
Cardiac surgery Allen (2014)
Multiple organ failure (MOF) Swank and Deitch (1996)
Sepsis due to surgery MacFie (2004); Puleo et al. (2011)
∗‘Sepsis’ is widely used to imply living microbes, but as is now well known it can also occur in the absence of any culturable microbes, including those incapable of
proliferation due to antibiotic activity. Sepsis may commonly result simply from the effects of molecules such as LPS on the generation of inammatory cytokines
(Kotsaki and Giamarellos-Bourboulis 2012; Balakrishnan et al. 2013).
suggested that a changed gut microbiota represents the initial
site of autoimmunity generation, and might be a critical epige-
netic factor in autoimmune diseases such as rheumatoid arthri-
tis (Scher and Abramson 2011; Luckey et al. 2013; Brusca, Abram-
son and Scher 2014; Catrina, Deane and Scher 2014;C
´
enit et al.
2014; Taneja 2014). There is also evidence that regulatory T cells
in the gut are inuenced by microbial factors, and that a changed
microbiota (dysbiosis) may inuence the induction and suppres-
sor functions of these cells, in turn leading to a changed gut mu-
cosal immunity (Kinoshita and Takeda 2014).
We have earlier reviewed the literature that suggests that
dysbiosis can cause gut epithelial barrier dysfunction, and
thereby provide a point of entry into the body, including the
blood, resulting in atopobiosis. This is supported by recent re-
search that has suggested that blood microbiota might be impli-
cated in various (cardiovascular and other) diseases. Sequence-
based techniques provided evidence for the presence of such
a blood microbiome. The question now arises as to whether
such a microbiome’s presence can be directly measured by e.g.
ultrastructural (microscopic) methods, since a consequence of
any translocation of microbes between the gut microbiome and
blood is that they should then be observable in blood. The next
sections will provide visual evidence of the presence of such
a microbiota in Alzheimer’s disease (AD) and PD. As shown in
Tab le 3, these conditions are known to be associated with the
presence of dysbiosis.
Direct measurement by ultrastructural (microscopic)
methods
Direct measurement by ultrastructural (microscopic)methods of
analysis shows that microbes are in fact common constituents
of blood in inammatory diseases [previously seen in PD—Fig. 8
in (Pretorius et al. 2014a and in AD—Fig. 2in (Lipinski and Pre-
torius 2013). We show and annotate selected micrographs from
these papers in Fig. 4]. An important concern that needs to be
addressed, as is also the case with sequence-based methods, is
whether the presence of microbiota in whole blood is indeed
not the result of introduced external contamination. There is
in fact considerable evidence in the literature that bacteria as
well as other microorganisms can reside inside RBCs (e.g. Mi-
nasyan 2014), and thus able to cross the RBC membrane some-
how (see Table 5). Transmission electron microscopy (TEM) anal-
ysis showing bacteria inside cells would also tend to imply that
the bacteria were not externally introduced artefactually during
the preparation of the samples.
For the current paper, we have revisited our AD and PD
samples and gures from Pretorius et al. (2014a) and Lipinski
and Pretorius (2013) and noted the prevalence of bacteria in al-
most all of the AD and PD samples, in numbers much in ex-
cess of those seen in our database of thousands micrographs
from healthy individuals. Here we show additional micrographs
from the previously published samples (see Figs 5and 6). In
both conditions (see Figs 5AD and 6PD), microbes were noted
in close proximity to RBCs, and in some cases RBCs extended
pseudopodia-like projections towards the microbiota. SEM anal-
ysis of AD whole blood (Fig. 5) shows that mostly coccus-shaped
bacteria are present. White blood cells are seen in close proxim-
ity to these bacteria in AD patients (see Fig. 5A–C). SEM anal-
yses of PD patients (Fig. 6) show both coccus- and bacillus-
shaped bacteria in close proximity to RBCs. We also observed
that RBCs extend pseudopodia towards these bacteria and this
might be part of the mechanism by which the bacteria en-
ter the RBCs (see Fig. 6C–F). We also note possibly dividing
coccus-shaped bacteria in both these conditions, indicated with
blue arrows on Fig. 5A (AD patient) and Fig. 6D (PD patient).
This might suggest that these bacteria may be(come) cultur-
able under appropriate conditions (see also Soina et al. 2012;
Epstein 2013).
TEM analysis of the samples from Lipinski and Pretorius
(2013) and Pretorius et al. (2014a) showed the presence in-
side RBCs of cells that appeared to be microbial in nature
(unpublished data). These internalized cells further provide
evidence for a sustained presence of such a blood micro-
biota (and one hardly explained by contamination) (see Fig. 7A
and B: AD and C and D: PD). Bacteria are shown with ar-
rows in the micrographs. No bacterial membrane was noted;
therefore, the bacteria may be L-forms. There seems to be
bacterial species selectivity for a given disease, as our pre-
liminary observations suggest a prevalence for bacillus-type

Potgieter et al.11
Figure 3. Schematic representation ofdysbiosis, bacterial translocation and atopobiosis. (A) When intestinal microbiota are associated with dysbiosis,(B) the gut barrier
(1 and 2) becomes compromised; this leads to (C), a route of entry via the gut epithelia causing (D) bacterial translocation. Bacterial translocation is also associated with
a compromised systemic immune system barrier (3). Therefore, intestinal microbiota dysbiosis (A) followed by bacterial translocation (D) results in (E) atopobiosis. (F)
The results of bacterial translocation are seen in various conditions (see Table 4).
bacteria in AD, but both coccus- and bacillus-shaped bacteria
in PD patients.
Our observations suggest that the presence of bacteria in
these two diseases occurs in only a small fraction of the RBC
population, which is why we had not really noted them in our
previous studies (e.g. Bester et al. 2013; Pretorius et al. 2013,
2014a,b; Pretorius and Kell 2014), and SEM and TEM analysis con-
rms this observation. We have never (or not yet) found bacte-
ria inside RBCs from healthy controls (these without overt, diag-
nosed diseases) when studying blood smears using TEM analy-
sis. The microscopy preparation methods involve a washing pro-
cess, and this may wash away some of the bacteria, or RBCs and
white blood cells associated with bacteria. Therefore, the actual
quantication of the bacteria can only be done by other means;
however, dormancy and viability versus non-viability issues per-
tain (as discussed above).
We found a denite association between RBCs and bacteria,
with RBCs (see Figs 6and 7) forming pseudopodia-like extension,
as if in the process of engulng bacteria. Both coccoid (round)
and bacillary (elongated) bacteria were found in PD whole blood
SEM micrographs, but only coccoid forms in AD whole blood
SEM micrographs. Samples from 25 diagnosed AD patients were
studied and bacteria were detected in 14 individuals from this
AD sample, while samples from 30 PD patients were studied,
in 21 of whom we detected bacteria. Obviously, the type of bac-
teria cannot be identied from ultrastructural observations. As
with the timeline of established cases such as the role of H.
pylori in ulcers and colon cancer, the next tasks are to bring

12 FEMS Microbiology Reviews
Figure 4. Micrographs taken from previously published manuscripts. (A–C)Bac-
terial presence in PD, originally shown in Fig. 8A, C and Gin Pretorius et al.
(2014a). (D) Bacterial presence in AD, originally shown in Fig. 2in Lipinski and
Pretorius (2013).
these microscopically observed bacteria into culture and to carry
out sequence-based studies to establish their role (if any) in
non-communicable diseases. However, to illustrate that the bac-
teria may indeed be engulfed by the RBCs, and to conrm that
the phenomenon is not due to external contamination, we show
TEM micrographs from both of the studied diseases (see Fig. 7,
AD and PD).
CONCLUDING REMARKS AND PROSPECTIVE
EXPERIMENTS
‘Non-culturable’ (which should be called ‘not-easily-culturable’
or ‘not-yet-cultured’) microbes are commonplace in the ‘envi-
ronmental microbiology’ of soil and water, and the blood cer-
tainly represents an ‘environment’. As we show here, there is
a large and scattered literature, increasing in size, to the effect
that there might be a (mainly dormant) microbial component
in a variety of chronic diseases that are normally considered to
be non-microbial or non-communicable in nature, even when
microbes appear absent by culturability criteria. Our previous
Tab le 5. Some microorganisms that are known to invade red blood cells.
Pathogen Type of microorganism Mechanism of invasion References
Anaplasma marginale A tick-borne pathogen that causes
the disease anaplasmosis in cattle.
Via major surface protein 1a (MSP1a) Kocan et al.
(2004)
Bartonella bacilliformis
B.quintana
Bartonella species are fastidious
Gram-negative bacteria, which
belong to the alpha group of the
domain Proteobacteria.
The Trw T4SS mediates attachment of Bartonella to
red blood cells in Bartonella lineage 4. Bartonella is
collected in pits and trenches that form as a result
of deformation factor. Invaginations supposedly
pinch off to carry the content in a vacuole structure
to the cytoplasm of the red blood cell where the
organism persists.
Iwaki-Egawa and
Ihler (1997);
Coleman and
Minnick (2001);
Rolain et al.
(2003); Eicher
and Dehio (2012)
Brucella melitensis Facultative intracellular
Gram-negative coccobacilli.
Invasion shown in mouse erythrocytes. Mechanism
to be identied.
Vitry et al. (2014)
Francisella tularensis Highly infectious bacterium,
which can cause severe disease
tularemia with an infection of
fewer than 10 bacteria
Via serum complement-dependent and
independent mechanisms.
Conlan (2011);
Horzempa et al.
(2011)
Mycoplasma suis A member or the hemotrophic
mycoplasma group that parasitize
erythrocytes in pigs.
Invasion occurs in a similar manner to that of P.
falciparum and B. bacilliformis. Attachment via MSG1
(GAPDH) protein.
Groebel et al.
(2009); Zhang
et al. (2014c)
M. bovis Small cell wall-less bacterium that
contributes to a number of chronic
inammatory diseases in dairy
and feedlot cattle.
Undetermined. van der Merwe,
Prysliak and
Perez-Casal
(2010)
M. gallisepticum Mycoplasmas are small cell
wall-less prokaryotes.
Not known. Vogl et al. (2008)
Plasmodium falciparum The main malaria parasite, part of
whose life cycle involves
inhabiting RBCs.
Recognition of surface receptors precedes a
reorientation where the apical end is adjusted to
the erythrocyte. A tight junction that involves
high-afnity ligand receptor interactions is formed.
The tight junction moves from the apical to
posterior pole and is powered by the actin-myosin
motor of the parasite. The adhesive proteins at the
junction are proteolytically removed when the
posterior pole is reached, most likely by a rhomboid
resident protease in a process that facilitates
membrane resealing. The invasion process
produces a parasitophorous vacuole containing the
merozoite.
Cowman and
Crabb (2006)

Potgieter et al.13
Tab le 5. (Continued.)
Pathogen Type of microorganism Mechanism of invasion References
Streptococcus pneumoniae Gram-positive bacterium which
causes infection-related diseases.
LPXTG motif-containing pneumococcal proteins,
erythrocyte lipid rafts and erythrocyte actin
remodeling are involved in the invasion mechanism.
Yam agu ch i et al.
(2013)
Theileria sporozites Intracellular protozoan
transmitted by ixodid ticks. Infect
wild and domesticated ruminants.
Phylogenetically most closely
related to Babesia.
Occurs in a similar manner to sporozoite entry. Shaw (2003);
Bishop et al.
(2004)
Figure 5. RBCs with microbiota from patients with diagnosed AD (additional mi-
crographs from sample used in Lipinski and Pretorius 2013). These micrographs
are representative of bacteria found in smears of 14 of the 30 AD individuals.
(Aand B) coccus-shaped bacteria associated with white blood cell; (B) coccus-
shaped bacteria associated with an erythrocyte and white blood cell; (C)two
white blood cells associated with coccus-shaped bacteria; (D) a string of cocci-
blue arrow shows possibly dividing coccoid bacteria; (E) an erythrocyte associ-
ated with coccus-shaped bacteria; (F) a high machine magnication of a coccus-
shaped bacteria associated with a dense matted brin deposit. Scale bar: 1 μm.
work (e.g. Bester et al. 2013; Pretorius et al. 2013,2014a;Kelland
Pretorius 2014,2015; Pretorius and Kell 2014) has implied iron
dysregulation as a regular accompaniment to, and probable con-
tributory factor for, a variety of similar diseases, all of which
have an inammatory component. We argue here that there is
also a microbial contribution to this in the blood, and it is not un-
reasonable that the microbial requirement for iron means that,
despite the oxidative stress it can entail (Touati 2000;Kell2009,
2010), microbes may be anticipated to increase in prevalence
when iron is free (e.g. Ratledge 2007; Clifton, Corrent and Strong
2009; Sia, Allred and Raymond 2013; Chu et al. 2014) and avail-
able (D’Onofrio et al. 2010), probably behaving in a social manner
(Kell, Kaprelyants and Grafen 1995; West and Buckling 2003;Dig-
gle et al. 2007; Harrison and Buckling 2009).
Figure 6. RBCs with microbiota from patients with diagnosed PD (additional mi-
crographs from sample used in Pretorius et al. 2014a). These micrographsare rep-
resentative of bacteria found in smears of 21 of the 30 PD individuals. (A) A col-
lection of coccus- and bacillus-shaped bacteria; (B) coccus- and bacillus-shaped
bacteria associated with erythrocyte; (C) bacillus-shaped bacteria in close prox-
imity with erythrocyte. Erythrocyteforms extensions towards bacteria; (Dand E)
bacillus-shaped bacteria associated with elongated erythrocytes; (F) coccus- and
bacillus-shaped bacteria close to erythrocyte that extends pseudopodia towards
the bacteria. Coccus-shaped bacteria shown with pink arrows; bacillus-shaped
bacteria shown with white arrows. Dividing coccus-shaped bacteria shown with
blue arrow. Scale bar: 1 μm.
We have here pointed up the likelihood of a steady crop
of effectively dormant microbes being a feature of blood bi-
ology in chronically diseased humans, including those with
non-communicable diseases. As with any complex system, the
magnitude of any component is affected by the kinetics of every
relevant step; while the precise nature of all the interactions is
uncertain, Fig. 8describes the general network—the rst step in
any systems analysis (Kell 2006; Kell and Knowles 2006).
Consequently, we recognize that the analysis above has
largely been qualitative (the ‘presence’ of a microbial compo-
nent in a specic disease is a qualitative statement). However,
chronic, non-communicable diseases are very far from being

14 FEMS Microbiology Reviews
Figure 7. TEM conrming the presence of bacteria inside erythrocytes of (Aand
B)AD,(Cand D) PD. (Additional micrographs from sample used in Lipinski and
Pretorius (2013) and Pretorius et al. (2014a). Arrows in each micrograph show the
presence of cellular inclusions, without visible membranes. Inclusions are not
typically noted in erythrocytes. We suggest that these inclusions are bacteria,
possibly as L-forms. Scale bar =1μm (A, C, D); 200 nm (D).
static (and thousands of human genes change their expression
at least 2-fold even on a diurnal basis; Zhang et al. 2014b). Thus, a
clear further issue is to seek to understand how the blood micro-
biome may co-vary with the day-to-day dynamics of chronic dis-
eases. For example, rheumatoid arthritis has circadian rhythms
(Straub and Cutolo 2007) and is well known to provide signi-
cant variations (‘ares’; Flurey et al. 2014) in severity at different
times. A reasonable strategy is thus to look for changes in a de-
tectable blood microbiome in this and other diseases that show
such ares. As with H. pylori and stomach ulcers (and cancer),
the simple prediction is that bactericidal antibiotics should be
of value in the treatment of such supposedly non-communicable
diseases. Indeed, this prediction is borne out for diseases such
as rheumatoid arthritis (Ogrendik 2009a,2013a; Kwiatkowska
and Ma´
sli ´
nska 2012) and multiple sclerosis (Ochoa-Rep´
araz et al.
2009;2011), while antipneumococcal vaccination has shown ef-
cacy in preventing stroke (Vila-Corcoles et al. 2014). Of course,
events such as heart attacks and strokes (and see Table 4)may
also be seen as sudden increases in severity of an underlying
condition, and in some cases (such as the much increased like-
lihood of strokes after subarachnoid haemorrhages; McMahon
et al. 2013), analysis of changes in the blood microbiome might
prove predictive.
The obvious next tasks are thus to relate the number and
nature of blood microbes observed in cases such as the above
to microbial sequences and antigens that can be detected in
aliquots of the same samples (e.g. Salipante et al. 2013,2015),
to determine the physiological state of the various microbes (in-
cluding e.g. whether they are L-forms), and to establish methods
to bring them (back) into culture. Since microbes, inammation
and various syndromes are such common co-occurrences (as
are coagulopathies; Kell and Pretorius 2015), longitudinal stud-
ies will have a specially important role, as they will both show
the dynamics and be able to help discriminate cause and ef-
fect during the time evolution of chronic, non-communicable
diseases in ageing populations. The immunogenicity of per-
sisters, and their ability to induce various kinds of inamma-
tion, must be rather different from that of replicating organ-
isms, and this must be investigated. Armed with such collec-
tive knowledge, we might be better placed to develop thera-
peutics such as pre- and probiotics and bactericidal antibiotics
for use in such cases previously thought to lack a microbial
contribution.
Figure 8. Relationships between a dormant blood microbiome and chronic disease dyamics.

Potgieter et al.15
FUNDING
We thank the Biotechnology and Biological Sciences Research
Council (grant BB/L025752/1) as well as the National Re-
search Foundation (NRF) of South Africa for supporting this
collaboration.
Conict of interest. None declared.
GLOSSARY
16S ribosomal RNA: a component of the 30S small subunit of
prokaryotic ribosomes. The 16S rRNA gene is found in all bacte-
ria and archaea and consists of nine short hypervariable regions
that may be used to distinguish bacterial taxa.
Anabiosis: when an organism is in a state of very low
metabolic activity to the extent where it is hardly measurable
and in some cases come to a standstill. The physiological and
biochemical processes are arrested for different periods of time
but can be reversed.
Atopobiosis (Greek ,
ατooπoςor atopos) appearance of the gut
or other microbiome in the wrong place.
Bacterial translocation: the passage of viable resident bac-
teria from the gastrointestinal tract to normally sterile tissues
such as the mesenteric lymph nodes and the other internal
organs.
Cryptobiosis: refers to latent life or a state where an organism
lacks any visible signs of life but is not dead in that it may revert
to a state of aliveness as usually dened. Its metabolic activity
becomes hardly measurable, or comes reversibly to a standstill.
Culturability: the ability of a cell to reproduce.
Direct viable count: the original method comprises incuba-
tion of samples with nutrients (yeast extract) and a single an-
timicrobial agent that specically inhibits DNA synthesis but not
RNA synthesis (nalidixic acid). Cell division ceases as a result
of DNA synthesis inhibition but other cellular metabolic activ-
ities remain unaffected and therefore cells continue to metab-
olize nutrients and grow in size, which allows their detection
microscopically in situ.
Dormant:notviableinthesenseofnotbeingmoreorlessim-
mediately culturable, but may be returned to a state of viability
or culturability by preincubation under suitable conditions.
Dysbiosis: derangement of the species distribution in the
normal microbiome.
L-forms: these bacteria are cell wall-decient forms of nor-
mal bacteria. They are able to proliferate as sphaeroplasts or
protoplasts under certain conditions.
Metagenomics: direct genetic analysis of a collection of
genomes contained in an environmental sample.
Microbiome: the genetic sum of the ecological community
of commensal, symbiotic and pathogenic microorganisms that
lives on and inside our bodies.
‘Most Probable Number’ technique: is a method used to
quantify the concentration of viable microorganisms in a sam-
ple. It involves replicate liquid broth growth in 10-fold dilutions.
When a dilution lacks growth, it is assumed not to have any or-
ganisms. Back-calculation via a Poissonian distribution leads to
the ‘most probable number’ in the original sample
Non-axenic culture: contains more than one species, variety
or strain of organism.
Non-viable: incapable of observable replication by any stated
means normally capable of effecting replication in the relevant
organism.
Phylogenetics: a discipline of evolutionary biology that stud-
ies the relationships between organisms based on how closely
similar some of their macromolecular sequences are.
Pleomorphic: possessing the ability to change shape or size
in response to environmental stimuli.
Resuscitation: induction of apparently non-culturable cells to
a state of culturability.
Sterile: refers to an absence of operationally viable organ-
isms.
Viable: capable of observable replication, i.e. culturable, by
any stated means.
REFERENCES
Aboelatta YA, Abd-Elsalam AM, Omar AH, et al. Selective diges-
tive decontamination (SDD) as a tool in the management of
bacterial translocation following major burns. Ann Burns Fire
Disasters 2013;26:182–8.
Abrahamsson TR, Jakobsson HE, Andersson AF, et al. Low diver-
sity of the gut microbiota in infants with atopic eczema. J
Allergy Clin Immun 2012;129:434–40,440 e431–32.
Abrahamsson TR, Jakobsson HE, Andersson AF, et al. Low gut mi-
crobiota diversity in early infancy precedes asthma at school
age. Clin Exp Allergy 2014;44:842–50.
Abrahamsson TR, Wu RY, Jenmalm MC. Gut microbiota and al-
lergy: the importance of the pregnancy period. Pediatr Res
2015;77:214–9.
Adams JB, Johansen LJ, Powell LD, et al. Gastrointestinal
ora and gastrointestinal status in children with autism—
comparisons to typical children and correlation with autism
severity. BMC Gastroenterol 2011;11:22.
Alam MZ, Alam Q, Kamal MA, et al. A possible link of gut micro-
biota alteration in type 2 diabetes and Alzheimer’s disease
pathogenicity: an update. CNS Neurol Disord-Dr 2014;13:383–
90.
Allan EJ, Hoischen C, Gumpert J. Bacterial L-forms. Adv Appl Mi-
crobiol 2009;68:1–39.
Allen SJ. Gastrointestinal complications and cardiac surgery. J
Extra-Corp Technol 2014;46:142–9.
Allen-Vercoe E. Bringing the gut microbiota into focus through
microbial culture: recent progress and future perspective.
Curr Opin Microbiol 2013;16:625–9.
Almenoff PL, Johnson A, Lesser M, et al. Growth of acid fast L
forms from the blood of patients with sarcoidosis. Thorax
1996;51:530–3.
Amann RI, Ludwig W, Schleifer KH. Phylogenetic identication
and in situ detection of individual microbial cells without
cultivation. Microbiol Rev 1995;59:143–69.
Amar J, Lange C, Payros G, et al. Blood microbiota dysbiosis
is associated with the onset of cardiovascular events in
a large general population: the D.E.S.I.R. study. PLoS One
2013;8:e54461.
Amar J, Serino M, Lange C, et al. Involvement of tissue bacteria
in the onset of diabetes in humans: evidence for a concept.
Diabetologia 2011;54:3055–61.
Amar S, Engelke M. Periodontal innate immune mecha-
nisms relevant to atherosclerosis. Mol Oral Microbiol 2014,
DOI:10.1111/omi.12087.
Amato SM, Orman MA, Brynildsen MP. Metabolic control of
persister formation in Escherichia coli.Mol Cell 2013;50:
475–87.
Anders HJ, Andersen K, Stecher B. The intestinal microbiota, a
leaky gut, and abnormal immunity in kidney disease. Kidney
Int 2013;83:1010–6.

16 FEMS Microbiology Reviews
Anthony RM, Brown TJ, French GL. Rapid diagnosis of bacteremia
by universal amplication of 23S ribosomal DNA followed
by hybridization to an oligonucleotide array. J Clin Microbiol
2000;38:781–8.
Aronov PA, Luo FJ, Plummer NS, et al. Colonic contribution to
uremic solutes. J Am Soc Nephrol 2011;22:1769–76.
Arumugam M, Raes J, Pelletier E, et al. Enterotypes of the human
gut microbiome. Nature 2011;473:174–80.
Bakhtiar SM, LeBlanc JG, Salvucci E, et al. Implications of the hu-
man microbiome in inammatory bowel diseases. FEMS Mi-
crobiol Lett 2013;342:10–7.
Bakken LR, Olsen RA. The relationship between cell size and vi-
ability of soil bacteria. Microb Ecol 1987;13:103–14.
Balaban NQ, Merrin J, Chait R, et al. Bacterial persistence as a
phenotypic switch. Science 2004;305:1622–5.
Balagopal A, Philp FH, Astemborski J, et al. Human immunode-
ciency virus-related microbial translocation and progression
of hepatitis C. Gastroenterology 2008;135:226–33.
Balakrishnan A, Marathe SA, Joglekar M, et al. Bacterici-
dal/permeability increasing protein: a multifaceted protein
with functions beyond LPS neutralization. Innate Immun
2013;19:339–47.
Balamurugan R, Mary RR, Chittaranjan S, et al. Low levels of fae-
cal lactobacilli in women with iron-deciency anaemia in
south India. Brit J Nutr 2010;104:931–4.
Balzan S, de Almeida Quadros C, de Cleva R, et al. Bacterial
translocation: overview of mechanisms and clinical impact.
J Gastroen Hepatol 2007;22:464–71.
Bennett BJ, de Aguiar Vallim TQ, Wang Z, et al. Trimethylamine-
N-oxide, a metabolite associated with atherosclerosis, ex-
hibits complex genetic and dietary regulation. Cell Metab
2013;17:49–60.
BererK,MuesM,KoutrolosM,et al. Commensal microbiota and
myelin autoantigen cooperate to trigger autoimmune de-
myelination. Nature 2011;479:538–41.
Berg RD. Bacterial translocation from the gastrointestinal tract.
Trends Microbiol 1995;3:149–54.
Bergen WG. Small-intestinal or colonic microbiota as a potential
amino acid source in animals. Amino Acids 2014;47:251–8.
Bergman E. Energy contributions of volatile fatty acids from
the gastrointestinal tract in various species. Physiol Rev
1990;70:567–90.
Bested AC, Logan AC, Selhub EM. Intestinal microbiota, probi-
otics and mental health: from Metchnikoff to modern ad-
vances: part III - convergence toward clinical trials. Gut Pathog
2013;5:4.
Bester J, Buys AV, Lipinski B, et al. High ferritin levels have ma-
jor effects on the morphology of erythrocytes in Alzheimer’s
disease. Front Aging Neurosci 2013;5:00088.
Bieghs V, Trautwein C. Innate immune signaling and gut-liver
interactions in non-alcoholic fatty liver disease. Hepatobiliary
Surg Nutr 2014;3:377–85.
Bingham CO III,Moni M. Periodontal disease and rheuma-
toid arthritis: the evidence accumulates for complex
pathobiologic interactions. Curr Opin Rheumatol 2013;25:
345–53.
Bishop R, Musoke A, Morzaria S, et al. Theileria: intracellular pro-
tozoan parasites of wild and domestic ruminants transmit-
ted by ixodid ticks. Parasitology 2004;129:S271–83.
Blaser M, Bork P, Fraser C, et al. The microbiome explored: recent
insights and future challenges. Nat Rev Microbiol 2013;11:213–
7.
Blaser MJ. The microbiome revolution. J Clin Invest 2014;124:
4162–5.
Bowdish DM, Davidson DJ, Hancock R. A re-evaluation of the role
of host defence peptides in mammalian immunity. Curr Pro-
tein Pept Sc 2005;6:35–51.
Bravo JA, Forsythe P, Chew MV, et al. Ingestion of Lactobacillus
strain regulates emotional behavior and central GABA recep-
tor expression in a mouse via the vagus nerve. P Natl Acad Sci
2011;108:16050–5.
Brown K, DeCoffe D, Molcan E, et al. Diet-induced dysbiosis of
the intestinal microbiota and the effects on immunity and
disease. Nutrients 2012;4:1095–119.
Brusca SB, Abramson SB, Scher JU. Microbiome and mucosal in-
ammation as extra-articular triggers for rheumatoid arthri-
tis and autoimmunity. Curr Opin Rheumatol 2014;26:101–7.
Bruzzese E, Callegari ML, Raia V, et al. Disrupted intestinal mi-
crobiota and intestinal inammation in children with cys-
tic brosis and its restoration with Lactobacillus GG: a ran-
domised clinical trial. PLoS One 2014;9:e87796.
Burcelin R, Serino M, Chabo C, et al. Metagenome and
metabolism: the tissue microbiota hypothesis. Diabetes Obes
Metab 2013;15 (Suppl 3):61–70.
Calcott PH, Calvert TJ. Characterization of 3’: 5’ -cyclic
AMP phosphodiesterase in Klebsiella aerogenes and its role
in substrate-accelerated death. J Gen Microbiol 1981;122:
313–21.
Calcott PH, Postgate JR. On substrate-accelerated death in Kleb-
siella aerogenes.J Gen Microbiol 1972;70:115–22.
Cani PD, Bibiloni R, Knauf C, et al. Changes in gut micro-
biota control metabolic endotoxemia-induced inammation
in high-fat diet-induced obesity and diabetes in mice. Dia-
betes 2008;57:1470–81.
Cani PD, Osto M, Geurts L, et al. Involvement of gut microbiota
in the development of low-grade inammation and type 2
diabetes associated with obesity. Gut Microbes 2012;3:279–88.
Caporaso JG, Lauber CL, Costello EK, et al. Moving pictures of the
human microbiome. Genome Biol 2011;12:R50.
Cardona F, Andres-Lacueva C, Tulipani S, et al. Benets of
polyphenols on gut microbiota and implications in human
health. J Nutr Biochem 2013;24:1415–22.
Casades ´
us J. Bacterial L-forms require peptidoglycan synthesis
for cell division. Bioessays 2007;29:1189–91.
Catrina AI, Deane KD, Scher JU. Gene, environment, micro-
biome and mucosal immune tolerance in rheumatoid arthri-
tis. Rheumatology 2014, DOI:10.1093/rheumatology/keu469.
Cavanagh JP, Hjerde E, Holden MT, et al. Whole-genome se-
quencing reveals clonal expansion of multiresistant Staphy-
lococcus haemolyticus in European hospitals. J Antimicrob
Chemoth 2014;69:2920–7.
C´
enit MC, Matzaraki V, Tigchelaar EF, et al. Rapidly expanding
knowledge on the role of the gut microbiome in health and
disease. Biochim Biophys Acta 2014;1842:1981–92.
Chang SS, Hsu HL, Cheng JC, et al. An efcient strategy for
broad-range detection of low abundance bacteria without
DNA decontamination of PCR reagents. PLoS One 2011;6:
e20303.
Chen P, Schnabl B. Host-microbiome interactions in alcoholic
liver disease. Gut Liver 2014;8:237–41.
Chien LN, Chi NF, Hu CJ, et al. Central nervous system infections
and stroke—a population-based analysis. Acta Neurol Scand
2013;128:241–8.
Cho I, Blaser MJ. The human microbiome: at the interface of
health and disease. Nat Rev Genet 2012;13:260–70.
Chu BC, Otten R, Krewulak KD, et al. The solution structure, bind-
ing properties, and dynamics of the bacterial siderophore-
binding protein FepB. J Biol Chem 2014;289:29219–34.

Potgieter et al.17
Clark MA, Jepson MA. Intestinal M cells and their role in bacterial
infection. Int J Med Microbiol 2003;293:17–39.
Claudi B, Sprote P, Chirkova A, et al. Phenotypic variation of
Salmonella in host tissues delays eradication by antimicro-
bial chemotherapy. Cell 2014;158:722–33.
Clegg JS. Cryptobiosis—a peculiar state of biological organiza-
tion. Comp Biochem Phys B 2001;128:613–24.
Clemente JC, Ursell LK, Parfrey LW, et al. The impact of the
gut microbiota on human health: an integrative view. Cell
2012;148:1258–70.
Clifton MC, Corrent C, Strong RK. Siderocalins: siderophore-
binding proteins of the innate immune system. Biometals
2009;22:557–64.
Clingenpeel S, Schwientek P, Hugenholtz P, et al. Effects of sam-
ple treatments on genome recovery via single-cell genomics.
ISME J 2014;8:2546–9.
Coleman SA, Minnick MF. Establishing a direct role for the
Bartonella bacilliformis invasion-associated locus B (IalB)
protein in human erythrocyte parasitism. Infect Immun
2001;69:4373–81.
Conlan JW. Francisella tularensis: a red-blooded pathogen. JInfect
Dis 2011;204:6–8.
Conte MP, Schippa S, Zamboni I, et al. Gut-associated bacterial
microbiota in paediatric patients with inammatory bowel
disease. Gut 2006;55:1760–7.
Cooke G, Behan J, Costello M. Newly identied vitamin K-
producing bacteria isolated from the neonatal faecal ora.
Microb Ecol Health D 2006;18:133–8.
Corr SC, Gahan CC, Hill C. M-cells: origin, morphology and role
in mucosal immunity and microbial pathogenesis. FEMS Im-
munol Med Mic 2008;52:2–12.
Cowman AF, Crabb BS. Invasion of red blood cells by malaria par-
asites. Cell 2006;124:755–66.
Craciun S, Balskus EP. Microbial conversion of choline to
trimethylamine requires a glycyl radical enzyme. P Natl Acad
Sci USA 2012;109:21307–12.
Dalager-Pedersen M, Sogaard M, Schonheyder HC, et al. Risk for
myocardial infarction and stroke after community-acquired
bacteremia: a 20-year population-based cohort study. Circu-
lation 2014;129:1387–96.
Davey HM, Kell DB. Flow cytometry and cell sorting of heteroge-
neous microbial populations: the importance of single-cell
analyses. Microbiol Rev 1996;60:641–96.
De Angelis M, Piccolo M, Vannini L, et al. Fecal microbiota
and metabolome of children with autism and pervasive
developmental disorder not otherwise specied. PLoS One
2013;8:e76993.
de Goffau MC, Fuentes S, van den Bogert B, et al. Aberrant gut mi-
crobiota composition at the onset of type 1 diabetes in young
children. Diabetologia 2014;57:1569–77.
De Preter V, Verbeke K. Metabolomics as a diagnostic tool in gas-
troenterology. World J Gastrointest Pharmacol Ther 2013;4:97–
107.
Dedysh SN. Cultivating uncultured bacteria from northern wet-
lands: knowledge gained and remaining gaps. Front Microbiol
2011;2:184.
Degnan PH, Taga ME, Goodman AL. Vitamin B12 as a mod-
ulator of gut microbial ecology. Cell Metab 2014;20:
769–78.
Deitch EA. Gut-origin sepsis: evolution of a concept. Surgeon
2012;10:350–6.
Delzenne NM, Cani PD. Interaction between obesity and the gut
microbiota: relevance in nutrition. Annu Rev Nutr 2011;31:15–
31.
Delzenne NM, Neyrinck AM, B¨
ackhed F, et al. Targeting gut mi-
crobiota in obesity: effects of prebiotics and probiotics. Nat
Rev Endocrinol 2011;7:639–46.
Demoruelle MK, Deane KD, Holers VM. When and where does in-
ammation begin in rheumatoid arthritis? Curr Opin Rheuma-
tol 2014;26:64–71.
den Besten G, van Eunen K, Groen AK, et al. The role of short-
chain fatty acids in the interplay between diet, gut micro-
biota, and host energy metabolism. J Lipid Res 2013;54:2325–
40.
Desbonnet L, Garrett L, Clarke G, et al. The probiotic Bidobacte-
ria infantis: an assessment of potential antidepressant prop-
erties in the rat. J Psychiatr Res 2008;43:164–74.
Detert J, Pischon N, Burmester GR, et al. The association between
rheumatoid arthritis and periodontal disease. Arthritis Res
Ther 2010;12:218.
Devaraj S, Hemarajata P, Versalovic J. The human gut micro-
biome and body metabolism: implications for obesity and di-
abetes. Clin Chem 2013;59:617–28.
Devine KM. Bacterial L-forms on tap: an improved methodology
to generate Bacillus subtilis L-forms heralds a new era of re-
search. Mol Microbiol 2012;83:10–3.
Didelot X, Bowden R, Wilson DJ, et al. Transforming clinical mi-
crobiology with bacterial genome sequencing. Nat Rev Genet
2012;13:601–12.
Diggle SP, Grifn AS, Campbell GS, et al. Cooperation and
conict in quorum-sensing bacterial populations. Nature
2007;450:411–4.
Dinakaran V, Rathinavel A, Pushpanathan M, et al. Elevated lev-
els of circulating DNA in cardiovascular disease patients:
metagenomic proling of microbiome in the circulation. PLoS
One 2014;9:e105221.
Ding PH, Jin LJ. The role of lipopolysaccharide-binding pro-
tein in innate immunity: a revisit and its relevance to
oral/periodontal health. J Periodontal Res 2014;49:1–9.
Ding T, Schloss PD. Dynamics and associations of micro-
bial community types across the human body. Nature
2014;509:357–60.
Domingue GJ. Demystifying pleomorphic forms in persistence
and expression of disease: Are they bacteria, and is peptido-
glycan the solution? Discov Med 2010;10:234–46.
Domingue GJ, Schlegel JU. Novel bacterial structures in human
blood: cultural isolation. Infect Immun 1977;15:621–7.
Domingue GJ, Sr, Woody HB. Bacterial persistence and expres-
sion of disease. Clin Microbiol Rev 1997;10:320–44.
Dom´
ınguez-Cuevas P, Mercier R, Leaver M, et al. The rod to L-
form transition of Bacillus subtilis is limited by a requirement
for the protoplast to escape from the cell wall sacculus. Mol
Microbiol 2012;83:52–66.
D’Onofrio A, Crawford JM, Stewart EJ, et al. Siderophores from
neighboring organisms promote the growth of uncultured
bacteria. Chem Biol 2010;17:254–64.
Dostal A, Baumgartner J, Riesen N, et al. Effects of iron sup-
plementation on dominant bacterial groups in the gut, fae-
cal SCFA and gut inammation: a randomised, placebo-
controlled intervention trial in South African children. Brit
JNutr2014;112:547–56.
Dostal A, Chassard C, Hilty FM, et al. Iron depletion and repletion
with ferrous sulfate or electrolytic iron modies the compo-
sition and metabolic activity of the gut microbiota in rats. J
Nutr 2012;142:271–7.
Dowd SE, Sun Y, Secor PR, et al. Survey of bacterial diversity in
chronic wounds using pyrosequencing, DGGE, and full ribo-
some shotgun sequencing. BMC Microbiol 2008;8:43.

18 FEMS Microbiology Reviews
Drennan M. What is ‘Sterile Blood’? Brit Med J 1942;2:526.
Duda VI, Suzina NE, Polivtseva VN, et al. Ultramicrobacteria: for-
mation of the concept and contribution of ultramicrobacteria
to biology. Mikrobiologiia 2012;81:415–27.
Duytschaever G, Huys G, Bekaert M, et al. Cross-sectional and
longitudinal comparisons of the predominant fecal micro-
biota compositions of a group of pediatric patients with cys-
tic brosis and their healthy siblings. Appl Environ Microb
2011;77:8015–24.
Duytschaever G, Huys G, Bekaert M, et al. Dysbiosis of bidobac-
teria and Clostridium cluster XIVa in the cystic brosis fecal
microbiota. JCystFibros2013;12:206–15.
Ebringer A, Rashid T. Rheumatoid arthritis is caused by a Proteus
urinary tract infection. APMIS 2014;122:363–8.
Eicher SC, Dehio C. Bartonella entry mechanisms into mam-
malian host cells. Cell Microbiol 2012;14:1166–73.
Eilers H, Pernthaler J, Glockner FO, et al. Culturability and In situ
abundance of pelagic bacteria from the North Sea. Appl Env-
iron Microb 2000;66:3044–51.
Eisenreich W, Dandekar T, Heesemann J, et al. Carbon
metabolism of intracellular bacterial pathogens and
possible links to virulence. Nat Rev Microbiol 2010;8:
401–12.
Eleftheriadis T, Liakopoulos V, Leivaditis K, et al. Infections in
hemodialysis: a concise review—Part 1: bacteremia and res-
piratory infections. Hippokratia 2011;15:12–7.
ElRakaiby M, Dutilh BE, Rizkallah MR, et al. Pharmacomicro-
biomics: the impact of human microbiome variations on sys-
tems pharmacology and personalized therapeutics. Omics
2014;18:402–14.
Emsley HC, Chamorro A. Stroke bugs: current and emerging con-
cepts relevant to infection in cerebrovascular disease. Infect
Disord Drug Targets 2010;10:65–6.
Emsley HC, Hopkins SJ. Acute ischaemic stroke and infection:
recent and emerging concepts. Lancet Neurol 2008;7:341–53.
Emsley HC, Smith CJ, Gavin CM, et al. An early and sustained pe-
ripheral inammatory response in acute ischaemic stroke:
relationships with infection and atherosclerosis. J Neuroim-
munol 2003;139:93–101.
Emsley HC, Tyrrell PJ. Inammation and infection in clinical
stroke. J Cerebr Blood F Met 2002;22:1399–419.
Epstein SE, Zhou YF, Zhu J. Infection and atherosclerosis: emerg-
ing mechanistic paradigms. Circulation 1999;100:e20–8.
Epstein SE, Zhu J, Naja AH, et al. Insights into the role of in-
fection in atherogenesis and in plaque rupture. Circulation
2009;119:3133–41.
Epstein SS. The phenomenon of microbial uncultivability. Curr
Opin Microbiol 2013;16:636–42.
Erridge C. The roles of pathogen-associated molecular patterns
in atherosclerosis. Trends Cardiovas Med 2008;18:52–6.
Errington J. L-form bacteria, cell walls and the origins of life. Open
Biol 2013;3:120143.
Everard A, Matamoros S, Geurts L, et al. Saccharomyces boulardii
administration changes gut microbiota and reduces hepatic
steatosis, low-grade inammation, and fat mass in obese
and type 2 diabetic db/db mice. MBio 2014;5:e01011–4.
Faure M, Mettraux C, Moennoz D, et al. Specic amino acids in-
crease mucin synthesis and microbiota in dextran sulfate
sodium-treated rats. JNutr2006;136:1558–64.
Feeley JC, Gorman GW, Weaver RE, et al. Primary isolation
media for Legionnaires disease bacterium. J Clin Microbiol
1978;8:320–5.
Fern ´
andez-Cruz A, Marin M, Kestler M, et al. The value of com-
bining blood culture and SeptiFast data for predicting com-
plicated bloodstream infections caused by Gram-positive
bacteria or Candida species. J Clin Microbiol 2013;51:1130–6.
FestiD,SchiumeriniR,EusebiLH,et al. Gut microbiota and
metabolic syndrome. World J Gastroenterol 2014;20:16079–94.
Finegold SM, Dowd SE, Gontcharova V, et al. Pyrosequencing
study of fecal microora of autistic and control children.
Anaerobe 2010;16:444–53.
Flores GE, Caporaso JG, Henley JB, et al. Temporal variability is a
personalized feature of the human microbiome. Genome Biol
2014;15:531.
Flurey CA, Morris M, Richards P, et al. It’s like a juggling
act: rheumatoid arthritis patient perspectives on daily life
and are while on current treatment regimes. Rheumatology
2014;53:696–703.
Fredricks DN. The Human Microbiota: How Microbial Communities
Affect Health and Disease. Hoboken, NJ: Wiley, 2013.
Fremont M, Coomans D, Massart S, et al. High-throughput 16S
rRNA gene sequencing reveals alterations of intestinal mi-
crobiota in myalgic encephalomyelitis/chronic fatigue syn-
drome patients. Anaerobe 2013;22:50–6.
French AL, Evans CT, Agniel DM, et al. Microbial translocation
and liver disease progression in women coinfected with HIV
and hepatitis C virus. J Infect Dis 2013;208:679–89.
Fricke WF, Rasko DA. Bacterial genome sequencing in the
clinic: bioinformatic challenges and solutions. Nat Rev Genet
2014;15:49–55.
Fugate JE, Lyons JL, Thakur KT, et al. Infectious causes of stroke.
Lancet Infect Dis 2014;14:869–80.
Fukuda S, Hase K, Ohno H. Application of a mouse ligated Peyer’s
patch intestinal loop assay to evaluate bacterial uptake by M
cells. JVisExp2011;e3225–30.
Gaibani P, Mariconti M, Bua G, et al. Development of a broad-
range 23S rDNA real-time PCR assay for the detection and
quantication of pathogenic bacteria in human whole blood
and plasma specimens. Biomed Res Int 2013;2013:264651.
G´
erard P. Metabolism of cholesterol and bile acids by the gut mi-
crobiota. Pathogens 2013;3:14–24.
Gerdes K, Maisonneuve E. Bacterial persistence and toxin-
antitoxin loci. Annu Rev Microbiol 2012;66:103–23.
Germain E, Castro-Roa D, Zenkin N, et al. Molecular mech-
anism of bacterial persistence by HipA. Mol Cell 2013;52:
248–54.
Gest H. The Modern Myth of ‘Unculturable’ Bacteria: Scotoma of Con-
temporary Microbiology. Faculty Research (Bloomington), 2008,
http://hdl.handle.net/2022/3149 (19 March 2015, date last ac-
cessed).
Geurts L, Neyrinck AM, Delzenne NM, et al. Gut microbiota
controls adipose tissue expansion, gut barrier and glucose
metabolism: novel insights into molecular targets and inter-
ventions using prebiotics. Benef Microbes 2014;5:3–17.
Giannelli V, DiGregorio V, Iebba V, et al. Microbiota and the gut-
liver axis: bacterial translocation, inammation and infec-
tion in cirrhosis. World J Gastroenterol 2014;20:16795–810.
Gich F, Schubert K, Bruns A, et al. Specic detection, isola-
tion, and characterization of selected, previously uncultured
members of the freshwater bacterioplankton community.
Appl Environ Microb 2005;71:5908–19.
Glaros TG, Chang S, Gilliam EA, et al. Causes and consequences
of low grade endotoxemia and inammatory diseases. Front
Biosci 2013;5:754–65.
Gonzalez A, Stombaugh J, Lozupone C, et al. The mind-body-
microbial continuum. Dialogues Clin Neurosci 2011;13:55–62.
Grau AJ, Urbanek C, Palm F. Common infections and the risk of
stroke. Nat Rev Neurol 2010;6:681–94.

Potgieter et al.19
Gribbin JR. In Search of Schr¨
odinger’s Cat: Quantum Physics and Re-
ality. London: Bantam Books, 1985.
Grif K, Fille M, W ¨
urzner R, et al. Rapid detection of bloodstream
pathogens by real-time PCR in patients with sepsis. Wien Klin
Wochenschr 2012a;124:266–70.
Grif K, Heller I, Prodinger WM, et al. Improvement of detection
of bacterial pathogens in normally sterile body sites with
a focus on orthopedic samples by use of a commercial 16S
rRNA broad-range PCR and sequence analysis. J Clin Microbiol
2012b;50:2250–4.
Groebel K, Hoelzle K, Wittenbrink MM, et al. Mycoplasma suis
invades porcine erythrocytes. Infect Immun 2009;77:576–84.
Guo ZZ, Wang P, Yi ZH, et al. The crosstalk between gut inam-
mation and gastrointestinal disorders during acute pancre-
atitis. Curr Pharm Des 2014;20:1051–62.
Harrison F, Buckling A. Cooperative production of siderophores
by Pseudomonas aeruginosa.Front Biosci 2009;14:4113–26.
Havey TC, Fowler RA, Daneman N. Duration of antibiotic therapy
for bacteremia: a systematic review and meta-analysis. Crit
Care 2011;15:R267.
Hevia A, Milani C, Lopez P, et al. Intestinal dysbiosis associated
with systemic lupus erythematosus. MBio 2014;5:e01548–14.
Hill MJ. Intestinal ora and endogenous vitamin synthesis. Eur J
Cancer Prev 1997;6(Suppl 1):S43–5.
Hold GL, Smith M, Grange C, et al. Role of the gut microbiota
in inammatory bowel disease pathogenesis: what have we
learnt in the past 10 years? World J Gastroenterol 2014;20:1192–
210.
Holden DW. Microbiology. Persisters unmasked. Science
2015;347:30–2.
Holmes E, Li JV, Athanasiou T, et al. Understanding the role of
gut microbiome-host metabolic signal disruption in health
and disease. Trends Microbiol 2011;19:349–59.
Hopkins PN. Molecular biology of atherosclerosis. Physiol Rev
2013;93:1317–542.
Horzempa J, O’Dee DM, Stolz DB, et al. Invasion of erythrocytes
by Francisella tularensis.J Infect Dis 2011;204:51–9.
Huang Y, Fan XG, Tang ZS, et al. Detection of Helicobacter pylori
DNA in peripheral blood from patients with peptic ulcer or
gastritis. APMIS 2006;114:851–6.
Hugenholtz P. Exploring prokaryotic diversity in the genomic
era. Genome Biol 2002;3:0003.1–0003.8.
Isberg RR. Discrimination between intracellular uptake and
surface adhesion of bacterial pathogens. Science 1991;252:
934–8.
Iwaki-Egawa S, Ihler GM. Comparison of the abilities of proteins
from Bartonella bacilliformis and Bartonella henselae to de-
form red cell membranes and to bind to red cell ghost pro-
teins. FEMS Microbiol Lett 1997;157:207–17.
Jepson MA, Clark MA. Studying M cells and their role in infection.
Trends Microbiol 1998;6:359–65.
Jiang W, Lederman MM, Hunt P, et al. Plasma levels of bacterial
DNA correlate with immune activation and the magnitude
of immune restoration in persons with antiretroviral-treated
HIV infection. J Infect Dis 2009;199:1177–85.
Joyce SA, MacSharry J, Casey PG, et al. Regulation of host weight
gain and lipid metabolism by bacterial bile acid modication
in the gut. P Natl Acad Sci USA 2014;111:7421–6.
Jun DW, Kim KT, Lee OY, et al. Association between small in-
testinal bacterial overgrowth and peripheral bacterial DNA
in cirrhotic patients. Dig Dis Sci 2010;55:1465–71.
Jung C, Hugot JP, Barreau F. Peyer’s patches: the immune sensors
of the intestine. Int J Inam 2010;2010:823710.
Kahle K, Kraus M, Scheppach W, et al. Studies on apple and blue-
berry fruit constituents: do the polyphenols reach the colon
after ingestion? Mol Nutr Food Res 2006;50:418–23.
Kakiyama G, Pandak WM, Gillevet PM, et al. Modulation of the
fecal bile acid prole by gut microbiota in cirrhosis. J Hepatol
2013;58:949–55.
Kamagata Y, Tamaki H. Cultivation of uncultured fastidious mi-
crobes. Microbes Environ 2005;20:85–91.
Kang DW, Park JG, Ilhan ZE, et al. Reduced incidence of Prevotella
and other fermenters in intestinal microora of autistic chil-
dren. PLoS One 2013;8:e68322.
Kaprelyants AS, Gottschal JC, Kell DB. Dormancy in non-
sporulating bacteria. FEMS Microbiol Rev 1993;10:271–85.
Kaprelyants AS, Kell DB. Dormancy in stationary-phase cul-
tures of Micrococcus luteus: ow cytometric analysis of
starvation and resuscitation. Appl Environ Microb 1993;59:
3187–96.
Kaprelyants AS, Mukamolova GV, Davey HM, et al. Quantitative
analysis of the physiological heterogeneity within starved
cultures of Micrococcus luteus by ow cytometry and cell
sorting. Appl Environ Microb 1996;62:1311–6.
Kaprelyants AS, Mukamolova GV, Kell DB. Estimation of dor-
mant Micrococcus luteus cells by penicillin lysis and by resus-
citation in cell-free spent medium at high dilution. FEMS Mi-
crobiol Lett 1994;115:347–52.
Kaprelyants AS, Mukamolova GV, Kormer SS, et al. Intercellular
signalling and the multiplication of prokaryotes: bacterial cy-
tokines. Symp Soc Gen Microbi 1999;57:33–69.
Karlsson FH, Fak F, Nookaew I, et al. Symptomatic atherosclero-
sis is associated with an altered gut metagenome. Nat Com-
mun 2012;3:1245.
Karlsson FH, Tremaroli V, Nookaew I, et al. Gut metagenome in
European women with normal, impaired and diabetic glu-
cose control. Nature 2013;498:99–103.
Karri S, Martinez VA, Coimbatore G. Effect of dihydrotestos-
terone on gastrointestinal tract of male Alzheimer’s disease
transgenic mice. 2010;48:453–65.
Kau AL, Ahern PP, Grifn NW, et al. Human nutrition, the gut
microbiome and the immune system. Nature 2011;474:327–
36.
Keilin D. The problem of anabiosis or latent life: history and cur-
rent concept. PRoySocLondBBio1959;150:149–91.
Kell DB. Metabolomics, modelling and machine learning in sys-
tems biology: towards an understanding of the languages of
cells. The 2005 Theodor B ¨
ucher lecture. FEBS J 2006;273:873–
94.
Kell DB. Iron behaving badly: inappropriate iron chelation as a
major contributor to the aetiology of vascular and other pro-
gressive inammatory and degenerative diseases. BMC Med
Genomics 2009;2:2.
Kell DB. Towards a unifying, systems biology understanding of
large-scale cellular death and destruction caused by poorly
liganded iron: Parkinson’s, Huntington’s, Alzheimer’s, pri-
ons, bactericides, chemical toxicology and others as exam-
ples. Arch Toxicol 2010;84:825–89.
Kell DB, Kaprelyants AS, Grafen A. Pheromones, social behaviour
and the functions of secondary metabolism in bacteria.
Trends Ecol Evol 1995;10:126–9.
Kell DB, Kaprelyants AS, Weichart DH, et al. Viability and activity
in readily culturable bacteria: a review and discussion of the
practical issues. Anton Leeuw 1998;73:169–87.
Kell DB, Knowles JD. The role of modeling in systems biology.
In: Szallasi Z, Stelling J, Periwal V (eds). System Modeling in

20 FEMS Microbiology Reviews
Cellular Biology: From Concepts to Nuts and Bolts. Cambridge:
MIT Press, 2006, 3–18.
Kell DB, Mukamolova GV, Finan CL Resuscitation of ‘uncultured’
microorganisms. In: Bull AT, et al. (ed). Microbial Diversity and
Bioprospecting. Washington, DC: American Society for Micro-
biology, 2003, 100–8.
Kell DB, Oliver SG. How drugs get into cells: tested and testable
predictions to help discriminate between transporter-
mediated uptake and lipoidal bilayer diffusion. Front Pharma-
col 2014;5:231.
Kell DB, Pretorius E. Serum ferritin is an important inamma-
tory disease marker, as it is mainly a leakage product from
damaged cells. Metallomics 2014;4:748–73.
Kell DB, Pretorius E. The simultaneous occurrence of both hyper-
coagulability and hypobrinolysis in blood and serum dur-
ing systemic inammation, and the roles of iron and b-
rin(ogen). Integr Biol 2015;7:24–52.
Kell DB, Ryder HM, Kaprelyants AS, et al. Quantifying hetero-
geneity: ow cytometry of bacterial cultures. Anton Leeuw
1991;60:145–58.
Keller M, Zengler K. Tapping into microbial diversity. Nat Rev Mi-
crobiol 2004;2:141–50.
Keren I, Kaldalu N, Spoering A, et al. Persister cells and tolerance
to antimicrobials. FEMS Microbiol Lett 2004a;230:13–8.
Keren I, Shah D, Spoering A, et al. Specialized persister cells and
the mechanism of multidrug tolerance in Escherichia coli.J
Bacteriol 2004b;186:8172–80.
Kern ´
eis S, Bogdanova A, Kraehenbuhl JP, et al. Conversion by
Peyer’s patch lymphocytes of human enterocytes into M cells
that transport bacteria. Science 1997;277:949–52.
Kibru D, Gelaw B, Alemu A, et al. Helicobacter pylori infection
and its association with anemia among adult dyspeptic pa-
tients attending Butajira Hospital, Ethiopia. BMC Infect Dis
2014;14:656.
Kimura I, Ozawa K, Inoue D, et al. The gut microbiota suppresses
insulin-mediated fat accumulation via the short-chain fatty
acid receptor GPR43. Nat Commun 2013;4:1829.
Kinoshita M, Takeda K. Microbial and dietary factors modu-
lating intestinal regulatory T cell homeostasis. FEBS Lett
2014;588:4182–7.
Kishino S, Takeuchi M, Park S-B, et al. Polyunsaturated fatty acid
saturation by gut lactic acid bacteria affecting host lipid com-
position. P Natl Acad Sci USA 2013;110:17808–13.
Klatt NR, Funderburg NT, Brenchley JM. Microbial transloca-
tion, immune activation, and HIV disease. Trends Microbiol
2013;21:6–13.
Kocan KM, de la Fuente J, Blouin EF, et al. Anaplasma marginale
(Rickettsiales: Anaplasmataceae): recent advances in den-
ing host-pathogen adaptations of a tick-borne rickettsia. Par-
asitology 2004;129 (Suppl):285–300.
Koch AL. The adaptive responses of Escherichia coli to a feast and
famine existence. Adv Microb Physiol 1971;6:147–217.
Koch AL. The variability and individuality of the bacterium. In:
Neidhardt FC, et al. (eds). Escherichia coli and Salmonella Ty-
phimurium: Cellular and Molecular Biology. Washington, DC:
American Society for Microbiology, 1987, 1606–14.
Koeth RA, Wang Z, Levison BS, et al. Intestinal microbiota
metabolism of L-carnitine, a nutrient in red meat, promotes
atherosclerosis. Nat Med 2013;19:576–85.
Kogure K, Simidu U, Taga N. A tentative direct microscopic
method for counting living marine bacteria. Can J Microbiol
1979;25:415–20.
Koren O, Knights D, Gonzalez A, et al. A guide to enterotypes
across the human body: meta-analysis of microbial commu-
nity structures in human microbiome datasets. PLoS Comput
Biol 2013;9:e1002863.
Koren O, Spor A, Felin J, et al. Human oral, gut, and plaque mi-
crobiota in patients with atherosclerosis. P Natl Acad Sci USA
2011;108:4592–8.
Kostic AD, Xavier RJ, Gevers D. The microbiome in inammatory
bowel disease: current status and the future ahead. Gastroen-
terology 2014;146:1489–99.
Kotsaki A, Giamarellos-Bourboulis EJ. Emerging drugs for the
treatment of sepsis. Expert Opin Emerg Dr 2012;17:379–91.
Kozarov E, Sweier D, Shelburne C, et al. Detection of bacterial
DNA in atheromatous plaques by quantitative PCR. Microbes
Infect 2006;8:687–93.
Koziel J, Mydel P, Potempa J. The link between periodontal
disease and rheumatoid arthritis: an updated review. Curr
Rheumatol Rep 2014;16:408.
Kwiatkowska B, Ma´
sli ´
nska M. Macrolide therapy in chronic in-
ammatory diseases. Mediat Inamm 2012;2012:636157.
Kyrpides NC, Hugenholtz P, Eisen JA, et al. Genomic encyclopedia
of bacteria and archaea: sequencing a myriad of type strains.
PLoS Biol 2014;12:1001920.
Lagier JC, Edouard S, Pagnier I, et al. Current and past strategies
for bacterial culture in clinical microbiology. Clin Microbiol Rev
2015a;28:208–36.
Lagier JC, Hugon P, Khelaia S, et al. The rebirth of culture in
microbiology through the example of culturomics to study
human gut microbiota. Clin Microbiol Rev 2015b;28:237–64.
Lanter BB, Sauer K, Davies DG. Bacteria present in carotid arterial
plaques are found as biolm deposits which may contribute
to enhanced risk of plaque rupture. MBio 2014;5:e01206–14.
Larsen N, Vogensen FK, van den Berg FW, et al. Gut microbiota in
human adults with type 2 diabetes differs from non-diabetic
adults. PLoS One 2010;5:e9085.
Lasken RS. Genomic sequencing of uncultured microorganisms
from single cells. Nat Rev Microbiol 2012;10:631–40.
Lasken RS, McLean JS. Recent advances in genomic DNA se-
quencing of microbial species from single cells. Nat Rev Genet
2014;15:577–84.
Le Chatelier E, Nielsen T, Qin J, et al. Richness of human
gut microbiome correlates with metabolic markers. Nature
2013;500:541–6.
LeBlanc JG, Milani C, de Giori GS, et al. Bacteria as vitamin sup-
pliers to their host: a gut microbiota perspective. Curr Opin
Biotechnol 2013;24:160–8.
Leli C, Cardaccia A, Ferranti M, et al. Procalcitonin better than C-
reactive protein, erythrocyte sedimentation rate, and white
blood cell count in predicting DNAemia in patients with sep-
sis. Scand J Infect Dis 2014;46:745–52.
Lelouard H, Henri S, De Bovis B, et al. Pathogenic bacteria and
dead cells are internalized by a unique subset of Peyer’s
patch dendritic cells that express lysozyme. Gastroenterology
2010;138:173–84, e171–73.
Leslie JL, Young VB. The rest of the story: the microbiome and
gastrointestinal infections. Curr Opin Microbiol 2015;23c:121–
5.
Lewis K. Persister cells. Annu Rev Microbiol 2010;64:357–72.
Lin W, Weinberg EM, Chung RT. Pathogenesis of accelerated -
brosis in HIV/HCV co-infection. J Infect Dis 2013;207 (Suppl
1):S13–8.
Ling LL, Schneider T, Peoples AJ, et al. A new antibi-
otic kills pathogens without detectable resistance. Nature
2015;517:455–9.

Potgieter et al.21
Lipinski B, Pretorius E. The role of iron-induced brin in the
pathogenesis of Alzheimer’s disease and the protective role
of magnesium. Front Hum Neurosci 2013;7:735.
Loman NJ, Constantinidou C, Chan JZ, et al. High-throughput
bacterial genome sequencing: an embarrassment of choice,
a world of opportunity. Nat Rev Microbiol 2012;10:599–606.
Lozupone CA, Li M, Campbell TB, et al. Alterations in the gut
microbiota associated with HIV-1 infection. Cell Host Microbe
2013;14:329–39.
Lozupone CA, Stombaugh JI, Gordon JI, et al. Diversity, sta-
bility and resilience of the human gut microbiota. Nature
2012;489:220–30.
Luckey D, Gomez A, Murray J, et al. Bugs, us: the role of the gut
in autoimmunity. Indian J Med Res 2013;138:732–43.
Lysak LV, Lapygina EV, Konova IA, et al. Quantity and taxo-
nomic composition of ultramicrobacteria in soils. Microbiol-
ogy 2010;79:408–12.
Ma HD, Wang YH, Chang C, et al. The intestinal microbiota and
microenvironment in liver. Autoimmun Rev 2015;14:183–91.
McColl BW, Allan SM, Rothwell NJ. Systemic infection, in-
ammation and acute ischemic stroke. Neuroscience
2009;158:1049–61.
McDermott AJ, Huffnagle GB. The microbiome and regulation of
mucosal immunity. Immunology 2014;142:24–31.
Macdonell MT, Hood MA. Isolation and characterization of ultra-
microbacteria from a gulf coast estuary. Appl Environ Microb
1982;43:566–71.
MacFie J. Current status of bacterial translocation as a cause of
surgical sepsis. Brit Med Bull 2004;71:1–11.
McHardy IH, Li X, Tong M, et al. HIV Infection is associated with
compositional and functional shifts in the rectal mucosal
microbiota. Microbiome 2013;1:26.
Macintire DK, Bellhorn TL. Bacterial translocation: clinical im-
plications and prevention. Vet Clin N Am-Small 2002;32:
1165–78.
McLaughlin RW, Vali H, Lau PCK, et al. Are there naturally occur-
ring pleomorphic bacteria in the blood of healthy humans? J
Clin Microbiol 2002;40:4771–5.
McMahon CJ, Hopkins S, Vail A, et al. Inammation as a predic-
tor for delayed cerebral ischemia after aneurysmal subarach-
noid haemorrhage. J Neurointerv Surg 2013;5:512–7.
Madan JC, Koestler DC, Stanton BA, et al. Serial analysis of the
gut and respiratory microbiome in cystic brosis in infancy:
interaction between intestinal and respiratory tracts and im-
pact of nutritional exposures. MBio 2012;3:e00251–12.
Maisonneuve E, Castro-Camargo M, Gerdes K. (p)ppGpp con-
trols bacterial persistence by stochastic induction of toxin-
antitoxin activity. Cell 2013;154:1140–50.
Maisonneuve E, Gerdes K. Molecular mechanisms underlying
bacterial persisters. Cell 2014;157:539–48.
Maiwald M, Relman DA. Whipple’s disease and Tropheryma
whippelii: secrets slowly revealed. Clin Infect Dis 2001;32:457–
63.
Maiwald M, von Herbay A, Fredricks DN, et al. Cultivation of
Tropheryma whipplei from cerebrospinal uid. J Infect Dis
2003;188:801–8.
Malaguarnera G, Giordano M, Nunnari G, et al. Gut microbiota
in alcoholic liver disease: pathogenetic role and therapeutic
perspectives. World J Gastroenterol 2014;20:16639–48.
Mancini N, Carletti S, Ghidoli N, et al. The era of molecular and
other non-culture-based methods in diagnosis of sepsis. Clin
Microbiol Rev 2010;23:235–51.
Manichanh C, Borruel N, Casellas F, et al. The gut microbiota in
IBD. Nat Rev Gastroentero 2012;9:599–608.
Mar´
ın L, Migu´
elez EM, Villar CJ, et al. Bioavailability of dietary
polyphenols and gut microbiota metabolism: antimicrobial
properties. BioMed Res Int 2015;2015:905215.
Mariotti S, Pardini M, Gagliardi MC, et al. Dormant Mycobac-
terium tuberculosis fails to block phagosome maturation and
shows unexpected capacity to stimulate specic human T
lymphocytes. JImmunol2013;191:274–82.
Marshall B. Helicobacter connections. ChemMedChem
2006;1:783–802.
Marshall BJ, Warren JR. Unidentied curved bacilli in the stom-
ach of patients with gastritis and peptic ulceration. Lancet
1984;1:1311–5.
Martin F-PJ, Dumas M-E, Wang Y, et al. A top-down systems biol-
ogy view of microbiome-mammalian metabolic interactions
in a mouse model. Mol Syst Biol 2007;3:112.
Mart´
ınez I, Perdicaro DJ, Brown AW, et al. Diet-induced alter-
ations of host cholesterol metabolism are likely to affect the
gut microbiota composition in hamsters. Appl Environ Microb
2013;79:516–24.
Mart´
ınez I, Wallace G, Zhang C, et al. Diet-induced metabolic im-
provements in a hamster model of hypercholesterolemia are
strongly linked to alterations of the gut microbiota. Appl En-
viron Microb 2009;75:4175–84.
Maslowski KM, Vieira AT, Ng A, et al. Regulation of inammatory
responses by gut microbiota and chemoattractant receptor
GPR43. Nature 2009;461:1282–6.
Mason CA, Hamer G, Bryers JD. The death and lysis of mi-
croorganisms in environmental processes.FEMS Microbiol Rev
1986;39:373–401.
MathiasA,PaisB,FavreL,et al. Role of secretory IgA in
the mucosal sensing of commensal bacteria. Gut Microbes
2014;5:688–95.
Mattman L. Cell WallDecient Forms: Stealth Pathogens. Boca Raton,
FL: CRC Press, 2001.
Mercier R, Kawai Y, Errington J. Excess membrane synthe-
sis drives a primitive mode of cell proliferation. Cell
2013;152:997–1007.
Mercier R, Kawai Y, Errington J. General principles for the forma-
tion and proliferation of a wall-free (L-form) state in bacteria.
Elife 2014;3:e04629.
Meyer RD. Legionella infections: a review of ve years of re-
search. Rev Infect Dis 1983;5:258–78.
Mifkovic A, Skultety J, Pindak D, et al. Specic aspects of acute
pancreatitis. Bratisl Lek Listy 2009;110:544–52.
Mifkovic A, Skultety J, Sykora P, et al. Intra-abdominal hyperten-
sion and acute pancreatitis. Bratisl Lek Listy 2013;114:166–71.
Minasyan H. Erythrocyte and blood antibacterial defense. Eur J
Microbiol Immunol 2014;4:138–43.
Montassier E, Batard E, Gastinne T, et al. Recent changes in
bacteremia in patients with cancer: a systematic review of
epidemiology and antibiotic resistance. Eur J Clin Microbiol
2013;32:841–50.
Montes-de-Oca M, Blanco MJ, Marquez M, et al. Haemodynamic
derangement in human immunodeciency virus-infected
patients with hepatitis C virus-related cirrhosis: the role of
bacterial translocation. Liver Int 2011;31:850–8.
Morgan XC, Huttenhower C. Meta’omic analytic techniques
for studying the intestinal microbiome. Gastroenterology
2014;146:1437–48.
Morgan XC, Tickle TL, Sokol H, et al. Dysfunction of the intestinal
microbiome in inammatory bowel disease and treatment.
Genome Biol 2012;13:R79.
Morita RY. Bacteria in Oligotrophic Environments: Starvation-Survival
Lifestyle. New York: Chapman and Hall, 1997.

22 FEMS Microbiology Reviews
Muegge BD, Kuczynski J, Knights D, et al. Diet drives convergence
in gut microbiome functions across mammalian phylogeny
and within humans. Science 2011;332:970–4.
Mukamolova GV, Kaprelyants AS, Young DI, et al. A bacterial cy-
tokine. P Natl Acad Sci USA 1998a;95:8916–21.
Mukamolova GV, Kormer SS, Kell DB, et al. Stimulation of the
multiplication of Micrococcus luteus by an autocrine growth
factor. Arch Microbiol 1999;172:9–14.
Mukamolova GV, Turapov OA, Kazarian K, et al. The rpf gene of
Micrococcus luteus encodes an essential secreted growth fac-
tor. Mol Microbiol 2002a;46:611–21.
Mukamolova GV, Turapov OA, Young DI, et al. A family of au-
tocrine growth factors in Mycobacterium tuberculosis.Mol Mi-
crobiol 2002b;46:623–35.
Mukamolova GV, Yanopolskaya ND, Kell DB, et al. On resuscita-
tion from the dormant state of Micrococcus luteus.Anton Leeuw
1998b;73:237–43.
Mu ˜
noz P, Cruz AF, Rodr´
ıguez-Cr´
eixems M, et al. Gram-negative
bloodstream infections. Int J Antimicrob Ag 2008;32 (Suppl
1):S10–14.
Munteanu D, Negru A, Radulescu M, et al. Evaluation of bacterial
translocation in patients with chronic HCV infection. Rom J
Intern Med 2014;52:91–6.
Murray PR. The clinician and the microbiology laboratory. In:
Bennett JE, Dolin R, Blaser MJ (eds). Mandell, Douglas and Ben-
nett’s Principles and Practice of Infectious Diseases. Philadelphia:
Saunders Elsevier, 2015.
Mylotte JM, Tayara A. Blood cultures: clinical aspects and con-
troversies. Eur J Clin Microbiol 2000;19:157–63.
Nagalingam NA, Lynch SV. Role of the microbiota in inamma-
tory bowel diseases. Inamm Bowel Dis 2012;18:968–84.
Nagata E, de Toledo A, Oho T. Invasion of human aortic en-
dothelial cells by oral viridans group streptococci and induc-
tion of inammatory cytokine production. Mol Oral Microbiol
2011;26:78–88.
Narihiro T, Kamagata Y. Cultivating yet-to-be cultivated
microbes: the challenge continues. Microbes Environ
2013;28:163–5.
Natarajan N, Pluznick JL. From microbe to man: the role of mi-
crobial short chain fatty acid metabolites in host cell biology.
Am J Physiol-Cell Ph 2014;307:C979–85.
Neuman Y. Cryptobiosis: a new theoretical perspective. Prog Bio-
phys Mol Bio 2006;92:258–67.
Nichols D, Cahoon N, Trakhtenberg EM, et al. Useofichipfor
high-throughput in situ cultivation of ‘uncultivable’ micro-
bial species. Appl Environ Microb 2010;76:2445–50.
Nielsen HH, Qiu J, Friis S, et al. Treatment for Helicobacter pylori
infection and risk of Parkinson’s disease in Denmark. Eur J
Neurol 2012;19:864–9.
Nikkari S, McLaughlin IJ, Bi W, et al. Does blood of healthy
subjects contain bacterial ribosomal DNA? J Clin Microbiol
2001;39:1956–9.
O’Mahony SM, Clarke G, Borre YE, et al. Serotonin, tryptophan
metabolism and the brain-gut-microbiome axis. Behav Brain
Res 2015;277:32–48.
Ochoa-Rep´
araz J, Mielcarz DW, Begum-Haque S, et al. Gut, bugs,
and brain: role of commensal bacteria in the control of cen-
tral nervous system disease. Ann Neurol 2011;69:240–7.
Ochoa-Rep´
araz J, Mielcarz DW, Ditrio LE, et al. Role of gut com-
mensal microora in the development of experimental au-
toimmune encephalomyelitis. JImmunol2009;183:6041–50.
Ogrendik M. Efcacy of roxithromycin in adult patients with
rheumatoid arthritis who had not received disease-
modifying antirheumatic drugs: a 3-month, random-
ized, double-blind, placebo-controlled trial. Clin Ther
2009a;31:1754–64.
Ogrendik M. Rheumatoid arthritis is linked to oral bacteria: eti-
ological association. Mod Rheumatol 2009b;19:453–6.
Ogrendik M. Antibiotics for the treatment of rheumatoid arthri-
tis. Int J Gen Med 2013a;7:43–7.
Ogrendik M. Rheumatoid arthritis is an autoimmune disease
caused by periodontal pathogens. Int J Gen Med 2013b;6:383–
6.
Ol´
ah A, Romics L, Jr. Enteral nutrition in acute pancreati-
tis: a review of the current evidence. World J Gastroenterol
2014;20:16123–31.
Owen JL, Mohamadzadeh M. Microbial activation of gut dendritic
cells and the control of mucosal immunity. J Interf Cytok Res
2013;33:619–31.
Owyang C, Wu GD. The gut microbiome in health and disease.
Gastroenterology 2014;146:1433–6.
Page EE, Nelson M, Kelleher P. HIV and hepatitis C coinfec-
tion: pathogenesis and microbial translocation. Curr Opin HIV
AIDS 2011;6:472–7.
Pallen MJ, Loman NJ, Penn CW. High-throughput sequencing
and clinical microbiology: progress, opportunities and chal-
lenges. Curr Opin Microbiol 2010;13:625–31.
Parracho HM, Bingham MO, Gibson GR, et al. Differences between
the gut microora of children with autistic spectrum disor-
ders and that of healthy children. J Med Microbiol 2005;54:987–
91.
Percival SL, Hill KE, Williams DW, et al. A review of the scien-
tic evidence for biolms in wounds. Wound Repair Regen
2012;20:647–57.
Petersen C, Round JL. Dening dysbiosis and its inuence on host
immunity and disease. Cell Microbiol 2014;16:1024–33.
Petriz BA, Castro AP, Almeida JA, et al. Exercise induction of gut
microbiota modications in obese, non-obese and hyperten-
sive rats. BMC Genomics 2014;15:511.
Pughoeft KJ, VersalovicJ. Human microbiome in health and dis-
ease. Annu Rev Pathol 2012;7:99–122.
Pham VH, Kim J. Cultivation of unculturable soil bacteria. Trends
Biotechnol 2012;30:475–84.
Postgate JR. Viability measurements and the survival of mi-
crobes under minimum stress. Adv Microb Physiol 1967;1:1–
23.
Postgate JR. Viable counts and viability. Methods Microbiol
1969;1:611–28.
Postgate JR. Death in Microbes and Macrobes. Cambridge: Cam-
bridge University Press, 1976.
Power SE, O’Toole PW, Stanton C, et al. Intestinal microbiota, diet
and health. Brit J Nutr 2014;111:387–402.
Prajsnar TK, Hamilton R, Garcia-Lara J, et al. A privileged in-
traphagocyte niche is responsible for disseminated infection
of Staphylococcus aureus in a zebrash model. Cell Microbiol
2012;14:1600–19.
Pretorius E, Bester J, Vermeulen N, et al. Profound morphological
changes in the erythrocytes and brin networks of patients
with hemochromatosis or with hyperferritinemia, and their
normalization by iron chelators and other agents. PlosOne
2014a;9:e85271.
Pretorius E, Kell DB. Diagnostic morphology: biophysical in-
dicators for iron-driven inammatory diseases. Integr Biol
2014;6:486–510.
Pretorius E, Swanepoel AC, Buys AV, et al. Eryptosis as a marker
of Parkinson’s disease. Aging 2014b;6:788–818.
Pretorius E, Vermeulen N, Bester J, et al. A novel method for
assessing the role of iron and its functional chelation in

Potgieter et al.23
brin bril formation: the use of scanning electron mi-
croscopy. Toxicol Mech Method 2013;23:352–9.
Primas H. Chemistry, Quantum Mechanics and Reductionism. Berlin:
Springer, 1981.
Proal AD, Albert PJ, Marshall TG. Autoimmune disease and the
human metagenome. In: Nelson KE (ed). Metagenomics of the
Human Body. New York: Springer Science and Business Media,
2011.
Proal AD, Albert PJ, Marshall TG, et al. Immunostimulation in
the treatment for chronic fatigue syndrome/myalgic en-
cephalomyelitis. Immunol Res 2013;56:398–412.
Proal AD, Albert PJ, Marshall TG. Inammatory disease and the
human microbiome. Discov Med 2014;17:257–65.
Puddu A, Sanguineti R, Montecucco F, et al. Evidence for
the gut microbiota short-chain fatty acids as key patho-
physiological molecules improving diabetes. Mediat Inamm
2014;2014:162021.
Puleo F, Arvanitakis M, Van Gossum A, et al. Gut failure in the
ICU. Semin Respir Crit Care 2011;32:626–38.
Puspita DI, Kamagata Y, Tanaka M, et al. Are unculti-
vated bacteria really uncultivable? Microbes Environ 2012;27:
356–66.
Puspita DI, Uehara M, Katayama T, et al. Resuscitation promoting
factor (Rpf) from Tomitella biformata AHU 1821(T) promotes
growth and resuscitates non-dividing cells. Microbes Environ
2013;28:58–64.
QinJ,LiY,CaiZ,et al. A metagenome-wide association study of
gut microbiota in type 2 diabetes. Nature 2012;490:55–60.
Radajewski S, Ineson P, Parekh NR, et al. Stable-isotope probing
as a tool in microbial ecology. Nature 2000;403:646–9.
Ram´
ırez JH, Parra B, Gutierrez S, et al. Biomarkers of cardiovascu-
lar disease are increased in untreated chronic periodontitis:
a case control study. Aust Dent J 2014;59:29–36.
Ratledge C. Iron metabolism and infection. Food Nutr Bull
2007;28:S515–23.
Rechner AR, Kuhnle G, Hu H, et al. The metabolism of dietary
polyphenols and the relevance to circulating levels of conju-
gated metabolites. Free Radical Res 2002;36:1229–41.
Renesto P, Crapoulet N, Ogata H, et al. Genome-based design of
a cell-free culture medium for Tropheryma whipplei. Lancet
2003;362:447–9.
Renko J, Lepp PW, Oksala N, et al. Bacterial signatures in
atherosclerotic lesions represent human commensals and
pathogens. Atherosclerosis 2008;201:192–7.
Rhee SH. Lipopolysaccharide: basic biochemistry, intracellular
signaling, and physiological impacts in the gut. Intest Res
2014;12:90–5.
Rinke C, Schwientek P, Sczyrba A, et al. Insights into the phy-
logeny and coding potential of microbial dark matter. Nature
2013;499:431–7.
Robles-Alonso V, Guarner F. From basic to applied research:
lessons from the human microbiome projects. J Clin Gastroen-
terol 2014;48 (Suppl 1):S3–4.
Rogler G, Rosano G. The heart and the gut. Eur Heart J
2014;35:426–30.
Rolain JM, Maurin M, Mallet MN, et al. Culture and antibiotic sus-
ceptibility of Bartonella quintana in human erythrocytes. An-
timicrob Agents Ch 2003;47:614–9.
Ronco C. Endotoxin removal: history of a mission. Blood Purif
2014;37 (Suppl 1):5–8.
Rooks MG, Veiga P, Wardwell-Scott LH, et al. Gut microbiome
composition and function in experimental colitis during
active disease and treatment-induced remission. ISME J
2014;8:1403–17.
Rosenfeld ME, Campbell LA. Pathogens and atherosclerosis: up-
date on the potential contribution of multiple infectious
organisms to the pathogenesis of atherosclerosis. Thromb
Haemostasis 2011;106:858–67.
Ross R, Mills S, Hill C, et al. Specic metabolite production by
gut microbiota as a basis for probiotic function. Int Dairy J
2010;20:269–76.
Sabatino A, Regolisti G, Brusasco I, et al. Alterations of intestinal
barrier and microbiota in chronic kidney disease. Nephrol Dial
Transpl 2014, DOI:10.1093/ndt/gfu287.
Sacchi P, Cima S, Corbella M, et al. Liver brosis, microbial
translocation and immune activation markers in HIV and
HCV infections and in HIV/HCV co-infection. Dig Liver Dis
2015;47:218–25.
Sahin N, Gonzalez JM, Iizuka T, et al. Characterization of two
aerobic ultramicrobacteria isolated from urban soil and a de-
scription of Oxalicibacterium solurbis sp. nov. FEMS Microbiol
Lett 2010;307:25–9.
Saito A, Rolfe RD, Edelstein PH, et al. Comparison of liquid
growth media for Legionella pneumophila. J Clin Microbiol
1981;14:623–7.
Salipante SJ, Roach DJ, Kitzman JO, et al. Large-scale ge-
nomic sequencing of extraintestinal pathogenic Escherichia
coli strains. Genome Res 2015;25:119–28.
Salipante SJ, Sengupta DJ, Rosenthal C, et al. Rapid 16S rRNA
next-generation sequencing of polymicrobial clinical sam-
ples for diagnosis of complex bacterial infections. PLoS One
2013;8:e65226.
Salter SJ, Cox MJ, Turek EM, et al. Reagent and laboratory contam-
ination can critically impact sequence-based microbiome
analyses. BMC Biol 2014;12:87.
S´
anchez-Calvo JM, Garc´
ıa-Castillo M, Lamas A, et al. Gut mi-
crobiota composition in cystic brosis patients: molecu-
lar approach and classical culture. JCystFibros2008;7:
S50.
Sandler NG, Douek DC. Microbial translocation in HIV infection:
causes, consequences and treatment opportunities. Nat Rev
Microbiol 2012;10:655–66.
Sanz Y, Moya-P´
erez A. Microbiota, inammation and obesity.
Adv Exp Med Biol 2014;817:291–317.
Sato J, Kanazawa A, Ikeda F, et al. Gut dysbiosis and detection of
‘live gut bacteria’ in blood of Japanese patients with type 2
diabetes. Diabetes Care 2014;37:2343–50.
Sato S, Kiyono H, Fujihashi K. Mucosal immunosenescence in
the gastrointestinal tract: a mini-review. Gerontology 2014.
Sawa T. The molecular mechanism of acute lung injury caused
by Pseudomonas aeruginosa: from bacterial pathogenesis to
host response. J Intensive Care 2014;2:10.
Sayin SI, Wahlstrom A, Felin J, et al. Gut microbiota regulates
bile acid metabolism by reducing the levels of tauro-beta-
muricholic acid, a naturally occurring FXR antagonist. Cell
Metab 2013;17:225–35.
Scanlan PD, Buckling A, Kong W, et al. Gut dysbiosis in cystic
brosis. JCystFibros2012;11:454–5.
Scheperjans F, Aho V, Pereira PA, et al. Gut microbiota are related
to Parkinson’s disease and clinical phenotype. Movement Dis-
ord 2015;30:350–8.
Scher JU, Abramson SB. The microbiome and rheumatoid arthri-
tis. Nat Rev Rheumatol 2011;7:569–78.
Schnabl B, Brenner DA. Interactions between the intestinal mi-
crobiome and liver diseases. Gastroenterology 2014;146:1513–
24.
Schroeter J, Wilkemeyer I, Schiller RA, et al. Validation of
the microbiological testing of tissue preparations using the

24 FEMS Microbiology Reviews
BACTEC blood culture system. Transfus Med Hemoth 2012;39:
387–90.
Schulz MD, Atay C, Heringer J, et al. High-fat-diet-mediated dys-
biosis promotes intestinal carcinogenesis independently of
obesity. Nature 2014;514:508–12.
Seksik P, Rigottier-Gois L, Gramet G, et al. Alterations of the dom-
inant faecal bacterial groups in patients with Crohn’s disease
of the colon. Gut 2003;52:237–42.
Seringec N, Guncu G, Arihan O, et al. Investigation of hemorhe-
ological parameters in periodontal diseases. Clin Hemorheol
Micro 2014, DOI:10.3233/CH-141892.
Severance EG, Gressitt KL, Stallings CR, et al. Discordant pat-
terns of bacterial translocation markers and implications for
innate immune imbalances in schizophrenia. Schizophr Res
2013;148:130–7.
Severance EG, Yolken RH, Eaton WW. Autoimmune dis-
eases, gastrointestinal disorders and the microbiome in
schizophrenia: more than a gut feeling. Schizophr Res 2014,
DOI:10.1016/j.schres.2014.06.027.
Sharma BR. Infection in patients with severe burns: causes and
prevention thereof. Infect Dis Clin N Am 2007;21:745–59.
Shaw MK. Cell invasion by Theileria sporozoites. Trends Parasitol
2003;19:2–6.
Sheedy JR, Wettenhall RE, Scanlon D, et al. Increased d-lactic
acid intestinal bacteria in patients with chronic fatigue syn-
drome. In Vivo 2009;23:621–8.
Sia AK, Allred BE, Raymond KN. Siderocalins: siderophore bind-
ing proteins evolved for primary pathogen host defense. Curr
Opin Chem Biol 2013;17:150–7.
Singh S, Eldin C, Kowalczewska M, et al. Axenic culture of fas-
tidious and intracellular bacteria. Trends Microbiol 2013;21:
92–9.
Soina VS, Lysak LV, Konova IA, et al. Study of ultramicrobacte-
ria (nanoforms) in soils and subsoil deposits by electron mi-
croscopy. Eurasian Soil Sci 2012;45:1048–56.
Steinberg SM. Bacterial translocation: what it is and what it is
not. Am J Surg 2003;186:301–5.
Stevenson BS, Eichorst SA, Wertz JT, et al. New strategies for
cultivation and detection of previously uncultured microbes.
Appl Environ Microb 2004;70:4748–55.
Stewart EJ. Growing unculturable bacteria. J Bacteriol
2012;194:4151–60.
Straub RH, Cutolo M. Circadian rhythms in rheumatoid arthritis:
implications for pathophysiology and therapeutic manage-
ment. Arthritis Rheum 2007;56:399–408.
Swank GM, Deitch EA. Role of the gut in multiple organ fail-
ure: bacterial translocation and permeability changes. Worl d
JSurg1996;20:411–7.
Syrj¨
anen J, Valtonen VV, Iivanainen M, et al. Preceding infection
as an important risk factor for ischaemic brain infarction in
young and middle aged patients. Brit Med J 1988;296:1156–60.
Tanaka T, Kawasaki K, Daimon S, et al. Ahiddenpitfallinthe
preparation of agar media undermines microorganism cul-
tivability. Appl Environ Microb 2014;80:7659–66.
Taneja V. Arthritis susceptibility and the gut microbiome. FEBS
Lett 2014;588:4244–9.
TangWH,WangZ,LevisonBS,et al. Intestinal microbial
metabolism of phosphatidylcholine and cardiovascular risk.
New Engl J Med 2013;368:1575–84.
Tedeschi GG, Bondi A, Paparelli M, et al. Electron microscopical
evidence of the evolution of corynebacteria-like microorgan-
isms within human erythrocytes. Experientia 1978;34:458–60.
Thaiss CA, Zeevi D, Levy M, et al. Transkingdom control of micro-
biota diurnal oscillations promotes metabolic homeostasis.
Cell 2014;159:514–29.
The Integrative HMP (iHMP) Research Network Consortium.
The Integrative Human Microbiome Project: dynamic anal-
ysis of microbiome-host omics proles during periods
of human health and disease. Cell Host Microbe 2014;16:
276–89.
Thwaites GE, Gant V. Are bloodstream leukocytes Trojan Horses
for the metastasis of Staphylococcus aureus?Nat Rev Microbiol
2011;9:215–22.
Tojo R , S u ´
arez A, Clemente MG, et al. Intestinal microbiota in
health and disease: role of bidobacteria in gut homeosta-
sis. World J Gastroenterol 2014;20:15163–76.
Tomas-Barberan F, Garcia-Villalba R, Quartieri A, et al. In vitro
transformation of chlorogenic acid by human gut microbiota.
Mol Nutr Food Res 2014;58:1122–31.
Touati D. Iron and oxidative stress in bacteria. Arch Biochem Bio-
phys 2000;373:1–6.
Trøseid M, Manner IW, Pedersen KK, et al. Microbial translocation
and cardiometabolic risk factors in HIV infection. AIDS Res
Hum Retrov 2014;30:514–22.
Tsujimoto H, Ono S, Mochizuki H. Role of translocation of
pathogen-associated molecular patterns in sepsis. Digest
Surg 2009;26:100–9.
Turnbaugh PJ, Hamady M, Yatsunenko T, et al. A core gut micro-
biome in obese and lean twins. Nature 2009;457:480–4.
Turnbaugh PJ, Ley RE, Mahowald MA, et al. An obesity-associated
gut microbiome with increased capacity for energy harvest.
Nature 2006;444:1027–131.
Vaarala O. Human intestinal microbiota and type 1 diabetes. Curr
Diabetes Rep 2013;13:601–7.
Vajro P, Paolella G, Fasano A. Microbiota and gut-liver axis: their
inuences on obesity and obesity-related liver disease. JPe-
diatr Gastr Nutr 2013;56:461–8.
Valencia-Shelton F, Loeffelholz M. Nonculture techniques for
the detection of bacteremia and fungemia. Future Microbiol
2014;9:543–59.
van der Merwe J, Prysliak T, Perez-Casal J. Invasion of bovine
peripheral blood mononuclear cells and erythrocytes by My-
coplasma bovis. Infect Immun 2010;78:4570–8.
van Duynhoven J, Vaughan EE, Jacobs DM, et al. Metabolic fate
of polyphenols in the human superorganism. P Natl Acad Sci
USA 2011;108 (Suppl 1):4531–8.
Varani S, Stanzani M, Paolucci M, et al. Diagnosis of blood-
stream infections in immunocompromised patients by real-
time PCR. JInfect2009;58:346–51.
Vartoukian SR, Palmer RM, Wade WG. Strategies for culture
of ‘unculturable’ bacteria. FEMS Microbiol Lett 2010;309:
1–7.
V´
azquez-Castellanos JF, Serrano-Villar S, Latorre A, et al. Al-
tered metabolism of gut microbiota contributes to chronic
immune activation in HIV-infected individuals. Mucosal Im-
munol 2014, DOI:10.1038/mi.2014.107.
Venkatesh M, Mukherjee S, Wang H, et al. Symbiotic bacte-
rial metabolites regulate gastrointestinal barrier function via
the xenobiotic sensor PXR and Toll-like receptor 4. Immunity
2014;41:296–310.
Vila-Corcoles A, Ochoa-Gondar O, Rodriguez-Blanco T, et al. Eval-
uating clinical effectiveness of pneumococcal vaccination in
preventing stroke: the CAPAMIS Study 3-year follow-up. J
Stroke Cerebrovasc 2014;23:1577–84.
Vincent JL, Rello J, Marshall J, et al. International study of the
prevalence and outcomes of infection in intensive care units.
JAMA 2009;302:2323–9.
Vitry MA, Hanot Mambres D, Deghelt M, et al. Brucella melitensis
invades murine erythrocytes during infection. Infect Immun
2014;82:3927–38.

Potgieter et al.25
Vizcarra JA, Wilson-Perez HE, Espay AJ. The power in num-
bers: gut microbiota in Parkinson’s disease. Movement Disord
2015;30:296–8.
Vogl G, Plaickner A, Szathmary S, et al. Mycoplasma gallisep-
ticum invades chicken erythrocytes during infection. Infect
Immun 2008;76:71–7.
Votyakova TV, Kaprelyants AS, Kell DB. Inuence of vi-
able cells on the resuscitation of dormant cells in Mi-
crococcus luteus cultures held in an extended stationary
phase: the population effect. Appl Environ Microb 1994;60:
3284–91.
Vujkovic-Cvijin I, Dunham RM, Iwai S, et al. Dysbiosis of the gut
microbiota is associated with HIV disease progression and
tryptophan catabolism. Sci Transl Med 2013;5:193–1.
Walker AW, Duncan SH, Louis P, et al. Phylogeny, culturing, and
metagenomics of the human gut microbiota. Trends Microbiol
2014;22:267–74.
Wallet F, Loiez C, Herwegh S, et al. Usefulness of real-time PCR
for the diagnosis of sepsis in ICU-acquired infections. Infect
Disord Drug Targets 2011;11:348–53.
Wang Y, Chen Y, Zhou Q, et al. A culture-independent approach
to unravel uncultured bacteria and functional genes in a
complex microbial community. PLoS One 2012c;7:e47530.
Wang Z, Klipfell E, Bennett BJ, et al. Gut ora metabolism of
phosphatidylcholine promotes cardiovascular disease. Na-
ture 2011;472:57–63.
Wang Z, Zhang L, Guo Z, et al. A unique feature of iron loss via
close adhesion of Helicobacter pylori to host erythrocytes. PLoS
One 2012b;7:e50314.
Wang ZW, Li Y, Huang LY, et al. Helicobacter pylori infection con-
tributes to high risk of ischemic stroke: evidence from a
meta-analysis. JNeurol2012a;259:2527–37.
Weinstein MP. Current blood culture methods and systems: clin-
ical concepts, technology, and interpretation of results. Clin
Infect Dis 1996;23:40–6.
Weinstock GM. Genomic approaches to studying the human mi-
crobiota. Nature 2012;489:250–6.
West SA, Buckling A. Cooperation, virulence and siderophore
production in bacterial parasites. Proc R Soc B 2003;270:37–44.
Wiest R, Garcia-Tsao G. Bacterial translocation (BT) in cirrhosis.
Hepatology 2005;41:422–33.
Wiest R, Lawson M, Geuking M. Pathological bacterial transloca-
tion in liver cirrhosis. J Hepatol 2014;60:197–209.
Wiest R, Rath HC. Gastrointestinal disorders of the critically
ill. Bacterial translocation in the gut. Best Pract Res Cl Ga
2003;17:397–425.
Wikoff WR, Anfora AT, Liu J, et al. Metabolomics anal-
ysis reveals large effects of gut microora on mam-
malian blood metabolites. P Natl Acad Sci USA 2009;106:
3698–703.
Williams BB, Van Benschoten AH, Cimermancic P, et al. Discov-
ery and characterization of gut microbiota decarboxylases
that can produce the neurotransmitter tryptamine. Cell Host
Microbe 2014;16:495–503.
Williams BL, Hornig M, Buie T, et al. Impaired carbohydrate
digestion and transport and mucosal dysbiosis in the in-
testines of children with autism and gastrointestinal distur-
bances. PLoS One 2011;6:e24585.
Williams BL, Hornig M, Parekh T, et al. Application of novel
PCR-based methods for detection, quantitation, and phy-
logenetic characterization of Sutterella species in intestinal
biopsy samples from children with autism and gastrointesti-
nal disturbances. MBio 2012;3:00261–11.
Williams TA, Foster PG, Cox CJ, et al. An archaeal origin of eu-
karyotes supports only two primary domains of life. Nature
2013;504:231–6.
Wilson ML, Weinstein MP. General principles in the labora-
tory detection of bacteremia and fungemia. Clin Lab Med
1994;14:69–82.
Winter SE, Thiennimitr P, Winter MG, et al. Gut inammation
provides a respiratory electron acceptor for Salmonella. Na-
ture 2010;467:426–9.
Woese CR, Fox GE. Phylogenetic structure of the prokary-
otic domain: the primary kingdoms. P Natl Acad Sci USA
1977;74:5088–90.
Woese CR, Kandler O, Wheelis ML. Towards a natural sys-
tem of organisms: proposal for the domains Archaea,
Bacteria, and Eucarya. P Natl Acad Sci USA 1990;87:
4576–79.
Xayarath B, Freitag NE. Optimizing the balance between host and
environmental survival skills: lessons learned from Listeria
monocytogenes. Future Microbiol 2012;7:839–52.
Yamaguchi M, Terao Y, Mori-Yamaguchi Y, et al. Streptococcus
pneumoniae invades erythrocytes and utilizes them to evade
human innate immunity. PLoS One 2013;8:e77282.
Yang J, Feng L, Ren J, et al. Correlation between the severity of
periodontitis and coronary artery stenosis in a Chinese pop-
ulation. Aust Dent J 2013;58:333–8.
Yarza P, Spr ¨
oer C, Swiderski J, et al. Sequencing orphan species
initiative (SOS): lling the gaps in the 16S rRNA gene
sequence database for all species with validly published
names. Syst Appl Microbiol 2013;36:69–73.
Yatsunenko T, Rey FE, Manary MJ, et al. Human gut micro-
biome viewed across age and geography. Nature 2012;486:
222–7.
Young D, Stark J, Kirschner D. Systems biology of persistent
infection: tuberculosis as a case study. Nat Rev Microbiol
2008;6:520–8.
Yu RQ, Yuan JL, Ma LY, et al. Probiotics improve obesity-
associated dyslipidemia and insulin resistance in high-fat
diet-fed rats. Zhongguo Dang Dai Er Ke Za Zhi 2013;15:1123–7.
Zengler K, Toledo G, Rappe M, et al. Cultivating the uncultured.
P Natl Acad Sci USA 2002;99:15681–6.
Zhang H, Liao X, Sparks JB, et al. Dynamics of gut microbiota in
autoimmune lupus. Appl Environ Microb 2014a;80:7551–60.
Zhang R, Lahens NF, Ballance HI, et al. A circadian gene ex-
pression atlas in mammals: implications for biology and
medicine. P Natl Acad Sci USA 2014b;111:16219–24.
Zhang Y, Yew WW, Barer MR. Targeting persisters for tuberculo-
sis control. Antimicrob Agents Ch 2012;56:2223–30.
Zhang Y, Zou Y, Ma P, et al. Identication of Mycoplasma suis
MSG1 interaction proteins on porcine erythrocytes. Arch Mi-
crobiol 2014c;80:7551–60.
Zhu Y, Jameson E, Crosatti M, et al. Carnitine metabolism to
trimethylamine by an unusual Rieske-type oxygenase from
human microbiota. P Natl Acad Sci USA 2014;111:4268–73.
Zimmermann MB, Chassard C, Rohner F, et al. The effects of
iron fortication on the gut microbiota in African children:
a randomized controlled trial in Cote d’Ivoire. Am J Clin Nutr
2010;92:1406–15.