Studying bacterial infections through culture-
Geraint B. Rogers,1Mary P. Carroll2and Kenneth D. Bruce1
Geraint B. Rogers
1Molecular Microbiology Research Laboratory, Pharmaceutical Science Division, Franklin-Wilkins
Building, King’s College London, 150 Stamford Street, London SE1 9NH, UK
2Cystic Fibrosis Unit, Southampton University Hospitals NHS Trust, Tremona Road, Southampton
SO16 6YD, UK
The ability to characterize accurately the cause of infection is fundamental to effective treatment.
The impact of any antimicrobial agents used to treat infection will, however, always be constrained
by both the appropriateness of their use and our ability to determine their effectiveness. Traditional
culture-based diagnostic microbiology is, in many cases, unable to provide this information.
Molecular microbiological approaches that assess the content of clinical samples in a culture-
independent manner promise to change dramatically the types of data that are obtained routinely
from clinical samples. We argue that, in addition to the technical advance that these
methodologies offer, a conceptual advance in the way that we reflect on the information generated
is also required. Through the development of both of these advances, our understanding of
infection, as well as the ways in which infections can be treated, may be improved. In the analysis
of the microbiological content of certain clinical samples, such as blood, cerebrospinal fluid, brain
and bone biopsy, culture-independent approaches have been well documented. Herein, we
discuss how extensions to such studies can shape our understanding of infection at the many
sites of the human body where a mixed flora, or in more ecological terms, a community of
microbes, is present. To do this, we consider the underlying principles that underpin diagnostic
systems, describe the ways in which these systems can be applied to community characterization,
and discuss the significance of the data generated. We propose that at all locations within the
human body where infection is routinely initiated within the context of a community of microbes,
the same principles will apply. To consider this further, we take insights from areas such as the
gut, oral cavity and skin. The main focus here is understanding respiratory tract infection, and
specifically the infections of the cystic fibrosis lung. The impact that the use of culture-
independent, molecular analyses will have on the way we approach the treatment of infections is
The need to establish congruence between a pathological
condition and a single causative agent, as required by
Koch’s postulates, has shaped the development of dia-
gnostic microbiology. As such, this has been of great
importance in characterizing many aetiological agents.
Moreover, this approach defined the underlying philo-
sophy of how microbes from many different environments
have been studied. A process that isolates a single organism
in axenic culture can be appropriate as an assay for certain
types of infection. For instance, where a disease state results
from the entry of a single causative agent into the
bloodstream, as is the case with members of the genus
Borrelia and Lyme disease (Coulter et al., 2005). However,
there is a growing need to consider infection within the
context of a complex microbial milieu. This is reflected in
the fact that bacteria infecting human tissues often
comprise mixed communities, particularly when mucosal
barriers have been compromised (Brogden et al., 2005).
Furthermore, the expression of social behaviour by
bacteria, such as the organization of structured bacterial
communities in biofilms (Xavier & Foster, 2007; Nadell
et al., 2009) has significant, and typically adverse,
consequences for therapy (Socransky & Haffajee, 2002;
Costerton, 2005; Chavez de Paz, 2007; Christensen et al.,
2007; Tre-Hardy et al., 2009). These findings alone already
challenge the appropriateness of the study of bacterial
infections based on single strains isolated through in vitro
This may be further emphasized through the physiological
states, such as viable but non-culturable, in which bacterial
cells can exist (Colwell, 2000; Anderson et al., 2004).
Moreover, from many body sites, the number of cells
that grow is typically less than those detected. This
Journal of Medical Microbiology (2009), 58, 1401–1418
013334G2009 SGM Printed in Great Britain1401
phenomenon is illustrated by work carried out by
Socransky et al. (1963), where it was estimated that
approximately 50% of the cells in the debris from the
human gingival crevice remained uncultured despite the
use of chemically diverse growth media, and both aerobic
and anaerobic conditions. The impact of physiological
status, together with the impact of immune response and
antibiotic therapy, on bacterial cells will be considered later
in this work. However, to fully understand the process of
an infection developing in areas that contain bacterial
communities, such as the gut, the respiratory tract and
skin, it must be considered within the complex microbial
An encounter with an organism capable of causing disease
will not necessarily result in frank infection. The expression
of a particular type of activity by the pathogen, the host or
both may be necessary for infection to occur, with a wide
range of factors, not least the composition and behaviour
of the microbial community, potentially important. The
complexities are illustrated by a number of phenomena,
such as the ability of pathogenic bacteria to exist in a
carrier state, where the host does not develop disease, but
harbours an infective organism that may cause disease in
those to whom it is transmitted. For example, Neisseria
meningitidis or Vibrio cholerae, cause disease in some
individuals but not others (Yazdankhah & Caugant, 2004;
Vanden Broeck et al., 2007). A further example is the
ability of pathogenic bacteria to turn on or off disease-
producing genes depending on circumstance, such as the
production of exotoxins by Clostridium difficile (Voth &
Ballard, 2005). Interactions between bacteria and host
surfaces, therefore, represent a continuum, from transient
contact, through colonization to pathogenic activity and
Such considerations of course bring a far greater complex-
ity to the examination of causative agents and their role in
pathogenesis. Despite this, the possibility that a more
extensive range of factors influence the development or the
exacerbation of a disease cannot be ignored. Identifying
these factors and understanding their impact may in itself
provide an opportunity to predict, and ultimately modify,
infection. For these reasons, molecular microbiological
approaches that allow the elucidation of the mechanisms
involved will result in a fundamental shift in the way in
which infection is studied and understood. Before
considering examples of infective processes, it is important
to describe the molecular microbiological approaches that
allow a culture-free means of analysis of clinical samples.
Development of bacterial community profiling
We know that natural environments contain a diverse array
and number of microbes (Whitman et al., 1998).
Speculation over the association of microbes with disease
also goes back thousands of years, with early reference to
this made by Marcus Terentius Varro in 36 BC with a
warning over the danger of locating homesteads near
swamps due to the presence of organisms that could cause
disease, but which were too small to be seen (Cato & Varro,
1935). In 1546, Girolamo Fracastoro speculated about the
existence of contagious particles (Fracastoro, 1930).
However, it was not until the advances made in microscopy
and microbiology by scientists such as Van Leeuwenhoek,
Pasteur and ultimately Koch that identification of specific
micro-organisms could be performed. These advances led
to the idea that particular aetiological agents were
associated with certain diseases. Furthermore, in setting
out his postulates, Koch produced guidelines for establish-
ing this relationship between a micro-organism and the
causation of an infectious disease (Koch, 1884).
The principle that to isolate a microbe it must be separated
from the bacterial milieu by enrichment culture – a process
of selection using conditions favourable to its growth,
whilst excluding other species – was further developed by
Martinus Beijerinck (Brock et al., 1994). This principle
forms the basis of traditional, culture-based diagnostic
microbiology, and persists to the present. Despite the
effectiveness of this, even at the beginning of the 20th
century there was a recognition that certain infections were
in fact polymicrobial in nature (Loux & Coplin, 1902;
However, without a theoretical basis on which to construct
models of polymicrobial activity, the identification of
specific bacterial species in infections is limited in its
predictive ability, and provides microbiologists with few
insights (Prosser et al., 2007). The process of developing
such a basis is further hindered by the absence of the tools
and disciplines of ecological theory from the contemporary
mindset in microbiology (Prosser et al., 2007). However,
the potential importance of the polymicrobial nature of
infections led to an investigation of these interactions, with
parallels drawn from many areas of microbial ecology
(Smith, 1982; Rotstein et al., 1985; Brogden et al., 2005;
Brown & Buckling, 2008).
Culture-based diagnostic microbiology relies on our
detection of aetiological agents through our ability to
provide the conditions they require to grow in vitro. In
many cases, even where the involvement of a specific
pathogen is suspected, it can be extremely difficult to
cultivate it in artificial media. This was illustrated by the
difficulty encountered in identifying a causative agent for
Whipple’s disease. Here, despite Whipple reporting in 1907
the presence of rod-shaped micro-organisms in the vacuole
of macrophages after silver-staining (Whipple, 1907), the
agent remained obscure until 1999. Although Tropheryma
whipplei was propagated in human fibroblast cells in that
year (Raoult et al., 2000), it was only through genomics-
based design of a cell-free culture medium that T. whipplei
was finally grown in axenic culture (Renesto et al., 2003).
Being able to characterize accurately which bacterial species
are involved in an infection becomes more difficult still
when many different species are present. In such
circumstances, the size of the pool of unknown species
G. B. Rogers, M. P. Carroll and K. D. Bruce
1402 Journal of Medical Microbiology 58
cannot easily be determined and the ability to associate
particular species with particular conditions is challenging.
In gut microbial ecosystems, culture-independent surveys
of 16S rRNA gene diversity have indicated that more than
75% of the phylotypes detected in the human large
intestine do not correspond closely to known cultured
species (Suau et al., 1999; Eckburg et al., 2005; Flint et al.,
2007), an even greater proportion than reported by
Socransky et al. (1963) using culture-based approaches.
The question of whether unculturable species exist is the
subject of debate. Gest (2008) has pointed out that with
sufficient effort and at least some cells in the correct
physiological state, there is no fundamental boundary to in
vitro cultivation. Indeed, as our understanding of niches
such as the cystic fibrosis (CF) lung increases, so does our
ability to replicate it as a growth environment in vitro
(Palmer et al., 2007a). However, it must also be recognized
that where the effort required is prohibitive, some species
become effectively unculturable in routine diagnostic
microbiology. More fundamentally, where species cannot
be cultured using standard media, under standard condi-
tions, there may be no indication of their presence in a
sample. In contrast to traditional culture-based diagnostics,
molecular genetic approaches avoid the need for in vitro
cultivation. In particular, the advent of PCR amplification
provided a ready basis for the development of assays to
exploit differences in DNA sequences. The importance of
PCRto clinical microbiology
Searching the PubMed database, the numbers of ‘hits’ for
the annual number of publications linking those terms
more than doubled in the decade since 1998. The range of
applications of PCR (reviewed by Whelen & Persing, 1996)
and real-time PCR (reviewed by Espy et al., 2006) in
clinical microbiology is equally increasing.
One of the greatest advantages conferred by PCR is that it
allows detection of bacterial species to be based on nucleic
acids extracted directly from clinical samples. The process
typically involves the extraction of total nucleic acids from
a clinical sample, followed by the amplification of the
region of interest using specific oligonucleotide primers. In
this way, the need for culture prior to detection is removed.
Species-specific, culture-independent, PCR assays have
been developed for the detection of a wide range of
bacterial pathogens – Clostridium difficile, group B
Streptococcus, Bacillus anthracis (Bergseng et al., 2007;
Kane et al., 2008; Sloan et al., 2008). By basing species
identification on DNA sequence, the accuracy of iden-
tification is increased. Spilker et al. (2008) illustrated this in
the context of CF lung infections by showing the high
frequency of misidentification of Bordetella spp. by
diagnostic microbiology laboratories based on culture
analysis – a situation that has previously disguised the
relatively high prevalence of these species in CF airway
infections. Furthermore, these species-specific PCR assays
confer a greater level of sensitivity of detection compared
to conventional culture-based diagnostics (Chia et al.,
2004; Azzari et al., 2008; Chiba et al., 2009). This increased
sensitivity can be important, for example, when attempting
to detect fastidious bacteria (Fenollar & Raoult, 2004).
Equally, where the available specimen has been collected
after antibiotic therapy, or where transportation conditions
have been poor, this increased sensitivity may be crucial
(Davies et al., 2006; Rosey et al., 2007). The more recent
development of real-time PCR has not only provided a
means to determine the bacterial load in a sample with a
high degree of speed and accuracy, but also further
increased the sensitivity of these assays (Rosey et al., 2007).
Although these culture-independent approaches provide
significant improvements in accuracy, the use of species-
specific PCR techniques is equivalent to the use of selective
media in culture-dependent approaches. They require a
prediction to be made as to which agent is likely to be
associated with a particular sample, and have a practical
limit to the number of species-specific assays that can be
performed. In partial response to this, multiplex PCR
systems that can detect multiple species of interest have
been developed for certain contexts. For example, Benson
et al. (2008) developed an assay that detects gene-specific
DNA sequences of six respiratory bacterial species
(Streptococcus pneumoniae, N. meningitidis, Haemophilus
influenzae, Legionella pneumophila, Mycoplasma pneumo-
niae and Chlamydophila pneumoniae). Systems such as this
can be useful in rapidly detecting the presence of a number
of known pathogens using a single assay. However, as with
single-target PCR assays, multiplex PCR systems require
predetermination of the bacterial species likely to be
present in a given infection. Furthermore, these tests are
used often with the assumption that detecting the presence
of an agent is sufficient for pathology to be inferred. This
theme will be reconsidered later; however, it is worth
stressing again here that bacteria rarely exist in mono-
cultures and that species–species interactions can pro-
foundly affect the behaviour of individual species (Wuertz
et al., 2004; Dubey et al., 2006; Andersson et al., 2008; Van
der Heijden et al., 2008).
The identification of regions of particular phylogenetically
informative genes that are conserved across large sections
of the domain Bacteria offers an alternative approach.
These conserved regions can be used to amplify sequences
from any bacterial content of a sample, rather than just a
single species. This represents a fundamentally different
process to either culture-based assays, species-specific PCR
or multiplex PCR. Also key to this process is that between
the conserved regions there is sufficient sequence variation
to allow species discrimination, allowing detection of
bacterial species without the need to predict which
pathogen(s) may be present. Whilst a number of genes
have been used for such ‘broad-range PCR’, the most
important of these phylogenetically informative regions is
the 16S rRNA gene, which contains both the highly
conserved and highly variable regions required (Clarridge,
2004). This can be be performed either on strains of
bacteria already isolated, or as will form the focus later,
directly on clinical samples. This process has become
Molecular approaches to the study of infections
increasingly important to clinical microbiology – since
their introduction, ribosomal gene sequencing studies have
allowed the identification of novel bacterial species from
human samples (Woo et al., 2008). Arguably, one of the
main reasons to use PCR though has been in relation to the
analysis of samples regarded as culture negative. Here,
studies have shown the importance of broad-range PCR in
securing a diagnosis where traditional culture has not been
successful (Harris & Hartley, 2003).
Working with samples containing many different species of
course means that a mix of PCR products will be generated.
To resolve information from these products, one or more
strategies can be applied. Of these, the most commonly
used include single strand conformation polymorphism,
denaturing gradient gel electrophoresis, temperature gra-
dient gel electrophoresis, 16S rRNA gene sequencing and
terminal RFLP (T-RFLP) profiling (Nocker et al., 2007;
Juste et al., 2008; Malik et al., 2008). Each of these
techniques exploit the variable internal regions directly or
indirectly to resolve the different PCR products into
separate species or groups of species. The selection of the
technique to be used and the sequence to be targeted
depend on both the characteristics of the community to be
analysed and the type of data that is required (Nocker et al.,
2007; Juste et al., 2008). Such broad-range PCR-based
assays have been widely used to detect bacteria in samples
as clinically diverse as heart-valve material, cerebrospinal
fluid and synovial fluid (Jalava et al., 2001; Rothman et al.,
2002; Gauduchon et al., 2003; Podglajen et al., 2003;
Saravolatz et al., 2003; Schuurman et al., 2004), and have
been shown to provide an increased level of sensitivity
(Rantakokko-Jalava et al., 2000). The coupling of universal
PCR with community profiling techniques therefore allows
the direct characterization of the bacterial community in a
clinical sample, and provides the theoretical ability to
identify all bacterial species present, including those
refractory to cultivation.
The ability to characterize accurately bacterial communities
may be crucial if pathogenesis is related to changes in
community composition. Such scenarios may arise in a
number of contexts. For example, the development of
vaginosis is believed to involve the modification of a
normal bacterial flora to one that is associated with
pathogenesis. This process involves the depletion of
Lactobacillus populations, which are usually dominant in
the vagina of healthy women, and an increase in a mixture
of other bacterial species, often including Gardnerella
Peptostreptococcus species, Gram-negative anaerobic rods
such as Prevotella species, Mycoplasma hominis and
Ureaplasma urealyticum, and sometimes Mobiluncus spe-
cies (Hill, 1993; Pybus & Onderdonk, 1999; Persson et al.,
2009). Another example is in the development of human
dental caries. Here, the expression of certain virulence
factors by Streptococcus mutans, an important aetiological
agent, has been shown to be inhibited by other species of
oral bacteria through interference with their cell–cell
Furthermore, reports of the role of interactions associated
with the development of disease in the oral cavity are
numerous (Grenier & Mayrand, 1986; Liljemark &
Bloomquist, 1996). Moreover, multispecies bacterial infec-
tions can occur in regions normally free from bacteria,
such as in the development of chronic respiratory
infections that occur in CF or chronic obstructive
pulmonary disease (COPD) airways (Rogers et al., 2003,
2004; Katznelson, 2006, Sibley et al., 2006; Veeramachaneni
& Sethi, 2006; Harris et al., 2007). Not all ‘invasions’ are
detrimental to the host though, as, for example, probiotic
bacteria can be exploited to prevent the pathogenic effects
of bacteria (Cremonini et al., 2001; Hamilton-Miller, 2003;
Lutgendorff et al., 2008).
& Kuramitsu, 2005).
Combined, this means that an incomplete, and possibly
distorted, picture of infection is obtained when only
selective assays are employed. Knowing that alone,
however, raises the question of what types of data are
most clinically informative. To be able to understand this,
we need first to understand more about the bacterial
community through intensive study of a number of model
systems such as the CF lung.
Why study the bacterial communities associated
with the CF lung?
The lower respiratory tract of CF patients represents an
ideal habitat for investigating the processes that are
involved in the development and dynamics of polymicro-
bial infections (Table 1). In CF, the basic defect that results
in abnormal functioning of the CF transmembrane
conductance regulator protein principally relates to
abnormal ion transfer across epithelial cell surfaces,
resulting in impaired mucociliary clearance (Clunes &
Boucher, 2007; Coakley & Boucher, 2007). The lungs of
patients with CF are normal in utero and, before the onset
of infection and inflammation, represent as favourable an
anatomical niche for bacterial colonization as those
without CF. After birth the lower airways are exposed to
a diverse range of bacteria, amongst other material,
through their constant ventilation and close proximity to
the communities of the upper respiratory tract and oral
cavity. In healthy individuals the mucociliary escalator
helps to remove bacteria from the lower airways. However,
when these systems fail to function efficiently, as is the case
in CF (Matsui et al., 1998; Boucher, 2004), bacteria that
gain access to these regions may begin the process of
colonization (Accurso, 1997). Although poorly understood
at present, this process may begin shortly after birth, with a
typical progression towards chronic infection and inflam-
Once colonization has occurred, lung infections in CF
patients are characterized by periods of relative stability,
punctuated by infective exacerbations (Goss & Burns,
2007). These exacerbations have a significantly negative
G. B. Rogers, M. P. Carroll and K. D. Bruce
1404Journal of Medical Microbiology 58
effect on both quality of life (Britto et al., 2002) and
survival (Liou et al., 2001), with a median predicted
survival in the UK of 35.2 years (CFT, 2007). During
exacerbations, antibiotics are administered to reduce
sputum bacterial load (Ramsey, 1996) with an expectation
of an improvement of pulmonary symptoms (Regelmann
et al., 1990).
For many years, culture-based diagnostic microbiology has
been employed to characterize the microbial content of CF
sputum in an attempt to gain a better understanding of the
relationship between infection and disease. Based on this
process, a relatively small number of bacterial species have
been considered as having an important role in CF lung
disease (Razvi & Saiman, 2007), most notably Pseudomonas
aeruginosa, Burkholderia cepacia complex, Staphylococcus
aureus, H. influenzae and Stenotrophomonas maltophilia
(Gilligan, 1991; Heijerman, 2005). Several studies have
documented additional species in the CF lung, albeit less
frequently, and include among others Achromobacter spp.,
Pandoraea spp., Ralstonia and non-tuberculous mycobac-
teria (Gilligan, 1991; Coenye et al., 2002; Lambiase et al.,
Our original working hypothesis was that, in common with
the findings of these culture-based studies, a range of
species wider than those traditionally associated with the
CF lung would be detected using culture-independent
approaches. Given the importance of maintaining lung
function in these patients, we also hypothesized that any
bacterial species might be significant in the progression of
lung disease. An ability to manage more effectively chronic
bacterial infections in the CF lung could dramatically
improve both the longevity of CF patients and their quality
of life. Some of the first information on this came in 2003
through the culture-independent approach of T-RFLP
profiling, which involves electrophoretic resolution of PCR
products based on the relative position of restriction sites.
This technique was used to analyse the bacterial content of
92 sputum samples from adult CF patients attending a
single CF clinic in the UK. On average, more than 14
separate terminal restriction fragment (T-RF) bands,
representing one or more bacterial species, were detected
in each of the patients. Nearly 80% of the five most
common species detected were not any of the five key CF
species (P. aeruginosa, Burkholderia cepacia complex,
Staphylococcus aureus, H. influenzae and Stenotropho-
monas maltophilia). Furthermore, extensive 16S rRNA
gene clone sequence analysis showed that these additional
species came from a wide range of different phylogenetic
branches of the domain Bacteria (Heijerman, 2005).
It is difficult to deal succinctly with such a wide diversity
of species. To give some insight though, the ‘key’
CF pathogens fall into two divisions of bacteria –
Proteobacteria (H. influenzae, P. aeruginosa, Stenotropho-
monas maltophilia, Burkholderia cepacia, Alcaligenes xylo-
soxidans) and Firmicutes (Staphylococcus aureus). Culture-
independent studies have shown that these bacterial
divisions are also represented by species of the genera
(Proteobacteria). However, culture-independent studies
have further shown that species belonging to three other
bacterial divisionsare also
Actinobacteria (including Actinomyces spp., Rothia spp.),
Bacteroides/Chlorobi (including Prevotella spp., Porphyro-
monas spp., Capnocytophaga spp., Treponema spp.) and
Fusobacteria (Fusobacterium spp.). With the ever increas-
ing capacity to sequence in depth the species in any clinical
sample, the diversity observed will only rise further.
An unexpected finding of this first study was that genera such
as the anaerobes Veillonella and Prevotella formed a
Table 1. Rationale for studying chronic lower respiratory infections (LRIs)
LRIs are thought to be the third most important cause of mortality globally accounting for more than 4
million deaths annually (Murray & Lopez, 1997). In 2004, the World Health Organization estimated
that LRIs were responsible for 6.8% of deaths worldwide (WHO, 2004).
The treatment of LRIs represents a significant proportion of total healthcare expenditure (Monte et al.,
Chronic LRIs are polymicrobial (Sibley et al., 2006; Sethi & Murphy, 2008), and can involve higher
bacterial interactions both within species, for instance, in the formation of biofilms (Kobayashi, 2005),
and between species, resulting in changes to virulence levels (Sibley et al., 2008a).
The lower respiratory tract provides a range of environmental niches that can be colonized by bacteria,
including, in some disease states, anaerobic environments (Worlitzsch et al., 2002; Yoon et al., 2002).
The number of bacterial species associated with chronic respiratory infections is increasing, with the
role of the majority as yet unknown. However, it must be assumed that each has the potential to
adversely affect patient health.
Despite repeated high dose courses of intravenous antibiotics, the impact on colonizing flora is modest,
and, at best, results in a management of infective exacerbations.
LRIs, such as CF, can be used as models to investigate bacterial behaviour in infection, generating
knowledge that can be transferred to other polymicrobial infections.
Complex bacterial communities
Diverse array of niches
Lack of understanding of the role
of infective bacteria
Lack of effective treatment
Transfer of knowledge
Molecular approaches to the study of infections
surprising once the nature of the CF respiratory tract is
considered in more detail.Once colonized, the CF airways are
both chemically and physically diverse, containing complex
nutrients and carbon sources. Importantly, they also contain
regions with a range of oxygen potentials allowing both
aerobic and anaerobic growth (Yoon et al., 2002).
The process of determining the identities of all the species
detected in studies such as these is ongoing, with the degree
to which the bacteria in different individuals are similar yet
to emerge. The need to determine the significance to CF
lung disease of individual species is, however, clear. Initial
studies on a limited number of adult CF patients indicated
that the same set of species could be detected over the
course of a year in each patient, despite the occurrence of
periods of infective exacerbation and the extensive use of
intravenous antibiotic treatment (F. A. Stressmann, A.
Walker, G. B. Rogers, T. V. W. Daniels, A. Lilley, M. P.
Carroll, C. J. van der Gast & K. D. Bruce, unpublished
results). As such, this emphasizes the importance of
determining the individual roles that these species play.
We also asked more directly how culture-based microbio-
logy would compare to culture independent T-RFLP
profiling of the bacterial communities present in the CF
lung. For the purposes of this experiment, each band
visualized represents a species. Examples of two such
comparative analyses are shown in the profiles in Fig. 1.
Here, two CF sputum samples have been divided, with one
aliquot being used to inoculate the range of media
routinely used for CF diagnostic microbiology, the other
being subjected to culture-independent analysis. All the
material cultured after 40 h was subjected to nucleic acid
extraction. DNA was also extracted, without cultivation,
from the second aliquot. Once the ribosomal genes were
amplified and analysed by T-RFLP profiling, some marked
and fundamental differences were seen. Discrepancies in
the ability to detect bands derived from different species
using the two approaches, or differences in the band
intensities obtained, could be due to a number of factors.
Firstly, it must be recognized that like all techniques,
culture-independent approaches themselves can introduce
bias, e.g. in terms of primer selectivity, threshold over
background and differential cell lysis (Schutte et al., 2008).
This topic has beenreviewed
Wintzingerode et al., 1997). Whilst this issue needs to be
further addressed in relation to clinical microbiology, other
factors may in fact be more significant. Clearly, certain
bacterial species grow particularly well in the culture
conditions used, for example Staphylococcus aureus.
Moreover, species that require particular growth condi-
tions that are not part of routine CF sputa diagnostic
microbiology were detected in the culture-independent
approach, for example those requiring anaerobic condi-
tions most notably here again within the genus Prevotella.
Many species that fall into this second group are present at
levels comparable to, or often greater than, those of species
considered previously to be the key CF pathogens.
Currently, however, the presence of these species is not
taken into account, with treatment strategies designed
around the small number believed to be of clinical
significance. Considering this further, conventional cul-
ture-based diagnostics has in certain studies shown little
correlation with clinical parameters (Gibson et al., 2003) or
with culture-independent studies (Rogers et al., 2009). For
this reason, many of the current microbiological data
generated are often viewed as being of little value by
Furthermore, diagnostic analysis, as currently employed,
is unlikely to identify pathogens that are emerging, such as
Burkholderia pseudomallei (Holland et al., 2002; O’Carroll
et al., 2003) and the ‘Streptococcus milleri’ group (Parkins
et al., 2008), let alone those not previously described. This
last point has been highlighted through the wide range of
through the use of broad-range molecular analyses
(Rogers et al., 2004; Bittar et al., 2008; Sibley et al., 2008b).
So far, this review has discussed some of the disadvantages
of the use of conventional culture-based approaches in the
characterization of polymicrobial infections. Furthermore,
the development of the culture-independent techniques,
which have the potential to circumvent many of those
flaws, has been described. However, it is now important to
consider how the application of these approaches can best
be used to develop our understanding of the processes
Fig. 1. Two sets of T-RFLP profiles that have each been
generated from both nucleic acids extracted directly from sputum
samples and from cultures derived from those samples. Culture-
based profiles involved DNA extraction from pooled cultures
generated on blood agar, cystine-lactose-electrolyte-deficient
agar, Columbia CNA agar, chocolate agar, Pseudomonas-
selective agar and Burkholderia cepacia complex-selective media
in aerobic culture at 37 6C. Arrows indicate notable differences in
the T-RF banding patterns.
G. B. Rogers, M. P. Carroll and K. D. Bruce
1406Journal of Medical Microbiology 58
involved in infection, again, using CF lung infections as a
Healthy and diseased airway flora – sampling and
In healthy, non-CF individuals, bacteria are cleared from
the airways, and are, therefore, denied the opportunity to
colonize them. Despite this, induced sputum samples taken
from healthy individuals were found to contain far higher
numbers of bacterial species, albeit at much lower loads,
than had been obtained for expectorated CF sputa (Rogers
et al., 2005b). Setting aside the difference in the sample
collection method, the apparent difference in diversity
between the two sample sets is likely to be due to the
presence in the CF airways of some species in very high
numbers. This will have the effect of masking the presence
of other less prevalent, non-colonizing species by pushing
them effectively below a threshold of detection.
Furthermore, the exposure of the airways to inhaled
bacteria, as well as the existence of bacterial flora in the
upper airways and oral cavity, may affect the composition
of CF clinical samples. As described above, this seems to be
the case with certain oral anaerobes being detected in the
CF lung. Whilst, in comparison to established populations
in the lower airways, inhaled bacteria are likely to be
present in very low numbers, the potential for contamina-
tion of CF respiratory samples with upper airway flora was
a concern. Sputum passes through the upper airways and
oral cavity during expectoration, and a significant propor-
tion of the bacteria isolated by conventional microbiology
are classified as ‘oral flora’ in diagnostic reports. The
presence of such species as a clinically significant factor
would require a fundamental change in the way routine
microbiological data are interpreted. For this reason, early
reports of anaerobic species, known to colonize the oral
cavity, in CF sputum were somewhat controversial.
However, as more evidence of a substantive role of
anaerobic species in CF lung infections is generated
(Sibley et al., 2008b; Tunney et al., 2008) the need to
consider the implications of their presence in CF sputa
increases. For these reasons it is essential to establish
whether anaerobic species are genuinely colonizing the
lower airways. There are a number of strategies by which
this can be achieved.
One way to address the problem of contamination is to
analyse samples with minimized exposure to upper airway
bacteria. These include bronchoalveolar lavage (BAL)
samples, where material can be collected from the lower
airways using a protective brush to prevent contamination
during the introduction and removal of the bronchoscope.
A limited study of BAL samples has indicated the presence
of ‘oral flora’, including Prevotella spp. and Veillonella spp.,
in the lower airways of CF infants (G. B. Rogers, M. S.
Payne, J. P. Legg, G. J. Connett, F. A. Stressmann, A.
Walker, T. V. W. Daniels, M. P. Carroll & K. D. Bruce,
unpublished results). Another approach is by the analysis
of samples that have not passed though the upper airways
and oral cavity, and are, therefore, less exposed to
contamination, such as samples obtained as trans-tracheal
aspirates (TTA). TTA analysis is, however, rarely per-
formed due to the highly invasive nature of the sample
collection procedure. Despite this, aerobic and anaerobic
culture of TTA samples from paediatric patients has
revealed the presence of anaerobes, including Veillonella
species, in the majority of cases (Brook & Fink, 1983).
Furthermore, comparison of sputum and mouthwash
samples from CF patients using molecular profiling
techniques showed sputum samples not to be subject to
profound contamination by oral cavity bacteria, and
provided further evidence of colonization of the CF lung
by oral bacterial species (Rogers et al., 2006). Despite
studies of this type, further work is required to establish the
size and nature of these populations of anaerobic bacteria
in the lower airways. More generally, however, we are still
left with communities of CF lung bacteria that are present
but not as mere contaminants of sampling methodology.
How are the bacterial communities in the CF lung
The difficulty of treating established bacterial communities
in CF patients has led to great importance being attached
to determining the mechanisms that drive community
development in paediatric patients. A better understanding
of the mechanisms key to this process would offer an
opportunity to disrupt it. Attempts to elucidate these
mechanisms are, however, hampered by the fact that
infants rarely produce sputum, and material collected from
higher up the respiratory tract may not predict lower
respiratory pathogens (Ramsey et al., 1991; Armstrong
et al., 1995).
Whilst limited in the depth of data that they can provide,
and remaining focused on species of known clinical
significance, culture-dependent analyses have given some
insights into the mechanisms that may be involved in
airway colonization. For example, data derived using this
approach indicate that Staphylococcus aureus and non-
encapsulated H. influenzae are isolated by culture-based
microbiology early in life, whereas nearly all CF patients
become infected with P. aeruginosa over time (Renders
et al., 2001). Furthermore, as patients acquire P. aeruginosa,
Staphylococcus aureus tends to be detected less frequently
(Machan et al., 1992; CFF, 2007), although both species are
commonly co-isolated (Hoffman et al., 2006). A mech-
anism for this relationship has been suggested involving the
production by P. aeruginosa of an anti-staphylococcal 4-
hydroxy-2-heptylquinoline-N-oxide (HQNO) when co-
infecting CF airways with Staphylococcus aureus (Machan
et al., 1992). However, this compound also protects
Staphylococcus aureus during co-culture from commonly
used aminoglycoside antibiotics such as tobramycin
(Hoffman et al., 2006). Furthermore, it has been shown
that prolonged growth of Staphylococcus aureus with P.
Molecular approaches to the study of infections
aeruginosa selects for typical Staphylococcus aureus small-
colony variants, which have stable aminoglycoside resist-
ance (Miller et al., 1980) and are persistent in chronic
infections, including those found in CF (Proctor et al.,
2006). It has, therefore, been suggested that Staphylococcus
aureus density within CF airways reflects a balance between
the suppressive effects of antibiotics and HQNO, and
(Hoffman et al., 2006).
Many of the processes likely to be important in the bacterial
colonization of CF airways, either within or between species,
are mediated by both concentration-dependent auto-
inducer-mediated signalling systems (quorum sensing)
(Waters & Bassler, 2005) and concentration-independent
signalling systems (Lee et al., 2007). Quorum-sensing
systems, in particular, have been implicated in a number
of activities that have a significant clinical impact in chronic
respiratory infections, for example, the formation of
biofilms (Parsek & Singh, 2003; Kobayashi, 2005), the
production of virulence factors such as pyocyanin and
elastase (Winstanley & Fothergill, 2009), and the production
of bacteriocins (Fontaine et al., 2007).
In addition to determining the behaviour of particular
species in CF (Molina et al., 2008; Moreau-Marquis et al.,
2008), there is also evidence from some culture-based
studies that quorum sensing is implicated in the poly-
microbial nature of these infections. Cross-talk between the
quorum-sensing systems, involving the recognition of the
signal produced by a different species, has been shown to
be capable of affecting the behaviour of CF pathogens.
Unidirectional cross-talk occurs between P. aeruginosa and
Burkholderia cepacia complex, involving the recognition by
Burkholderia cepacia of the C4 3-oxo-C12 homoserine
lactones produced by P. aeruginosa at low concentrations
to activate its cep quorum-sensing system (Eberl &
Tu ¨mmler, 2004). Since P. aeruginosa colonization often
precedes Burkholderia cepacia in CF infections, a non-
specific quorum sensing could be one mechanism by which
the latter is able to develop as a multispecies community
with P. aeruginosa, and thereby colonize the lower airways
(Jayaraman & Wood, 2008). Furthermore, quorum-sensing
systems are known to be employed by a wide range of
bacterial species (Jayaraman & Wood, 2008). This is
suggestive not only of a potential mechanism by which
CF bacterial communities might be structured, but also of
a possible means by which the development of infections
could be disrupted (Janssens et al., 2008; Kiran et al., 2008).
In addition to direct interaction through quorum-sensing
systems, diverse bacterial communities also provide an
opportunity for horizontal gene transfer. This is a process
that allows for rapid transfer of genes under strong
selection, such as genes that encode antibiotic resistance
(Salyers et al., 2004). The facilitation of horizontal gene
transfer has clear implications for bacterial communities in
infections, particularly those that have high exposure to
antibiotics, as in the CF lung.
Succession, the orderly process of community development
moving towards a state of equilibrium (Odum, 1969,
Cotgreave & Forseth, 2002) may also play a role. There is
evidence for the involvement of succession in the
establishment of bacterial communities in non-CF clinical
contexts. For example, the colonization of the neonatal gut
(Favier et al., 2002; Palmer et al., 2007b), the establishment
of bacterial flora in the oral cavity (Nyvad & Kilian, 1990;
Li et al., 2004; Jenkinson & Lamont, 2005; Kolenbrander
et al., 2006) and the development of infections in the root
canal system (Fabricius et al., 1982; Tani-Ishii et al., 1994;
It is clear is that these are dynamic processes. The presence
of non-invasive micro-organisms in the airways can lead to
damage to the respiratory mucosa due to the production of
exotoxins, leading to inhibition of ciliary function and
damage to the bronchial epithelium (Denny, 1974; Wilson
& Cole, 1988; Kanthakumar et al., 1993), inhibition of
mucociliary transport (Munro et al., 1989; Read et al.,
1992), alteration of respiratory epithelial ion transport
(Stutts et al., 1986; Graham et al., 1993), and stimulation of
mucus secretion (Somerville et al., 1992). These processes
have been extensively reviewed by Cole (1997).
A model of the process by which infection is established in
skin wounds has been developed (Edwards & Harding,
2004) and parallels can be drawn between skin wounds and
the CF airways. Both of these contexts represent a hitherto
sterile niche, but one that is proximal to an existing
bacterial flora present on the surface of the skin, or the
upper airways in the case of CF. As such, many of the key
components of the model developed for colonization of
skin wounds may be reflected in chronic lower airway
In this model, the path to the establishment of infections
involves a number of separate stages: contamination – the
presence of non-replicating organisms; colonization – the
presence of replicating micro-organisms in the absence of
tissue damage; and finally infection – the presence of
replicating organisms with subsequent host injury (Dow
et al., 1999). The transition to infection is influenced by a
number of factors, particularly the number of bacteria
(g tissue)21, the virulence and pathogenicity of the
organism, and the ability of the host to mount an effective
immune response (Wysocki, 2002). The infective dose
required for infection varies from species to species, and is
influenced by the organism’s interactions with surrounding
microflora (Bowler, 2003). In an analogous CF model,
factors such as the ability to clear bacteria from the airways,
and the impairment of the immune response, may
influence the point at which a threshold bacterial load is
reached, thus, making colonization possible.
The timescale over which a bacterial community develops
in the CF airways appears to be short. Studies of the
microbial content of BAL samples have indicated that, by 3
months of age, nearly 40% of infants identified by a
neonatal CF screening program had a lower respiratory
G. B. Rogers, M. P. Carroll and K. D. Bruce
1408Journal of Medical Microbiology 58
tract infection (Davis, 1999). Furthermore, culture-inde-
pendent T-RFLP analysis of BAL samples from sputum-
producing paediatric patients showed that, even at an early
age, there were no significant differences between the
bacterial communities detected and those found in samples
from CF adults in terms of diversity (Rogers et al., 2005a).
These findings are of critical importance given the fact that
current treatment practice for these patients is aimed at
preventing initial colonization and eradication of popula-
tions of particular species believed to be of key clinical
significance (Balfour-Lynn & Elborn, 2007).
Whilst there is evidence that the diversity of bacterial
communities in the airways of CF patients increases during
early life, the division of patients into ‘paediatric’ and
‘adult’ is not necessarily relevant when considering airway
infection. The age at which the bacterial communities in
these infections reach a given level of complexity could be
influenced by a range of factors and may vary considerably
between individuals. It is not yet clear whether a ‘climax
community’ exists, or whether the community is in a
continued state of flux coupled with a decline in
respiratory function. However, it is possible that particular
species might be representative of particular stages in the
process of development, and as such, may be clinically
Can a community be regarded as pathogenic?
In a thoughtful paper, Jenkinson and Lamont raised the
possibility of a microbial community being pathogenic
(Jenkinson & Lamont, 2005). This would be the most
potentially alarming consequence for the human host –
that the community together is more damaging than any
individual component species alone. The evidence is
increasing that this may be more than just a hypothetical
The ability of members of the bacterial flora to influence
the virulence of P. aeruginosa in CF respiratory infections
has been investigated using a Drosophila melanogaster
infection model. Sibley et al. (2008a) demonstrated that in
this model environment oropharyngeal species could be
classed into three distinct groups – in addition to species
that were either virulent, or avirulent regardless of the
presence or absence of P. aeruginosa, there was a further
group that were not pathogenic alone, but in combination
with P. aeruginosa dramatically reduced the survival rates
of the host organism. These data clearly suggest that the
dismissal of oropharyngeal flora as clinically insignificant
when identified in sputum samples could be short sighted.
Therefore, the scope for such interactions to influence the
virulence of key pathogens, such as P. aeruginosa, is greatly
increased in a bacterial community containing many
different species. This ability of ‘non-pathogens’ to
influence the behaviour of pathogens again underlines
the inappropriateness of considering, in isolation, indi-
vidual members of polymicrobial infections.
A further level of complexity is provided by the fact that,
with time, the process of interaction with the host may lead
to changes in the genomes of infecting organisms. For
example, it has been shown that P. aeruginosa strains
present in advanced CF infections differ systematically
from those of ‘wild-type’ P. aeruginosa (Smith et al., 2006;
Yang et al., 2008). This process involved the mutation of
many genes that code for virulence factors important in the
process of establishment of infection, including genes
functioning in O-antigen biosynthesis, type III secretion,
twitching motility, exotoxin A regulation, multidrug efflux,
osmotic balance, phenazine biosynthesis, quorum sensing
and iron acquisition (Smith et al., 2006). The reasons for
this are not fully clear. However, strong conflict and
selection pressures can arise among multiple species and
strains in biofilms, and spontaneous mutation can generate
conflict even within biofilms initiated by genetically
identical cells (Diggle et al., 2007; Nadell et al., 2009). In
this way, the same bacterial species may result in very
different clinical outcomes at different stages of infection.
Again, further insights into the processes involved in CF
infections can be gained from examination of polymicro-
bial infections in other contexts. For example, important
parallels can be drawn for the study of chronic wounds.
Wounds, such as those associated with venous leg
ulceration, are often colonized by bacterial communities
comprising more than 20 separate species (Bowler &
Davies, 1999; Davies et al., 2004; Gjodsbol et al., 2006;
Dowd et al., 2008). Significantly, the diversity of the
bacterial communities in these wounds influences their size
and healing time (Robson et al., 1999; Bowler et al., 2001;
Ryan, 2007). Crucially, it is the number of different
bacterial species present that relates to wound healing,
rather than certain individual bacterial species. Specifically,
it has been shown that the presence of four or more
bacterial species in such wounds correlates positively with
non-healing (Hill et al., 2003). This significance of
polymicrobial nature of the communities suggests key
roles for synergistic processes (Bowler, 2003).
Therefore, through greater characterization of the bacterial
communities present in CF airway infections, it may be
possible to establish links between particular community
traits, relating to either their composition or their activity,
and poor clinical prognosis. Furthermore, these traits
may originate not from the presence of particular species in
the bacterial community, but from the interactions
between different community members, and between the
community and the host. As such, it may be helpful to
consider such communities as being, in themselves,
pathogenic. Extending this, of course, is the realization
that community pathogenicity is not ‘fixed’, and may be
differentially realized at different times due to host,
microbial, or other, factors. This suggests, therefore, that
greater efforts will be required to understand pathogenic
community dynamics. We consider this to be a particularly
pressing issue given its potential clinical significance.
Molecular approaches to the study of infections
Tailoring molecular profiling to the study of
dynamic systems – a second generation of
Generating snapshots of bacterial communities may help to
develop a clearer picture of what constitutes healthy or
disease community characteristics, or to identify species
whose presence may be clinically significant. However,
such information is of relatively little interest if our aim is
to be able to predict and prevent infections, or ameliorate
exacerbations. For this, we need to apply culture-
independent profiling longitudinally.
When studying dynamic systems, it is essential that what is
measured changes as rapidly as the community that it
represents. Bacterial community profiling has typically
involved using total DNA extracted directly from a sample
as a template for PCR amplification. However, this has
disadvantages when used to characterize short-term
changes in community structure. Firstly, measurements
based on DNA respond only slowly to changes in bacterial
populations. DNA-based signals generated from bacteria
that are highly metabolically active, senescent or dead can
be of equal intensity. This is a serious concern. For
example, following antibiotic treatment for patients with
infective endocarditis, the process of clearing bacterial
DNA can be slow (Rovery et al., 2005). Rovery et al. (2005)
also showed DNA from different species differing in the
length of persistence, with for example, streptococcal DNA
Furthermore, due to the stability of extracellular DNA in
the CF airways, there is potential for signals to be generated
from DNA released from lysed bacterial cells long after they
have ceased to be viable. In the same way, the transition
between a senescent population and a rapidly growing one
can only be detected once the population has expanded.
For these reasons, attempts to detect the impact of
therapies using DNA-based systems are severely hampered,
and the diagnostic value of techniques of this kind are
However, the contribution of both extracellular DNA and
DNA that is derived from bacterial cells that are no longer
viable can be minimized. Propidium monoazide (PMA)
added to the clinical sample prior to nucleic extraction, is
able to enter cells whose structural integrity has been lost
and intercalate into their DNA. Exposure to a bright light
source causes covalent cross-links to form, rendering the
DNA unable to act as a PCR template. In contrast, PMA is
unable to enter cells whose cell wall is intact. Treatment of
samples in this way results in bacterial community profiles
being generated solely from DNA contained within viable
bacteria (Fig. 2).
Application of this approach to CF sputum samples
suggests that that the presence of dead bacterial cells can
significantly bias profiles from samples that are untreated
with PMA (Rogers et al., 2008). This is of clear importance
in scenarios such as the CF lung where the impact on
bacteria of both the host immune response and antibiotics
The use of procedures such as these to base community
profiling on only those bacteria that have the potential to
have a clinical impact offers many new opportunities. For
example, they could reveal the degree to which antibiotic
therapies are bactericidal towards different populations
within the overall community. However, what they cannot
do is indicate the degree to which therapies have a
bacteriostatic impact. Furthermore, they will not differ-
entiate between a population that is limited in some way,
and therefore has a low metabolic rate, and one that is
highly metabolically active. This is an issue that can be
addressed, however, by switching of the basis of the
bacterial community profiling from rRNA genes to rRNA.
In this way, it is possible to limit bacterial community
profiles to only those cells that are metabolically active.
By reverse transcribing 16S rRNA extracted directly from
samples, a pool of complementary DNA (cDNA) is
generated that can then be used as template for PCR.
Whilst the number of rRNA gene copies per cell is fixed for
a given species, the number of rRNA copies varies with
metabolic activity – the higher the metabolic rate the more
ribosomes required for transcription. Therefore, by per-
forming DNA- and RNA-based profiling on a sample in
parallel, it is possible to determine both the relative
Fig. 2. Two sets of T-RFLP profiles that have each been
generated from untreated and PMA-treated CF sputum sample
aliquots. Arrows indicate notable differences in the T-RF band
G. B. Rogers, M. P. Carroll and K. D. Bruce
1410Journal of Medical Microbiology 58
abundance of a bacterial species, and its relative metabolic
activity. This strategy has been successfully applied to CF
sputum samples and has revealed that, within individual
samples, different populations of bacteria range from
senescent to highly active (Fig. 3) (Rogers et al., 2005b).
Such an approach could be particularly valuable in
identifying early positive or negative responses to anti-
By providing an indication of the relative metabolic rates of
different community members, comparative profiling of
ribosomal genes and their RNA transcripts offers the
potential to be informative regarding a number of key
processes. Firstly, this approach can indicate where
populations are senescent, and as such represent organisms
that, whilst possibly being of reduced clinical significance at
the time of profiling, have the potential to rapidly become
highly significant over a short period of time were
circumstances to change. Secondly, where highly metabo-
lically active populations are identified, the likely expan-
sion of a population can be predicted. In this way, it may
be possible to intervene early and thereby reduce the
clinical impact of an exacerbation. Finally, it is known that
metabolic activity levels can influence the impact of
antimicrobial therapies (Pamp et al., 2008); therefore,
determining the levels of metabolic activity of populations
of bacteria within the community could inform of the
likely degree of success that different therapeutic interven-
tions would have. Furthermore, such data concerning the
dynamics of polymicrobial infections would
advantage attempts at their treatment in a wide range of
clinical contexts, and the need to include such approaches
in the more general study of infection is clear.
Comparative profiling approaches are not without their
problems. The most significant of these is ensuring that
comparisons are made like with like. Whilst rRNA levels
are determined by metabolic activity levels, rRNA gene
levels are typically constant for a given species; however,
they are not constant between species. The number of
ribosomal gene copies can range from 1 to 15 (Bercovier et
al., 1986; Andersson et al., 1995; Rainey et al., 1996);
therefore, having a significant effect on the size of the
template available for amplification. Furthermore, whilst
rRNA is relatively unstable and is broken down rapidly,
DNA is able to persist in a sample long after the cell that
produced it has died. This is likely to result in some
bacterial populations appearing to be present but inactive,
when in fact no viable population is present.
However, by combining some of the advances that have
been made recently in community profiling, such as basing
analysis on cDNA, and the use of PMA treatment to
prevent the contribution of DNA from non-viable cells, it
may be possible to overcome these issues and generate a
bacterial community profile that accurately characterizes
species prevalence, viability and metabolic activity. The
development of such a strategy would greatly improve our
ability to understand the underlying processes that govern
the dynamics of polymicrobial infections. The issue of
variation in DNA signals due to variation in ribosomal
operon number is significant. However, it can be argued
that it would be sufficient to base profiling on rRNA alone.
Since levels of rRNA are proportional to activity of a given
population, they can be equated with the clinical impact of
Characterization of CF airway infections
For the potential of molecular microbiological analysis to
be realized, it is important that it is applied to the study of
chronic CF lung infections in a focused way. To be of
greatest use in the treatment of patients, such investigations
should be targeted towards answering three key sets of
(1) The treatment of established chronic bacterial
infections in the CF lung is difficult to manage
effectively. Ultimately, the prevention of such infec-
tions from becoming established should be a fun-
damental research goal. Drawing from this, we need to
understand how bacterial communities develop. In
other systems this occurs through a process of
succession, to a ‘climax community’, in which a state
of equilibrium is reached where the community
changes little unless the environment is disrupted
(Cotgreave & Forseth, 2002). Such climax communities
have been reported in the human body, e.g. in gut
microbiology (Falk et al., 1998) and root canal
infections (Siqueira et al., 2002). Analogously, are we
able to characterize an ordered progression towards a
climax community in CF infections starting from the
Fig. 3. Regions of two T-RFLP profiles generated from a single
sample. The profile on the left was generated from rDNA, whereas
the profile on the right was generated from reverse transcribed
rRNA. The relative intensities of T-RF bands produced by a given
bacterial population indicate that there is a range of levels of
metabolic activity in the community, ranging from inactive to highly
Molecular approaches to the study of infections
initial bacterial colonization in infants? If so, can points
be identified at which this process might be disrupted?
(2) If such climax communities exist in CF infections,
what are their characteristics? Is there more than one
type, and if so, what are their implications for disease
severity and the treatment of infection? Currently, the
presence of certain species is used as an indicator of
both prognosis and response to certain interventions,
for example, the presence of Burkholderia cenocepacia is
associated with poor survival rates following lung
transplantation (Boussaud et al., 2008; Murray et al.,
2008). By extension, in CF infections, what character-
istics relate to particular clinical markers and out-
comes? This may be particularly important in relation
to infective exacerbations.
(3) Can these data inform clinicians about the impact
made by a course of treatment? Currently, the efficacy
of the use of antibiotics to treat bacterial infections in
the CF airways can only be assessed indirectly,
primarily through measures such as lung function
and general well-being. The degree to which interven-
tions are successful in interfering with bacterial growth
– their primary role – is not addressed and so cannot be
used to shape treatment.
The success of the application of molecular microbiological
profiling to CF lung infections will be determined by the
degree to which it can address these questions.
The application of the molecular approaches outlined here
to the study of chronic bacterial respiratory infections
promises to greatly increase our understanding of the
mechanisms of disease and provide a basis for improving
therapeutic interventions. However, there are three key
areas that should be addressed to ensure the advantages
that they confer are fully exploited.
Firstly, in parallel to the use of molecular profiling to
further our understanding of the basic processes of
infection, it is important that they are also developed to
provide frontline clinical diagnostics. For this to happen, a
translational strategy is required to identify features of
infection that are of high clinical significance and develop
diagnostic protocols that can provide clinicians with data
that are useful in the design of therapeutic regimes.
Secondly, a realization of the implications for treatment of
the polymicrobial nature of CF airway disease must occur.
Rather than being based on the clinical significance of
species studied in isolation, a model of disease where the
bacterial community as a whole is considered as the
‘pathogen’ would be helpful. As recognized in polymicro-
bial infections in other areas of the body, it not necessarily
possible to achieve eradication of infection, and instead the
aim should be to achieve a host manageable bioburden
(Bowler, 2003). As such, the suitability of treatment
practices such as the administration of 14 day courses of
intravenous antibiotics in response to infective exacerba-
tions should be reviewed. Originally designed to achieve
eradication of bacteria in infections, such approaches may
serve to increase antibiotic resistance in the bacterial
community and drug-related side-effects in the patient, but
do not necessarily provide substantial advantages over
shorter antibiotic courses. Furthermore, the use of
antibiotic prophylaxis, in an attempt to manage popula-
tions of bacteria between exacerbations, may be ineffective,
and possibly counter-productive.
Finally, the development and refinement of molecular
profiling technologies is constant. For example, more
effective means of resolving mixed bacterial PCR products
generated from multispecies templates are being devised.
These include MS-based approaches, such as the Ibis T5000
system (Ecker et al., 2006, 2008), that are able to derive
species identities based on base composition signatures of
the sequences amplified (Ecker et al., 2006, 2008). In
addition, ultra-high throughput sequencing (Braslavsky
et al., 2003; Margulies et al., 2005; Shendure et al., 2005;
Bentley et al., 2008) now offers the opportunity to generate
highly detailed bacterial community composition data. It is
vital that advances such as these are fully exploited in the
characterization of CF lung infections.
Meta-community analysis has already been employed to
determine the resistance reservoir in soil communities
(Schmitt et al., 2006; Allen et al., 2008), and a similar
approach could allow the determination of the total pool of
resistance markers present in chronic respiratory infection
or group of infections. This is particularly important given
the mobile nature of many of the sequences that confer
antibiotic resistance (Butaye et al., 2003; Bennett, 2008)
and the reduction in treatment efficacy that can result from
their spread. Whilst this review has focused primarily on
bacterial infection, clinical situations will often be further
complicated by the presence of viruses or fungal species.
The truly polymicrobial nature of many infections also
needs to be recognized. Ways of studying this are
increasing. For example, the development of culture-
independent strategies for the characterization of fungal
diversity (Bouchara et al., 2009), as well as the increasing
recognition of the value of molecular diagnostics in the
detection of respiratory viruses (Mahony, 2008), offers an
opportunity to examine more closely the relationships
between these groups of pathogens. Whilst adding further
complexity to our understanding of community patho-
genicity, this process will, however, be necessary for the
most appropriate means of treatment to be given. Other
areas of interest include changing the level of phylogenetic
resolution of total community profiling approaches, by
target sequence selection, the profiling of strains within
species of interest would be possible, or using the ability to
determine changes in levels of expression of specific
virulence genes in vivo using quantitative PCR to
characterize responses to changes in patient health or
G. B. Rogers, M. P. Carroll and K. D. Bruce
1412 Journal of Medical Microbiology 58
Data derived from molecular microbiological investi-
gations of CF respiratory infections have led to the
realization that they involve bacterial communities, with
multiple diverse bacterial species identified in individual
samples (Rogers et al., 2004; Sibley et al., 2008b). Clearly if
some or all of the species new to CF lung microbiology are
of relevance to the progression of lung disease then they
need to be factored into clinical consideration. However,
based on the evidence from both CF and wider contexts, it
may be necessary to view the bacterial community as a
pathogenic entity in its own right that can express different
degrees of virulence at different times. There is an
increasing recognition of the need to introduce novel
systems to deal with many of the problems inherent to
culture-dependent systems. A conceptual shift – towards an
understanding of more complex diagnostic scenarios – is
needed just as much as the technical means by which to
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