Journal of Infectious Diseases Advance Access published March 14, 2014

1  Carbapenem-Resistant Klebsiella pneumoniae exhibit variability in

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capsular polysaccharide and capsule associated virulence traits

E. Diago-Navarro1, L. Chen2, V. Passet3,4, S. Burack1, A. Ulacia-Hernando1, R. P. Kodiyanplakkal1, M. H. Levi5, S. Brisse3,4, B. N Kreiswirth2, B. C.

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Dept of Medicine Infectious Disease Division Albert Einstein College of

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Medicine and Montefiore Medical Center, Bronx, NY, USA

Public Health Research Institute Tuberculosis Center, NJMS-Rutgers

University, Newark, NJ, USA

Institut Pasteur, Microbial Evolutionary Genomics, Paris, France

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CNRS, UMR3525, Paris, France

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Dept of Clinical Microbiology Montefiore Medical Center, Bronx, New York

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Address correspondence to: Bettina C. Fries, M.D. Tel: 718-430-2365. Fax:

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718-430-8968. E-mail: [email protected]

© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of  America. All rights reserved. For Permissions, please e‐mail: [email protected]

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Fries1,#

2  ABSTRACT BACKGROUND. Novel therapies are urgently needed to treat Carbapenem-

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resistant Klebsiella pneumoniae (CR-Kp)-mediated infection, which constitute a major health threat in the US. In order to assess if it is feasible to develop anti-capsular antibodies as a potential novel therapy it is crucial to first

systematically characterize capsular polysaccharide (CPS) and virulence traits

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METHODS. Forty CR-Kp were genotyped by Pulsed Field Gel Electrophoresis, Multilocus Sequence typing (MLST) and molecular capsule typing (C-patterns

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and wzi sequencing). Their biofilm formation, serum resistance, macrophagemediated killing, and virulence in Galleria mellonella were compared. MAb

was investigated.  

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(1C9) was generated by co-immunization with two CPSs and cross-reactivity

RESULTS. MLST assigned 80% of CR-Kp isolates to the ST258-clone.

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Molecular capsule typing identified new C-patterns; including C200/wzi-154, which was widely represented and associated with blaKPC-3-bearing strains. Heterogeneity was detected in biofilm formation and macrophage-mediated killing. Differences in serum resistance correlated with virulence in G.

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mellonella. ST258 strains carrying blaKPC-3 were less virulent than those with

blaKPC-2. MAb 1C9 cross-reacted with 58% of CR-Kp CPSs.

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CONCLUSIONS. CR-Kp ST258 strains exhibit variability of virulenceassociated traits. Differences were associated with the type of KPC gene and CPS. Identification of cross-reacting anti-CPS mAbs encourages their development as adjunctive therapy.

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in these strains. 



INTRODUCTION

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In the past decades carbapenem-resistant K. pneumoniae (CR-Kp) strains have emerged in the United States since 2001 and worldwide [1]. Currently, the most common carbapenemase in the US is K. pneumoniae carbapenemase (KPC), an Ambler molecular class A enzyme that facilitates hydrolysis of a broad

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prevalence of CR-Kp in the United States among health-care-associated

infections increased from 1.6% in 2001 to 10.4% in 2011 [2]. The majority of

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clinical CR-Kp isolates in the US are of MLST–defined clonal background ST258 that carry KPCs (blaKPC-2 or blaKPC-3) [3]. CR-Kp infections have high

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mortality rates (40-50%), and result in increased treatment and hospitalization costs [4, 5]. With no novel antimicrobials for emerging CR-Kp in sight, efforts to explore alternative treatment options and prevention of global dissemination are

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warranted [6].

One of the main virulence factors of Kp is its capsular polysaccharide (CPS) [7]. CPS is expressed in vivo, promotes biofilm formation and exerts an anti-opsonic effect, all of which evade the host immune response. Strategies targeting the

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CPS have been successful both in vaccine development as well as passive immunotherapy for other encapsulated pathogens. For Kp protective efficacy of anti-capsular antibodies (Ab) has been demonstrated in animal models, further

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supporting efforts to develop antibodies as adjunctive therapy [8]. CPS genes in Kp strains are chromosomally encoded and clustered in the cps genomic locus [9, 10]. Over 77 capsular (K) serotypes have been described. However, strains of ST258 have not been extensively characterized for their K-serotype or

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variety of β-lactams. Recent CDC surveillance data estimates that the

4  molecular methods of cps cluster analysis such as C-pattern [10] and wzi sequencing [11]. In this study we characterized 40 CR-Kp strains from the

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Bronx with respect to their CPS, biofilm formation, resistance to serum and macrophage killing as well as virulence in a Galleria mellonella and mouse

model. This study is the first to document significant CPS-associated variability including novel C-patterns and wzi alleles among CR-Kp strains of the ST258

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significant variability was documented with respect to virulence-associated

Abs are discussed.

Kp strains

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MATERIAL AND METHODS

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traits. The implications of these findings for efforts of developing anti-capsular

CR-Kp strains were collected from inpatients at Montefiore Medical Center

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(MMC) in Bronx, New York that presented with CR-Kp bacteremia between December 2010-November 2012. Retrospective chart review of patient data was done with IRB approval. For comparison 8 carbapenem-susceptible Kp strains (CS-Kp) were collected during the same time period. Kp was cultured in

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Luria-Bertani (LB) broth or agar plates at 30 ºC or 37 ºC. Hypermucoviscosity phenotype was determined with the string test as described [12]. Determination of genetic relatedness

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PFGE-typing of K. pneumoniae isolates was performed according to the PulseNet protocol (http://www.cdc.gov/pulsenet/protocols.htm) analyzing XbaI restriction enzyme patterns with a CHEF-DR II system (Bio-Rad, USA). MLST was carried out following the guidelines of the Institut Pasteur K. pneumoniae

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clone. Despite variability, cross-reactive Abs could be generated. In addition

5  MLST Database (www.pasteur.fr/mlst) [13]. Novel wzi alleles were incorporated into the K. pneumoniae sequence typing database at bigsdb.web.pasteur.fr

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CPS typing and Glycosyl composition analyses HincII restriction enzyme pattern of the PCR-amplified cps cluster, C-typing [10] and typing by wzi sequencing, which is strongly associated with K-type [11]

was performed as described [10, 11]. K-serotyping was performed at Statens

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15] with minor modifications (Supplemental methods). Carbohydrate

composition and linkage analysis was performed at the Complex Carbohydrate

Biofilm formation (BF) assays

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Research Center (Athens, GA) as previously described [16, 17].

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BF assays were performed at 37°C as described [18, 19] (Supplemental Methods). Data obtained were used to classify the strains as high (OD > 0.6), median (OD ≤0.6 and >0.4) or low producers (OD ≤ 0.4).

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Serum resistance assays

In vitro virulence assays were performed as published [12, 20] and described (Supplemental Methods). Kp strains were categorized into three different groups: no serum resistance meaning unable to grow (survival ratio ≤1),

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moderate serum resistance those strains with moderate growth (survival ratio >1 and ≤5) or high serum resistance which included Kp strains that exhibited high rate of replication (survival ratio >5).

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Macrophage-mediated killing In vitro killing of CR-Kp strains was investigated in the J774.16 macrophage cell line as published [21] and described (Supplemental methods). Intracellular

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Serum Institute (Copenhagen, Denmark). CPS was purified as described [14,

6  killing was based on the decrease of viable bacteria 30 min after initial coincubation relative to time 0.

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G. mellonella and murine infection models Virulence of CR-Kp strains was assessed in G. mellonella by injecting 20 larvae with 104 CFU of Kp in 10 l PBS. Control animals were injected with PBS only. Larvae were kept at 37ºC in the dark on sterile Petri plates and survival was

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strains, 5 l of hemolymph were pooled from 20 larvae at different time points. CFU were calculated from 50 l of hemolymph [22]. Intratracheal and

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intravenous infection in mice were performed as published [23] (Supplemental methods) and approved by the Animal Care and Use Committee.

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mAb generation and agglutination assays

MAb 1C9 to CPS C200 and C186 was generated by immunization with CPS in complete Freund’s adjuvant (CFA) followed by booster of CPS in incomplete

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Freund’s adjuvant (IFA) of BALB/c mice. Fusion and cloning was performed as described [24]. Agglutination was carried out as previously described on glass slides [8].

Statistical analysis

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Data are presented in mean ± standard deviation or median, range. Differences between patient data were analyzed by a Fisher’s exact test. Survival data were

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analyzed with a log-rank test. Statistical tests were performed with GraphPad Prism 6 for Mac.

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assessed for 3 weeks. To compare in vivo replication dynamics of CR-Kp

7  RESULTS Patient characteristics

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Fourty CR-Kp strains were derived from blood of 38 septicemic patients, who

were hospitalized at MMC. Patients were elderly with average age of 64 years and 71% resided in nursing homes. Mortality was high (52%) (Table 1).

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percent patients identified to be of Caucasian race and with diabetes. Days until effective treatment was started were variable in both groups and ranged from -

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33 to 18 days. Four CR-Kp infected patients never received effective treatment prior to death. Antibiotic susceptibility of the strains was performed by the microbiology laboratory, which identified CR in 40 strains by standard CLSI

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laboratory practice [25], 87% were also resistant to ciprofloxacin, 53% to amikacin, 36% to gentamicin and 16% to polymyxin B.

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Strain typing

MLST and PFGE were performed on the 40 CR-Kp strains and 8 concomitantly collected CS-Kp strains. MLST identified co-infection with distinct isolates in 4 patients. Two patients were infected with 2 distinct CR-Kp isolates, and 2

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patients with a CR- and CS-Kp isolate. Most (32/40, 80%) of CR-Kp strains belonged to ST258. Other sequence types were identified including ST37 (2/40,

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5%), ST502 (1/40, 2.5%), and ST14 (1/40, 2.5%) (Table S1) and were unrelated. Also 3 new types, ST1403 (2/40), ST1404 (1/40), and ST1406 (1/40) were identified of which the latter differs from ST258 only in the phoE allele. High (76%) similarity among PFGE patterns confirmed relatedness of CR-Kp strains assigned to the ST258 cluster. The other sequence types clustered into

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Characteristics of survivors and non-survivors were comparable except for

8  different PFGE profiles. Of the 8 CS-Kp strains 3 belonged to ST258 (33%), and ST23, ST111, ST15, ST502 and the new ST1405 each included one isolate.

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Sequence analysis identified blaKPC-2 in 20/40 strains and the blaKPC-3 variant in 17/40 isolates. No significant correlation between type of blaKPC gene and patient outcome was found. Capsule typing (C-typing)

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digestion of the PCR-amplified capsule gene region [10] and by wzi sequencing [11]. C-typing identified 17 different C-patterns (Figure 2,Table S1). The largest

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cluster included 11 CR-Kp strains that exhibited a novel C-pattern, C200 which is related to C14a, the C-type of reference strain 138 with a K14 serotype [10].

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Five CR-Kp strains with C-patterns C193, C194, C195, C196 and C197, had a highly similar C-pattern to C200 (75%). Strains with these C-patterns and C200 contain in common, the same and newly identified wzi154 allele. In addition, ten

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unique novel C-patterns were identified in this study and now named C186 (wzi50), C184 (wzi154), C189 (wzi50), C102-like (wzi29), C190 (wzi29), C191 (wzi29), C192 (wzi29), C201 and C202 (both wzi150). Five other previously described C-types were identified in 9 other CR-Kp strains. They include C102-

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like, which could be further differentiated by wzi sequencing (wzi29, wzi153, wzi150), C23a (wzi83), C51a (wzi50), C15a (wzi50), and C16a (wzi16),

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respectively. Even among CS-Kp strains 2 novel C-types C189 (wzi50), C198 (wzi151) were identified in addition to the already described C200/wzi154, C102-like/wzi29, C15a/wzi50, C51a/wzi29, and C1a/wzi1. Classical Kserotyping was performed in 4 CR-Kp strains. One C200 strain was identified as K34, whereas the other C200 and a C186 were untypable, and a C102 strain

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Molecular typing of the cps gene cluster was performed by restriction enzyme

9  was typed as K48. As expected a hypermucoviscous capsule (string test positive) was documented in a CS-Kp strain (#20, Table S1) with a C1a/wzi1

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molecular serotype that belongs to the hypervirulent clone ST23 of serotype K1 [26]. Interestingly 3 CR-Kp strains, two with a C200 C-pattern (#2,and #40, Table S1) and one with a C15a C-pattern (#9) also expressed a

hypermucoviscous capsule phenotype. Furthermore this study found a

(11/16 vs 4/20 Fisher’s test, p-value 0.0021). Taken together substantial

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3 gene

variability in CPS was documented in CR-Kp strains, including ST258 strains. In

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addition, the lack of typability of some isolates with molecular CPS variants highlights the limitations of traditional K-typing.

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Agglutination of Klebsiella by Anti-CPS IgM

Mab (1C9) an IgM was produced by mouse immunized with 2 purified CPSs

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(C200, the one typed as K34, and C186) for which monosaccharide composition analysis demonstrated distinct sugar content (Table 2). ELISA confirmed that 1C9 binds with high affinity to both C200 and C186 and not to bovine serum albumin (data not shown). To assess the cross-reactivity of mAb1C9 its ability to agglutinate CR-Kp strains expressing different CPSs was

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investigated. MAb 1C9 agglutinated 7 of the 14 distinct C-types and 26 of the

40 tested CR-Kp strains. Specifically, agglutination was observed in all (16/16)

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strains expressing C200 and related C-patterns, and in all 3 CR-Kp strains with C186 C-pattern. In addition the mAb-1C9 agglutinated all C23a strains, C189, C192 and C184 and C102-like CR-Kp strains (Figure 3). Seven C-types were not agglutinated. Agglutination was consistent for all isolates of a specific Cpattern except the C102a group, of which 1 isolate agglutinated and 5 did not.

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significant association of C200 or related C-patterns and the presence of blaKPC-

10  Phenotypic traits analyses Planktonic growth and biofilm formation

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Growth of Kp strains in suspension varied only slightly (median doubling time of 23.7 min ranging from 16.7 to 33.9 min, Table S1) however biofilm formation

varied considerably (Figure 4A and Table S1). The highest biofilm producer was CS-Kp#20 (ST23), which exhibited the C1a/wzi1-K1 molecular serotype and

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(ST258). Biofilm production did not correlate with doubling time of bacteria in

Killing experiments

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suspension.

CPS shields Kp bacteria from phagocytosis and killing by phagocytic cells.

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Marked differences among Kp strains with respect to resistance to killing by the J744.2 macrophage cell line (Figure 4B and Table S1) were found. Although killing of individual strains was highly variable and ranged from 26-86%, no

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consistent correlation with a C-pattern, wzi allele or clonal background was established.

Serum Resistance Assays

Resistance to human serum was compared, as it constitutes an important

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virulence trait that allows Kp to persist in vivo. Again considerable variability in serum resistance was documented for CR-Kp strains (Table S1). Among CR-Kp

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strains 37.5% were highly resistant, 52.5% moderate and 10% not resistant to serum. Growth dynamics in human serum confirmed impaired growth of CR-Kp

strains classified as highly serum resistant (Figure 4C). Furthermore, significant association of blaKPC-2-bearing strains (non-C200 and related) and high serum resistance was established (unpaired t-test, p-value 0.05).

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hypermucoviscous phenotype, followed by CS-Kp#32 (ST15) and CR-Kp#34

11  Virulence in in vivo infection models. Virulence of CR-Kp strains was compared in the moth G. mellonella infection

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model and for selected strains also tested in a murine tracheal and blood stream infection (BSI) model. Infection with 104 cfu from CR-Kp strains resulted in profound variability of G. mellonella waxworm survival ranging from

avirulence to death within days (Figure 4D, Table S1). Specifically, 9/40 of the

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17 days, PBS control 14 days), whereas 12/40 CR-Kp strains induced rapid

death (median survival 1-2 days). Interestingly strains exhibiting high virulence

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in G. mellonella and high resistance to human serum more frequently exhibit wzi50, wzi150 alleles compared to strains exhibiting low resistance and less

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virulence (9/14 vs 2/34, Fisher’s test p-value

Carbapenem-resistant Klebsiella pneumoniae exhibit variability in capsular polysaccharide and capsule associated virulence traits.

Novel therapies are urgently needed to treat carbapenem-resistant Klebsiella pneumoniae (CR-Kp)-mediated infection, which constitute a major health th...
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