Epidemiological and microbiological characteristics of an outbreak 1. caused by OXA-48-producing Enterobacteriaceae in a neonatal 2

JCM Accepts, published online ahead of print on 26 June 2013 J. Clin. Microbiol. doi:10.1128/jcm.01049-13 Copyright 2013, American Society for Microbiology. All Rights Reserved. 1 Epidemiological and microbiological characteristics of an outbreak 1 caused by OXA-48-producing Enterobacteriaceae in a neonatal 2 intensive care unit in Jerusalem 3 4 Amos Adler 1^*, Ester Solter 1^, Samira Masarwa 1, Tamar Miller-Roll 1, Bassam 5 Abu-Libdeh 2, Hatem Khammash 2, Khalil Najem 2, Susan Dekadek 2, Chen Stein- 6 Zamir 3, Nafez Nubani 3, Amin Kunbar 3, Marc Victor Assous 4, Yehuda Carmeli 1 7 and Mitchell J. Schwaber 1. 8 9 1 -National Center for Infection Control, Ministry of Health, Tel-Aviv 10 2 -Makassed Hospital, Jerusalem 11 3 -Jerusalem District Health Office, Ministry of Health, Jerusalem 12 4 -Shaare-Zedek Medical Center, Microbiology and Immunology Laboratory, 13 Jerusalem, Israel 14 15 ^Both authors contributed equally to the study. 16 17 Running title: OXA-48 producing Enterobacteriaceae outbreak in a NICU 18 19 *Corresponding author: Amos Adler 20 Tel-Aviv Sourasky Medical Center 21 6 Weizmann St., Tel-Aviv, Israel 22 Tel: 972-3-6947551, fax: 972-3-6947586 23 Email: amosa@tlvmc.gov.il 24 25

2 Abstract 1 This study describes the course of an OXA-48-producing Enterobacteriaceae (OPE) 2 outbreak that started in March 2012 in a neonatal intensive care unit (NICU) in 3 Jerusalem. During the peak of the outbreak (January-August 2012), there were 49 4 patients who had proven or suspected acquisition of OPE in the NICU, including 16 5 with invasive infections, out of a total of 156 patients hospitalized during that period. 6 Three children hospitalized in the Pediatric ICU were identified as carriers of OPE. 7 Three patients with a previous stay in the affected NICU were identified as OPE 8 carriers upon admission to another hospital. The Ministry of Health was notified and 9 intervened in July 2012. The intervention included cohorting colonized patients, 10 frequent rectal surveillance and improving the implementation of infection control 11 practices. As a result, the incidence of OPE acquisition declined to 5 cases in the first 12 4 months with no new cases in the following 3 months. 31 patient-unique isolates 13 were available for analysis: 29 K. pneumoniae isolates, all belonging to a single clone 14 (ST39), and 2 isolates of Enterobacter cloacae. All isolates possessed bla OXA-48 and 15 bla CTX-M-14, located on the same plasmid. This plasmid, similar to the global bla OXA-48-16 harboring vector, has now acquired bla CTX-M-14, leading to resistance to all β-lactam 17 agents. 18 19 20 21 22 23 24

3 Introduction 1 The OXA-48 carbapenemase was first discovered in various Enterobacteriaceae 2 species isolated in Turkey and other countries in the Middle-East (5). Within years, 3 several sporadic cases of OXA-48 producing Enterobacteriaceae (OPE) infections 4 have been reported from across Europe. Most of these cases were related to patients 5 with previous exposure to healthcare systems in the Middle East and North Africa 6 (25). In Israel, the first cases of OPE were described in patients coming from the 7 Palestinian Authority (PA) or from neighboring countries (3). As KPC-producing 8 Enterobacteriaceae were already endemic in Israel at that time (23), OPE were 9 suspected based on their susceptibility to 3 rd -generation cephalosporins, a unique 10 feature of OPE (19). Another unique feature of OPE in Israel and worldwide, is the 11 location of bla OXA-48 inside a Tn1999 transposon, harbored by a unique, 62 kb IncL/M 12 plasmid, that is capable of self inter-species conjugation (3, 18). In recent years, 13 outbreaks of OPE were reported from several European countries (8, 16, 20, 26). 14 These outbreaks were caused mainly by monoclonal spread of K. pneumoniae strains 15 that co-produced CTX-M-15, conferring resistance also to all cephalosporins. bla CTX- 16 M-15 was located on a separate plasmid in the outbreak isolates; it was found inside the 17 same transposon in a single case report (21). All of these outbreaks, as well as other 18 sporadic cases, were reported in adults only. In the present study, we describe the first 19 case of a large-scale OPE outbreak in a Neonatal Intensive Care Unit (NICU), the 20 intervention and its results. The outbreak was propagated by a combination of both 21 monoclonal spread of a single K. pneumoniae strain and by horizontal transfer of the 22 modified bla OXA-48 -harboring plasmid, co-carrying the bla CTX-M-14 gene. 23 24 25

4 Methods 1 Setup and design 2 Makassed Hospital (MH) is a 250-bed hospital, affiliated with the School of Medicine 3 of Al-Quds University and representing its main teaching hospital. Located in East 4 Jerusalem, it provides secondary and tertiary medical services for the local Palestinian 5 population, and functions as a referral center for patients from the Palestinian 6 Authority and the Gaza Strip. The Neonatal Intensive Care Unit (NICU) is a 27-bed 7 unit, caring for premature (including very-low birthweight babies) and sick babies 8 who were born there or transferred from other hospitals in the Palestinian territories. It 9 contains intensive care (15 beds) and intermediate care (12 beds) sections. 10 The intervention team of the Israeli Ministry of Health (MoH) was composed of 11 personnel from the offices of the National Center for Infection Control (NCIC) and 12 the Jerusalem District Health Office (JDHO). Data were collected prospectively from 13 the initiation of the intervention (June 2012) and retrospectively from that point back 14 to the time the first cases were identified (March 2012). 15 General overview of the OPE outbreak and Infection control practices at Makassed 16 Hospital 17 Prior to the OPE outbreak, infection control practices that included hand hygiene and 18 isolation procedures were implemented in the NICU and throughout the hospital, but 19 with suboptimal compliance. Surveillance for carbapenem-resistant 20 Enterobacteriaceae (CRE) was performed on admission in all high-risk patients 21 (defined as patients either being transferred from another medical center or arriving 22 from the Palestinian territories) and as part of contact investigation. 23 The OPE NICU outbreak lasted from March 2012 with the last case of OPE carriage 24 identified in December 2012, resulting in a total of 57 affected patients, including 16 25

5 with invasive infections. Aside from this outbreak, the incidence of CRE at MH 1 during this period was low - between July 2012 until April 2013, 5 new cases were 2 identified upon admission and only one case was detected in a hospitalized patient. 3 Rectal carbapenem-resistant Enterobacteriaceae surveillance cultures and 4 phenotypic characterization of isolates 5 MH lab: Rectal CRE surveillance culture swabs were streaked onto MacConkey agar 6 plates supplemented with either cefotaxime (10 mg/l) or meropenem (0.5 mg/l), per 7 local protocol. Pink colonies growing around the disks were picked and processed. 8 Bacterial identification was done by manual biochemical tests and antimicrobial 9 susceptibility testing was done by disk diffusion (7). Complete phenotypic and 10 molecular characterization was performed by the NCIC lab on available isolates as 11 part of the investigation but was not available before the intervention. 12 NCIC lab: Rectal CRE surveillance cultures were transported within 4 h to the NCIC 13 lab. Swabs were inoculated into BHI broth and incubated overnight prior to subculture 14 onto MacConkey agar with imipenem 1mg/L (4). Suspicious (pink) colonies were 15 selected for further evaluation. Identification and antimicrobial susceptibility testing 16 were performed by the VITEK-2 system (biomérieux, Marcy l Etoile, France). 17 Ertapenem, imipenem and meropenem MIC were verified by the agar dilution assay 18 (6). Phenotypic characterization included the modified Hodge test (MHT), and 19 synergy testing of ertapenem with and without EDTA (EDTAST).(10) 20 Molecular characterization of isolates 21 Isolates were screened by PCR for bla KPC,(22) bla NDM-1 and bla OXA-48 (19). bla CTX-M, 22 bla TEM and bla SHV were identified by PCR and sequencing (14, 26). Pulsed-field gel 23 electrophoresis (PFGE) was performed for K. pneumoniae and Enterobacter cloacae 24 isolates as previously described (12). Multi-locus sequence typing (MLST) was 25

6 performed for representative PFGE types of the K. pneumoniae (9) isolates. Since 1 only 29/57 patient-unique isolates were available for molecular and phenotypic 2 analyses (from the latter part of the outbreak), we referred to the isolates as either 3 OPE or CRE, depending on whether full characterization was done or not, 4 respectively. 5 Plasmid analysis 6 Plasmid DNA, purified from a representative K. pneumoniae isolate and the 2 E. 7 cloacae isolates, was transformed into E. coli DH10B. Transformants were selected 8 on LB agar supplemented with cefazolin (8 mg/l) and confirmed by bla OXA-48 -PCR. 9 Determination of plasmid size and restriction-fragment length polymorphism (RFLP) 10 were done as previously described (11). The genetic environment of bla OXA-48 was 11 studied by PCR and sequencing (5). The presence of the repa, para and trau was 12 tested by PCR (18). The genetic environments up- and downstream to bla CTX-M-14 13 were studied by sequencing of the bla OXA-48 /bla CTX-M-14 -carrying plasmid, using 14 primers directed to the 5' and 3' ends of bla CTX-M-14, respectively. The sequence was 15 analyzed using the ISfinder website (https://www-is.biotoul.fr/) (24). 16 17 Results 18 Epidemiological features of the outbreak 19 The first clinical cases of CRE in the NICU were noted in mid-march, 2012. Two 20 babies had CRE bacteremia, one with Klebsiella spp. and one with Serratia 21 marcescens, in both cases more than 72 hours from admission. In the second case, the 22 baby was admitted flowing a short stay in a Palestinian Authority hospital. 23 Subsequently, Klebsiella spp. were isolated in CRE surveillance cultures in 6 babies. 24 In the following 17 weeks, until the beginning of the NCIC intervention, 27 new cases 25

7 were identified (figure 1a: cases identified in the MH NICU are marked in grey, and 1 cases identified in other MH wards and additional hospitals are marked in black), with 2 incidence reaching 9 cases/week. All isolates were identified as either Klebsiella spp. 3 or K. pneumoniae, except for 1 isolate of E. coli and 2 of Enterobacter cloacae (see 4 text below and table 1). 5 Intervention and outcome 6 The NCIC and the JDHO were notified in early July 2012, and an intervention was 7 launched. The measures taken included: a) cohorting of colonized patients in a 8 completely separate, interim location, with complete separation of staff and 9 equipment from the unaffected babies; b) closing the NICU to new admissions (this 10 was done for 3 weeks); c) reinforcement of infection control practices (including hand 11 hygiene, equipment and environmental cleaning, injection safety, standard and 12 isolation precautions); d) frequent rectal surveillance cultures of all CRE-negative 13 babies in the NICU (at least once a week); e) CRE surveillance of exposed patients 14 hospitalized in other pediatric wards in the hospital; f) CRE surveillance and close 15 monitoring of admissions of exposed infants to other hospitals (via the MoH reporting 16 system) and g) submission of all available CRE isolates for molecular studies at the 17 NCIC lab. Implementation of the intervention was overseen in frequent site visits to 18 the hospital undertaken by staff of the NCIC and JDHO. Surveillance of hospital staff 19 and the mothers of the NICU babies were not performed as colonization among them 20 was not believed to be related to the propagation of the outbreak. 21 During the first week of the intervention, 8 new cases were identified in the NICU 22 (surveillance-7, clinical-1). Two additional new carriers were identified in the next 23 month, all previously exposed (figure 1a). The NICU was re-opened for new 24 admissions after 3 weeks with the previously identified carriers cohorted in a separate 25

8 location. A previously exposed infant readmitted to the Pediatric Intensive Care unit 1 was found to be a carrier of OXA-48-producing K. pneumoniae, and a survey of that 2 unit identified 2 additional carriers, with the infant presumably serving as the index 3 case in that unit. Three previously-exposed infants admitted to another hospital were 4 found to be carriers of OPE (figure 1b: black and grey circles represent acquisition of 5 OPE during or after the initial NICU outbreak (3-7.2012), respectively; a dashed 6 arrow represents home stay prior to re-admission). During the peak of the outbreak 7 (January-August 2012), 49 patients were identified with proven or suspected 8 acquisition of OPE in the NICU, out of a total of 156 patients hospitalized in the 9 NICU during that period (31%). Invasive infections (blood and/or CSF) were detected 10 in 16 patients and 4 of these patients died within 14 days of isolation of the pathogen. 11 One patient carrying the outbreak K. pneumoniae clone, with no known exposure to 12 other carriers or core staff members (i.e., nurses and physicians), was identified in the 13 Pediatrics ward. Following the re-opening of the NICU, 5 additional patients carrying 14 the epidemic K. pneumoniae clone were identified (figure 1b). Additional site visits 15 by the MoH teams were conducted and these patients were cohorted in separate rooms 16 with dedicated nursing staff. Weekly CRE surveillance was continued in the NICU 17 until present with no new cases of OPE identified. 18 In addition to the OPE outbreak in the NICU, 5 additional isolates of carbapenem- 19 resistant K. pneumoniae were detected in the Pediatrics ward and the PICU of MH: 3 20 isolates of OPE were detected by surveillance culture upon admission from Gaza and 21 the PA, all presenting different PFGE patterns than that of the epidemic strain (data 22 not shown); 1 isolate of KPC-producing K. pneumoniae was detected by surveillance 23 culture upon admission to the PICU of a patient transferred from a hospital in central 24 Israel; an additional isolate was later discovered in another patient in that unit. 25

9 Microbiological and molecular features of OXA-48-producing Enterobacteriaceae 1 isolates 2 A total of 31 outbreak-related isolates from 29 patients were submitted for analysis to 3 the NCIC laboratory. K. pneumoniae was isolated from all patients, and all isolates 4 belonged to a single PFGE type, identified as ST39 (table 1). Two patients also 5 carried OXA-48 producing E. cloacae, belonging to two different PFGE types. All 6 isolates were resistant to ampicillin, cefazolin, piperacillin-tazobactam, ceftriaxone 7 and ertapenem, but had MIC values below 1 mg/l for imipenem and meropenem 8 (table 1). The E. coli DH10B transformant isolates from both the K. pneumoniae and 9 E. cloacae donor strains had similar resistance patterns for carbapenems and were also 10 resistant to ampicillin, cefazolin, piperacillin-tazobactam and ceftriaxone but not to 11 any other class of antimicrobials. All isolates tested positive by MHT and carried 12 bla OXA-48 and bla CTX-14 ; they tested negative by the EDTAST, bla KPC and bla NDM PCR. 13 The K. pneumoniae isolates also carried bla CTX-15, bla TEM-1 and bla SHV-1. The bla OXA-48-14 harboring plasmids were ~62 kb in size and carried the repa, para and trau genes, as 15 did our previously described bla OXA-48 -harboring plasmids (3). Unlike these plasmids, 16 they also carried bla CTX-14 and had an identical restriction-fragment length 17 polymorphism pattern that differed from the pattern(s) of these plasmids (data not 18 shown). bla CTX-14 was surrounded by ISEcp1 (AJ242809) (figure 2). The entire 3.1 kb 19 structure was inserted in position 26950, corresponding to the poxa-48a plasmid 20 (JN626286.1) (18). 21 22 Discussion 23 In the present study, we describe the first case of OPE in the Neonatal and Pediatric 24 population. Moreover, this is the first report of an outbreak caused by CRE of any 25

10 kind in this population. This population has not been spared nosocomial outbreaks 1 caused by other types of antimicrobial-resistant bacteria, e.g., extended-spectrum β- 2 lactamase producing Enterobacteriaceae (17), corresponding with the occurrence in 3 other patient populations. It is noteworthy however, that although sporadic cases of 4 OPE in adults have been described in Israel (3), the first and so far the only OPE 5 outbreak in Israel occurred in the pediatric population. The data regarding the 6 prevalence of OPE in the PA and Gaza are limited, but based on this study and our 7 previous report (3), it appears likely that OPE are endemic in these areas, similar to 8 the situation in other Middle-Eastern countries (5, 14). 9 Due to the fact that several months elapsed between the onset of the outbreak and the 10 involvement of the MoH, we were unable to obtain the microbiological data necessary 11 to determine its origin. During the investigation of this outbreak we identified carriers 12 of additional, unrelated OPE who were admitted to other pediatric services from the 13 PA or Gaza. It is therefore likely that the outbreak strain was introduced in a similar 14 way, following an admission of an unrecognized carrier, either directly to the NICU 15 or to one of the other pediatric services. The monoclonal nature of the outbreak and 16 the spread of the outbreak strain to other ward lead to the conclusion that although 17 initial introduction of the strain via maternal carriage could not be excluded, this 18 mechanism is unlikely to have played a role in the subsequent propagation of the 19 outbreak. 20 The epidemic strain presumably spread initially in the NICU, leading to colonization 21 of at least 49 patients with 16 invasive infections. From there, we were able to trace 22 its spread to the PICU following the re-admission of a former NICU-exposed baby 23 that led to transmission of the strain to two additional children. Following the 24 intervention, the incidence of new colonization or infection significantly declined. 25

11 However, despite the cohorting of the OPE carriers and the allocation of a dedicated 1 nursing staff to care for them, the epidemic strain re-appeared in the re-opened NICU, 2 presumably due to a single exposed infant who had previously tested negative, who 3 transmitted the strain to 4 additional infants. The strain was also detected in one 4 patient in the Pediatrics ward not known to be exposed to any of the carriers (figure 5 1b), suggesting transmission via either contaminated equipment or other hospital staff. 6 Surveillance of OPE colonization among hospital staff was not performed as staff 7 carriage has not been shown to be an important factor in nosocomial transmission of 8 multi-drug-resistant Enterobacteriaceae (1). Indeed, implementation of proper 9 infection control policies, including hand hygiene and ongoing surveillance of patients 10 at risk, proved to be sufficient to contain the resurgence of the outbreak. 11 One of the dreaded threats to public health in any local outbreak is its potential to 12 spread via unrecognized carriers to other healthcare centers (13). The establishment of 13 our nationally-coordinated registry of CRE (23) enabled us to alert and communicate 14 with the neighboring hospitals, allowing detection of carriers upon admission and 15 their prompt isolation, resulting in outbreak containment. 16 Similar to previous reports from Europe [7-10], the main mechanism of this outbreak 17 was monoclonal spread of K. pneumoniae. The epidemic clone, ST-39, was 18 previously reported as a VIM-producing strain in a nosocomial outbreak in Spain 19 [26], but has never been associated with OPE outbreaks. In addition, we identified 20 two cases of presumed horizontal plasmid transfer in two patients, who carried OXA- 21 48-producing E. cloacae in addition to the epidemic strain. The bla OXA-48 -harboring 22 plasmid in these isolates was similar in size to the poxa-48a plasmid and possessed 23 its characteristic genes (18), but differed from it by the presence of bla CTX-M-14 carried 24 along the ISEcp1 transposon. Although additional β-lactamase-encoding genes, 25

12 including bla CTX-M-15, were also present in the outbreak strain as in previous reports 1 (26), this is only the second report describing the integration of an additional 2 resistance gene into the poxa-48a plasmid (21). This unique characteristic allowed 3 us to track the route of horizontal plasmid transfer and to differentiate the outbreak 4 strain from non-related OPE strains that were introduced to the hospital during the 5 outbreak. The addition of another resistance element to this plasmid is an ominous 6 finding due to the plasmid's demonstrated high degree of transmissibility (3, 5); the 7 added resistance gene in this case renders all recipient strains resistant to 8 cephalosporins, thus limiting therapeutic options even further. 9 In conclusion, the study of this outbreak teaches us that while standard infection 10 control measures are effective for initial containment, continuous vigilant monitoring 11 is essential in order to detect and respond to a subsequent resurgence. It also 12 demonstrates the value of a centralized coordinating body, allowing for prompt 13 identification of unidentified carriers on admission to other hospitals. In areas where 14 CRE of various types are endemic, a thorough molecular analysis that includes 15 identification of resistance genes, clones and plasmids is of paramount importance in 16 an outbreak setting for accurate assessment of the outbreak's course, containment and, 17 should it occur, resurgence. 18 19 20 21 22 23 24

13 Figure 1. Temporal and spatial analysis of the OXA-48 producing 1 Enterobacteriaceae (OPE) outbreak in the NICU. A. Incidence of suspected and 2 proven OPE infections (surveillance and clinical cultures). Cases initially identified in 3 the MH NICU (grey lines), other MH wards and additional hospital (black lines) are 4 included. B. Spatial dissemination of the OPE outbreak outside the NICU. OPE 5 outbreak was ongoing in the NICU from March 2012 until July 2012 (solid line); the 6 NICU was opened to new admissions only, after 3 weeks (dashed line). Filled and 7 empty black circles represent acquisition of OPE during the initial NICU outbreak (3-8 7.2012), with first detection during or following NICU stay, respectively. Grey circles 9 represent acquisition of OPE outside of the initial NICU outbreak. A dashed arrow 10 represents home stay prior to re-admission. A solid arrow represents transmission of 11 the outbreak strain. 12 13 Figure 2. Schematic structure of the bla CTXM-14 -containing insert. The orientation 14 of the insert is presented in relation to the poxa-48a plasmid (JN626286.1). 15 16 17 18 19 20 21 22 23 24 25

14 References 1 1. A. Adler, M. Gniadkowski, A. Baraniak, R. Izdebski, J. Fiett, A. Salvia, 2 J.V. Samso, C. Lawrence, J. Salomon, M. Paul, Y. Schwartzberg, E. 3 Mordechai, A. Rossini, J. Fierro, C. Lammens, S. Malhotra-Kumar, H. 4 Goossens, W. Hryniewicz, C. Brun-Buisson YC. 2013. A multinational study 5 of colonisation with extended-spectrum beta-lactamases (ESBL)-producing 6 Enterobacteriaceae in HCW and family members of patients admitted to 7 rehabilitation centres. 23rd European Congress of Clinical Microbiology and 8 Infectious Diseases (ECCMID). Berlin. 9 2. A. Valverde, P. Ruiz-Garbajosa, T. Curiao, M. Tato, D. Gijón, F. Baquero, 10 T.M. Coque RC. 2011. Klebsiella pneumoniae sequence types originally 11 associated with specific ESBLs might act as substrates for recently emerged 12 metallo-beta-lactamases or KPC enzymes.21st European Congress of Clinical 13 Microbiology and Infectious Diseases (ECCMID). Milan. 14 3. Adler a., Shklyar M, Schwaber MJ, Navon-Venezia S, Dhaher Y, Edgar R, 15 Solter E, Benenson S, Masarwa S, Carmeli Y. 2011. Introduction of OXA- 16 48-producing Enterobacteriaceae to Israeli hospitals by medical tourism. 17 Journal of Antimicrobial Chemotherapy 66:2763 2766. 18 4. Adler A, Navon-Venezia S, Moran-Gilad J, Marcos E, Schwartz D, 19 Carmeli Y. 2011. Laboratory and clinical evaluation of screening agar plates 20 for detection of carbapenem-resistant enterobacteriaceae from surveillance 21 rectal swabs. Journal of clinical microbiology 49:2239 42. 22 5. Carrër A, Poirel L, Yilmaz M, Akan OA, Feriha C, Cuzon G, Matar G, 23 Honderlick P, Nordmann P. 2010. Spread of OXA-48-encoding plasmid in 24 Turkey and beyond. Antimicrobial agents and chemotherapy 54:1369 73. 25 6. Clinical and Laboratory Standards Institute. 2012. Methods for Dilution 26 Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically ; 27 Approved Standard Ninth Edition. Clinical and Laboratory Standards 28 Institute, Wayne, PA. 29 7. Clinical and Laboratory Standards Institute. 2011. Performance Standards 30 for Antimicrobial Susceptibility Testing; Twenty-First Informational 31 Supplement. Approved standard MS100-S20. Clinical and Laboratory 32 Standards Institute, Wayne, PA. 33 8. Cuzon G, Ouanich J, Gondret R, Naas T, Nordmann P. 2011. Outbreak of 34 OXA-48-positive carbapenem-resistant Klebsiella pneumoniae isolates in 35 France. Antimicrobial agents and chemotherapy 55:2420 3. 36 9. Diancourt L, Passet V, Verhoef J, Grimont PAD, Brisse S. 2005. Multilocus 37 sequence typing of Klebsiella pneumoniae nosocomial isolates. Journal of 38 clinical microbiology 43:4178 82. 39

15 10. Giske CG, Gezelius L, Samuelsen Ø, Warner M, Sundsfjord A, Woodford 1 N. 2011. A sensitive and specific phenotypic assay for detection of metallo-β- 2 lactamases and KPC in Klebsiella pneumoniae with the use of meropenem 3 disks supplemented with aminophenylboronic acid, dipicolinic acid and 4 cloxacillin. Clinical microbiology and infection 17:552 6. 5 11. Goren MG, Carmeli Y, Schwaber MJ, Chmelnitsky I, Schechner V, 6 Navon-Venezia S. 2010. Transfer of carbapenem-resistant plasmid from 7 Klebsiella pneumoniae ST258 to Escherichia coli in patient. Emerging 8 infectious diseases 16:1014 7. 9 12. Leavitt A, Carmeli Y, Chmelnitsky I, Goren MG, Ofek I, Navon-Venezia 10 S. 2010. Molecular epidemiology, sequence types, and plasmid analyses of 11 KPC-producing Klebsiella pneumoniae strains in Israel. Antimicrobial agents 12 and chemotherapy 54:3002 6. 13 13. Lopez J a, Correa a, Navon-Venezia S, Correa a L, Torres J a, Briceño 14 DF, Montealegre MC, Quinn JP, Carmeli Y, Villegas M V. 2011. 15 Intercontinental spread from Israel to Colombia of a KPC-3-producing 16 Klebsiella pneumoniae strain. Clinical microbiology and infection : the official 17 publication of the European Society of Clinical Microbiology and Infectious 18 Diseases 17:52 6. 19 14. Matar GM, Dandache I, Carrër A, Khairallah M-T, Nordmann P, Sabra 20 A, Araj GF. 2010. Spread of OXA-48-mediated resistance to carbapenems in 21 Lebanese Klebsiella pneumoniae and Escherichia coli that produce extended 22 spectrum beta-lactamase. Annals of tropical medicine and parasitology 23 104:271 4. 24 15. Navon-Venezia S, Chmelnitsky I, Leavitt A, Carmeli Y. 2008. 25 Dissemination of the CTX-M-25 family beta-lactamases among Klebsiella 26 pneumoniae, Escherichia coli and Enterobacter cloacae and identification of the 27 novel enzyme CTX-M-41 in Proteus mirabilis in Israel. The Journal of 28 antimicrobial chemotherapy 62:289 95. 29 16. Paño-Pardo JR, Ruiz-Carrascoso G, Navarro-San Francisco C, Gómez-Gil 30 R, Mora-Rillo M, Romero-Gómez MP, Fernández-Romero N, García- 31 Rodríguez J, Pérez-Blanco V, Moreno-Ramos F, Mingorance J. 2013. 32 Infections caused by OXA-48-producing Klebsiella pneumoniae in a tertiary 33 hospital in Spain in the setting of a prolonged, hospital-wide outbreak. The 34 Journal of antimicrobial chemotherapy 68:89 96. 35 17. Paterson DL, Bonomo RA. 2005. Extended-spectrum beta-lactamases: a 36 clinical update. Clinical microbiology reviews 18:657 86. 37 18. Poirel L, Bonnin R a, Nordmann P. 2012. Genetic features of the widespread 38 plasmid coding for the carbapenemase OXA-48. Antimicrobial agents and 39 chemotherapy 56:559 62. 40

16 19. Poirel L, He C, Tolu V, Nordmann P. 2004. Emergence of Oxacillinase- 1 Mediated Resistance to Imipenem in Klebsiella pneumoniae. Antimicrobial 2 agents and chemotherapy 48:15 22. 3 20. Potron A, Kalpoe J, Poirel L, Nordmann P. 2011. European dissemination of 4 a single OXA-48-producing Klebsiella pneumoniae clone. Clinical 5 microbiology and infection : the official publication of the European Society of 6 Clinical Microbiology and Infectious Diseases 17:E24 6. 7 21. Potron A, Nordmann P, Rondinaud E, Jaureguy F, Poirel L. 2013. A 8 mosaic transposon encoding OXA-48 and CTX-M-15: towards pan-resistance. 9 The Journal of antimicrobial chemotherapy 68:476 7. 10 22. Schechner V, Straus-Robinson K, Schwartz D, Pfeffer I, Tarabeia J, 11 Moskovich R, Chmelnitsky I, Schwaber MJ, Carmeli Y, Navon-Venezia S. 12 2009. Evaluation of PCR-based testing for surveillance of KPC-producing 13 carbapenem-resistant members of the Enterobacteriaceae family. Journal of 14 clinical microbiology 47:3261 5. 15 23. Schwaber MJ, Lev B, Israeli A, Solter E, Smollan G, Rubinovitch B, Shalit 16 I, Carmeli Y. 2011. Containment of a country-wide outbreak of carbapenem- 17 resistant Klebsiella pneumoniae in Israeli hospitals via a nationally 18 implemented intervention. Clinical infectious diseases 52:848 55. 19 24. Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M. 2006. ISfinder: 20 the reference centre for bacterial insertion sequences. Nucleic acids research 21 34:D32 6. 22 25. Tzouvelekis LS, Markogiannakis a., Psichogiou M, Tassios PT, Daikos GL. 23 2012. Carbapenemases in Klebsiella pneumoniae and Other 24 Enterobacteriaceae: an Evolving Crisis of Global Dimensions. Clinical 25 Microbiology Reviews 25:682 707. 26 26. Voulgari E, Zarkotou O, Ranellou K, Karageorgopoulos DE, Vrioni G, 27 Mamali V, Themeli-Digalaki K, Tsakris A. 2013. Outbreak of OXA-48 28 carbapenemase-producing Klebsiella pneumoniae in Greece involving an ST11 29 clone. The Journal of antimicrobial chemotherapy 68:84 8. 30 27. Woodford N, Fagan EJ, Ellington MJ. 2006. Multiplex PCR for rapid 31 detection of genes encoding CTX-M extended-spectrum (beta)-lactamases. The 32 Journal of antimicrobial chemotherapy 57:154 5. 33 34 35

Table 1. Microbiological and molecular characteristics of OXA-48 producing Enterobacteriaceae isolates. Variable K. pneumoniae E. cloacae No. of isolates 29 2 Source 1 B-4, S-1, R-24 R Strains 2, no. 1 (ST39) 2 β-lactamase-encoding genes Ertapenem MIC, mg/l (median, range) Imipenem MIC, mg/l (median, range) Meropenem MIC, mg/l (median, range) bla OXA-48, -CTX-M-14, -CTX-M- bla OXA-48, -CTX-M-14 15, TEM-1, SHV-1 2, 2->16 3, 2-4 0.5, 0.25->8 0.75, 0.5-1 0.5, 0.25->8 0.5, 0.25-1 bla OXA-48 plasmid, kb ~62 ~62 bla OXA-48 plasmid, genes bla CTX-M-14, repa, para, trau bla CTX-M-14, repa, para, trau Tn1999 type 1999.2 1999.2 1 -source: B-blood, S-sputum, R-rectal; 2 -typing was done by PFGE and MLST for K. pneumoniae and by PFGE for E. cloacae.