Multiclonal dispersal of KPC genes following the emergence of non-st258 KPC-producing Klebsiella pneumoniae clones in Madrid, Spain

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1 J Antimicrob Chemother 2013; 68: doi: /jac/dkt237 Advance Access publication 20 June 2013 Multiclonal dispersal of KPC genes following the emergence of non-st258 KPC-producing Klebsiella pneumoniae clones in Madrid, Spain Patricia Ruiz-Garbajosa 1 *, Tania Curiao 1,2, Marta Tato 1, Desirèe Gijón 1, Vicente Pintado 3,Aránzazu Valverde 1,4, Fernando Baquero 1,2,5, María Isabel Morosini 1, Teresa M. Coque 1,2,5 and Rafael Cantón 1,5 1 Servicio de Microbiología, Hospital Universitario Ramón y Cajal and Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain; 2 CIBER en Epidemiología y Salud Pública (CIBERESP), Madrid, Spain; 3 Servicio de Enfermedades Infecciosas, Hospital Universitario Ramón y Cajal and Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain; 4 Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense, Madrid, Spain; 5 Unidad de Resistencia a Antibióticos y Virulencia Bacteriana asociada al Consejo Superior de Investigaciones Científicas, Madrid, Spain *Corresponding author: Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Madrid, Spain. Tel: ; Fax: ; pruizg@salud.madrid.org Received 15 January 2013; returned 21 February 2013; revised 16 May 2013; accepted 21 May 2013 Objectives: To analyse the ongoing epidemiology of KPC-producing Enterobacteriaceae after a non-st258 KPC-3- producing Klebsiella pneumoniae outbreak in a university hospital in Madrid, Spain. Methods: Enterobacterial isolates (one per patient based on bacterial identification and typing patterns) with carbapenem MICs higher than the EUCAST epidemiological cut-off values, a positive modified Hodge test and carbapenem/boronic acid combination disc test results were studied (16 March 2010 to 31 January 2012) and compared with KPC-producing isolates previously described in our institution (September 2009 to February. The bacterial population structure (PFGE and multilocus sequence typing), carbapenemase genes and KPC plasmids were studied. Patients clinical records were reviewed. Results: Twenty-four KPC-producing Enterobacteriaceae (20 K. pneumoniae,2escherichia coli and 2 Enterobacter cloacae) from 23 patients (13 males, median age 72 years) were studied. Most KPC-producer strains were considered as colonizers. Six K. pneumoniae clones (ST11, ST20, ST384, ST454, ST659 and ST971), two E. coli clones (ST224 and ST357) and one E. cloacae clone were identified. bla KPC-3 was located on IncFII K plasmids of 100 kb (E. coli ST357 and K. pneumoniae ST384, ST454, ST659 and ST971 clones) and on IncN plasmids of 40 kb (K. pneumoniae ST11 clone). Non-typeable plasmids of 20 kb containing bla KPC-2 were detected in scarcely represented clones (K. pneumoniae ST20, E. coli ST224 and E. cloacae). Conclusions: During the 2 year period following the emergence of non-st258 KPC-3-producing K. pneumoniae isolates in our institution, the bla KPC-3 and bla KPC-2 genes efficiently penetrated other Enterobacteriaceae lineages. Non-ST258 K. pneumoniae isolates were mostly responsible for the dissemination of KPC enzymes, producing a complex epidemiological picture. Keywords: carbapenemase-producing Enterobacteriaceae, carbapenem resistance, IncFII K plasmids Introduction Genes coding for KPC enzymes are predominantly identified among Klebsiella pneumoniae isolates and, increasingly, among other Enterobacteriaceae and in Pseudomonas spp. and Acinetobacter baumannii. 1 Until recently, the global predominance of KPC-producing K. pneumoniae was associated with clonal group (CG) 258, compromising the globally spread sequence type (ST) 258, and a diversity of single- and double-locus variants including ST11. 1,2 Other widespread (e.g. ST437) and local STs have been sporadically identified in areas with a high prevalence of KPC isolates. 3 These data indicate that bla KPC genes have a potentially high risk of dissemination among different clones and species. 4 6 We previously described the emergence of KPC-3-producing K. pneumoniae in Spain that was not associated with CG258 clones. 7 During a 5 month period (September 2009 to February, KPC-3-producing ST384 and ST388 K. pneumoniae clones were recovered from seven and one patient, respectively, in # The Author Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please journals.permissions@oup.com 2487

2 Ruiz-Garbajosa et al. different hospital wards. 7 In this study, we describe the ongoing epidemiology of KPC-producing Enterobacteriaceae in our institution, showing the dissemination of KPC enzymes in different K. pneumoniae, Escherichia coli and Enterobacter cloacae clones. Methods Bacterial isolates and study design From 16 March 2010 through 31 January 2012, a total of 98 Enterobacteriaceae isolates with decreased carbapenem susceptibility according to the EUCAST epidemiological cut-off values ( a positive modified Hodge test (MHT) and carbapenem/boronic acid combination disc test result were recovered from 23 patients. Clinical records of infected or colonized patients were retrospectively reviewed. Infection control measures were implemented after the detection of each new case as previously described. 7 Surveillance samples (, pharyngeal and wound swabs) were plated on chromid ESBL agar (biomérieux, Marcy l Étoile, France). Growing colonies were sought for carbapenemase production. 7 Bacterial identification and antibiotic susceptibility testing Bacterial identification was performed with a matrix-assisted laser desorption ionization time of flight mass spectrometry method (Bruker Daltonics GmbH, Leipzig, Germany). Antibiotic susceptibility was determined by standard microdilution and Etest (biomérieux), and interpreted according to EUCAST criteria. The screening for carbapenemase production included the MHT and combined-disc test using meropenem plus boronic acid or EDTA. 7 Extended-spectrum b-lactamase (ESBL) production was screened by double-disc synergy testing. 7 Characterization of antibiotic resistance genes Characterization of genes encoding carbapenemases (bla KPC, bla VIM, bla NDM and bla OXA-48 ) and ESBLs (bla TEM, bla SHV and bla CTX-M ) was carried out by PCR and sequencing. 7,8 qnr genes were sought by multiplex PCR. 7 Transferability of bla KPC and plasmid characterization The transfer of KPC plasmids was assessed by filter mating using E. coli K-12 BM21 as the recipient. 7 Transconjugants were selected on Luria Bertani agar plates containing imipenem (0.5 mg/l), ceftazidime (2 mg/l) and rifampicin (100 mg/l). Plasmid DNAwas extracted using a modified alkalinelysis protocol, 7 and the incompatibility group was determined using the PCR-based replicon typing scheme. 9 The size of the KPC plasmids and the association with replicon type were confirmed by hybridization of S1 nuclease-digested genomic DNA from E. coli transconjugants (or wild-type strains in the absence of transfer) with appropriate probes. 10 Similarity between the KPC plasmids was assessed by comparing EcoRI-, PstI- and HpaI-digested plasmid DNA profiles in all transconjugants. 7 Clonal relatedness Isolates were typed by PFGE of XbaI-digested DNA. 7 K. pneumoniae and E. coli isolates were also characterized by multilocus sequence typing (MLST) (see and Results Patients and bacterial isolates Twenty-three patients were infected and/or colonized by KPCproducing Enterobacteriaceae isolates. The patients were mostly elderly (median age 72 years, range years) with multiple underlying illnesses (Table 1). Infection was documented in eight cases, the most prevalent being intra-abdominal infection (Table 1). Nineteen patients were admitted to different wards and had a prolonged hospital stay (median 38 days, range days). The median number of days between admission and the first positive culture with KPC-producing isolates was 23 (range 9 70 days). In the case of the remaining four patients, three were attended in the emergency room and one was an outpatient at the time of collection of the first positive sample. All of them had had a previous recent hospitalization in our institution. None of the patients had travelled abroad or been transferred from another hospital. Overall, 98 KPC-producing Enterobacteriaceae isolates were identified (79 K. pneumoniae, 16E. coli and 3 E. cloacae). In 15 patients, isolates were recovered either from multiple sites or consecutively from different samples. K. pneumoniae isolates were recovered from 20 patients either from clinical (n¼15) or surveillance samples (n¼64), while E. coli and E. cloacae were isolated only from surveillance sampling sites (n¼19). All Enterobacteriaceae consecutively recovered from the same patient exhibited indistinguishable PFGE profiles. Consequently, 24 enterobacterial isolates (20 K. pneumoniae, 2E. coli and 2 E. cloacae) corresponding to the first isolate of each clone identified per patient were selected for further molecular studies. Antimicrobial susceptibility and phenotypic carbapenemase detection All isolates were resistant to penicillin/inhibitor combinations, extended-spectrum cephalosporins and aztreonam, but were susceptible to amikacin and colistin (MIC,2 mg/l) (Table 2). The MIC range of tigecycline was mg/l (one isolate falling within the intermediate category). Carbapenem MICs varied from 0.25 mg/l to.32 mg/l (imipenem, MIC 50 /MIC 90 1/8 mg/l; meropenem, MIC 50 /MIC /6 mg/l; ertapenem, MIC 50 /MIC 90 1/4 mg/l). The MHT and the carbapenem/boronic acid combination disc test were positive for all isolates. The disc diffusion assay with EDTA was negative, as was that for ESBL detection. Clonal relatedness and distribution over time XbaI-PFGE identified six pulsotypes of K. pneumoniae, two of E. coli and one of E. cloacae (Table 2). MLST demonstrated that each K. pneumoniae PFGE type was associated with a different ST (ST11, ST20, ST384, ST454, ST659 and ST971). K. pneumoniae (Kpn)-A/ST384 and Kpn-G/ST659 (six isolates each) were the most frequently detected clones, followed by Kpn-F/ST454 (four isolates). With the exception of ST11, a single-locus variant of ST258, none of the K. pneumoniae belonged to the CG258 group. The distribution of KPC-producing clones over time since the first detection of KPC in our institution in September 2009 is shown in Figure S1 (available as Supplementary data at JAC Online). Only three K. pneumoniae clones (Kpn-A/ST384, Kpn-F/ST454 and 2488

3 2489 Table 1. Characteristics of the patients infected and/or colonized by KPC-producing Enterobacteriaceae Case no. a Age (gender) Hospital ward Underlying diseases LOS LOS until first positive culture First isolation site (date of isolation) 1 78 (F) Vascular Surgery (7th diabetes mellitus (16 March 2 33 (F) Outpatient breast carcinoma and surgical drainage sclerosing cholangitis (22 March 3 77 (F) General Surgery-ICU (9th hiatus hernia respiratory (24 March 4 63 (M) General Surgery-ICU (9th intra-abdominal sepsis (31 March 5 42 (M) Gastroenterology (11th acute pancreatitis (6 May 6 36 (F) Cardio-Paediatric-ICU congenital cardiopathy (29 June (2nd 7 24 (M) Emergency Crohn s disease abscess (7 July 8 86 (F) Internal Medicine (3rd diarrhoea 18 9 (05 August 9 72 (M) Vascular Surgery (7th pulmonary carcinoma (09 August (M) Internal Medicine (3rd hepatocellular (5 August carcinoma (M) Oncology (3rd adenocarcinoma (9 August (M) Internal Medicine (4th bronchiectasis and (07 pneumonia September (F) Internal Medicine-ICU septic shock wound (07 (1st October 10) (13 October (M) Internal Medicine-ICU subarachnoid (11 October (1st haemorrhage (M) General Surgery-ICU (9th colon carcinoma respiratory (25 October (M) General Surgery (11th (M) General Surgery-ICU (9th adenocarcinoma respiratory (03 December caecal carcinoma peritoneal fluid (13 December Bacterial species Infection site Colonization site E. coli not detected K. pneumoniae not detected wound K. pneumoniae respiratory tract and intra-abdominal infection pharyngeal and K. pneumoniae not detected pharyngeal and K. pneumoniae not detected K. pneumoniae not detected K. pneumoniae intra-abdominal infection not detected E. cloacae not detected E. cloacae not detected pharyngeal and K. pneumoniae not detected K. pneumoniae not detected K. pneumoniae not detected K. pneumoniae respiratory tract and surgical site infection pharyngeal and E. coli not detected pharyngeal and K. pneumoniae not detected pharyngeal and K. pneumoniae respiratory tract infection pharyngeal and K. pneumoniae not detected respiratory tract and K. pneumoniae intra-abdominal infection not detected Continued KPC-producing non-st258 Klebsiella pneumoniae clones JAC

4 Ruiz-Garbajosa et al. Table 1. Continued Bacterial species Infection site Colonization site First isolation site (date of isolation) LOS until first positive culture LOS Case Age no. a (gender) Hospital ward Underlying diseases K. pneumoniae not detected (M) Emergency ulcerative colitis (16 December K. pneumoniae not detected and wound (M) Traumatology (1st hip fracture wound (31 December 10) K. pneumoniae catheter-related bloodstream infection acute diverticulitis blood (13 June (F) General Surgery (10th K. pneumoniae intra-abdominal infection not detected caecal carcinoma abscess (17 June (F) General Surgery (11th K. pneumoniae surgical site infection pharyngeal and (M) Emergency necrotizing vasculitis wound (22 June K. pneumoniae not detected (F) Nephrology (7th kidney transplant (31 October a Cases are numbered in chronological order. In Case 13, the patient was infected by a KPC-3-producing K. pneumoniae and simultaneously colonized by a KPC-3-producing K. pneumoniae and a KPC-3-producing E. coli. ICU, intensive care unit; LOS, length of stay. Kpn-G/ST659) were found during more than a 1 month period, while other clones were only occasionally detected. Molecular characterization of antibiotic resistance genes and plasmids The presence of bla KPC-3 was demonstrated in five K. pneumoniae and in one E. coli clone (Eco-I/ST357) (Table 2). The remaining K. pneumoniae (Kpn-E/ST20), E. coli (Eco-C/ST224) and E. cloacae (Eclo-H) clones harboured bla KPC-2. None of the clones harboured bla ESBL, other bla carbapenemase or qnr genes. The bla KPC-3 gene was only transferable for isolates belonging to three K. pneumoniae clones (Kpn-F/ST454, Kpn-G/ST659 and Kpn-J/ST971) and one E. coli clone (Eco-I/ST357). In these clones, bla KPC-3 was located on an 100 kb plasmid showing a highly similar restriction fragment length polymorphism (RFLP) pattern in all transconjugants (Table 2). The nucleotide sequence of these replicons was 99% identical to those of pkpqil-itand pkpqil plasmids (GenBank accession numbers JN and GU595196), which belong to the FII K group. This plasmid was identified in one K. pneumoniae clone (Kpn-G/ST659) and one E. coli clone (Eco-I/ ST357), which were sequentially recovered in the same patient (Table 2). On the other hand, bla KPC-3 was located on nontransferable 100 kb IncFII K and 40 kb IncN plasmids in Kpn-A/ST384 and Kpn-D/ST11 clones, respectively. The bla KPC-2 gene was associated with non-typeable and non-transferable 20 kb plasmids (Table 2). Discussion The epidemiology of KPC producers has been mainly associated with the global expansion of multidrug-resistant K. pneumoniae ST258 and the dissemination of plasmids of different incompatibility groups. 3,4 The most remarkable finding from our local epidemiology is that the most prevalent KPC-K. pneumoniae clones (ST384, ST454 and ST659) have not been previously linked with KPC production and are not clustered with any of the high-risk clones previously associated with these enzymes. 3 In the USA and Israel, where KPC K. pneumoniae ST258 is already endemic, the later dissemination of bla KPC genes was also associated with non-epidemic genetic lineages. 5,6 In Spain, the prevalence of KPC is still low since most of the carbapenemases identified are VIM and more recently OXA-48. 2,11 In this scenario, bla KPC-3 genes emerged in genetic lineages that were probably circulating as part of our local clonal pool. This was in fact the case for Kpn-ST388, which was found in 2009 in our hospital carrying bla KPC-3 and bla CTX-M-10, but had been present since 1998 only carrying bla CTX-M However, we occasionally found ST11, a single-locus variant of ST258, which is also a worldwidespread clone, but linked to different predominant b-lactamase genes such as bla CTX-M-14, bla CTX-M-15, bla KPC-2, bla VIM, bla NDM and, recently, bla OXA-48. 2,4,12 14 In our institution, ST11 was not associated with either ESBL or VIM producers prior to the detection of KPC producers, but it has been responsible for the emergence of isolates carrying bla OXA-48 during 2012 (D. Gijón, unpublished results). These observations suggest that the spread of the so-called high-risk clones might be constrained by the local populations of K. pneumoniae. 2490

5 KPC-producing non-st258 Klebsiella pneumoniae clones JAC Table 2. Characteristics of the KPC-producing enterobacterial clones Plasmids a Bacterial species (no.) PFGE type/st Case no. bla KPC size (kb) Inc group Co-resistance profile b K. pneumoniae (n¼20) Kpn-A/ST384 15, 16, 17, 20, 21, 22 KPC FII K (CIP), (TGC) c Kpn-D/ST11 2 KPC-3 40 N GEN, TOB, CIP, SXT, FOS Kpn-E/ST20 3, 4 KPC-2 20 NT Kpn-F/ST454 5, 7, 10, 11 KPC FII K CIP, SXT, FOS Kpn-G/ST659 6, 12, 13, 14, 18, 19 KPC FII K CIP, MIN, (FOS) Kpn-J/ST KPC FII K CIP E. coli (n¼2) Eco-C/ST224 1 KPC-2 20 NT CIP, FOS Eco-I/ST KPC FII K E. cloacae (n¼2) Eclo-H 8, 9 KPC-2 20 NT CIP, (FOS) NT, non-typeable. a Plasmids from Kpn-F/ST454, Kpn-G/ST659, Kpn-J/ST971 and Eco-I/ST357 clones were transferred by conjugation and showed a similar RFLP pattern. The others were non-transferable plasmids. Clones Kpn-G/ST659 and Eco-I/ST357 were sequentially recovered in case 13 and harboured the same Inc FII K plasmid. b Parentheses indicate that not all isolates studied showed a resistance phenotype. CIP, ciprofloxacin; FOS, fosfomycin; GEN, gentamicin; MIN, minocycline; TGC, tigecycline; TOB, tobramycin; SXT, trimethoprim/sulfamethoxazole. c One isolate presented an MIC of 2 mg/l, which corresponds to intermediate susceptibility. In addition to the successful clones, previous studies have demonstrated that the spread of KPC genes is also linked to Tn4401 and different types of transferable IncF(k), IncL/M and IncX plasmids. 4,12,15 We reported the recovery of conjugative 100 kb IncFII k plasmids exhibiting the same RFLP pattern from K. pneumoniae and E. coli clones. IncFII k plasmids have greatlycontributed to the spread of antibiotic resistance genes such as bla CTX-M-15 or bla KPC among K. pneumoniae, but they also have the ability to transfer between other enterobacterial species. 16,17 bla KPC -harbouring IncFII k plasmids have been mainly described in K. pneumoniae ST258 in different countries including Israel, Italy, 12,17 19 Poland, Greece and the USA. The results of our work demonstrated, as in other studies, that IncFII k plasmids harbouring bla KPC genes were not restricted to ST258 clones. 12,17 Although the in vitro conjugative efficiency of this IncFII k plasmid was low (data not shown), we identified the same plasmid in a K. pneumoniae (Kpn-G/ST659) and in an E. coli (Eco-I/ST357) clone sequentially recovered from the same patient, suggesting an in vivo plasmid transfer. This phenomenon has previously been reported involving the transfer of a KPC-3-encoding IncFII k plasmid from K. pneumoniae ST258 to E. coli, which might act as an accidental recipient present in patients microbiota. 20 In this sense, the intestinal tract of colonized patients might playan important role in the clonal dissemination and horizontal transfer of genetic platforms between multiple K. pneumoniae clones (not only ST258) and other species, thus favouring the ecological adaptation and persistence of KPCgenes inthe hospital and the surrounding nosocomial environment. In summary, we have described the rapid penetration of bla KPC genes to different unrelated K. pneumoniae clones in the absence of the classic epidemic K. pneumoniae ST258 high-risk clone. These findings highlight the importance of horizontal gene transfer in the dissemination of bla KPC genes and the role of the local clonal pool as potential substrate for the acquisition of these genes. Funding This work wassupported byresearch grants from the European Commission (TROCAR-FP7-HEALTH-F , R-GNOSIS-FP7-HEALTH-F , EvoTAR- FP7-HEALTH-F ) and by the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III, Spain co-financed by the European Development Regional Fund A way to achieve Europe ERDF, the Spanish Network for Research in Infectious Diseases (REIPI RD12/0015). P. R.-G. is currently supported by a research contract from the European Commission (R-GNOSIS-FP7-HEALTH-F ). T. C. is supported by a fellow research contract grant FI09/ and A. V. by a post-doctoral contract Juan de la Cierva both contracts from the Ministerio de Economía y Competitividad, Instituto Carlos III, Spain. Transparency declarations None to declare. Supplementary data Figure S1 is available as Supplementary data at JAC Online ( oxfordjournals.org/). References 1 Nordmann P, Naas T, Poirel L. Global spread of carbapenemaseproducing Enterobacteriaceae. Emerg Infect Dis 2011; 17: Cantón R,Akóva M, Carmeli Y et al. Rapid evolution and spread of carbapenemases among Enterobacteriaceae in Europe. Clin Microbiol Infect 2012; 18: Woodford N, Turton JF, Livermore DM. Multiresistant Gram-negative bacteria: the role of high-risk clones in the dissemination of antibiotic resistance. FEMS Microbiol Rev 2011; 35:

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