EPIDEMIOLOGY

MICROBIAL DRUG RESISTANCE Volume 00, Number 00, 2013 ª Mary Ann Liebert, Inc. DOI: 10.1089/mdr.2013.0107

Virulence and Transferability of Resistance Determinants in a Novel Klebsiella pneumoniae Sequence Type 1137 in China Yang Liu,1 Xiang-Yang Li,2 La-Gen Wan,1 Wei-Yan Jiang,2 Jing-Hong Yang,2 and Fang-Qu Li 2

A study was designed to characterize three carbapenemase-producing Klebsiella pneumoniae isolated from pediatric patients in China. Molecular characterization was done using polymerase chain reaction and sequencing for blaVIM, blaNDM, blaIMP, blaKPC, blaCTX-Ms, blaOXAs, blaTEMs, and blaSHV; plasmid-mediated quinolone resistance determinants; aminoglycoside resistance determinants; multilocus sequencing typing; plasmid replicon typing; addiction; and virulence factors. Kp32 belonged to the newly described sequence type 1137, were positive for aac(6¢)-Ib-suzhou, qnrA1, qnrB4, qnrS1, aac(6¢)-Ib, rmtB, armA, blaSHV-12, blaCTX-M-15, blaKPC-2, and blaIMP-4; contained IncA/C plasmids that tested positive for K1 capsular antigens, the ccdAB (coupled cell division locus) addiction system and the wabG, ureA, rmpA, magA, allS, fimH, and the aerobactin virulence factors. However, the others belonged to clone ST11, and were positive for aac(6¢)-Ib-cr, qnrB4, blaCTX-M-14, blaSHV-11, aac(6¢)-Ib, rmtB, and blaKPC-2; contained IncFIA plasmids that tested positive for K2 capsular antigens, the vagCD addiction system and the uge, wabG, ureA, kfuBC, rpmA, and fimH virulence factors. ST1137 had more virulence factors than the comparative strains ST11. The blaKPC-2 gene was located on the IncFIA and IncA/C replicon groups of plasmids. An analysis of the genetic environment of blaKPC-2 gene has demonstrated that the blaKPC-2 gene was always associated with one of the Tn4401 isoforms (a or b). Our study suggested that K. pneumoniae carbapenemases being found in virulent K. pneumoniae should be emphasized, as this will eventually become a global health threat.

ST1137 that used two ST11 strains as comparative strains. The addiction and virulence factors associated with plasmids that encode for KPCs were also determined.

Introduction

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arbapenemase-producing Klebsiella pneumoniae has emerged as an important nosocomial pathogen. Three types of acquired carbapenemases have been reported, including the two major types: the molecular class A K. pneumoniae carbapenemases (KPCs) and the class B metallo-b-lactamases (MBLs). Previously, a K. pneumoniae strain carrying two types of carbapenemase, blaIMP-4 and blaKPC-2, was only reported in Wuhan province.17 Although virulence determinants have been well described in K. pneumoniae, very little is known about the virulence of KPC-2 and IMP-4 coproducing K. pneumoniae. A particular clonal lineage of carbapenemase-producing K. pneumoniae, sequence type (ST)11, is commonly associated with nosocomial outbreaks in China.14 Different virulence factors have been detected notably in strains of ST11 and ST258 types, which harbored a type 4-secretion system gene cluster.8 Therefore, a study was designed to characterize a novel K. pneumoniae

Materials and Methods Antimicrobial susceptibilities Three nonduplicate K. pneumoniae isolates were recovered from the pediatric patients admitted in our hospital between June 2010 and September 2010. The BacT/Alert 3D system (Sysmex bioMe´rieux) was used for the blood culture, and identification of the bacterial species was performed with the MicroScan WalkAway 96 SI system (Siemens Healthcare Diagnostics). The MICs of the following drugs were determined: amoxicillin/clavulanic acid (AMC), piperacillin-tazobactam (TZP), cefoxitin (FOX), ceftriaxone (CRO), ceftazidime (CAZ), aztreonam (AZT), imipenem (IPM), ertapenem (ERT), amikacin (AMK), gentamicin (GEN), tobramycin (TOB), ciprofloxacin (CIP), and trimethoprim-

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Department of Bacteriology, First Affiliated Hospital of Nanchang University, Nanchang University, Nanchang, People’s Republic of China. Department of Clinical Microbiology, Second Affiliated Hospital of Wenzhou Medical College, Wenzhou Medical College, Wenzhou, People’s Republic of China. 2

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2 sulfamethoxazole (SXT). Additional susceptibilities for meropenem (MER) were performed using E-test methodology with the manufacturer’s instructions (AB BioDisk). Throughout this study, results were interpreted using the CLSI criteria for broth dilution.4 b-Lactamase identification The presence of MBLs and carbapenemase production was determined in the K. pneumoniae with the MBL E-test (AB BioDisk) and the modified Hodge test using the manufacturer’s instructions. Polymerase chain reaction (PCR) amplification for blaVIM, blaNDM, blaIMP, blaKPC, blaCTX-Ms, blaOXAs, blaTEMs, and blaSHV was carried out on the isolates with a GeneAmp 9700 ThermoCycler instrument (Applied Biosystems) using PCR conditions and primers as previously described.10 The PCR products were sequenced using an ABI 3730 sequencer (Applied Biosystems), and the sequences were then compared with the reported sequences from GenBank. Detection and sequencing of plasmid-mediated quinolone resistance determinants and aminoglycoside resistance determinants genes The amplification of the quinolone resistance determinants (QRDs) and aminoglycoside resistance determinants (ARDs) genes was undertaken with PCR using custom primers and conditions as previously described.7 Multilocus sequence typing and pulsed-field gel electrophoresis Multilocus sequence typing was performed using seven conserved housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) (www.pasteur.fr/recherche/genopole/PF8/ mlst/K.pneumoniae.html). Pulsed-field gel electrophoresis (PFGE) was performed by using XbaI (GE Healthcare) as described. XbaI-macrorestriction patterns were interpreted according to the recommendations of Tenover et al.16

LIU ET AL. lated from the blood from a patient admitted with a community-acquired sepsis (Kp32). The patient was a 5-year-old boy from a small town outside Wenzhou, who presented to the local emergency department with a fever of 40C and abdominal pain. The diagnosis of community-acquired sepsis was made. Blood culture revealed hypermucoviscous K. pneumoniae Kp32 as the sole organism. During his stay in the intensive care unit (ICU), he received several courses of antibiotics that included etimicin, MER, and AZT. Active surveillance was performed after the isolation of Kp32 and included rectal swabs from patients in the same ICU; however, no additional cases were identified. Among the other two isolates, one was isolated from the urine from a patient with a hospital-acquired urinary tract infection (Kp28), and the other strain was isolated from the blood from a patient with a hospital-acquired bacteremia (Kp15). The isolates were tested by PFGE of XbaI-digested DNA. The PFGE patterns are shown in Figure 1. MLST of the three isolates resulted in two distinct allelic profiles: ST11 (allelic profile 3-3-1-1-1-1-4) corresponding to isolates Kp28 and Kp15; ST1137 (allelic profile 3-4-6-4-7-1-38) corresponding to isolates Kp32. These results matched with the PFGE results. Among the three isolates, two ST11 isolates harboring blaKPC-2 gene were isolated from burns unit as comparative strains, whereas the other isolate coproducing IMP-4 and KPC-2 carbapenemase was isolated from ICU. All patients were exposed to health-care environments in the 90-days before isolation, including prior hospitalization as well as invasive devices; indwelling central venous catheters; and mechanical intubation. The mean hospital length of stay from admission to positive culture was 21.7 days (median 22, range 15–28). Due to limited therapeutic options and poor outcomes associated with the resistant organism, the patient infected by the strain ST1137 died within 30 days after initial carbapenemse-producing K. pneumoniae isolation. However, the other two patients infected by the strains ST11 were successfully treated with IPM and AMK.

Plasmid analysis and replicon typing

Antimicrobial susceptibilities

Plasmid sizes were determined as previously described2 and assigned to plasmid families by PCR-based replicon typing.3 Conjugation experiments were performed by mating-out assays with nutrient agar containing MER 5 mg/ml and using Escherichia coli J53 (azide 100 mg/ml) as recipient.

Results with Vitek 2 showed that three carbapenemaseproducing K. pneumoniae were resistant to various antibiotics except for SXT. The MICs are illustrated in Table 1.

Serotype, addiction systems and virulence factors Serotype, plasmid addiction systems, and virulence factors were determined using PCR as recently described.5,15 Genetic environment of blaKPC-2 gene Genetic structures surrounding the blaKPC-2 gene were determined according to the Tn4401 PCR-mapping scheme as previously described.13 Results Patients and bacterial isolates Three unique K. pneumoniae isolates were obtained from three pediatric patients. These isolates were cultured from urine (n = 1) and blood (n = 2) samples. One strain was iso-

b-Lactamase identification All K. pneumoniae tested positive for carbapenemase production using the modified Hodge test, while only the Kp32 tested positive for MBL production using the MBL E-test. PCR and sequencing results showed that ST11 clones Kp15 and Kp28 were positive for blaKPC-2, blaCTX-M-14, blaTEM-1, and blaSHV-11. However, ST1137 clone Kp32 was positive for blaKPC-2, blaIMP-4, blaCTX-M-15, blaTEM-1, and blaSHV-12. Molecular characterization of K. pneumoniae Two K. pneumoniae Kp15 and Kp28, which belonged to ST11, were positive for aac(6¢)-Ib-cr, qnrB4, aac(6¢)-Ib, and rmtB, while Kp32 was positive for aac(6¢)-Ib-suzhou, qnrA1, qnrB4, qnrS1, aac(6¢)-Ib, rmtB, and armA, but belonged to new ST1137 (Table 1). Notably, all the three carbapenem-resistant K. pneumoniae strains included in this study were found to co-carry several QRDs and ARDs. They were shown to be

CARBAPENEMASE-PRODUCING K. PNEUMONIAE

3 Two ST11 isolates harbored five plasmids of 10, 25, 35, 60, and 120 kb in size, while Kp32 harbored four plasmids of 15, 30, 50, and 150 kb in size (data not shown). Transconjugants obtained with the three isolates tested positive with the modified Hodge test; plasmid analysis showed that Kp15-T and Kp28-T harbored the 120-kb plasmid and Kp32-T harbored the 150-kb plasmid. PCR confirmed that Kp15-T and Kp28-T contained blaKPC-2, qnrB4, and acc(6¢)-Ib-cr; while the Kp32-T harbored blaKPC-2, qnrS1, qnrB4, and acc(6¢)-Ib-suzhou. Kp15-T and Kp28-T belonged to the narrow host range IncFIA replicon group, while Kp32-T belonged to the broad host range IncA/C replicon type (Table 1). Characterization of the genetic environment of the blaKPC-2 gene The analysis of the genetic environment of blaKPC-2 by amplification of Tn4401, comparison of the restriction fragment length polymorphism patterns obtained by digestion of amplicons with PstI and HindIII, and sequencing of variants representative of distinct types revealed the presence of Tn4401 variants known as ‘‘a’’ (ST11 isolate) and ‘‘b’’ (ST1137 isolate). While Tn4401a was located on a 120-kb IncFIA plasmid, Tn4401b was detected in plasmids belonging to the incompatibility group IncA/C (150 kb) (data not shown). All those of IncFIA and IncA/C were transferred by conjugation. Tn4401 was inserted on different-sized plasmids that belonged to different incompatibility groups. Downstream of the blaKPC-2 gene was located a putative ISKpn6 element, and a truncated TEM-1 gene was inserted between them. The inverted repeats of ISKpn6 are 19 bp long, and its transposition generated a 5-bp target site duplication and without target site specificity. Discussion

FIG. 1. Pulsed-field gel electrophoresis profiles of Klebsiella pneumoniae isolates after restriction with XbaI. Lanes 1 and 5 correspond to a Salmonella serotype Braenderup strain (h9812). Lane 3 corresponds to a reference sequence type (ST)1137 K. pneumoniae strain coproducing K. pneumoniae carbapenemase-2 (KPC-2) and IMP-4. Lanes 2 and 4 correspond to ST11 K. pneumoniae strains producing KPC-2. resistant to both quinolones (CIP and LEV) and aminoglycosides (GM, AMK, and TOB). Most importantly, this is the first report of the co-expression of aac(6¢)-Ib-suzhou, qnrA1, qnrB4, qnrS1, aac(6¢)-Ib, rmtB, armA with blaKPC-2, and blaIMP-4 carbapenemases in a novel K. pneumoniae ST. Kp15 and Kp28 tested positive for K2 capsular antigens, the vagCD addiction system and the uge, wabG, ureA, kfuBC, and rpmA, and fimH virulence factors; while Kp32 tested positive for K1 capsular antigens, the ccdAB (coupled cell division locus) addiction system and the wabG, ureA, rmpA, magA, allS, and fimH, and the aerobactin virulence factors. The distribution of virulence factors is shown in Table 1.

Wei Z.Q. recently provided compelling evidence that KPC-2 and IMP-4 coproducing K. pneumoniae were widespread in southern China.18 The patients presented with a variety of hospital- and community-associated infections, with bacteremia being the most common clinical syndrome. In our study, a novel ST (ST1137) was detected that was different from the report by Wang et al.,17 which involved discovery of ST476 KPC-2 and IMP-4 coproducing K. pneumoniae in Wuhan, Hubei Province. The dominant clone of KPC-producing K. pneumoniae in China is ST11, which is closely related to ST258, which has been reported worldwide. However, ST1137 is a completely different variant of ST11 and ST476, indicating a long-distance relationship between them. To date, there are no reports of KPC-2 and IMP4 coproducing isolates that belong to this ST. Interestingly, two other KPC-2-producing K. pneumoniae from the burns unit of our hospital, which was used as comparative strains, were classified as ST11. A previous study from our hospital also showed evidence of a common KPC-2 carrying plasmid among the ST11 K. pneumoniae (data not shown). These observations indicate that horizontal acquisition of resistance is driving the KPC-2-producing K. pneumoniae phenotype at our institution. It was subsequently demonstrated that KPC2 type enzymes resided on mobile genetic elements and were transferable among the different STs. These different clones are also responsible for hosting plasmid-mediated QRDs and ESBLs, and it is possible that they play an important role in the intercontinental spread of antimicrobial resistance.

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LIU ET AL.

Table 1. Susceptibilities and Molecular Characteristics of Carbapenemase-Producing Klebsiella pneumoniae Kp15

Kp28

Kp32

> 16/8 > 64 > 16 > 32 > 16 > 16 >8 >8 > 32 > 32 >8 >8 >2 £ 2/38 Blood Burn unit K2 KPC-2, CTX-M-14, SHV-11, TEM-1

> 16/8 > 64 > 16 > 32 > 16 > 16 >8 >8 > 32 > 32 >8 >8 >2 £ 2/38 Urine Burn unit K2 KPC-2, CTX-M-14, SHV-11, TEM-1

QRDs

aac(6¢)-Ib-cr, qnrB4

aac(6¢)-Ib-cr, qnrB4

Multilocus sequence typing Plasmids (kb) Replicon typing ARDs Addiction systemsb pemKI ccdAB relE parD/E vagC/D hok-sok pndA/C srnB/C Virulence factorsc uge wabG ureA magA mrkD allS kfuBC rpmA fimH Aerobactin

ST11 10, 25, 35, 60, 120 IncFIA aac(6¢)-Ib, rmtB

ST11 10, 25, 35, 60, 120 IncFIA aac(6¢)-Ib, rmtB

> 16/8 > 64 > 16 > 32 > 16 > 16 >8 >8 > 32 > 32 >8 >8 >2 > 2/38 Blood ICU K1 KPC-2, IMP-4, CTX-M-15, SHV-12, TEM-1 aac(6¢)-Ib-suzhou, qnrS1, qnrA1, qnrB4 ST1137 15, 30, 50, 150 IncA/C aac(6¢)-Ib, rmtB, armA

Neg Neg Neg Neg Pos Neg Neg Neg

Neg Neg Neg Neg Pos Neg Neg Neg

Neg Pos Neg Neg Neg Neg Neg Neg

Pos Pos Pos Neg Neg Neg Pos Pos Pos Neg

Pos Pos Pos Neg Neg Neg Pos Pos Pos Neg

Pos Pos Pos Pos Neg Pos Neg Pos Pos Pos

a

Susceptibility to AMC TZP FOX CRO CAZ AZT MER IPM ERT AMK GEN TOB CIP SXT Sample Ward Capsular serotype/genotype b-Lactamases

a

MICs using the Microscan NEG MIC 30 panel. Addiction systems: pemKI, plasmid emergencymaintenance; ccdAB, coupled cell division locus; relE, relaxed control of stable RNA synthesis; parD/E, vagC/D, virulence-associated proteins; hok-sok, pndA/C, srnB/C, plasmid antisense RNA-regulated system. c Virulence factors: uge, encoding uridine diphosphate galacturonate 4-epimerase; wabG, involved in the biosynthesis of the outer core lipopolysaccharide; urea, related to urease operon; magA, mucoviscosity-associated gene A; mrkD, type 3 fimbriae adhesion; alls, activator of the allantoin regulon; kfuBC, iron-uptake system; rpmA, regulator of mucoid phenotype; fim, fimbrial gene encoding type 1 fimbrial adhesion. KPC-2, K. pneumoniae carbapenemase-2; ST, sequence type; MIC, minimum inhibitory concentration; AMC, amoxicillin/clavulanic acid; TZP, piperacillin-tazobactam; FOX, cefoxitin; CRO, ceftriaxone; CAZ, ceftazidime; AZT, aztreonam; IPM, imipenem; ERT, ertapenem; AMK, amikacin; GEN, gentamicin; TOB, tobramycin; CIP, ciprofloxacin; SXT, trimethoprim-sulfamethoxazole; MER, meropenem; QRD, quinolone resistance determinant; ARD, aminoglycoside resistance determinant; ICU, intensive care unit. b

Many carbapenemase-producing K. pneumoniae are highly multiresistant, but may remain susceptible to one or more aminoglycosides.11,12 In this study, three common ARDs genes (acc(6¢)-Ib, rmtB, and armA) and four common QRDs genes (qnrA1, qnrB4, qnrS1, and acc(6¢)-Ib-suzhou) were de-

tected in one novel K. pneumoniae ST1137 co-producing KPC2 and IMP-4 in a Chinese hospital. The three characterized strains showed that blaKPC-2 genes were generally located on large plasmids of various sizes. The plasmid present in the ST1137 strain carrying blaKPC-2 gene belonged to Inc groups

CARBAPENEMASE-PRODUCING K. PNEUMONIAE A/C. However, among the ST11 strains, the plasmid associated with KPC-2 belonged to the FIA group (Table 1). Similarly, Tn4401 is a Tn3-like transposon that is associated with blaKPC genes. This transposon was identified in isolates from different geographical origins, and of different STs, in K. pneumoniae.1 We have demonstrated here that Tn4401 (a or b) was inserted on plasmids varying in size and incompatibility group. In addition, 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. We showed here that an increase of virulence of KPC-2 and IMP-4 coproducing strain belonging to ST1137 (compared with ST11 strains) may be due to the plasmid type. KPC-2 and IMP-4 coproducing isolates are likely to be resistant to other classes of antibiotics except for SXT, limiting therapeutic options for such infections resulting in higher mortality rates and treatment failures.11 Fuursted et al.6 evaluated the presence of virulence factors in a single strain of carbapenemase-producing K. pneumoniae carrying the New-Delhi metallo-b-lactamase-1 (NDM-1) and compared it with noncarbapenemase-producing strains. Serotypes K1 and K2 have been generally considered the predominant virulent strains of K. pneumoniae and are highly resistant to phagocytosis.9 In our study, ST1137 K. pneumoniae was positive for K1 capsular antigens, different from the K2 capsular antigens of ST11 isolates. K1 isolated in South East Asia was the predominant serotype causing pyogenic liver abscess. K2 serotype is among the most common capsule types isolated from patients with UTI, pneumonia, or bacteremia. It is apparent from Table 1 that the KPC-2 and IMP4 coproducing strain (ST1137) possessed a higher virulence trait compared with the other ST11 strains. It would be of interest to extend these virulence studies to other published KPC-2 and IMP-4 coproducing isolates, and to compare and evaluate more virulence mechanisms both in vitro and in other animal models. In summary, our study suggested that the carbapenemaseproducing K. pneumoniae might have different virulence factors among various STs. Its intrinsic virulence potential in coexistence with its antibiotic resistance mechanism could promote and partly explain its epidemiological successful rapid global spread. However, further dissemination of the KPC plasmids into virulent K. pneumoniae need to be studied further in the future. Efforts to assess the factors that contribute to attributable mortality in KPC K. pneumoniae infections should be continued, including the study of other virulence determinants, antibiotic resistance, host responses, and their relationship with clinical characteristics and outcomes.

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Acknowledgment All authors equally participated in study design, data analysis and interpretation, and writing of this article.

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Disclosure Statement All authors have no conflicts of interest.

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Address correspondence to: Yang Liu, MD Department of Bacteriology First Affiliated Hospital of Nanchang University Nanchang University Yong Wai Zheng Jie No. 17 Nanchang 330006 People’s Republic of China E-mail: [email protected]