Virulence and Plasmid Transferability of KPC Klebsiella pneumoniae at the Veterans Affairs Healthcare System of New Jersey

Abstract

Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae infections are associated with high mortality; however, little is known about the virulence determinants of KPC-producing K. pneumoniae. At the Veterans Affairs New Jersey Healthcare System (VA NJHCS), we investigated the virulence and plasmid transferability of 60 clinically unique KPC-containing K. pneumoniae isolates. All 60 isolates were negative for known virulence factors K1, K2, and K5 capsular antigens; rmpA; and the aerobactin gene by polymerase chain reaction. Isolates varied in their susceptibility to neutrophil phagocytosis, but were less resistant than the virulent serotype K1 isolate. Additionally, no deaths were seen on murine lethality studies. Conjugation results of this study showed that the bla_KPC gene can be transferred into an Escherichia coli J-53 strain but not to E. coli JP-995. However, the stability is very limited as E. coli J-53 does not retain bla_KPC-containing plasmids for any period of time. The lack of virulence factors in the set of KPC-producing K. pneumoniae studied suggests that morbidity and mortality may be due to detection issues or lack of effective antibiotics.

Introduction

K lebsiella pneumoniae (KP) is a pathogen associated with community-acquired and nosocomial infections.^15,25 For the past decade, KP has become increasingly resistant to conventional antibiotics. The increasing incidence of patients infected with extended-spectrum beta-lactamase (ESBL)-producing KP is driving the use of carbapenems because of their effectiveness against ESBL producers. Because carbapenems are usually the last line of defense for the treatment of resistant Gram-negative pathogens, the development of carbapenemase-producing KP is of great concern. KPC-producing KP was first reported in the United States in 2001.³¹ The rapid spread of KPC, particularly in New York, has been described as a threat, limiting treatment options.³ KPC has become an increasingly prevalent type of carbapenemase in the United States¹¹ and Israel¹⁷ and also an emerging concern in South America, Europe, and China.^{7,17, 27} KPC-producing isolates are likely to be resistant to other classes of antibiotics, limiting therapeutic options for such infections resulting in higher mortality rates and treatment failures.^10,28 Whether virulence contributes to the high attributable mortality in KPC-producing KP has not been investigated. However, in KPC-negative KP, virulence has been well documented.^25,32 K1, K2, and K5 capsular antigens along with the mucoid capsular phenotype are considered the main virulence factors in KP. Other virulence factors associated with KP include the extracapsular polysaccharide synthesis regulator gene (rmpA) related to the hypermucoviscosity phenotype, the ferric iron uptake system gene (kfu) required for the metabolism of iron to sustain growth in the host, and aerobactin, an iron siderophore that can increase murine lethality by 100-fold.^12,19,29 Although virulence determinants have been well described in KP, very little is known about the virulence of KPC-producing KP. The objective of this study is to investigate virulence factors of KPC-producing KP, the lethality in the murine model, its conjugation and plasmid stability, and neutrophilic phagocytosis rates.

Materials and Methods

From January 2007 to June 2010, 60 clinically unique, ertapenem-resistant KP isolates were acquired from the clinical microbiology laboratory at the Veteran Affairs New Jersey Health Care System (VA NJHCS) for this Institutional Review Board (IRB)-approved study (MIRB 00937). Virulence and conjugation testing was performed at the National Health Research Institutes of Taiwan.

Susceptibility testing

Antimicrobial susceptibility was determined by the broth microdilution method,¹ according to the Clinical and Laboratory Standards Institute's (CLSI) guidelines published in 2011. The following antimicrobial agents were used: cefazolin, amoxicillin/clavulanic acid, cefoxitin, cefotaxime, ceftazidime, imipenem, amikacin, gentamicin, ciprofloxacin, and trimethoprim-sulfamethoxazole.

Pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) was performed as described previously.⁹ The resulting genomic DNA profiles, or fingerprints, were interpreted according to established guidelines.^20,24

Confirmation of KPC production and KPC types by sequencing

All isolates selected were confirmed for KPC production by polymerase chain reaction (PCR). A gene-specific primer set, KPC-F (5′-ATG TCA CTG TAT CGC CGT CT-3′) and KPC-R (5′-TTT TCA GAG CCT TAC TGC CC-3′), was used to determine the presence of the KPC β-lactamase sequence.² The amplicons (893 bp) were sequenced using an automated sequencer by the Sanger method (ABI Prism 377 sequencer; Perkin-Elmer).²¹ The generated sequences were compared to the National Center for Biotechnology Information (NCBI) database at www.ncbi.nlm.nih.gov/blast/

Conjugation experiments

In this part of the study, two consecutive conjugation experiments were performed. In the first set of experiments, the sodium azide-resistant strain Escherichia coli J-53 (kindly provided by Prof. Jacoby, Division of Infectious Diseases, Lahey Clinic, Burlington, MA) or a rifampin-resistant strain of E. coli JP-995 (kindly supplied by J. Pittard, Department of Microbiology, University of Melbourne, Melbourne, Australia) was used as the recipient.^23,26 None of the donor strains or wild-type KP with KPC could grow on MacConkey agar with sodium azide or rifampin (100 μg/ml). The recipient and donor strains were inoculated separately into a brain–heart infusion broth (Oxoid) and were incubated at 37°C for 4 hours. They were then mixed together at a ratio of 1:10 (by volume) and were incubated overnight at 37°C. A 0.1-ml volume of the overnight broth mixture was then spread onto a MacConkey agar plate containing azide or rifampin (100 μg/ml) and imipenem (1 μg/ml). Transconjugants were then selected from the agar plate.

In the second phase of conjugation experiments, the transconjugant of E. coli J-53 containing the KPC plasmid acted as a donor. A serotype K2 KP with intrinsic resistance only to ampicillin was used as a recipient. Mating procedure was described as above except by using specific selection brilliant green agar-containing inositol-nitrate-deoxycholate (BIND) for KP.³⁰ Serotype K2 KP transconjugants were selected using the Klebsiella-selective medium BIND supplemented with 2 μg/ml of imipenem. None of the donors, including E. coli J-53 with KPC, recipient, and serotype K2 K. pneumoniae, could grow on the selective BIND medium with imipenem. The only colonies that could grow on BIND with imipenem were the serotype K2 KP strain harboring the imipenem-resistant KPC plasmid. Transconjugants from the first- and second-round conjugation experiments were subcultured onto MacConkey or Mueller Hinton agar (BBL) for 14 days to check for KPC plasmid stability in E. coli and KP after conjugation.

PCR detection of virulence-associated genes rmpA and aerobactin, and serotypes K1, K2, and K2

PCR experiments to determine the presence of the specific genes for serotype K1, K2, and K5; rmpA; and the aerobactin gene were performed as previously described.²⁵

Virulence studies

In vitro and in vivo assays to assess virulence were performed by neutrophil phagocytosis and mice lethality experiments. Neutrophil phagocytosis was performed as previously described.¹⁸ A serotype K1 KP (STL-43) isolated from a liver abscess was used as a control strain. This strain is hypervirulent with a median lethal dose (LD50) <10² colony forming unit (CFU) and is highly phagocytic resistance (<10% of phagocytic rate 15 minutes). Two isogenic mutants from wild-type STL-43 with capsule deficiency and became highly susceptible to phagocytosis (∼90% of phagocytic rate at 15 minutes) were also used as controls.¹⁸ For the determination of LD50 in mice, pathogen-free, adult, 6–8-week-old, male BALB/c mice weighing 20–25 g were obtained from the National Laboratory Animal Center. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal care procedures and protocols were approved by the Institutional Animal Care and Use Committee of the National Health Research Institute (NHRI-IACUC-096004-A). Tenfold serial dilutions of KP were made from a starting concentration of 10⁶ CFU, and the adult BALB/c mice had intraperitoneal injections with 0.1 ml of each concentration. Six mice were used as a sample population for each bacterial concentration.⁴ Symptoms and signs of infection were observed for 14 days. Survival of the inoculated mice was recorded, and the LD50 was calculated using SigmaPlot version 7.0 from SPSS Inc.

Results

Antimicrobial susceptibility and KPC types

Among 60 clinically unique KPC-positive KP isolates, 100% resistance was found to piperacillin–tazobactam, cefazolin, cefotaxime, ceftazidime, ceftriaxone, and ciprofloxacin followed by 92% resistance to cefoxitin and 33% to gentamicin. Reduced susceptibility to imipenem and/or meropenem was seen in 95% of the isolates. Sequencing results showed two types of KPC (17 KPC-2 and 43 KPC-3 isolates).

Conjugation and bla_KPC plasmid stability test

On day 1 of conjugation experiments, none of the 13 rifampin-susceptible KP strains, including 7 KPC-2 and 6 KPC-3 isolates, could transfer the bla_KPC plasmid into recipient E. coli JP-995. On the contrary, plasmids carrying bla_KPC from a parental nonserotype K2 KP, in all 60 isolates, were successfully transferred into sodium azide-resistant E. coli J-53 on the day-1 conjugation experiment. E. coli J-53::bla_KPC transconjugants were obtained from the MacConkey agar plate containing azide (100 μg/ml) and imipenem (2 μg/ml) (Fig. 1A). Subculture E. coli J-53::bla_KPC into the same selective plates showed no growth of E. coli J-53::bla_KPC transconjugants, suggesting the loss of bla_KPC or instability of the bla_KPC plasmid in E. coli (Fig. 1B). By using E. coli J-53::bla_KPC transconjugants obtained directly from the day-1 selective agar plate for conjugation with recipient serotype K2 KP, transconjugants of serotype K2 K. pneumoniae::bla_KPC were obtained by the Klebsiella-selective medium BIND supplemented with 2 μg/ml of imipenem (Fig. 1C). In contrast to E. coli J-53:: bla_KPC, subculture of serotype K2 K. pneumoniae :: bla_KPC onto the same selective agar plate showed good growth in the same selective BIND agar plate (Fig. 1D). No plasmid loss after 14 days of subculture onto the MacConkey or Muller Hinton agar plate was observed for K2 K. pneumoniae :: bla_KPC, indicating the stability of the bla_KPC plasmid in KP (Fig. 1E). All strains were verified by PCR to ensure that no contamination occurred during each experiment for donor, recipient of E. coli J-53, transconjugants of E. coli J-53::bla_KPC, recipient of serotype K2 KP, or transconjugants K2 K. pneumoniae::bla_KPC (Fig. 1F).

FIG. 1.

Conjugation experiments of plasmids carrying bla_KPC. (A) Transconjugants, Escherichia coli J-53:: bla_KPC, were obtained on day 1 using MacConkey agar plates containing azide (100 μg/ml) and imipenem (2 μg/ml); (B) subculture of E. coli J-53:: bla_KPC by inoculating into the same selective agar plate; (C) second round of conjugation experiments using E. coli J-53:: bla_KPC obtained directly from the day-1 selective agar procedure and recipient serotype K2 Klebsiella pneumoniae. Transconjugants, K2 K. pneumoniae :: bla_KPC, were obtained by the Klebsiella-selective medium BIND supplemented with 2 μg/ml of imipenem; (D) subculture transconjugants K2 K. pneumoniae :: bla_KPC onto the same selective agar plate. (E) Subculture of K2 K. pneumoniae :: bla_KPC by inoculating onto MacConkey agar plates for 14 consecutive days; (F) PCR verification of donor K. pneumoniae with bla_KPC (lane 1), transconjugants E. coli J-53:: bla_KPC (lane 2), recipient serotype K2 K. pneumoniae (lane 3), and transconjugants K2 K. pneumoniae :: bla_KPC (lane 4).

Detection of virulence-associated determinants, neutrophil phagocytosis, and mice lethality experiments

Detection of K1, K2, and K5 virulence-associated serotypes, and rmpA and aerobactin genes, revealed that all 60 isolates were non-K1, -K2, or -K5 serotypes and did not carry rmpA and aerobactin genes. Twenty-five isolates were selected for neutrophil phagocytosis assays based on their diversity of PFGE profiles. These isolates varied in neutrophil phagocytic resistance (Fig. 2). Seven isolates were phagocytic resistant as determined by the phagocytosis rate calculated using the percentage of neutrophil-ingested fluorescein isothiocyanate-labeled bacteria over time. Phagocytic rates around 25%–35% at 15 minutes and the susceptible phagocytic rate >40% at 15 minutes, respectively, were selected for further mice lethality studies. Murine lethality studies by IP injection showed that LD50 were >10⁶ CFU for all seven isolates, indicating low virulence. In contrast, LD50 of the known virulent strain STL-43 was <10² CFU.

FIG. 2.

Phagocytosis assay for randomly selected bla_KPC isolates. The phagocytosis rate was calculated using the percentage of neutrophil-ingested fluorescein isothiocyanate-labeled bacteria over the time. The bla_KPC isolates are labeled as filled diamonds. Serotype K1 isolates obtained from previous studies³⁰ were used as a control for virulence assessment. STL-43 (open triangle) was obtained from patients with liver abscess. STL43ΔrfbP (open rectangle) and STL43ΔWzy_KPK1 (open circle) were capsule-deficient nonpolar-rfbP and -Wzy_KPK1 mutant strains derived from STL-43, respectively.⁴

Discussion

KPC KP has become an increasing prevalent pathogen and is now endemic in the Northeastern United States.^3,5,29 Clinical mortality due to KPC KP has been well documented.¹¹ However, virulence and plasmid transmissibility of KPC KP have not been well investigated. The findings in this study hope to increase the understanding of virulence and plasmid transmissibility of KPC-producing KP. KPC-producing KP infections are usually associated with resistance to multiple classes of antibiotics, and treatment options are very limited. Furthermore, testing for KPC has been problematic.¹⁴ Improper therapy may contribute to the increased morbidity and mortality seen in KPC KP infections.

After probing for known virulence factors, we found that these 60 KPC KP isolates did not contain genes for K1, K2, and K5 capsular antigens; rmpA; or the aerobactin gene. We selected these virulence markers as these have been found in hypervirulent strains of KP associated with liver abscess and metastatic pyogenic infections.²² In addition, intraperitoneal injections of KPC KP in murine lethality models did not cause mortality even at a very high dose of >10⁶ CFU of KP. The combination of the lack of KP virulence factors and no deaths seen in murine models suggests that mortality in KPC-producing KP infections may not be attributed to increased virulence.

Transmissibility of plasmids containing the bla_KPC gene to other Enterobacteriaceae has also been of great concern. Although the conjugation results of this study showed that the bla_KPC gene can be transferred into the E. coli J-53 strain, its stability is very limited as E. coli J-53 does not seem to be able to retain bla_KPC for any period of time. In addition, we were not able to transfer the bla_KPC gene into E. coli JP-995. The bla_KPC-containing plasmid is conjugable, but was most stable on a species-specific level. These results seem to agree with what we see at the VA NJHCS. Since the first reporting of KPC KP at VA NJHCS in 2007, we have not seen carbapenem-resistant E. coli. However, although rare, E. coli containing bla_KPC has been documented.¹⁶ This may be due to the difference in the strains of E. coli. A previous study has shown that stability and the frequency of plasmid conjugal transfer are varied due to genetic elements such as replicons and transposons.¹ The KPC gene is located in a Tn3-based transposon (Tn4401), which explains the rapid spread among Enterobacteriaceae.⁸ Cuzon et al. were able to demonstrate the great efficiency of integration and transpostion of Tn4401 among different plasmids without insertion-site specificity.⁸ In this study, transmission of the KPC plasmid could be obtained in the first day of co-culturing the donor KP and recipient E. coli. However, losing carbapenem resistance by single colony subculture of the transconjugant into the same medium after 24 hours suggests the lack of genetic elements required for maintaining the stability of the KPC plasmid in recipient E. coli. Further investigation on the transmissibility of these KPC-containing E. coli needs to be done.

An important finding in this study is that the KPC-producing KP does not have known virulence factors found in KP strains causing liver abscesses in Asia; however, other virulence factors have been recently described. Adhesins, biofilm production, and heat resistance (the clpK gene) have been associated with increased pathogenicity in K. pneumoniae isolates.¹³ Fuursted et al. evaluated the presence of virulence factors in a single strain of carbapenemase-producing KP carrying the New-Delhi metallo-beta-lactamase-1 (NDM-1) and compared it to noncarbapenemase-producing strains of KP.¹³ The NDM-1-producing KP was found to be a K2 serotype and a biofilm producer. In addition, the isolate was more virulent than the comparative strains in the murine sepsis model. We did not investigate these other potential virulence factors; however, no murine deaths on lethality studies suggest that mortality due to KPC KP is not due to increased virulence. Other contributing factors may include advanced age, procedures, and line placement, among other reported risk factors. However, further dissemination of the KPC plasmid into virulent types of KP cannot be excluded in the future. Efforts to assess the factors that contribute to attributable mortality in KPC KP infections should be continued, including the study of other virulence determinants, antibiotic resistance, host responses, and their relationship with clinical characteristics and outcomes.

Footnotes

Acknowledgment

This work was supported by grants from the National Science Council (NSC-98-2314-B-016-024-MY3), Taiwan Center for Diseases Control (DOH-100-DC-1025), and from the National Health Research Institutes, Taiwan.

Disclosure Statement

No competing financial interests exist.

References

Ambrozic

, Ostroversnik

, Starcic

, Kuhar

, Grabnar

et al. 1998. Escherichia coli CoIV plasmid pRK100: genetic organization, stability and conjugal transfer. Microbiology, 144,(Pt 2):343–352.

Bradford

P.A.

, Bratu

, Urban

, Visalli

, Mariano

et al. 2004. Emergence of carbapenem-resistant Klebsiella species possessing the class A carbapenem-hydrolyzing KPC-2 and inhibitor-resistant TEM-30 beta-lactamases in New York City. Clin. Infect. Dis., 39:55–60.

Bratu

, Landman

, Haag

, Recco

, Eramo

et al. 2005. Rapid spread of carbapenem-resistant Klebsiella pneumoniae in New York City: a new threat to our antibiotic armamentarium. Arch Intern. Med., 165:1430–1435.

Chen

J.H.

, Siu

L.K.

, Fung

C.P.

, Lin

J.C.

, Yeh

K.M.

et al. 2010. Contribution of outer membrane protein K36 to antimicrobial resistance and virulence in Klebsiella pneumoniae. J. Antimicrob. Chemother., 65:986–990.

Chiang

, Mariano

, Urban

, Colon-Urban

, Grenner

et al. 2007. Identification of carbapenem-resistant klebsiella pneumoniae harboring KPC enzymes in New Jersey. Microb. Drug. Resist., 13:235–239.

[CLSI] Clinical, Laboratory Standards Institute. 2011. Performance standards for antimicrobial susceptibility testing; 20th informational supplement M100-S17. Clinical and Laboratory Standards Institute: Wayne, PA.

Cuzon

, Naas

, Demachy

M.C.

, Nordmann

2008. Plasmid-mediated carbapenem-hydrolyzing beta-lactamase KPC-2 in Klebsiella pneumoniae isolate from Greece. Antimicrob. Agents Chemother., 52:796–797.

Cuzon

, Naas

, Nordmann

2011. Functional characterization of Tn4401, a Tn3-based transposon involved in blaKPC gene mobilization. Antimicrob. Agents Chemother., 55:5370–5373.

D'Agata

E.M.

, Gerrits

M.M.

, Tang

Y.W.

, Samore

, Kusters

J.G

. 2001. Comparison of pulsed-field gel electrophoresis and amplified fragment-length polymorphism for epidemiological investigations of common nosocomial pathogens. Infect. Control. Hosp. Epidemiol., 22:550–554.

10.

Elemam

, Rahimian

, Mandell

2009. Infection with panresistant Klebsiella pneumoniae: a report of 2 cases and a brief review of the literature. Clin. Infect. Dis., 49:271–274.

11.

Endimiani

, Hujer

A.M.

, Perez

, Bethel

C.R.

, Hujer

K.M.

et al. 2009. Characterization of blaKPC-containing Klebsiella pneumoniae isolates detected in different institutions in the Eastern USA. J. Antimicrob. Chemother, 63:427–437.

12.

Fang

C.T.

, Chuang

Y.P.

, Shun

C.T.

, Chang

S.C.

, Wang

J.T.

2004. A novel virulence gene in Klebsiella pneumoniae strains causing primary liver abscess and septic metastatic complications. J. Exp. Med., 199:697–705.

13.

Fuursted

, Schøler

, Hansen

, Dam

, Bojer

M.S.

et al. 2011. Virulence of a Klebsiella pneumoniae strain carrying the New Delhi metallo-beta-lactamase-1 (NDM-1) Microbes. Infect., 14:155–158.

14.

Gomez

, Urban

, Mariano

, Colon-Urban

, Eng

R.H.

et al. 2011. Phenotypic and Genotypic Screening and Clonal Analysis of Carbapenem-Resistant Klebsiella pneumoniae at a Single Hospital. Microb. Drug. Resist., 17:251–257.

15.

Keynan

, Rubinstein

2007. The changing face of Klebsiella pneumoniae infections in the community. Int. J. Antimicrob. Agents, 30:385–389.

16.

Landman

, Urban

, Backer

, Kelly

, Shah

et al. 2010. Susceptibility profiles, molecular epidemiology, and detection of KPC-producing Escherichia coli isolates from the New York City vicinity. J. Clin. Microbiol., 48:4604–4607.

17.

Leavitt

, Navon-Venezia

, Chmelnitsky

, Schwaber

M.J.

, Carmeli

2007. Emergence of KPC-2 and KPC-3 in carbapenem-resistant Klebsiella pneumoniae strains in an Israeli hospital. Antimicrob. Agents Chemother., 51:3026–3029.

18.

Lin

J.C.

, Chang

F.Y.

, Fung

C.P.

, Xu

J.Z.

, Cheng

H.P.

et al. 2004. High prevalence of phagocytic-resistant capsular serotypes of Klebsiella pneumoniae in liver abscess. Microbes. Infect., 6::1191–1198.

19.

L.C.

, Fang

C.T.

, Lee

C.Z.

, Shun

C.T.

, Wang

J.T

. 2005. Genomic heterogeneity in Klebsiella pneumoniae strains is associated with primary pyogenic liver abscess and metastatic infection. J. Infect. Dis., 192:117–128.

20.

McDougal

L.K.

, Steward

C.D.

, Killgore

G.E.

, Chaitram

J.M.

, McAllister

S.K.

et al. 2003. Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database. J. Clin. Microbiol., 41:5113–5120.

21.

Sanger

, Air

G.M.

, Barrell

B.G.

, Brown

N.L.

, Coulson

A.R.

et al. 1977. Nucleotide sequence of bacteriophage phi×174 DNA. Nature, 265:687–695.

22.

Siu

L.K.

, Fung

C.P.

, Chang

F.Y.

, Lee

, Yeh

K.M.

et al. 2011. Molecular Typing and Virulence Analysis among Serotype K1 Klebsiella pneumoniae Isolated from Liver Abscess Patients and Stool Carriage from Non-Infectious Subjects in Hong Kong, Singapore and Taiwan. J. Clin. Microbiol., 49:3761–3765.

23.

Siu

L.K.

, Ho

P.L.

, Yuen

K.Y.

, Wong

S.S.

, Chau

P.Y.

1997. Transferable hyperproduction of TEM-1 beta-lactamase in Shigella flexneri due to a point mutation in the pribnow box. Antimicrob. Agents Chemother., 41:468–470.

24.

Tenover

F.C.

, Arbeit

R.D.

, Goering

R.V.

, Mickelsen

P.A.

, Murray

B.E.

et al. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol., 33:2233–2239.

25.

Turton

J.F.

, Baklan

, Siu

L.K.

, Kaufmann

M.E.

, Pitt

T.L.

2008. Evaluation of a multiplex PCR for detection of serotypes K1, K2 and K5 in Klebsiella sp. and comparison of isolates within these serotypes. FEMS Microbiol. Lett., 284:247–252.

26.

Wang

, Tran

J.H.

, Jacoby

G.A.

, Zhang

, Wang

et al. 2003. Plasmid-mediated quinolone resistance in clinical isolates of Escherichia coli from Shanghai, China. Antimicrob. Agents Chemother., 47:2242–2248.

27.

Wei

Z.Q.

, Du

X.X.

, Yu

Y.S.

, Shen

, Chen

Y.G.

et al. 2007. Plasmid-mediated KPC-2 in a Klebsiella pneumoniae isolate from China. Antimicrob. Agents Chemother., 51:763–765.

28.

Weisenberg

S.A.

, Morgan

D.J.

, Espinal-Witter

, Larone

D.H.

2009. Clinical outcomes of patients with Klebsiella pneumoniae carbapenemase-producing K. pneumoniae after treatment with imipenem or meropenem. Diagn. Microbiol. Infect. Dis., 64:233–235.

29.

Woodford

, Tierno

P.M.

Jr. , Young

, Tysall

, Palepou

M.F.

et al. 2004. Outbreak of Klebsiella pneumoniae producing a new carbapenem-hydrolyzing class A beta-lactamase, KPC-3, in a New York Medical Center. Antimicrob. Agents Chemother., 48:4793–4799.

30.

Yeh

K.M.

, Lin

J.C.

, Yin

F.Y.

, Fung

C.P.

, Hung

H.C.

et al. 2010. Revisiting the importance of virulence determinant magA and its surrounding genes in Klebsiella pneumoniae causing pyogenic liver abscesses: exact role in serotype K1 capsule formation. J. Infect. Dis., 201:1259–1267.

31.

Yigit

, Queenan

A.M.

, Anderson

G.J.

, Domenech-Sanchez

, Biddle

J.W.

et al. 2001. Novel carbapenem-hydrolyzing beta-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrob. Agents Chemother., 45:1151–1161.

32.

W.L.

, Ko

W.C.

, Cheng

K.C.

, Lee

C.C.

, Lai

C.C.

et al. 2008. Comparison of prevalence of virulence factors for Klebsiella pneumoniae liver abscesses between isolates with capsular K1/K2 and non-K1/K2 serotypes. Diagn. Microbiol. Infect. Dis., 62:1–6.

Virulence and Plasmid Transferability of KPC Klebsiella pneumoniae at the Veterans Affairs Healthcare System of New Jersey

Abstract

Introduction

Materials and Methods

Susceptibility testing

Pulsed-field gel electrophoresis

Confirmation of KPC production and KPC types by sequencing

Conjugation experiments

PCR detection of virulence-associated genes rmpA and aerobactin, and serotypes K1, K2, and K2

Virulence studies

Results

Antimicrobial susceptibility and KPC types

Conjugation and blaKPC plasmid stability test

Detection of virulence-associated determinants, neutrophil phagocytosis, and mice lethality experiments

Discussion

Footnotes

Acknowledgment

Disclosure Statement

References

Conjugation and bla_KPC plasmid stability test