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Abstract

ABSTRACT Typical Klebsiella pneumoniae is an opportunistic pathogen, which mostly affects those with weakened immune systems and tends to cause nosocomial infections. A subset of hypervirulent K. pneumoniae serotypes with elevated production of capsule polysaccharide can affect previously healthy persons and cause life-threatening community-acquired infections, such as pyogenic liver abscess, meningitis, necrotizing fasciitis, endophthalmitis and severe pneumonia. K. pneumoniae utilizes a variety of virulence factors, especially capsule polysaccharide, lipopolysaccharide, fimbriae, outer membrane proteins and determinants for iron acquisition and nitrogen source utilization, for survival and immune evasion during infection. This article aims to present the state-of-the-art understanding of the molecular pathogenesis of K. pneumoniae.
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ISSN 1746- 0913
Future Microbiol. (2014) 9(9), 1071–10 81
part of
10.2217/FMB.14.48 © 2014 Future Medicine Ltd
REVIEW
Molecular pathogenesis of Klebsiella
pneumoniae
Bei Li‡,1, Yuling Zhao‡,2, Changting Liu3, Zhenhong Chen*,3 &DongshengZhou*,4
1Department o f Dermatology, Taihe Hospital, Hubei University of Med icine, Shiyan, Hubei, China
2The 89th hosp ital of People’s Liberation Army, Weifang, Shandong, China
3Nanlou Respir atory Diseases Depar tment, Chinese People’s Liber ation Army General Hospital, Beijing, China
4State Key Laborator y of Pathogen & Biosecurity, Beijing Instit ute of Microbiology & Epidemiology, Beijing, China
*Authors for correspondence: dongshengzhou1977@gmail.com; zhenhong_chen@hotmail.com
Authors contributed equally
ABSTRACT Typ ica l Klebsiella pneumoniae is an opportunistic pathogen, which mostly
aects those with weakened immune systems and tends to cause nosocomial infections.
A subset of hypervirulent K. pneumoniae serotypes with elevated production of capsule
polysaccharide can aect previously healthy persons and cause life-threatening community-
acquired infections, such as pyogenic liver abscess, meningitis, necrotizing fasciitis,
endophthalmitis and severe pneumonia. K.pneumoniae utilizes a variety of virulence factors,
especially capsule polysaccharide, lipopolysaccharide, mbriae, outer membrane proteins
and determinants for iron acquisition and nitrogen source utilization, for survival and immune
evasion during infection. This article aims to present the state-of-the-art understanding of
the molecular pathogenesis of K.pneumoniae.
KEYWORDS
Klebsiella pneumoniae
pathogenesis virulence
determinants
Klebsiella pneumoniae, a member of the family Enterobacteriaceae, is a rod-shaped, Gram-negative,
lactose-fermenting bacillus with a prominent capsule. Typical K. pneumoniae is an opportunistic
pathogen that is widely found in the mouth, skin and intestines, as well as in hospital settings and
medical devices. Opportunistic K. pneumoniae mostly affects those with compromised immune
systems or who are weakened by other infections. Colonization of the GI tract by opportunistic
K. pneumoniae generally occurs prior to the development of nosocomial infections, and K. pneu-
moniae colonization can be further found in the urinary tract, respiratory tract and blood [1] .
K. pneumoniae biofilms that form on medical devices (e.g., catheters and endotracheal tubes) pro-
vide a significant source of infection in catheterized patients [2] . Nosocomial infections caused by
K. pneumoniae tend to be chronic due to the two following major reasons: K. pneumoniae biofilms
formed in vivo protect the pathogen from attacks of the host immune responses and antibiotics [3] ;
and nosocomial isolates of K. pneumoniae often display multidrug-resistance phenotypes that are
commonly caused by the presence of extended-spectrum β-lactamases or carbapenemases, making
it difficult to choose appropriate antibiotics for treatment [4,5] .
At least 78 capsular (K antigen) serotypes have been recognized for K. pneumoniae [6 –8] . A few
serotypes (including predominantly K1 and K2) have a unique hypermucoviscous (hypervirulent)
phenotype due to increased production of capsule polysaccharide (CPS), which is recognized as
the most important virulence factor of K. pneumoniae (see below) and is defined by the appear-
ance of hypermucoviscous colonies grown on agar plates [9 ,10]. A string test indicates the presence
of hypermucoviscosity when a inoculation loop is able to generate a viscous string larger than
5 mm in length by stretching bacterial colonies on agar plate [1 0] . The degree of mucoidy appears
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to positively correlate with the successful estab-
lishment of invasive infections [11] . Nevertheless,
whether there is hypersecretion of capsule in the
hypervirulent strains remains questionable, as
the increased amount of polysaccharide observed
might be some exopolysaccharides rather than
‘truly organized’ CPS sugars.
Hypervirulent K. pneumoniae is highly
invasive and can affect previously healthy per-
sons, causing life-threatening and often com-
munity-acquired infections, such as pyogenic
liver abscess, meningitis, necrotizing fasciitis,
endophthalmitis and severe pneumonia [9,10].
In particular, serotype K1/K2-caused pyogenic
liver abscess, often complicated by metastatic
infections, has emerged worldwide in the past
two decades [12 ] . The ability to metastatically
spread from one organ to other organs is charac-
teristic of hypermucoviscous K. pneumoniae and
is uncommon for enteric Gram-negative bacilli
in the presence of host immune defense.
To the best of our knowledge, there are no
full reviews on the molecular pathogenesis of
K. pneumoniae. This article presents the state-
of-the-art understanding of the virulence deter-
minants of K. pneumoniae, with a particular
emphasis on hypervirulent K. pneumoniae.
Capsule polysaccharide
CPS gene loci
K. pneumoniae CPS is an acidic polysaccharide
generally composed of repeating units of three
to six sugars. K. pneumoniae CPS is synthesized
by the Wzy-dependent polymerization pathway,
which is well characterized for Escherichia coli
group I CPS [13 ,14], and the cps gene clusters
commonly consist of genes for sugar nucleo-
tide synthesis, capsule repeat-unit synthesis and
capsular repeat-unit assembly and export [15 ,16] .
The full length of the K. pneumoniae cps gene
clusters ranges from 21 to 30 kb, harboring
16–25 genes (see [15 ,16] for the gene structure
of different cps gene clusters). The 5´ terminal
regions of all known K. pneumoniae cps gene
clusters contain six conserved genes (in the fol-
lowing order: galF, orf2, wzi, wza, wzb and wzc)
and the 3´ end regions contain a conserved gene
gnd and are mostly terminated at the ugd gene,
while the central regions, which encode the
proteins for the polymerization and assembly of
CPS subunits, are highly divergent [15,16]. Both
wzy and wzi genes are present in all K types,
but each of them has a high level of sequence
variability among distinct K types. Accordingly,
wzy-targeting PCR assays have been established
to identify K1, K2, K3, K5, K20, K54 and K57
[17,18] , and moreover, wzi sequencing is able to
define the K types of most clinical K. pneumo-
niae isolates [19 ] . In addition, the capsular typ-
ing method based on wzc sequencing has been
used for the detection of novel capsular types
of K. pneumoniae [7] .
CPS synthesis & magA
CPS synthesis initiates with the assembly of indi-
vidual sugar-repeat units, which are catalyzed
by different glycosyltransferases in a sequential
manner [1 3, 14] . The resulting nascent repeats
are transferred across the inner membrane by a
flippase Wzx and undergo polymerization by a
Wzy polymerase in the periplasmic space [1 3 ,14] .
Further polymerization control and export of
mature CPS to the bacterial cell surface occur
under the combined action of Wza (an inner
membrane tyrosine autokinase), Wzb (an pro-
tein-tyrosine phosphatase) and Wzc (an inte-
gral outer membrane lipoprotein) [13 ,14] . magA
(wzyKpK1) encodes a capsular K1-specific Wzy
polymerase [20 ], but plays no role in lipopoly-
saccharide (LPS) synthesis [2 1] and acts as an
important virulence determinant in experi-
mental K. pneumoniae K1-induced metastatic
infections [22] .
rmpA/rmpA2
Plasmid-borne rmpA and its isoform rmpA2
encode the transcriptional activators of cps gene
transcription, CPS synthesis and hypermucovis-
ity in K. pneumoniae K1/K2 [23 –25] . In the K2
strain CG43, both rmpA and rmpA2 are acti-
vators of CPS biosynthesis and are important
for virulence in mice [23 ,24] . In the K1 strain
NTUH-K2044, besides plasmid-borne rmpA
and rmpA2, there is still an rmpA paralog on
the chromosome, and only plasmid-borne rmpA
enhances the cps gene transcription [25] .
Resistance to phagocytosis
The presence of a thick capsule at cell surface
protects K. pneumoniae from opsonization and
phagocytosis by macrophages [26 ], neutrophils
[27] , epithelial cells [28] and dendritic cells (DCs)
[29] by blocking binding and internalization
(Figure 1). Hypervirulent K. pneumoniae K1
shows significantly lower levels of interaction
with macrophages compared with nonhyper-
virulent strains [3 0] , and moreover, the unusual
feature of extensive pyruvation of glucuronic
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Figure 1. Klebsiella pneumoniae-mediated immune evasion. Klebsiella pneumoniae employs various surface structures (e.g., CPS, LPS,
OmpA, OmpK36 and AcrAB) to evade host immune defenses, enabling the bacteria to resist complement-mediated killing, the action
of host-derived antimicrobial peptides and the phagocytosis of epithelial cells, macrophages, neutrophils and DCs. This enables them
to escape from neutrophil-mediated intracellular killing of engulfed bacteria, to impair the production of the proinammatory cytokine
IL-8 and antimicrobial peptides hBD2 and hBD3 by airway epithelial cells and to inhibit the maturation of DCs.
CPS: Capsule polysaccharide; DC: Dendritic cell; LPS: Lipopolysaccharide; NK: Natural killer.
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acid and acetylation of C2-OH (or C3-OH)
of fucose in K1 CPS may help K. pneumo-
niae to avoid phagocytosis [31,3 2]. In addition,
hypervirulent K1 K. pneumoniae, after being
phagocytosed by neutrophils, can steadily
escape from neutrophil-mediated intracel-
lular killing and transport the K1 bacteria to
distant sites, such as the liver, causing abscess
formation (Figure 1) [30].
Suppression of early inammatory
response
Airway epithelial cells produce Toll-like recep-
tors (TLRs) in order to recognize conserved
molecules expressed by pathogens, which in
turn activate signaling pathways for produc-
ing antimicrobial molecules, such as human
β-defensins (hBDs; see below) and costimula-
tory molecules and for releasing cytokines and
chemokines (Figure 1). In sharp contrast to the
infection of wild-type capsulated K. pneumo-
niae strains that characteristically lacking the
above early inflammatory response, avirulent
CPS mutants activate a potent inflammatory
program [33 ,34] . Mechanistically, the anti-
inflammatory effect of CPS is characterized by
inhibition of IL-8 expression through inhib-
iting TLR2 and TLR4 signaling [33, 34] and
NOD1-dependent pathways [35] .
Resistance to antimicrobial peptides
CPS at the K. pneumoniae cell surface acts as
a protective shield against the access of host-
derived antimicrobial peptides, and free CPS
released from bacterial cells can trap antimi-
crobial polypeptides in order to reduce the
amount of antimicrobial polypeptides reaching
the bacterial surface (Figure 1) [36 ,37] . Moreover,
sublethal concentrations of antimicrobial pep-
tides in the airway induce cps gene expression,
which in turn protects bacteria against the
action of antimicrobial polypeptides [37] . hBDs
produced by airway epithelial cells are potent
antimicrobial peptides; hBD1 is constitutively
Action of host-derived
antimicrobial peptides
Production of hBD2
and hBD3 by airway
epithelial cells
Production of IL-8
by airway epithelial cells
Neutrophil chemotaxis
Phagocytosis of
epithelial cells Phagocytosis of
macrophages
Phagocytosis and
maturation of DC cells
TNF-αIL-12 and TNF-α
Phagocytes The adaptive immunity
Inflammatory cytokines
NK cell migration
Distant metastases
Intracellular killing
CPS, LPS
OmpA, OmpK36
CPS CPS Phagocytosis
of neutrophils
Complement-mediated
killing
CPS, LPS,
OmpA
CPS, LPS,
OmpA, AcrAB
Klebsiella pneumoniae
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expressed, whereas the expression of hBD2 and
hBD3 is inducible by pathogens and proinflam-
matory cytokines [3 8,39]. K. pneumoniae-bound
CPS decreases the express of hBD2 and hBD3
through preventing TLR-dependent responses
and stimulating the expression of CYLD and
MKP-1, which act as the negative regulators of
hBD expression (Figure1) [40].
Inhibition of DC maturation
K. pneumoniae CPS can impair DC maturation
(Figure 1) and thereby reduce the DC-mediated
production of pro-Th1 cytokines, such as IL-12
and TNF-α, which will lead to the destructive
function of immature DCs during K. pneu-
moniae antigen presentation (thereby impair-
ing T-cell activation), and moreover result in
reduced DC-mediated natural killer cell migra-
tion [29]. Taken together, inhibition of DC
maturation by K. pneumoniae CPS allows the
bacteria to avoid the host defenses and thus to
multiply in vivo more easily.
Lipopolysaccharide
LPS comprises three parts: the highly con-
served and hydrophobic lipid A anchored in the
outer membrane; the highly variable O-antigen
as the outermost component of LPS; and the
core polysaccharide connecting lipid A and
O-antigen.
O-antigen
At least nine O-antigen groups (O1, O2, O2ac,
O3, O4, O5, O7, O8 and O12) have been rec-
ognized in K. pneumoniae [4 1] . Biosynthesis
of O-antigen is executed by the enzymes
encoded by a six-gene wb cluster composed of
wzm, wzt, wbbM, glf, wbbN and wbbO with
respect to their transcriptional direction. The
wb cluster has a conserved gene organization,
but shows high genetic variation in correspond-
ing coding sequences, accounting for the high
chemical variability in different O-antigen
groups [4 2]. O1 is the most common serotype
among clinical K. pneumoniae isolates [4 1] , and
it is also more prevalent in hypermucoviscous
(invasive) strains than in nontissue-invasive
strains [42]. K. pneumoniae O-antigen pre-
vents access of complement components to
activators (e.g., porins and rough LPS) and
thus contributes to bacterial resistance against
complement-mediated killing (Figure 1) [43] ,
and there is a higher frequency of serum resist-
ance among O1-serotype isolates than among
non-O1-serotype isolates [42] . Interestingly, in
K. pneumoniae O-antigen-deficient strains, CPS
protects the microorganism against complement
killing (Figure 1) [44 ,45] . As shown in a murine
model of septicemia generated by intraperito-
neal injection, as well as in a murine model of
liver abscess by intragastric injection, the O1
antigen of hypermucoviscous K. pneumoniae
serotype O1:K1 plays a strong role in virulence
by conveying resistance to serum killing and
by promoting bacterial dissemination to and
colonization of internal organs after the onset of
bacteremia [42] . In a murine model of pneumo-
nia caused by hypermucoviscous K. pneumoniae
serotype O1:K2, both CPS and LPS O-antigens
are essential to blood passage of the bacteria
and the development of sepsis, but only CPS
is involved in the development of K. pneumo-
niae-induced pulmonary infections, because
CPS (but not LPS O-antigen) modulates the
deposition of C3 and protects the pathogen
against human alveolar macrophage-mediated
phagocytosis [26] . Nevertheless, O-antigen con-
tributes to lethality by increasing the propensity
for bacteremia and not by significantly chang-
ing the early course of intrapulmonary infection
[46] . The O-antigen of K. pneumoniae serotype
O5:K57 contributes to in vitro adhesion to
uroepithelial cells, and also colonization of uri-
nary tracts of rats infected by the transurethral
route [43]. As a factor regulating innate immune
responses in the lung, surfactant protein D plays
an important role in the effective inhibition of
the adhesion of K. pneumoniae to lung epithe-
lial cells through specific interaction with the
mannose-rich repeating units of the O-antigen
of this pathogen [47].
Core polysaccharide
Only two types (type 1 and type 2) of core
polysaccharide have been characterized for
K. pneumoniae, which were synthesized by
the products of two different 13-gene wa gene
clusters [48 ,4 9]. The type 2 wa gene cluster is
composed of hldD, waaF, waaC, wabK, waaL,
wabM, waaQ, wabG, wabH, orf10, waaA, waaE
and coaD [48] . The two wa gene clusters differ
by only two genes: type 1 has wabI and wabJ,
which encode 3-deoxy-d-manno-octulosonic
acid (Kdo) transferase and heptosyltransferase,
which are responsible for the incorporation
of the last two outer core residues Kdo and
l,d-HepI, respectively; type 2 presents the
two corresponding genes wabK and wabM,
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which are involved in the transfer of the last
two outer core Glc residues [4 8]. Accordi ngly,
the major structural difference between these
two core types occurs at the GlcN substituent
in the outer core-proximal disaccharide GlcN-
(1,4)-GalA: the GlcpN residue of the type 2
core is substituted at the O-4 position by the
disaccharide β-Glcp(1– 6)-α-Glcp(1), while
that of type 1 is replaced at the O-6 position
by a Kdo residue or by α-Hep (1– 4)- α-Kdo(2)
[48,49] . The single-gene mutants of K. pneumo-
niae LPS core synthease genes, such as waaC,
waaF, wabG and wabG, are greatly attenuated
when tested in different animal models [ 48 –5 1] ,
and show drastically reduced colonization abili-
ties in experimental urinary tract infections in
rats [50] . Hypermucoviscous K. pneumoniae
syntheses type 2 core polysaccharide [52] , and
the replacement of the type 1 core in a type 2
strain presents lower virulence than the wide-
type strain of core type 2 in a murine model
infected via the intraperitoneal route [48].
Lipid A
Nascent core lipid A is synthesized in the
cytoplasm by a set of conserved constitutive
enzymes, transported by the ABC transporter
MsbA and finally anchored in the outer mem-
brane. Occurring during lipid A transportation,
covalent modifications of lipid A are catalyzed
by various modification enzymes upon environ-
mental stimuli, which is involved in modulating
the virulence of a number of Enterobacteriaceae
pathogens [53] . K. pneumoniae lipid A modifi-
cation contributes to resistance to host innate
defenses, especially including resistance to anti-
bacterial peptides (Figure 1), and the mutation
of modification of the genes for this enzyme
leads to the attenuation of K. pneumoniae viru-
lence when tested in different animal models
[54 ,55] . Lipid A and core polysaccharide, but
not O-antigen are required for resistance to
phagocytosis by mouse alveolar macrophages
(Figure1) [45] , which plays an important role in
host defense against K. pneumoniae [5 6] .
Fimbriae
At least four types of fimbriae, namely type 1
fimbriae [57] , type 3 fimbriae [5 7] , Kpc fimbriae
[58] and KPF-28 adhesin [5 9] , have been char-
acterized experimentally for K. pneumoniae.
The former three, as opposed to KPF-28, are
synthesized by hypermucoviscous O1:K1 strain
NTUH-K2044 with determined complete
genome sequences [52], and NTUH-K2044 still
harbors six uncharacterized fimbrial gene loci:
kpa, kpb, kpd, kpe, kpf and kpe [5 8] .
Type 1 mbriae
K. pneumoniae chromosomes contain a con-
served fimbria-encoding region that is com-
posed of the regulatory gene locus mrkHIJ, the
type 3 fimbriae gene cluster mrkABCDF, a four-
gene interspacing region and the type 1 fim-
briae gene locus fimBEAICDFGHK [5 7] . Ty p e
1 fimbriae are thin, rigid, adhesive, thread-like
surface appendages on the outer membrane,
and the appendages are primarily composed
of repeating FimA subunits with an adhesin
molecule FimH at the tip [60] . Compared with
E. coli, the K. pneumoniae fim gene cluster has an
unique gene fimK located behind fimH. FimK
act as transcriptional regulator with a putative
DNA-binding region at its N-terminus and it
can specifically bind to a vegetative promoter
upstream of fimA in order to stimulate fimA
transcription [61] . A 314-bp region flanked by
inverted repeat sequences (fim switch), named
fimS, is located upstream of fimA, and moreo-
ver, fimS mediates the phase-variable expression
of K. pneumoniae type 1 fimbriae [62], which is
very similar to the expression of E. coli type
1 fimbriae [63] . K. pneumoniae type 1 fimbriae
extend beyond the capsule and mediate bacte-
rial adhesion to mannose-containing structures
on host cells or on extracellular matrices via the
adhesin FimH [64] . K. pneumoniae type 1 fim-
briae are essential for the initial establishment
of urinary tract infection, but have no effect
on the ability of K. pneumoniae to colonize the
intestine or to infect the lung [62] .
Type 3 mbriae
K. pneumoniae type 3 fimbriae are characterized
as 2–4-nm wide and 0.5–2-μm long append-
ages. mrkA encodes the fimbrial subunit, which
is polymerized to form the helical fimbrial shaft
[65] . The adhesive subunit, with the ability to
bind to collagen molecules, is encoded by mrkD
and located at the tip of the fimbriae [65] . mrkB,
mrkC and mrkF encode the chaperone, usher
and scaffolding proteins, respectively, which are
responsible for fimbrial assembly/stabilization
[65] . Type 3 fimbriae mediate in vitro adhesion
to epithelial cells and kidney and lung tissues,
most likely in a mannose-resistant manner [6 6] .
Type 3 fimbriae act as a major contributor to
K. pneumoniae biofilm formation, but play no
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role in intestine and pulmonary infections [5 7] .
K. pneumoniae-induced urinary tract infec-
tions are frequently associated with the forma-
tion of K. pneumoniae biofilms on indwelling
urinary catheters. Both type 1 and type 3 fim-
briae, functioning in a compensating manner,
enhance K. pneumoniae biofilm formation on
urinary catheters [67]. In addition to type 1
fimbriae, type 3 fimbriae act as another impor-
tant colonization factor for K. pneumoniae bio-
film-associated urinary infections due to the
indwelling of urinary catheters [6 8].
Kpc mbriae
The Kpc fimbriae are synthesized and assembled
by the products of the kpcABCD operon, which
is highly associated with hypermucoviscous
K. pneumoniae [58] . A site-specific recombinase-
encoding gene kpcI together with a promoter
element kpcS (a 302-bp intergenic DNA region
flanked by 11-bp inverted repeats) are located
upstream of kpcA [58] . KpcA is the major subunit
component of Kpc fimbriae, and KpcS in com-
bination with KpcI mediates the phase-variable
regulation of Kpc fimbriation [5 8] . Heterologous
expression of kpcABCD in a fimbriate E. coli
makes the recombinant bacterium present
Kpc fimbriae, and further confers on it higher
biofilm-forming activity, indicating that the
Kpc fimbriae may contribute to K. pneumoniae
biofilm formation [5 8] .
KPF-28 adhesin
As a polymer of a 28-kDa major fimbrin subunit,
KPF-28 is a long, thin and flexible fimbria that
is 4–5 nm in diameter and 0.5–2 mm in length,
and the structural gene of the KPF-28 major
subunit is located on a transferable R plasmid
encoding the CAZ-5/SHV-4 β-lactamase [59 ] .
The multidrug-resistant K. pneumoniae strains
producing plasmid-encoded CAZ-5/SHV-4 and
KPF-28 represent a epidemic clone of K. pneu-
moniae in the hospitals in Clermont-Ferrand,
France [69]. No KPF-28 expression can be
observed in E. coli transconjugants harboring
the CAZ-5/SHV-4-encoding plasmid alone,
and thus, besides the R plasmid, additional
factor(s) most likely encoded by the K. pneu-
moniae chromosome are required in order to
promote KPF-28 expression [59] . The KPF-28
fimbriae contribute to the adhesion of K. pneu-
moniae to human Caco-2 cell lines, indicating
that the fimbriae may be a colonization factor
within the mammalian intestine [59] .
Outer membrane proteins
OmpA
It is evidenced that CPS is necessary but not
sufficient to attenuate airway epithelial cell-
mediated inflammatory responses [35] . OmpA
is one of the major outer membrane proteins of
Gram-negative bacteria, and it is highly con-
served among Enterobacteriaceae. K. pneumo-
niae OmpA, independent of CPS, is important
for preventing the activation of airway epi-
thelial cells via acting on NF-κB-, p38- and
p44/42-dependent pathways and thus contrib-
utes to the attenuation of the airway epithelial
cell-mediated inflammatory response (Figure 1)
[70] . Loss of OmpA makes K. pneumoniae more
susceptible to antimicrobial peptides, but has
no effect on CPS production, which is known
to be responsible for resistance to antimicro-
bial peptides [ 71] . It is thought that OmpA is
involved in the activation of as-yet unknown
systems dedicated to ameliorating the cytotox-
icity of antimicrobial peptides. OmpA also con-
tributes to resistance to phagoyctosis by alveolar
macrophages (Figure 1) [45].
Outer membrane porins
K. pneumoniae produces two major outer mem-
brane porins – OmpK35 and OmpK36 – through
which hydrophilic molecules (e.g., nutrients
and cephalosporins/carbapenems) diffuse into
the bacteria [7 2]. In addition, K. pneumoniae
expresses alternative porins, such as KpnO [73]
and OmpK26 [74 ], in order to compensate for
the absence of OmpK35/36. Loss of any of
OmpK36, KpnO or OmpK26 leads to increased
resistance to cephalosporins/carbapenems and
reduced virulence in mouse models of acute
systemic infections, while loss of OmpK35 has
no effect on antibiotic resistance and virulence
[45,7 2–74 ]. Loss of OmpK36 remodels the surface
structure of K. pneumoniae and thereby alters
the binding of phagocytes, leading to increased
susceptibility to phagocytosis and thus an
attenuation in virulence (Figure 1) [45,72] .
Eux pumps
K. pneumoniae expresses the efflux pump AcrAB,
which contributes to the export of not only
antibiotics (e.g., quinolones and β-lactams),
but also host-derived antimicrobial agents (e.g.,
the antimicrobial agents present in human
bronchoalveolar lavage fluid and human anti-
microbial peptides; Figure 1), and AcrAB acts as a
determinant of K. pneumoniae resistance to host
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innate immune defenses [75] . The inactivation
of AcrAB not only leads to a multidrug resist-
ance phenotype, but also to a reduced capac-
ity to cause pneumonia in a murine model [75] .
The expression of another K. pneumoniae efflux
pump, namely EefABC, is not related to any
antibiotic resistance phenotype, but it confers
acid tolerance in vitro and high competition
potential in the host GI tract [76].
Iron acquisition
Iron is essential for bacterial growth both in
vitro and in vivo. At least 12 distinct iron uptake
systems can be identified in K. pneumoniae
NTUH-K2044, and they can be assigned into
four major classes: Fe2+ transporter Feo, ABC
transporter, hemophore-based uptake systems
and siderophore-based uptake systems ( Tabl e 1) .
Of these 12 iron uptake systems, two ABC trans-
porters (Kfu [77] and Sit [78]) and three sidero-
phore-based systems (Yersinia high-pathogenicity
island [79, 80] , Iuc [80, 81] and IroA [80] ) have been
shown to be required for the full virulence of
K. pneumoniae. The kfu, high-pathogenicity
island, iuc and iroA regions are highly associated
with hypervirulent K. pneumoniae, thereby repre-
senting horizontally acquired virulence loci dur-
ing the evolution of hypervirulent variants from
opportunistically pathogenic K. pneumoniae [82] .
Bacterial pathogens cope with the scarcity
of iron in their mammalian hosts (in which
iron is bound by various iron-binding proteins)
through synthesizing small iron-scavenging
molecules called hemophores or siderophores.
Enterobacteriaceae pathogens, including K. pneu-
moniae, produce a prototypical siderophore called
enterobactin, which has the highest iron affinity
compared with any other known iron chelators.
Mammals have evolved to secrete lipocalin 2 in
order to sequester enterobactin, leading to the
blocking of siderophore-based iron acquisition
as a part of their innate immune response [83] .
As a countermeasure, a subset of K. pneumoniae
isolates with a tendency to cause clinical respira-
tory or pulmonar y infections produces additional
siderophores, such as yersiniabactin (a phenolate-
type siderophore) and salmochelin (a glycosylated
form of enterobactin), in order to rejuvenate the
siderophore-based iron acquisition pathways,
allowing this pathogen to evade the lipocalin
2-based mechanism of the host innate immune
defense [84,85]. Notably, hypervirulent K. pneumo-
niae secrete quantitatively more and biologically
more active siderophore molecules than classical
K. pneumoniae, delineating an additional mech-
anism by which hypervirulent K. pneumoniae
increases its pathogenic potential [86] .
Nitrogen source utilization
Urease
Many gut pathogens, including K. pneumoniae,
can produce cytoplasmic urease in order to hydro-
lyze urea to ammonia and carbon dioxide as a
source of nitrogen for growth. The ureDABCEFG
operon encodes structural subunits (UreA,
UreB and UreC) of the metalloenzyme urease
Table 1. Iron uptake systems in Klebsiella pneumoniae NTUH-K2044.
Category System Substrate Gene name Gene ID
Feo Feo Fe2+ feoABC KP1_5110–KP1_5112
ABC transporter Sit Fe2+ sitABCD KP1_4347–KP1_4350
Kfu Fe3+ kfuABC K P1_19 80– KP1_19 82
Fec Ferric citrate fecBDEA KP1_3247–KP1_3250
Yiu Unknown yiuABC K P1_14 41– KP1_1439
Hemophore based Hmu Hemin/hemoprotein hmuRSTUV KP1_ 4356 –KP1_4362
Siderophore based Fep-Ent Enterobactin fepA-e ntD, fes-entF, fepDGC,
ybdA, fe pB, entCEBA
KP1_1547– KP1_15 46,
KP1_1548–KP1_1549,
KP1_1553–KP1_1551,
KP1_1555, KP1_1556,
KP1_1557–KP1_1560
Fhu Ferrichrome fhuABCD K P1_10 02–KP1_1005
IroA Salmochelin iroN, iroBCD KP1_3609, KP1_3610–K P1_3613
  iroBCDN KP1_p028–KP1_p025
Iuc Aerobactin iucABCD-iutA KP1_p319– KP1_p314
High-pathogenicity island Yersiniabactin ybtPQXS, ybtA-irp2-irp1-
ybtUTE-fyuA
KP1_3586 –KP1_3583,
KP1_3587–K P1_3593
Future Microbiol. (2014) 9(9)
1078
REviEW Li, Zhao, Liu, Chen & Zhou
future science group
and accessory nickel-binding proteins (UreD,
UreE, UreF and UreG) that are responsible for
the incorporation of nickel ions into the active
site of the urease enzyme [87] . The inactivation
of the K. pneumoniae urease-based metabolism
of urea will impair the growth of this pathogen
in the host GI tract, where urea is abundant [88] .
Allantoin metabolism
A 22-kb chromosomal all gene locus responsi-
ble for allantoin metabolism is highly associated
with hypervirulent K. pneumoniae [82] and plays
an important role in K. pneumoniae-induced liver
infection [89] . Primary liver abscesses caused by
hypervirulent K. pneumoniae frequently occur in
diabetes mellitus patients with an increased allan-
toin concentration. The allantoin utilization phe-
notype described in hypervirulent K. pneumoniae
elevates its capability to compete for allantoin as a
nitrogen source in mammalian hosts [8 9].
Conclusion
Ty pic a l K. pneumoniae is an opportunistic
pathogen commonly causing nosocomial infec-
tions, but a subset of hypervirulent serotypes
(including predominantly K1 and K2) due to
increased production of CPS affect previously
healthy persons to cause life-threatening invasive
infections. CPS is the most important virulence
factor of K. pneumoniae, which plays important
roles in resistance to phagocytosis, suppression of
early inflammatory response, resistance to anti-
microbial peptides, and inhibition of DC cell
maturation. Additional K. pneumoniae virulence
factors include LPS, fimbriae, outer membrane
proteins, and determinants for iron acquisition
and nitrogen source utilization.
Future perspective
The current understanding of K. pneumoniae
pathogenesis is mainly derived from studying
individual genes, although global screening
methods, such as signature-tagged mutagenesis
assays [90], have been used for identifying candi-
dates of virulence determination. Huge genetic
variability can be found among different types
of K. pneumoniae, which probably cause dra-
matic differences in pathogenicity [82] . The
regulatory networks governing complex cel-
lular pathways to operate in a concerted man-
ner, which enable the K. pneumoniae lifestyle
in mammalian hosts, are still poorly under-
stood. Genomics and transcriptomics studies
are promising to provide us with deeper under-
standing of the K. pneumoniae–host interac-
tions from a genome-scale view and to iden-
tify numerous candidates of virulence-related
genes for further hypothesis-based functional
characterization.
Financial & competing interests disclosure
Financial support was provided by the Foundation for
Innovative Research Team in Hubei University of Medicine
(2011 CXX01), the Fund for Fostering the National
Natural Science Foundation of Hubei University of
Medicine (2012JPY09), the National Key Program for
Infectious Disease of China (2013ZX10004216) and the
National Basic Research Program of China
(2014CB744400). The authors have no other relevant
affiliations or financial involvement with any organization
or entity with a financial interest in or financial conflict
with the subject matter or materials discussed in the
manuscript apart from those disclosed.
No writing assistance was utilized in the production of
this manuscript.
EXECUTivE SUMMARY
Klebsiella pneumoniae-induced infection
Typ ica l Klebsiella pneumoniae is an opportunistic pathogen that commonly causes nosocomial infections.
Due to their increased production of capsule polysaccharide, a subset of hypervirulent K.pneumoniae serotypes
(including predominantly K1 and K2) aect previously healthy persons and cause life-threatening invasive infections.
Virulence determinants of K.pneumoniae
Capsule polysaccharide is the most important virulence factor of K.pneumoniae that plays important roles in resistance
to phagocytosis, suppression of early inammatory responses, resistance to antimicrobial peptides and inhibition of
dendritic cell maturation.
Additional K.pneumoniae virulence factors include lipopolysaccharide, mbriae, outer membrane proteins and
determinants of iron acquisition and nitrogen source utilization.
The roles of these virulence factors in survival and immune evasion during infection are discussed in this article.
1079
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Molecular pathogenesis of Klebsiella pneumoniae REviEW
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... According to the European Center for Disease Prevention and Control (ECDC), MDR is defined as 'acquired non-susceptibility to at least one Indeed, the integrative conjugal elements and giant plasmids are the effective genetic elements which support the high virulence characteristics in HVKP strains [50][51][52]. K. pneumoniae encompasses four important and effective virulence factors, e.g., adhesive fimbriae (including type 1 type 3 fimbriae), capsule, lipopolysaccharide (LPS) and siderophores [5,23,[53][54][55]. ...
... The virulence factor of the capsule covers the K. pneumoniae bacterial cells against the host immune system responses such as phagocytosis, complement proteins, opsonophagocytosis, oxidative killing and antimicrobial peptides. In another word, the encapsulated bacterial cells of K. pneumoniae are capable of evading the host's immune system through their capsule antigens mimicking the host glycans to survive [27,49,54,55,58,59]. As aforementioned, the Kantigen belonging to K. pneumoniae capsule is an effective criterion for classification and serotyping of the pathogenic strain of K. pneumoniae. ...
... The virulence factor of the capsule covers the K. pneumoniae bacterial cells against the host immune system responses such as phagocytosis, complement proteins, opsonophagocytosis, oxidative killing and antimicrobial peptides. In another word, the encapsulated bacterial cells of K. pneumoniae are capable of evading the host's immune system through their capsule antigens mimicking the host glycans to survive [27,49,54,55,58,59]. As aforementioned, the K-antigen belonging to K. pneumoniae capsule is an effective criterion for classification and serotyping of the pathogenic strain of K. pneumoniae. ...
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Klebsiella pneumoniae is a Gram-negative opportunistic pathogen responsible for a variety of community and hospital infections. Infections caused by carbapenem-resistant K. pneumoniae (CRKP) constitute a major threat for public health and are strongly associated with high rates of mortality, especially in immunocompromised and critically ill patients. Adhesive fimbriae, capsule, lipopolysaccharide (LPS), and siderophores or iron carriers constitute the main virulence factors which contribute to the pathogenicity of K. pneumoniae. Colistin and tigecycline constitute some of the last resorts for the treatment of CRKP infections. Carbapenemase production, especially K. pneumoniae carbapenemase (KPC) and metallo-β-lactamase (MBL), constitutes the basic molecular mechanism of CRKP emergence. Knowledge of the mechanism of CRKP appearance is crucial, as it can determine the selection of the most suitable antimicrobial agent among those most recently launched. Plazomicin, eravacycline, cefiderocol, temocillin, ceftolozane-tazobactam, imipenem-cilastatin/relebactam, meropenem-vaborbactam, ceftazidime-avibactam and aztreonam-avibactam constitute potent alternatives for treating CRKP infections. The aim of the current review is to highlight the virulence factors and molecular pathogenesis of CRKP and provide recent updates on the molecular epidemiology and antimicrobial treatment options.
... Klebsiella (K.) pneumoniae is a Gram-negative opportunistic nosocomial bacterial pathogen. It is involved in several localized and disseminated hospital-acquired infections such as burns infections, sepsis, respiratory and gastrointestinal tract infections, urinary tract infections, pyogenic liver abscesses, and soft tissue and wound infection [1]. The emergence of carbapenem-resistant K. pneumoniae (CRKP) strains has become an ultimate challenge for public health globally due to their ability to disseminate rapidly in the hospital environment and their extended antibiotic resistance phenotypes [2]. ...
Article
Background: Carbapenem resistance in K. pneumoniae have been reported to emerge in developing and developed countries. Studies on molecular detection of Carbapenem resistance gene in Imipenem resistant K. pneumoniae isolated from urine of patients with suspected Urinary tract infection in General Hospital, Keffi, Nigeria. was carried out. Methods: A total of two hundred and ten (210) urine samples were collected from patients and K. pneumoniae was isolated and identified using standard microbiological method. The Antibiotic susceptibility test for the isolates was carried out and interpreted in accordance with Clinical and Laboratory Standard Institute (CLSI) protocol. The Molecular detection of Carbapenem resistance gene were carried out using Polymerase Chain Reaction (PCR) method. The occurrence of K. pneumoniae was 46(23.3%). Results: The highest occurrence of K. pneumoniae in relation to age was observed at age 21-30 (27.1%) and lowest was at age≥ 50 (15.00%). In relation to gender the occurrence was higher in females (32.6%) than male (15.7%). The antibiotic resistance of K. pneumoniae showed that the isolates were more resistant to Sulphamethoxazole/Trimethoprim (89.7%) and less resistant to Gentamicin (30.6%) and Imipenem (16.3%). The occurrence of Multidrug resistance (MDR) and pan drug resistance (PDR) isolates were in order of occurrence MDR (83.6%) >PDR (16.3) > XDR (0.0%). The percentage occurrence of Carbapenem resistance gene in K. pneumoniae isolates were blaKpc (100%) positive and blaVIM (33.3%) positive. Conclusion: The occurrence of K. pneumoniae isolates from urine of suspected urinary tract infections of patients in this study location was high and antibiotics such as Cefexime, Gentamicin and Imipenem were very effective against the K. pneumoniae isolates. Also, most of the isolates were multi-drug resistance (MDR). In addition, KPC and VIM genes were predominantly detected in imipenem resistant isolates.
... K. pneumoniae is another bacterium from the Enterobacteriaceae family. It is an opportunistic pathogen that causes serious diseases such as pneumonia, bloodstream infections, urinary tract infections or sepsis, mainly in immunocompromised patients [19,20]. In recent years, the number of antibiotic-resistant strains has increased [21]; thus, K. pneumoniae has become one of the major threats due to significant morbidity and mortality [8]. ...
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Background Bacterial genotyping is a crucial process in outbreak investigation and epidemiological studies. Several typing methods such as pulsed-field gel electrophoresis, multilocus sequence typing (MLST) and whole genome sequencing are currently used in routine clinical practice. However, these methods are costly, time-consuming and have high computational demands. An alternative to these methods is mini-MLST, a quick, cost-effective and robust method based on high-resolution melting analysis. Nevertheless, no standardized approach to identify markers suitable for mini-MLST exists. Here, we present a pipeline for variable fragment detection in unmapped reads based on a modified hybrid assembly approach using data from one sequencing platform. Results In routine assembly against the reference sequence, high variable reads are not aligned and remain unmapped. If de novo assembly of them is performed, variable genomic regions can be located in created scaffolds. Based on the variability rates calculation, it is possible to find a highly variable region with the same discriminatory power as seven housekeeping gene fragments used in MLST. In the work presented here, we show the capability of identifying one variable fragment in de novo assembled scaffolds of 21 Escherichia coli genomes and three variable regions in scaffolds of 31 Klebsiella pneumoniae genomes. For each identified fragment, the melting temperatures are calculated based on the nearest neighbor method to verify the mini-MLST’s discriminatory power. Conclusions A pipeline for a modified hybrid assembly approach consisting of reference-based mapping and de novo assembly of unmapped reads is presented. This approach can be employed for the identification of highly variable genomic fragments in unmapped reads. The identified variable regions can then be used in efficient laboratory methods for bacterial typing such as mini-MLST with high discriminatory power, fully replacing expensive methods such as MLST. The results can and will be delivered in a shorter time, which allows immediate and fast infection monitoring in clinical practice.
... The bacterium is found growing in the environment, specifically in areas such as soil, water, and plant matter [6]. K. pneumoniae strains are classified into at least 79 serotypes, of which serotypes K1, K2, K5, K20, K54, and K57 are closely related to bacterial pathogenesis [7][8][9]. Studies have also indicated that K1 and K2 are high-risk clones with high virulence and multidrug resistance [10]. The rising incidence of high-virulent and multidrug-resistant (MDR) K. pneumoniae is one of the major clinical and public health issues worldwide [11]. ...
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Klebsiella pneumoniae can cause serious pneumonitis in humans. The bacterium is also the common causative agent of hospital-acquired multidrug-resistant (MDR) infections. Here we for the first time reported the genetic diversity of K. pneumoniae strains in 14 species of edible aquatic animals sampled in the summer of 2018 and 2019 in Shanghai, China. Virulence-related genes were present in the K. pneumoniae strains (n = 94), including the entB (98.9%), mrkD (85.1%), fimH (50.0%), and ybtA (14.9%) strains. Resistance to sulfamethoxazole-trimethoprim was the most prevalent (52.1%), followed by chloramphenicol (31.9%), and tetracycline (27.7%), among the strains, wherein 34.0% had MDR phenotypes. Meanwhile, most strains were tolerant to heavy metals Cu2+ (96.8%), Cr3+ (96.8%), Zn2+ (91.5%), Pb2+ (89.4%), and Hg2+ (81.9%). Remarkably, a higher abundance of the bacterium was found in bottom-dwelling aquatic animals, among which mollusk Tegillarca granosa contained K. pneumoniae 8-2-5-4 isolate from serotype K2 (ST-2026). Genome features of the potentially pathogenic isolate were characterized. The enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR)–based genome fingerprinting classified the 94 K. pneumoniae strains into 76 ERIC genotypes with 63 singletons, demonstrating considerable genetic diversity in the strains. The findings of this study fill the gap in the risk assessment of K. pneumoniae in edible aquatic animals.
... Thus, KbvR contributes to the bacterial defense against macrophage phagocytosis in K. pneumoniae. Additionally, the outer membrane (OM) that acts as a barrier in some gram-negative bacteria, preventing toxic compounds such as antibiotics and detergents from entering the cell (21). The folding and insertion of -barrel proteins into the OM are mediated by the -barrel assembly machinery (BAM) complex, which is composed of the integral membrane protein Ba mA (YaeT) and four accessory lipoproteins BamB (YfgL), BamC (NIpB), BamD (YfiO), and BamE (YfiE) (SmpA)? ...
Article
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The immune system is a complicated, closely regulated mechanism that evolved to keep people healthy from infectious pathogens. Phagocytosis is important for both innate and acquired immunity, which is a critical process for microbial pathogens and apoptotic cells to be consumed and eliminated. However, several pathogens have evolved different strategies to escape detection and killing by phagocytosis. Recently, with the increase in infectious diseases and antibiotic resistance, it is significant for people to have a deep understanding of immune evasion, which may become an opportunity to explore new treatments and vaccination. Additionally, researchers mostly study immune evasion of a single pathogen but rarely summarize pathogens from the perspective of immune mechanisms. Here, we present the current understanding of phagocytosis and give a brief discussion of how pathogens control phagocytosis at different stages.
... Siderophores obtain iron, which exists the host, showing a vital function in the propagation process of the pathogen [11,12]. K. pneumoniae features a pathogenicity related to an assortment of harmful components (including the improvement of the capsule, lipopolysaccharide, hyper-mucoviscosity, and press-securing framework), all of which help the pathogen to overcome the human host's natural resistance and maintain the infection in the host [13]. Severe community-acquired infections were detected among moderately healthy individuals with hypervirulent K. pneumoniae (HvKp). ...
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To date, coronavirus disease 2019 (COVID-19) and its variants have been reported as a novel public health concern threatening us worldwide. The presence of Klebsiella pneumoniae in COVID-19-infected patients is a major problem due to its resistance to multiple antibiotics, and it can possibly make the management of COVID-19 in patients more problematic. The impact of co-infection by K. pneumoniae on COVID-19 patients was explored in the current review. The spread of K. pneumoniae as a co-infection among critically ill COVID-19 patients, particularly throughout hospitalization, was identified and recorded via numerous reports. Alarmingly, the extensive application of antibiotics in the initial diagnosis of COVID-19 infection may reduce bacterial co-infection, but it increases the antibiotic resistance of bacteria such as the strains of K. pneumoniae. The correct detection of multidrug-resistant K. pneumoniae can offer a supportive reference for the diagnosis and therapeutic management of COVID-19 patients. Furthermore, the prevention and control of K. pneumoniae are required to minimize the risk of COVID-19. The aim of the present review is, therefore, to report on the virulence factors of the K. pneumonia genotypes, the drug resistance of K. pneumonia, and the impact of K. pneumoniae co-infection with COVID-19 on patients through a study of the published scientific papers, reports, and case studies.
Article
Environmental pollution of antibiotic resistance genes (ARGs) has been a great public concern. Integrons, as mobile genetic elements, with versatile gene acquisition systems facilitate the horizontal gene transfer (HGT) and pollution disseminations of ARGs. However, little is understood about the characteristics of ARGs mediated by integrons, which hampers our monitoring and control of the mobile antimicrobial resistance risks. To address these issues, we reviewed 3,322 publications concerning detection methods and pipeline, ARG diversity and evolutionary progress, environmental and geographical distribution, bacterial hosts, gene cassettes arrangements, and based on which to identify ARGs with high risk levels mediated by integrons. Diverse ARGs of 516 subtypes attributed to 12 types were capable of being carried by integrons, with 62 core ARG subtypes prevalent in pollution source, natural and human-related environments. Hosts of ARG-carrying integrons reached 271 bacterial species, most frequently carried by opportunistic pathogens Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. Moreover, the observed emergence of ARGs together with their multiple arrangements indicated the accumulation of ARGs mediated by integrons, and thus pose increasing HGT risks under modern selective agents. With the concerns of public health, we urgently call for a better monitoring and control of these high-risk ARGs. Our identified Risk Rank I ARGs (aacA7, blaOXA10, catB3, catB8, dfrA5) with high mobility, reviewed key trends and noteworthy advancements, and proposed future directions could be reference and guidance for standard formulation.
Article
Objective. To study in vitro antimicrobial resistance and prevalence of the most clinically important carbapenemases genes in Klebsiella pneumoniae clinical isolates in Nizhny Novgorod. Materials and Methods. A total of 238 K. pneumoniae clinical isolates from upper and lower respiratory tracts, abdominal cavity, urogenital tract, and wound discharge were tested in this study. Species identification was done using WalkAway 96 analyzer (Siemens, Germany) with POS Combo Type 20 tablets (Beckman Coulter, USA) and Multiscan FC spectrophotometer (Thermo Scientific, Finland) with Microlatest tablets (PLIVA-Lachema, Czech Republic). Antimicrobial resistance was determined by discdiffusion method and using microbiological analyzer WalkAway 96 (Siemens, Germany). Minimal inhibitory concentrations for colistin were determined using the “MIC Colistin” kit (Erba Mannheim, Czech Republic). Detection of carbapenemases genes (KPC, OXA-48 group, IMP, VIM and NDM) was performed by RT-PCR using CFX-96 machine (Bio-Rad, USA) and commercial kits «MDR KPC/OXA-48-FL» and «MDR MBL-FL» (AmpliSens, Russia). Results. More than 90% of K. pneumoniae isolates in Nizhny Novgorod were resistant to III–V generation cephalosporins, 53.8% – to gentamicin, 71.2% – to ciprofloxacin, 81.2% – to co-trimoxazole, 88.1% – to ertapenem, 37.1% – to doripenem, 21.6% – to imipenem, 34.3% – to meropenem, 3.2% – to colistin. Genes of КРС-like carbapenemases were detected in 13.1% of isolates, OХA-48 – in 21.6%. Metallobeta-lactamases were not identified among tested isolates. Conclusions. Currently, there are no antimicrobials that active against all K. pneumoniae isolates in Nizhny Novgorod. Carbapenems and polymyxins remain active against more than 50% of isolates.
Article
To survive and establish a niche for themselves, bacteria constantly evolve. Toward that, they not only insert point mutations and promote illegitimate recombinations within their genomes but also insert pieces of ‘foreign’ deoxyribonucleic acid, which are commonly referred to as ‘genomic islands’ (GEIs). The GEIs come in several forms, structures and types, often providing a fitness advantage to the harboring bacterium. In pathogenic bacteria, some GEIs may enhance virulence, thus altering disease burden, morbidity and mortality. Hence, delineating (i) the GEIs framework, (ii) their encoded functions, (iii) the triggers that help them move, (iv) the mechanisms they exploit to move among bacteria and (v) identification of their natural reservoirs will aid in superior tackling of several bacterial diseases, including sepsis. Given the vast array of comparative genomics data, in this short review, we provide an overview of the GEIs, their types and the compositions therein, especially highlighting GEIs harbored by two important pathogens, viz. Acinetobacter baumannii and Klebsiella pneumoniae, which prominently trigger sepsis in low- and middle-income countries. Our efforts help shed some light on the challenges these pathogens pose when equipped with GEIs. We hope that this review will provoke intense research into understanding GEIs, the cues that drive their mobility across bacteria and the ways and means to prevent their transfer, especially across pathogenic bacteria.
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Cultured lung epithelial cells release antibacterial activity upon contact with Pseudomonas aeruginosa (PA), which is impaired in cystic fibrosis (CF). In order to identify the factors responsible for killing PA by a biochemical approach, we purified antimicrobial activity from supernatants of the A549 lung epithelial cell line, previously stimulated with PA bacteria, by subsequent high performance liquid chromatography. NH(2)-terminal sequencing of a major bactericidal compound revealed it to be identical with human beta-defensin-2 (hBD-2). A mucoid phenotype of PA, but not two nonmucoid PA strains, high concentrations (> 10 microg/ml) of PA lipopolysaccharide, tumor necrosis factor alpha, and interleukin (IL)-1beta, but not IL-6, dose-dependently induced hBD-2 messenger RNA in cultured normal bronchial, tracheal, as well as normal and CF-derived nasal epithelial cells. Genomic analysis of hBD-2 revealed a promoter region containing several putative transcription factor binding sites, including nuclear factor (NF) kappaB, activator protein (AP)-1, AP-2, and NF-IL-6, known to be involved in the regulation of inflammatory responses. Thus, hBD-2 represents a major inducible antimicrobial factor released by airway epithelial cells either on contact with mucoid PA or by endogenously produced primary cytokines. Therefore, it might be important in lung infections caused by mucoid PA, including those seen in patients with CF.
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Capsule is an important virulence factor in bacteria. A total of 78 capsular types have been identified in Klebsiella pneumoniae. However, there are limitations in current typing methods. We report here the development of a new genotyping method based on amplification of the variable regions of the wzc gene. Fragments corresponding to the variable region of wzc were amplified and sequenced from 76 documented capsular types of reference or clinical strains. The remaining two capsular types (reference strains K15 and K50) lacked amplifiable wzc genes and were proven to be acapsular. Strains with the same capsular type exhibited ≧94% DNA sequence identity across the variable region (CD1-VR2-CD2) of wzc. Strains with distinct K types exhibited <80% DNA sequence identity across this region, with the exception of three pairs of strains: K22/K37, K9/K45, and K52/K79. Strains K22 and K37 shared identical capsular polysaccharide synthesis (cps) genes except for one gene with a difference at a single base which resulted in frameshift mutation. The wzc sequences of K9 and K45 exhibited high DNA sequence similarity but possessed different genes in their cps clusters. K52 and K79 exhibited 89% wzc DNA sequence identity but were readily distinguished from each other at the DNA level; in contrast, strains with the same capsular type as K52 exhibited 100% wzc sequence identity. A total of 29 strains from patients with bacteremia were typed by the wzc system. wzc DNA sequences confirmed the documented capsular type for twenty-eight of these clinical isolates; the remaining strain likely represents a new capsular type. Thus, the wzc genotyping system is a simple and useful method for capsular typing of K. pneumoniae.
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Aim: To establish a PCR-based genotyping method for clinical Klebsiella pneumoniae. Materials & methods: The prevalence of six serotype markers, 41 large variably presented gene clusters, and seven additional virulence markers were screened by PCR in 327 clinical K. pneumoniae strains from China. Results: Detection of serotype markers enabled the identification of capsular serotypes K1, K2, K5, K20, K54 and K57. K. pneumoniae isolates of different origins gave distinct profiles of virulence loci, allowing us to gain a full overview of virulence gene distribution of the strains tested. A novel genotyping scheme was established to group clinical K. pneumoniae strains into distinct complexes based on the profiles of large variably presented gene clusters and virulence markers. Conclusion: This PCR-based genotyping method would be useful to not only characterize genetic diversity and virulence gene distribution, but also for genotyping, origin tracing and risk estimation of K. pneumoniae.
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Pathogens of the genus Klebsiella have been classified into distinct capsular (K) types for nearly a century. K typing of Klebsiella species still has important applications in epidemiology and clinical microbiology, but the serological method has strong practical limitations. Our objective was to evaluate the sequencing of wzi, a gene conserved in all capsular types of Klebsiella pneumoniae that codes for an outer membrane protein involved in capsule attachment to the cell surface, as a simple and rapid method for the prediction of K type. The sequencing of a 447-nucleotide region of wzi distinguished the K-type reference strains with only nine exceptions. A reference wzi sequence database was created by the inclusion of multiple strains representing K types associated with high virulence and multidrug resistance. A collection of 119 prospective clinical isolates of K. pneumoniae were then analyzed in parallel by wzi sequencing and classical K typing. Whereas K typing achieved typeability for 81% and discrimination for 94.4% of the isolates, these figures were 98.1% and 98.3%, respectively, for wzi sequencing. The prediction of K type once the wzi allele was known was 94%. wzi sequencing is a rapid and simple method for the determination of the K types of most K. pneumoniae clinical isolates.
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Klebsiella pneumoniae is one of the major pathogens causing hospital-acquired multidrug-resistant infections. The capsular polysaccharide (CPS) is an important virulence factor of K. pneumoniae. With 78 capsular types discovered thus far, an association between capsular type and the pathogenicity of K. pneumoniae has been observed. To investigate an initially non-typeable K. pneumoniae UTI isolate NTUH-K1790N, the cps gene region was sequenced. By NTUH-K1790N cps-PCR genotyping, serotyping and determination using a newly isolated capsular type-specific bacteriophage, we found that NTUH-K1790N and three other isolates Ca0507, Ca0421 and C1975 possessed a new capsular type, which we named KN2. Analysis of a KN2 CPS(-) mutant confirmed the role of capsule as the target recognized by the antiserum and the phage. A newly described lytic phage specific for KN2 K. pneumoniae, named 0507-KN2-1, was isolated and characterized using transmission electron microscopy. Whole-genome sequencing of 0507-KN2-1 revealed a 159 991 bp double-stranded DNA genome with a G+C content of 46.7% and at least 154 open reading frames. Based on its morphological and genomic characteristics, 0507-KN2-1 was classified as a member of the Myoviridae phage family. Further analysis of this phage revealed a 3738-bp gene encoding a putative polysaccharide depolymerase. A recombinant form of this protein was produced and assayed to confirm its enzymatic activity and specificity to KN2 capsular polysaccharides. KN2 K. pneumoniae strains exhibited greater sensitivity to this depolymerase than these did to the cognate phage, as determined by spot analysis. Here we report that a group of clinical strains possess a novel Klebsiella capsular type. We identified a KN2-specific phage and its polysaccharide depolymerase, which could be used for efficient capsular typing. The lytic phage and depolymerase also have potential as alternative therapeutic agents to antibiotics for treating K. pneumoniae infections, especially against antibiotic-resistant strains.
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Catheter associated urinary tract infections are a biofilm-mediated infection that cause a significant economic and health burden in nosocomial environments. Using a newly developed murine model of this type of infection, we investigated the role of fimbriae in implant-associated urinary tract infections by the Gram-negative bacterium Klebsiella pneumoniae, which is a proficient biofilm former and a commonly isolated nosocomial pathogen. Studies have shown that type 1 and type 3 fimbriae are involved in attachment and biofilm formation in vitro, and these fimbrial types are suspected to be important virulence factors during infection. To test this hypothesis the virulence of fimbrial mutants was assessed in independent challenges in which mice bladders were inoculated with the wild type or a fimbrial mutant, and in co-infection studies in which the wild type and fimbrial mutants were inoculated together to assess the results of a direct competition in the urinary tract. Using these experiments, we were able to show that both fimbrial types serve to enhance colonization and persistence. Additionally, a double mutant had an additive colonization defect under some conditions, indicating that both fimbrial types have unique roles in the attachment and persistence in the bladder and on the implant itself. All of these mutants were outcompeted by wild type in co-infection experiments. Using these methods we are able to show that type 1 and type 3 fimbriae are important colonization factors in the murine urinary tract when an implanted silicone tube is present.
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Klebsiella pneumoniae CG43, a heavy encapsulated liver abscess isolate, mainly expresses type 3 fimbriae. Type 1 fimbriae expression was only apparent in CG43S3ΔmrkA (the type 3 fimbriae-deficient strain). The expression of type 1 fimbriae in CG43S3ΔmrkA was reduced by deleting the fimK gene, but was unaffected by removing the 3'end of fimK encoding the C-termial EIL domain (EILfimK). Quantitative reverse-transcription PCR and promoter activity analysis showed that the putative DNA binding region at the N-terminus, but not the C-terminal EIL domain, of FimK positively affects transcription of type 1 fimbrial major subunit, fimA. An electrophoretic mobility shift assay demonstrated that the recombinant FimK could specifically bind to fimS, that is located upstream of fimA and contains a vegetative promoter for the fim operon, also reflecting possible transcriptional regulation. EILfimK was shown to encode a functional phosphodiesterase (PDE) via enhancing motility in Escherichia coli JM109 and in vitro using PDE activity assays. Moreover, EILfimK exhibited higher PDE activity than FimK implying that the N-terminal DNA binding domain may negatively affect the PDE activity of FimK. FimA expression was detected in CG43S3 expressing EILfimK or AILfimK suggesting that FimA expression is not directly influenced by the c-di-GMP level. In summary, FimK influences type 1 fimbriation by binding to fimS at the N-terminal domain, and thereafter, the altered protein structure may activate C-terminal PDE activity to reduce the intracellular c-di-GMP level.
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Klebsiella pneumoniae is an opportunistic pathogen, which causes a wide range of nosocomial infections. Recently, antibiotic resistance makes K. pneumoniae infection difficult to deal with. Investigation on virulence determinants of K. pneumoniae can provide more information about pathogenesis and unveil new targets for treatment or vaccine development. In this study, SitA, a Fur-regulated divalent cation transporter, was found significantly increased when K. pneumoniae was cultured in a nutrient-limited condition. A sitA-deletion strain (ΔsitA) was created to characterize the importance of SitA in virulence. ΔsitA showed higher sensitivity toward hydroperoxide than its parental strain. In a mouse intraperitoneal infection model, the survival rate of mice infected with ΔsitA strain increased greatly when compared with that of mice infected with the parental strain, suggesting that sitA deletion attenuates the bacterial virulence in vivo. To test whether ΔsitA strain is a potential vaccine candidate, mice were immunized with inactivated bacteria and then challenged with the wild-type strain. The results showed that using ΔsitA mutant protected mice better than using the wild-type strain or the capsule-negative congenic bacteria. In summary, SitA was found being important for the growth of K. pneumoniae in vivo and deleting sitA might be a potential approach to generate vaccines against K. pneumoniae.
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Klebsiella pneumoniae carbapenemases (KPCs) were originally identified in the USA in 1996. Since then, these versatile β-lactamases have spread internationally among Gram-negative bacteria, especially K pneumoniae, although their precise epidemiology is diverse across countries and regions. The mortality described among patients infected with organisms positive for KPC is high, perhaps as a result of the limited antibiotic options remaining (often colistin, tigecycline, or aminoglycosides). Triple drug combinations using colistin, tigecycline, and imipenem have recently been associated with improved survival among patients with bacteraemia. In this Review, we summarise the epidemiology of KPCs across continents, and discuss issues around detection, present antibiotic options and those in development, treatment outcome and mortality, and infection control. In view of the limitations of present treatments and the paucity of new drugs in the pipeline, infection control must be our primary defence for now.