International Journal of Antimicrobial Agents 31 (2008) 523–526 Carbapenem-resistant Escherichia coli and Klebsiella pneumoniae isolates from Turkey with OXA-48-like carbapenemases and outer membrane protein loss Dolunay Găulmez a,b, , Neil Woodford b , Marie-France I Palepou b , Shazad Mushtaq b , Gokhan Metan a,c , Yusuf Yakupogullari d,e , Sesin Kocagoz f , Omrum Uzun g , Gulsen Hascelik a , David M Livermore b a Hacettepe University Faculty of Medicine, Department of Microbiology and Clinical Microbiology, 06100 Sıhhiye, Ankara, Turkey b Antibiotic Resistance Monitoring and Reference Laboratory, Centre for Infections, Health Protection Agency, London, UK c Erciyes University Faculty of Medicine, Department of Infection and Clinical Microbiology, Kayseri, Turkey d Fırat University Faculty of Medicine, Department of Microbiology and Clinical Microbiology, Elazıg, Turkey e Malatya State Hospital, Department of Microbiology and Clinical Microbiology, Malatya, Turkey f Yeditepe University Faculty of Medicine, Department of Microbiology and Clinical Microbiology, Istanbul, Turkey g Hacettepe University Faculty of Medicine, Department of Internal Medicine, Section of Infectious Diseases, Ankara, Turkey Received December 2007; accepted 15 January 2008 Abstract Treatment options are limited in infections caused by extended-spectrum -lactamase (ESBL)-producing Enterobacteriaceae, with carbapenems generally preferred Disturbingly, however, carbapenem-resistant strains are emerging worldwide Here we report two clinical isolates, one Escherichia coli and one Klebsiella pneumoniae, each with high-level carbapenem resistance (imipenem minimum inhibitory concentration of 32 g/mL) They were isolated following imipenem therapy from two hospital patients who had received imipenem therapy in different regions of Turkey Both isolates produced OXA-48-like carbapenemases, enzymes so far reported only from Turkey Both isolates also had group CTX-M-type ESBLs and had lost major outer membrane proteins OXA-48-like carbapenemases appear to be scattered in Turkey and surveillance to determine their prevalence is warranted © 2008 Elsevier B.V and the International Society of Chemotherapy All rights reserved Keywords: Escherichia coli; Klebsiella pneumoniae; Carbapenem resistance; Carbapenemase; OXA-48; CTX-M ESBL Introduction Enterobacteriaceae producing extended-spectrum lactamases (ESBLs) are increasingly frequent agents of infection, with their spread driving greater use of carbapenems [1,2] Until recently, enzymatic carbapenem resistance was limited to infrequent pathogens with intrinsic carbapenemases such as Stenotrophomonas maltophilia, Aeromonas spp., Flavobacterium spp., Legionella gormanii, Bacillus cereus and a few Bacteroides fragilis isolates [3] However, ∗ Corresponding author Tel.: +90 312 305 1560; fax: +90 312 305 2084 E-mail addresses: dolunayglm@yahoo.com, dolunay@hacettepe.edu.tr (D Găulmez) acquired carbapenemases belonging to Ambler molecular classes A, B and D are increasingly being encountered [4] KPC carbapenemases belonging to class A may be plasmid or chromosomally encoded and are increasingly widespread; they are present in strains of Klebsiella pneumoniae that have spread across the USA from New York [4,5] and have recently also been reported in various Enterobacteriaceae from China, Colombia, France, Israel and the UK [4] Acquired class B metallo--lactamases (MBLs) are also globally distributed and principally comprise the IMP and VIM series [4,6] These are most often found in non-fermenters but have also become a serious public health concern in Enterobacteriaceae in a few locales, as, 0924-8579/$ – see front matter © 2008 Elsevier B.V and the International Society of Chemotherapy All rights reserved doi:10.1016/j.ijantimicag.2008.01.017 524 D Găulmez et al / International Journal of Antimicrobial Agents 31 (2008) 523–526 for example, with VIM enzymes among K pneumoniae in Greece [7,8] Class D (OXA-type) carbapenemases are mostly reported in Acinetobacter baumannii, occurring in several widely disseminated clones [9], with only a few reports of production by Enterobacteriaceae [10–12] Here we report the carbapenemase present in single carbapenem-resistant isolates of Escherichia coli and K pneumoniae from hospital patients in different regions of Turkey Materials and methods 2.1 Escherichia coli isolate Escherichia coli isolate 20079740 was isolated from a 45-year-old male patient in Hacettepe University Hospital, Ankara, Turkey, in July 2004 The patient had chronic renal insufficiency and suffered from bilateral pleural effusion He had variously received cefepime, cefoperazone/sulbactam, piperacillin/tazobactam and teicoplanin over a 6-week period After a pleural decortication operation, he was intubated and transferred to an intensive care unit (ICU), where he received imipenem 500 mg/day for 20 days following the clinical diagnosis of nosocomial sepsis, although without positive blood cultures Carbapenem-resistant E coli was then isolated from repeated pleural effusion cultures Although amikacin 300 mg/day was added to the regimen, the patient died 14 days later 2.2 Klebsiella pneumoniae isolate Klebsiella pneumoniae isolate CarR was isolated from a 61-year-old male patient in Fırat University Medical Centre, Elazıg (a city located in the eastern part of Turkey), in June 2005 He had been admitted to the ICU with a diagnosis of intracranial haemorrhage He received cefoperazone/sulbactam, cefepime and imipenem for different periods Klebsiella pneumoniae resistant to all -lactams, including carbapenems, was isolated from urine on the 29th day following admission This infection was treated successfully with amikacin monotherapy tions (MICs) were determined by agar dilution methodology following British Society for Antimicrobial Chemotherapy (BSAC) guidelines [15] 2.4 Polymerase chain reaction (PCR) and DNA sequencing Multiplex PCR was performed to detect genes encoding: (i) MBLs belonging to the IMP, VIM, GIM, SPM and SIM families [16]; (ii) CTX-M-type ESBLs, including those belonging to CTX-M groups 1, 2, 8, and 25 [17]; and (iii) plasmidic AmpC genes of the ACC, CIT, DHA, ENT/EBC, FOX and MOX groups [18] Single PCR reactions were used to screen for blaKPC [19], blaOXA-48 and insertion element IS1999 [10] as well as to amplify blaCTX-M orf [20] and blaCTX-M group [21] The blaOXA-48 , blaCTX-M orf and blaCTX-M group PCR products were cleaned using a Geneclean® Turbo kit (QBIO Gene, Cambridge, UK) and were sequenced on both strands by dye terminator chemistry on a Beckman Coulter CEQ8000 sequencer (Beckman Coulter, High Wycombe, UK) BioNumerics software (Applied Maths, St-MartensLatem, Belgium) was used to obtain consensus sequences, which were compared with those in the National Center for Biotechnology Information (NCBI) database using the BLAST program [22] 2.5 Transformation of β-lactam resistance Plasmid DNA was extracted from clinical isolates by alkaline lysis [23] and transformed into E coli DH5␣ by electroporation at 2.5 kV, 25 F and 200 using a Gene Pulser apparatus (Bio-Rad, Hemel Hempstead, UK) Transformants were selected on nutrient agar containing g/mL cefotaxime or 0.25 g/mL ertapenem 2.6 Outer membrane protein analysis The outer membrane proteins of E coli 20079740 and K pneumoniae CarR were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using membrane extracts from bacteria grown overnight in nutrient broth [24] 2.3 Antimicrobial susceptibility tests Susceptibilities were determined locally using the BD Phoenix System (Becton Dickinson, Franklin Lakes, NJ) for the E coli isolate 20079740 in Ankara and by disk diffusion for the K pneumoniae isolate CarR in Elazıg ESBL confirmation tests were done by disk diffusion according to Clinical and Laboratory Standards Institute recommendations [13] Inhibition zones around imipenem disks with and without ethylene diamine tetraacetic acid (EDTA) were compared to detect likely MBL production [14] On examination at the Antibiotic Resistance Monitoring and Reference Laboratory (London, UK), minimum inhibitory concentra- Results and discussion Escherichia coli 20079740 and K pneumoniae CarR were found to be resistant to imipenem and meropenem in routine susceptibility tests Each also gave a positive ESBL disk synergy result A phenotypic MBL test was tentatively interpreted as positive for E coli 20079740: there was no zone of inhibition around 10 g imipenem or meropenem disks, whereas hazy zones were observed when these disks were supplemented with L of 0.5 M EDTA (pH 8), suggesting potentiation MIC determination by BSAC methodology D Găulmez et al / International Journal of Antimicrobial Agents 31 (2008) 523–526 525 Table Minimum inhibitory concnetrations (MICs) for clinical isolates Escherichia coli and Klebsiella pneumoniae with OXA-48-like carbapenemase Antimicrobial agent Aztreonam Cefotaxime Cefotaxime/clavulanic acid Ceftazidime Ceftazidime/clavulanic acid Cefpirome Cefpirome/clavulanic acid Cefoxitin Piperacillin Piperacillin/tazobactam Imipenem Imipenem/EDTA (320 g/mL) Meropenem Ertapenem Ciprofloxacin Amikacin Gentamicin Tobramycin Minocycline Tigecycline Colistin MIC (g/mL) E coli 20079740 K pneumoniae CarR >64 >64 >32/4 256 >32/4 >64 >32/4 >64 >64 >64/4 32 16 16 >16 >8 >16 >16 ≤0.25 ≤0.5 >64 >256 >32/4 32 16/4 >64 >32/4 >64 >64 >64/4 32 16 16 >16 0.25 0.5 0.5 32 EDTA, ethylene diamine tetraacetic acid confirmed that E coli 20079740 and K pneumoniae CarR were broadly resistant to -lactams, including imipenem (MIC = 32 g/mL), meropenem (MIC = 16 g/mL) and ertapenem (MIC > 16 g/mL) (Table 1), but, in contrast with the disk results, did not indicate synergy between cephalosporins and clavulanic acid or between imipenem and EDTA PCR indicated that both E coli 20079740 and K pneumoniae CarR harboured blaOXA-48 -like genes and, in each case, partial sequencing of a 798-bp PCR product indicated identity with the prototype blaOXA-48 gene (GenBank accession no AY236073), described from a K pneumoniae isolate collected in Istanbul, Turkey [10] In addition, genes encoding group CTX-M ESBLs were detected in both isolates; partial sequencing was consistent with blaCTX-M-15 or blaCTX-M-3 in each case No genes encoding MBLs were detected, nor any encoding plasmidic AmpC enzymes or KPC carbapenemase The absence of MBL genes emphasises again the poor specificity of EDTA-based phenotypic tests; false-positive results are also frequent with Pseudomonas aeruginosa that lack MBLs and for A baumannii producing OXA-23-like -lactamases [25] Attempts were made to transfer resistance to E coli DH5␣ by transformation Although it was possible to transfer blaCTX-M -mediated cefotaxime resistance from both isolates, carbapenem resistance was not transferred in either case This suggests that the blaOXA-48 -like genes may have become chromosomally integrated The first reported Klebsiella strain with OXA-48 enzyme had a copy of insertion sequence IS1999 immediately upstream of blaOXA-48 [10], which is notable because this element has been associated Fig Outer membrane protein profiles of isolates with OXA-48-like carbapenemase Lane M, SDS-PAGE molecular weight standards, low range (Bio-Rad, UK); lane A, Klebsiella pneumoniae ATCC 10273 (control); lane B, K pneumoniae CarR; lane C, Escherichia coli NCTC 10418 (control); lane D, E coli 20079740 with increased expression of other -lactamases, including VEB-1 [26] Escherichia coli 20079740 carried IS1999 within its genome, as detected by PCR, but K pneumoniae CarR did not; the exact relationship between IS1999 and the blaOXA-48 -like gene in E coli 20079740 needs further investigation Both strains lacked at least one outer membrane porin (probably OmpC for E coli 20079740 and both OmpK35 and OmpK36 for K pneumoniae CarR) (Fig 1) Loss of these porins is known to augment carbapenem resistance conferred by -lactamases with weak or strong carbapenemase activity [27,28] The earlier K pneumoniae strain with an OXA-48 enzyme from Istanbul also lacked an outer membrane protein [10] Although carbapenemase activity of the blaOXA-48 -like gene product was not demonstrated, the identity of the gene to the prototype blaOXA-48 gene, together with the porin loss, is likely to be the reason for the carbapenem resistance detected in E coli 20079740 and K pneumoniae CarR To date, only a few carbapenem-resistant Enterobacteriaceae isolates have been reported from Turkey Gacar et al [29] reported an Enterobacter cloacae isolate with a VIM-5 enzyme, whilst at the Istanbul hospital where the first producer was collected, OXA-48 carbapenemase has since been found in several further isolates of K pneumoniae as well as in Citrobacter freundii [11], E coli (Laurent Poirel, personal communication) and, most recently, in 40 K pneumoniae isolates from a clonal outbreak [30] Carbapenems are the treatment of choice for serious infections caused by ESBL-producing Enterobacteriaceae and are critically important in this role For example, in Hacettepe Hospital where E coli 20079740 was isolated, 72% of bacteraemias caused by ESBL-producing E coli were treated with 526 D Găulmez et al / International Journal of Antimicrobial Agents 31 (2008) 523–526 a carbapenem [31] Any emergence of carbapenem resistance is therefore a serious concern and, with evidence of its scatter in Turkey, there is a clear need for a structured nationwide prevalence survey Acknowledgments Part of this work was presented at the 7th Febrile Neutropenia Symposium, 23–26 March 2006, Ankara, Turkey (poster no 072) The authors would like to thank Dr Mark A Toleman (Bristol, UK), Prof Kyungwon Lee (Seoul, South Korea) and Dr Ana Gales (Sao Paulo, Brazil) for providing the VIM-7-, SIM-1- and SPM-1-producing controls, respectively Funding: This study was supported by Federation of European Microbiological Societies (FEMS) Research 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present in single carbapenem- resistant isolates of Escherichia coli and K pneumoniae from hospital patients in different regions of Turkey Materials and methods 2.1 Escherichia. .. [27,28] The earlier K pneumoniae strain with an OXA- 48 enzyme from Istanbul also lacked an outer membrane protein [10] Although carbapenemase activity of the blaOXA -48 -like gene product was... 525 Table Minimum inhibitory concnetrations (MICs) for clinical isolates Escherichia coli and Klebsiella pneumoniae with OXA- 48- like carbapenemase Antimicrobial agent Aztreonam Cefotaxime Cefotaxime/clavulanic