Transcript Slides

Evolution of Klebsiella pneumoniae
Klebsiella pneumoniae is a Gramnegative, non-motile, encapsulated
(coated in polysaccharide), lactosefermenting, facultative anaerobic (able to
make ATP in presence of O2, but
converts to fermentation in anaerobic
conditions), rod-shaped bacterium.
Although found in the normal flora of the
mouth, skin, and intestines, it can cause
destructive changes to human lungs if
aspirated.
In clinical settings, it is the most significant member of the Klebsiella genus of
Enterobacteriaceae.
Klebsiellae have become important pathogens in hospital-acquired (nosocomial)
infections.
Klebsiella pneumoniae resistant strains
Klebsiella organisms with the ability to produce extended-spectrum blactamases (ESBL) are resistant to many antibiotics, including:
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All b-lactams (narrow and broad spectrum inhibitors of cell wall biosynthesis)
Aminoglycosides (target protein synthesis in gram-negative bacteria)
Fluoroquinolones (broad spectrum disruption of replication)
Tetracyclines (broad spectrum disruption of protein synthesis)
Chloramphenicol (broad spectrum disruption of protein synthesis)
Trimethoprim (DHFR inhibitor)
Sulfamethoxazole (Dihydropteroate synthetase inhibitor)
One of many carbapenem-resistant Enterobacteriaceae (CRE) is CarbapenemResistant Klebsiella pneumoniae (CRKP).
It was first described in North Carolina in 1996; since then CRKP has been
identified in 41 states. It is now the most common CRE species encountered
within the United States.
Over the past 10 years, a progressive increase in CRKP has been seen
worldwide.
Klebsiella pneumoniae spread
CRE spread worldwide
SUBURBAN CHICAGO
Klebsiella pneumoniae resistant strains
The most important mechanism of resistance by CRKP is the production of a
carbapenemase enzyme, blakpc.
The gene that encodes the blakpc enzyme is carried on a mobile piece of genetic
material (a transposon that can jump from chromosome to plasmid and back
called Tn4401, which increases the risk for dissemination.
CRE can be difficult to detect because some strains that harbor blakpc have
minimal inhibitory concentrations (MICs) that are elevated but still within the
susceptible range for carbapenems.
Because these strains are susceptible to carbapenems, they are not identified
as potential clinical or infection control risks using standard susceptibility testing
guidelines.
Patients with unrecognized CRKP colonization have been reservoirs for
transmission during nosocomial outbreaks.
KPC
Ten variants, KPC-2 through KPC-11 are
known, and they are distinguished by one
or two amino acid substitutions (KPC-1
was resequenced in 2008 and found to
be 100% identical to published
sequences of KPC-2).
KPC-1 was found in North Carolina, KPC2 in Baltimore and KPC-3 in New York.
They have only 45% homology with SME
and NMC/IMI enzymes and, unlike them,
can be encoded by self-transmissible
plasmids.
The class A Klebsiella pneumoniae
carbapenemase (KPC) is currently the
most common carbapenemase.
The sequence of KPC is quite different from other BLs
G (2a)
SHV-1 (2b)
TEM-1 (2b)
TEM-52 (2be)
NMC-A (2f)
SME-1 (2f)
KPC (2f)
KPC (2f)
SME-1 (2f)
NMC-A (2f)
TEM-52 (2be)
TEM-1 (2b)
SHV-1 (2b)
G (2a)
The sequence of KPC is quite different from other BLs
But its structure is fairly similar
G (2a)
SHV-1 (2b)
TEM-1 (2b)
TEM-52 (2be)
NMC-A (2f)
SME-1 (2f)
KPC (2f)
G (2a)
SHV-1 (2b)
TEM-1 (2b)
TEM-52 (2be)
NMC-A (2f)
SME-1 (2f)
KPC (2f)
KPC
SME-1
NMC-A
TEM-52
TEM-1
SHV-1
G
But its structure is fairly similar
Significance
Carbapenem-resistant Klebsiella pneumoniae has emerged globally as a
multidrug-resistant hospital pathogen for which there are few treatment options.
Clinical isolates classified by multilocus sequence typing (ST) as ST258 are the
most widespread.
The basis for the success of ST258 organisms above and beyond antibiotic
resistance is not known, nor is it clear whether infections are caused by a single
clone.
We used genome sequencing to reveal unexpected genetic diversity among
ST258 organisms (thus disproving the single-clone hypothesis) and identified a
recombination hotspot that accounts for the majority of divergence—and
presumably for serologic variation—among ST258 clinical isolates.
Our findings will facilitate the development of new clinical strategies designed to
prevent or treat infections caused by multidrug-resistant K. pneumoniae.
(more of the same) info on K. pneumonia
There is very high mortality (∼30–70%) among patients with bacteremia (blood)
or pulmonary (lung) infections caused by carbapenemase-producing K.
pneumoniae.
The large majority of these isolates are characterized genetically as multilocus
sequence type 258 (ST258) and are presumed to be clonally related by
descent.
Strains of ST258 are resistant to all β-lactam antibiotics and typically contain
plasmid-borne genes that encode aminoglycoside-modifying enzymes and
chromosomal mutations that confer fluoroquinolone resistance.
Carbapenem resistance in ST258 strains is predominantly encoded by K.
pneumoniae carbapenemase (KPC), and the gene, blakpc, is located on a
transposable element (Tn4401) that is integrated into many different plasmids.
Thus, blakpc (along with other plasmid-associated resistance elements) is
exchanged readily among K. pneumoniae and other Enterobacteriaceae, a
feature key to the spread and high prevalence of multidrug-resistant strains.
Multilocus sequence typing
Multilocus sequence typing (MLST) is a technique in molecular biology for the
typing of multiple loci.
The procedure characterizes isolates of microbial species using the DNA
sequences of internal fragments of multiple housekeeping genes.
Approximately 450-500 bp internal fragments of each gene are used, as these
can be accurately sequenced on both strands using an automated DNA
sequencer.
For each housekeeping gene, the different sequences present within a bacterial
species are assigned as distinct alleles and, for each isolate, the alleles at each
of the loci define the allelic profile or sequence type (ST).
Transmissibility and approach
Infections caused by CRKP occur primarily in individuals who have significant
clinical risk factors for acquiring these pathogens.
These attributes suggest that disease is caused by poor/defective host defense
rather than by enhanced bacterial virulence or transmissibility.
However, there are no data that bear on these issues, nor is there a clear
explanation for the predominance of ST258 globally.
We sequenced to closure the genomes of two KPC-producing ST258 K.
pneumoniae clinical isolates, and then performed comparative whole-genome
sequencing of 83 additional KP clinical isolates obtained from human infections
at diverse geographic locations worldwide.
Our data provide information that is important for our understanding of the
success of ST258 as a human pathogen, extending beyond antibiotic
resistance.
Transmissibility and approach
The two reference ST258 KP clinical isolates, one with mucoid and the other
with nonmucoid colony morphology, were obtained in 2010 from patients with
urinary tract infections at two separate healthcare institutions in New Jersey.
(Note: mucoid indicates a colony showing viscous or sticky growth typical of an
organism producing large quantities of a carbohydrate capsule.)
The genome sizes were 5,266,518 bp for the isolate named NJST258_1 and
5,293,301 bp for the isolate named NJST258_2, which is similar in length to
other K. pneumoniae genomes. Selected K. pneumoniae genomes were
compared with the two ST258 genomes, NJST258_1 and NJST258_2.
NJST258_1 has 8 putative prophages, 22 insertion sequences (IS), and 2
integrated conjugative elements (ICE). NJST258_2 lacks one of these
prophages (phage 6) and has three fewer IS, but the mobile genetic element
(MSG) content otherwise is similar to that of NJST258_1.
Whether the prophages or other MGEs present in ST258 lineage have
contributed to its recent success remains to be determined.
K. pneumoniae genome comparisons
Plasmid and chromosome-encoded antibiotic resistance
NJST258_1 and NJST258_2:
Are resistant to β-lactam antibiotics (including carbapenems) and β-lactamase
inhibitors (e.g., clavulanate and tazobactam), quinolones (ciprofloxacin and
levofloxacin), and aminoglycosides (amikacin and tobramycin).
NJST258_1:
Is also resistant to trimethoprim-sulfamethoxazole, gentamicin, minocycline,
colistin, and polymyxin B, but is susceptible to tigecycline.
Many of the antibiotic-resistance determinants are encoded on plasmids in
these two strains.
Tigecycline
Tigecycline is a glycylcycline antibiotic
developed by Francis Tally and marketed
by Wyeth under the brand name Tygacil.
It was given a U.S. Food and Drug
Administration (FDA) fast-track approval
and was approved on June 17, 2005.
It was developed in response to the growing prevalence of antibiotic resistance
in bacteria. The NDM-1 and KPC multidrug-resistant Enterobacteriaceae have
shown susceptibility to tigecycline.
It is structurally similar to the tetracyclines (which is an inhibitor of protein
synthesis), but has a substitution at the D-9 position which is believed to confer
broad spectrum activity.
Tigecycline is bacteriostatic and is a protein synthesis inhibitor by binding to the
30S ribosomal subunit of bacteria and thereby blocking entry of Aminoacyl-tRNA
into the A site of the ribosome during prokaryotic translation.
Plasmid and chromosome-encoded antibiotic resistance
Contain 5 (NJST258_1) vs 3 plasmids (NJST258_2) that vary in size from
10,925–142,768 bp and collectively encode resistance to multiple classes of
antibiotics and heavy metals.
The 3 plasmids identified in
NJST258_2 collectively
contained fewer antibiotic
and/or heavy metal
resistance determinants
than did the plasmids in
strain NJST258_1.
Plasmid and chromosome-encoded antibiotic resistance
The quinolone resistance-determining
regions in both genomes, including
gyrA, gyrB, parC, and parE genes,
contained several amino acid
replacements that likely explain
quinolone resistance in these strains.
Taken together, the data indicate that
NJST258_1 and NJST258_2 are
carbapenem-resistant K. pneumoniae
clinical isolates that encode
extensive resistance to other
classes of antibiotics, resulting in
the critical multidrug resistance
phenotype.
DNA Gyrase
Mutations observed in gyrase genes:
NJST258_1: S83I and D87G in GyrA,
and S80I in ParC
NJST258_2: S83I and D87Q in GyrA,
and S80I in ParC
The list of resistance genes in ST258 is staggering
Genome sequencing
To gain a better understanding of the emergence and evolution of the ST258
clone, we next performed whole-genome DNA sequencing of 83 additional
KP clinical isolates, and mapped the DNA sequence reads to reference strain
NJST258_1.
Isolates were selected based on the geographic and temporal distributions, KPC
variants, and Tn4401 patterns.
These clinical isolates were cultured from blood, urine, sputum, or skin of
infected individuals or from a rectal swab over a time period encompassing
2003–2012.
Isolates were obtained from healthcare facilities at diverse geographic locations
in the United States (Florida, Illinois, Pennsylvania, New Jersey, New York, and
Texas), Canada, Colombia, and Italy.
Seventy-five (89%) of these strains were ST258. The others were ST379 (4),
ST512 (4), or ST418 (1), which are all single-locus variants of ST258.
Genome sequencing
There were 2,436 unique SNPs in the core genome (defined here as all
nucleotide positions except those in plasmids and MGE) of all 84 isolates
(including NJST258_2) compared with reference isolate NJST238_1.
The 84 query isolates differed from NJST258_1 on average by 350 SNPs
(range, 116–784 SNPs) in the core genome, indicating that the isolates are
closely related. However, in comparison, we previously reported less core
genome genetic diversity—an average of ∼50 SNPs—among isolates of the
epidemic community-associated MRSA.
Of the 2,436 unique SNPs among all isolates, 310 (12.7%) were intergenic, 881
(36.2%) were synonymous, and 1,245 (51.1%) were nonsynonymous.
Phylogenetic tree
A phylogenetic tree, or evolutionary
tree, is a branching diagram or "tree"
showing the inferred evolutionary
relationships among various biological
species or other entities—their
phylogeny—based upon similarities and
differences in their physical or genetic
characteristics.
The taxa joined together in the tree are
implied to have descended from a
common ancestor.
Phylogenetic analysis in K. pneumoniae ST258
To estimate genetic relationships, we performed phylogenetic analyses using
concatenated SNP nucleotides present in the core genome. All isolates
segregated into two distinct subclades.
Isolates within clade 1 differed on average by 136 SNPs, and those within clade
2 differed by an average of 82 SNPs. In comparison, the two clades differed
from each other by an average of 529 SNPs.
96.3% of the KPC-producing isolates within clade 1 primarily contained
plasmids encoding KPC2, whereas 96.2% of those in clade 2 encoded KPC3.
The data indicate that the two lineages have had distinct evolutionary
histories and thus emerged independently. These findings were unexpected,
because the ST258 lineage has been considered to be a single clone or strain.
Concatenated SNP nucleotide sequences
Initial
alignment
Seq1
Seq2
Seq3
Seq4
ACTGTAAAGC…
ACTGTCCAGC…
AGGGTTCAGC…
ACGGTGCAGC…
Masked
alignment
Seq1
Seq2
Seq3
Seq4
xCTxxAAxxx…
xCTxxCCxxx…
xGGxxTCxxx…
xCGxxGCxxx…
Concatenated
SNP alignment
Seq1
Seq2
Seq3
Seq4
CTAA
CTCC
GGTC
CGGC
Divergence within K. pneumoniae ST258
To better understand the molecular
processes that underlie the distinct
evolution of clades 1 and 2, we
analyzed the distribution of SNPs in
specific genes and/or regions of the
core genome. Unexpectedly, we found
an ∼215-kbp region of the ST258
genome that had a disproportionate
number of SNPs.
Core phylogeny (-RD)
Phylogenetic analysis of the core
genome minus this region of
divergence (RD) and analysis of the
RD alone revealed that the genetic
differentiation in the core genomes
between the two clades is largely
attributed to SNPs in the RD.
RD chromosomal segment
RD phylogeny
Capsule polysaccharide
Capsule polysaccharide (CPS) is a well-known contributor to the success of K.
pneumoniae as a pathogen, in part because the variation in genes that encode
enzymes involved in CPS synthesis results in serologic diversity.
Notably, the CPS gene island, which encodes the capsule K-antigen in K.
pneumoniae, is located within the RD. Currently, more than 78 different Kserotypes have been identified, some of which are significantly associated with
serious human infections.
Unexpectedly, we identified two distinct cps gene clusters in the genomes of the
ST258 clinical isolates (ST258 cps 1 in clade 1 and cps 2 in clade 2).
The genetic differences between these two cps clusters are primarily
responsible for the segregation of ST258 isolates into two distinct clades.
Capsule polysaccharide
Concluding remarks
CRE—most notably ST258 K. pneumoniae—now are classified as an “Urgent
Threat” by the CDC.
Although the absolute number of estimated deaths caused by CRE is relatively
low (in the United States 610 deaths annually versus 11,000 caused by MRSA
and 14,000 caused by C. difficile), the potential exists for transfer of multidrug
resistance to Gram-negative community-associated pathogens such as E. coli.
As suggested by the CDC report on antibiotic resistance threats, a multi-tiered
approach is needed to prevent or moderate the threat posed by CRE.
Such an approach includes understanding the evolutionary genomic history of
the microbe as a means to gain new insight into its success as a human
pathogen, as we have done herein.
Unexpectedly, we discovered that ST258 is comprised of two distinct lineages or
clades rather than being a single clone as previously suggested, which are
differentiated largely by an RD that encodes CPS biosynthesis machinery.
Thus, independent acquisition of genetic material by an ancestral ST258
clone (rather than within-ST258 diversification) is the primary basis for
segregation of into two clades.
Concluding remarks
CRE—most notably ST258 K. pneumoniae—now are classified as an “Urgent
Threat” by the CDC.
Although the absolute number of estimated deaths caused by CRE is relatively
low (in the United States 610 deaths annually versus 11,000 caused by MRSA
and 14,000 caused by C. difficile), the potential exists for transfer of multidrug
resistance to Gram-negative community-associated pathogens such as E. coli.
As suggested by the CDC report on antibiotic resistance threats, a multi-tiered
approach is needed to prevent or moderate the threat posed by CRE.
Such an approach includes understanding the evolutionary genomic history of
the microbe as a means to gain new insight into its success as a human
pathogen, as we have done herein.
Unexpectedly, we discovered that ST258 is comprised of two distinct lineages or
clades rather than being a single clone as previously suggested, which are
differentiated largely by an RD that encodes CPS biosynthesis machinery.
Thus, independent acquisition of genetic material by an ancestral ST258
clone (rather than within-ST258 diversification) is the primary basis for
segregation of into two clades.