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Introduction

Carbapenems have increasingly been used in severe Enterobacteriaceae infections due to a worldwide increase of extended spectrum β-lactamases (ESBL)-producing organisms, which has resulted in the emergence of carbapenem resistances [1]. The aim of this study was to identify and characterize four carbapenem-non-susceptible isolates, three Klebsiella pneumoniae and one Escherichia coli collected from April through October 2008 at Gulhane Military Medical Academy Hospital (GMMAH), Ankara, Turkey.

Case 1A 3-year-old boy, diagnosed with neuroblastoma 2 years before, was admitted to the emergency department with complaints of a one week history of fever (39°C), cough, and vomiting. His cough had begun 7 days earlier and was productive of purulent sputum. On physical examination, there were rales and rhonchi in the left lower lung field by auscultation. Tracheal intubation was performed on the patient because of the respiratory distress. Laboratory evaluation at that time revealed a leukocyte count of 1000/mm3, a hemoglobin level of 8 g/dl, and erythrocyte sedimentation rate of 23.1 mm/h. Urine and throat cultures were negative. Chest radiography revealed bilateral interstitial lung infiltrates. The child was given meropenem for pneumonia. During hospitalization, imipenem-meropenem resistant Klebsiella pneumoniae was grown in blood cultures. Meropenem was changed to amikacin on the basis of the susceptibility results. The patient’s clinical condition deteriorated

Address correspondence and reprint requests to Abdullah Kilic, M.D., Departments of Microbiology and Clinical Microbiology, Gulhane Military Medical Academy and School of Medicine 06018, Ankara; tel +90 312 3043412; fax +90 312 3043402; e-mail abkilic@gata.edu.tr; or to Yi-Wei Tang, M.D., Ph.D., Molecular Infectious Disease Laboratory, Vanderbilt University Hospital, 4605 TVC, Nashville, TN 37232-5310; tel (615) 322-2035; fax (615) 343-8420; e-mail yiwei.tang@vanderbilt.edu

Identification and Characterization of OXA-48 Producing, Carbapenem-Resistant Enterobacteriaceae Isolates in Turkey

Abdullah Kilic1, Zerin Aktas2, Orhan Bedir1, Ramazan Gumral1, Yasemin Bulut3, Charles Stratton4, Yi-Wei Tang4, and A. Celal Basustaoglu1

1Department of Microbiology and Clinical Microbiology, Gulhane Military Medical Academy and School of Medicine 06018, Etlik, Ankara, Turkey; 2Departments of Microbiology and Clinical Microbiology Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey; 3Department of Microbiology, College of Medicine, Firat University, Elazig, Turkey; 4Departments of Pathology and Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA

Abstract. Carbapenem resistance in Enterobacteriaceae isolates has been reported from Turkey and is most often mediated by OXA-48 type carbapenemases. We report the identification and characterization of four carbapenem-resistant isolates (three Klebsiella pneumoniae and one Escherichia coli) among 515 clinical Enterobacteriaceae isolates collected during a 7-month study period in Ankara, Turkey. The four isolates were recovered from blood and urine specimens in patients with varied clinical manifestations. They had distinct pulsed-field gel electrophoresis patterns and harbored a variety of β-lactamases including blaTEM-1, blaSHV-12 genes, blaSHV-11, and/or blaCTX-M-15. PCR and sequencing analysis revealed that the blaOXA-48 gene was present in all four isolates. Our data indicated that the OXA-48-type carbapenemase was the only mechanism for carbapenem resistance in our hospital.

Keywords: Antibiotic resistance; Carbapenemase; OXA-48; Enterobacteriaceae

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over the first three days despite antibiotic treatment. He developed sudden cardiac arrest and was resuscitated, but the resuscitation was unsuccessful.

Case 2An 85-year-old woman presented with weakness in her legs and arms, sluggishness, and headache. She had been diagnosed in 2003 with Alzheimer disease and bladder cancer and had been followed over the past 8 years because of these two illnesses. She had had several episodes of headache and weakness during this 8-year period. On physical examination, the patient was un-cooperative. Her temperature was 38.7°C (oral); pulse, 92/minute; respirations, 20/minute; and blood pressure, 133/79 mm Hg. Her neck, cardiac and lung examinations were unremarkable. The blood hemoglobin level was 13.7 g/dl, the platelet count was 558000/mm3, and the leukocyte count was 23000/mm3. An abdominal ultrasound examination revealed that both kidneys exhibited grade III hydronephrosis and had solid masses. The liver and spleen ultrasound findings were unremarkable. Tracheal intubation was performed because of the difficulties with respiration. Urine and throat swab cultures for bacteria were negative. She was given meropenem the first day of admission. Blood cultures were performed on the second and third days after admission. Imipenem-meropenem resistant Escherichia coli was isolated from her blood cultures on the third day. Meropenem was changed to tigecycline on the basis of susceptibility results. Seven days after this antibiotic treatment, her blood cultures were negative.

Case 3A 23-year-old man was admitted to the general surgery department with severe abdominal pain and abdominal distension. An abdominal mass was detected by a computerized-assisted tomography scan of his abdomen. The incisional biopsy was diagnosed as Burkitt’s lymphoma. Based on these biopsy results, the patient was admitted to the hematology service with a diagnosis of Burkitt’s lymphoma. He then received six cycles of chemotherapy for this disease and was discharged. Six months later, he again presented

with abdominal pain and weakness. On physical examination, he was noted to have a mass in his right abdomen. His abdominal examination also revealed tenderness by palpation. His temperature was 37.2°C (oral); pulse, 100/minute; and blood pressure, 140/60 mm Hg. His laboratory investigations showed a white blood cell count of 300/mm3, hemoglobin of 11.5g/dL, and an erythrocyte sedimentation rate of 33.5 mm/h. His neck, cardiac, lung examinations were unremarkable. A bone marrow biopsy was performed and revealed marrow fibrosis with a dense infiltration of undifferentiated blast cells. He was started on an aggressive chemotherapy regimen with hyper-CVAD chemotherapy for 6 cycles. While receiving this chemotherapy, he developed a fever (38.2°C) and received antibiotic therapy with imipenem and teicoplanin. Blood cultures revealed an imipenem-meropenem resistant K. pneumoniae. The imipenem therapy was changed to tigecycline on the basis of susceptibility results. His fever abated within two days after initiation of the tigecycline therapy. The patient subsequently improved and was discharged.

Case 4A 23-year-old male patient presented to the urology department complaining of occasional dysuria, suprapubic pain, and lower abdominal discomfort. This patient had been previously diagnosed with urinary tuberculosis and had undergone augmentation cystoplasty (sigmoidocystoplasty and bilateral ureterosigmoidostomy). He was admitted as a patient due to his reduced bladder capacity because of bilateral vesicoureteral reflux (VUR). The physical examination was unremarkable except for mild tenderness in the supra-pubic region. On examination, his temperature was 37.3°C; pulse, 85/minute; respirations, 22/minute; and blood pressure, 117/63 mm Hg. Blood test results showed a hematocrit of 28.8%, an hemoglobin of 10.1 g/dl, an erythrocyte sedimentation rate of 28 mm/h, a platelet count of 376 000 /mm3, leukocytes of 6200/mm3, glucose of 66, a BUN of 169.3 and a creatinine of 1.11. Ultrasonography revealed normal kidneys and ureters. The patient then had surgery to correct his colovesical fistula and

OXA-48 producing Enterobacteriaceae 163

was given prophylactic therapy with ertapenem. During the hospitalization, he next developed an episode of cystitis and urine cultures grew imipenem-meropenem resistant K. pneumoniae. Based on susceptibility results, the patient was given tigecyclin. Urine cultures were negative after the antibiotic treatment. The patient was then discharged. Materials and Methods

Bacterial strains GMMAH is a 1,500-bed teaching hospital in Ankara, Turkey. All Enterobacteriaceae isolates were identified by conventional biochemical tests and confirmed by the Phoenix, an automated bacteriology system that performs bacterial identification (Becton Dickinson Diagnostic Systems, Sparks, MD, USA).

Susceptibility testing All isolates were tested against 19 antimicrobials by the Phoenix system (Becton Dickinson Diagnostic Systems). Interpretations of the test results were made using the CLSI criteria [2]. The susceptibility of the carbapenem-resistant isolates was also tested against sixteen antimicrobial agents by the CLSI reference broth microdilution using caution-adjusted Mueller-Hinton broth (BD Biosciences, Sparks, Md.) [2,3]. E. coli ATCC 25922 and K. pneumoniae ATCC 13883 were used during testing for quality assurance.

Screening for β-lactamases When strains were found to have reduced susceptibility to imipenem and/or meropenem, carbapenemase screening tests were assessed by the modified Hodge test [4], the imipenem-EDTA MBL Etest (AB BioDisk, Solna, Sweden), the imipenem-EDTA double-disk synergy test [5], the imipenem-EDTA combined disc test [6], broth based EDTA inhibition test (in-house assay), and Ken Thompson’s direct TE disk test at the CDC. ESBL production was tested by the Phoenix system and by the expanded double disc diffusion synergy test (discs of cefotaxime, ceftazidime, cefpodoxime and cefepime placed around amoxicillin/clavulanic acid). The synergic activity of clavulanate with ceftazidime was confirmed by means of the Etest strips containing

ceftazidime with or without clavulanate. Isolates were considered ESBL producers when clavulanate caused a 3 twofold-concentration decrease (ratio, 8) in the MIC of ceftazidime, in combination with a ceftazidime MIC 1 μg/ml. Additionally, a strain was considered ESBL positive if a phantom zone or a deformation of the ceftazidime zone could be observed, independent of the ratios or MICs [7].

PCR and sequencing DNA templates were prepared by the boiling method as described previously by Kilic et al. [8]. Carbapenem-resistance isolates were examined by PCR previously reported conditions with primers specific for blaKPC, blaIMP, blaVIM, blaTEM, blaSHV, blaCTX-M, and blaOXA-48

[9]. The nucleotide sequences were analyzed and multiple alignments were performed using the DNAMAN 4.1 Software (Lynnon BioSoft, Québec, Canada). ESBL genes, including blaTEM, blaSHV and blaCTX-M, were detected by PCR and sequenced using the Applied Biosystems sequencer (ABI Prism 310 genetic analyzer) as previously described [9].

Pulse field gel electrophoresis PFGE typing of XbaI-digested DNA for K. pneumoniae isolates was performed by a modification of a previously described method [10]. Electrophoresis was performed with a run time of 20 h under 5 to 30 sec linear ramped pulse times by the contour-clamped homogeneous electric field method with a Bio-Rad CHEF DR II system (Bio-Rad, Hercule, CA). The gels were stained with ethidium bromide (0.5 μg/ml) for 40 to 60 min and were photographed under UV transillumination and analyzed with the aid of Quantity One software (Bio-Rad). Results

During the study period, 215 K. pneumoniae and 300 E. coli isolates were recovered from inpatients and outpatients at the GMMAH. A total of three K. pneumoniae and one E. coli isolates producing carbapenem-hydrolyzing β-lactamase were recovered from four patients. All of the isolates were non-susceptible to ertapenem and meropenem based on MICs of ≥16 μg/ml

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KP-150 EC-155 KP-163 KP-177

Amikacin ≤8 (S) ≤8 (S) 4 (S) ≤1 (S)Ampicillin >32 (R) >32 (R) >32 (R) >32 (R)Aztreonam >64 (R) >64 (R) ≤2 (S) ≤2 (S)Cefazolin >32 (R) >32 (R) >32 (R) >32 (R)Cefepime >32 (R) >32 (R) 4 (S) 4 (S)Cefoxitin >32 (R) >32 (R) 16 (I) 32 (R)Ciprofloxacin >8 (R) >8 (R) 0.5 (S) 1 (S)Levofloxacin >8 (R) >8 (R) ≤0.25 (S) 2 (S)Colistin** 4 (R) ≤0.5 (S) ≤0.5 (S) ≤0.5 (S)Ertapenem >16 (R) >16 (R) 4 (I) >16 (R)Imipenem >32 (R) >32 (R) 32 (R) >32 (R)Meropenem 16 (R) >16 (R) >16 (R) >16 (R)Polymyxin B** 4 (R) ≤0.5 (S) ≤0.5 (S) ≤0.5 (S)Tetracycline 4 (S) >32 (R) ≤2 (S) 8 (I)Tigecycline 0.5 (S) ≤0.25 (S) 1 (S) 2 (S)Trimethoprim- >8/152 (R) ≤0.25/4.8 (S) ≤0.25/4.8 (S) 8/152 (R) sulfamethoxazole

Table 1. Antibiotic susceptibilities of carbapenem resistant three K. pneumoniae and one E. coli isolates by broth microdilution method*.

* Based on CLSI guidelines (CLSI, 2006; CLSI, 2008). S, susceptible; R, resistant; I, intermediate.** According to reference 20. S, susceptible; R, resistant.

AntibioticsMIC (µg/ml)

KP-150 EC-155 KP-163 KP-177

Identification K. pneumoniae E. coli K. pneumoniae K. pneumoniaeIsolation site Blood Blood Blood UrineblaTEM-1 + − − −blaSHV-12 + − − −blaSHV-11 − − + +blaCTX-M-15 − + − −blaOXA-48 + + + +KPC − − − −IMP − − − −VIM − − − −MHT + + + +Imipenem-EDTA MBL Etest − − − −ESBL production tests + + − −

Table 2. β-lactamase detection and characterization of the four carbapenem-resistant Enterobacteriaceae isolates.

KPC, K. pneumoniae carbapenemase; IMP, integron containing metallo-β-lactamase; VIM, Verona integron-encoded metallo-β-lactamase; MHT, Modified Hodge Test; +, detected; −, not detected.

VariableNumbers of isolates

OXA-48 producing Enterobacteriaceae 165

except isolates KP-163 (ertapenem MIC of 4 μg/ml), and more resistant to imipenem (MICs, ≥32 μg/ml) (Table 1). KP-150 and EC-155 isolates were inferred to produce an ESBL based on the criterion of a ≥8-fold reduction in the MICs of oxyimino-β-lactams by clavulanate.

Four carbapenem resistant isolates tested by the modified Hodge test, the imipenem-EDTA MBL Etest, the imipenem-EDTA double-disk synergy test, and the imipenem-EDTA combined disc test. All of these tests except the modified Hodge test were negative for all strains.

All of the isolates were negative for the genes encoding KPC, IMP, and VIM by PCR assay. PCR indicated that three K. pneumoniae and one E. coli isolates harbored the blaOXA-48 gene (Table 2). In addition, PCR with blaCTX-M, blaTEM and blaSHV-specific primers, followed by classic Sanger DNA sequencing, revealed identical blaTEM-1 and blaSHV-12 genes in KP-150, the blaSHV-11 gene in KP-163 and KP-177, while blaCTX-M-15 was found in EC-155 isolates.

The PFGE profile results demonstrated that three K. pneumoniae isolates producing OXA-48 carbapenemase had distinct PFGE patterns (Figure 1).

Discussion

Carbapenem resistance in Enterobacteriaceae occurs due to various mechanisms, including overproduction of Amp-C β-lactamases associated with loss of outer membrane porins and/or overexpression of efflux pumps [1]. OXA-type carbapenemases have been reported mainly in Acinetobacter spp. throughout many regions of the world and are divided into four phylogenetic subgroups: OXA-23 like, OXA-24 like, OXA-51 like and OXA-58 [11]. OXA-48 has been characterized from a K. pneumoniae isolate that was resistant to all β-lactams including carbapenems, with a strong activity against imipenem. The blaOXA-48 gene was not associated with a class 1 integron like most oxacillinase genes, but was plasmid located downstream from the insertion sequence element, IS1999 [12].

Enterobacteriaceae isolates that produce OXA-48 have rarely been reported from Turkey. The first case was reported in a K. pneumoniae isolate from Istanbul and was found to be resistant to all β-lactams, including carbapenems [12]. Subsequently, OXA-48 has been found in a Citrobacter freundii [13] isolate and an E. coli isolate as well as several K. pneumoniae isolates [9,14]. An outbreak of K. pneumoniae isolates producing OXA-48 was reported from Istanbul, having two different clones [15]. We identified four carbapenem-resistant Enterobacteriaceae isolates in blood and urine samples from 515 clinical K. pneumonia/E. coli isolates during a 7-month study period. In addition, we looked for other mechanisms related to carbapenem-resistance, including the KPC, IMP, and VIM genes; all were absent in the four isolates.

All of our four cases were serious patients with multiple underlying medical comorbidities and treated with carbapenems prior to the development of OXA-48 producing Enterobacteriaceae bloodstream and urinary tract infections. The mortality rate of infections caused by carbapenem resistant Enterobacteriaceae is very high [16]. Our patients’ mortality rate was 25%, which is even

Fig 1. Pulsed-field gel electrophoresis patterns of XbaI-digested genomic DNA of Klebsiella pneumoniae isolates. Lane 1, KP-150; lane 2, KP-163; lane 3, KP-177; lane 4, OXA-48 producing K. pneumoniae control strain was kindly provided from Istanbul University, Istanbul Medical Faculty. The molecular size is given in kilobases.

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lower compared with studies with 42.8%, 66%, and 71.9% mortality in especially bacteremic patients [17].

Enterobacteriaceae isolates producing OXA-48 are known to be highly resistant to carbapenems because of the combined effects of a high production of OXA-48, owing to the association of the blaOXA-48 gene with IS1999, and the lack of a 36 kDa porin [18]. Treatment of infections caused by OXA-producing K. pneumoniae is very difficult since most isolates are resistant to all β-lactams and fluoroqinolones. Tigecycline and polymyxins exhibit in vitro activity against many Gram-positive and Gram-negative microorganisms, including multiresistant strains [19]. In this study tigecycline and colistin exhibited good in vitro activity against carbapenem resistant Enterobacteriacaeae isolates. However, the KP-150 isolate was resistant to polymyxin B with an MIC=4 μg/ml. We suggest that tigecycline and colistin may serve as a promising therapeutic option for infection caused by carbapenem resistant Enterobacteriacaeae.

Acknowledgements:

We thank Jean Patel, J. Kamile Rasheed, and Karen Anderson from the US Centers for Disease Control and Prevention for their excellent discussion and assistance.

References

1. Deshpande LM, Jones RN, Fritsche TR, Sader HS. Occurrence and characterization of carbapenemase-producing Enterobacteriaceae: report from the SENTRY Antimicrobial Surveillance Program (2000-2004). Microb Drug Resist 2006;12:223-230.

2. Clinical and Laboratory Standards Institute. 2008. Performance standards for antimicrobial susceptibility testing; 18th informational supplement. CLSI document M100-S18. Clinical and Laboratory Standards Institute, Wayne, PA.

3. Clinical and Laboratory Standards Institute. 2006. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, 7th ed. CLSI document M7-A7. Clinical and Laboratory Standards Institute, Wayne, PA.

4. Anderson KF, Lonsway DR, Rasheed JK, Biddle J, Jensen B, McDougal LK, Carey RB, Thompson A, Stocker S, Limbago B, Patel JB. Evaluation of methods to identify the Klebsiella pneumoniae carbapenemase in Enterobacteriaceae. J Clin Microbiol 2007;45:2723-2725.

5. Lee K, Lim YS, Yong D, Yum JH, Chong Y. Evaluation of the Hodge test and the imipenem-EDTA double-disk synergy test for differentiating metallo-beta-lactamase-producing isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 2003;41:4623-4629.

6. Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y. Imipenem-EDTA disk method for differentiation of metallo-beta-lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 2002;40:3798-3801.

7. Apfalter P, Assadian O, Daxböck F, Hirschl AM, Rotter ML, Makristathis A. Extended double disc synergy testing reveals a low prevalence of extended-spectrum beta-lactamases in Enterobacter spp. in Vienna, Austria. J Antimicrob Chemother 2007;59:854-859.

8. Kilic A, Demiray T, Saracli MA, Bahar G, Aydogan H, Baysallar M, Doganci L. Bacteriological characterization of vancomycin-resistant enterococci ina pediatric hospital in Turkey. Ann Microbiol 2004;54:543-551

9. Aktas Z, Kayacan CB, Schneider I, Can B, Midilli K, Bauernfeind A. Carbapenem-hydrolyzing oxacillinase, OXA-48, persists in Klebsiella pneumoniae in Istanbul, Turkey. Chemotherapy 2008;54:101-106.

10. Durmaz R, Otlu B, Koksal F, Hosoglu S, Ozturk R, Ersoy Y, Aktas E, Gursoy NC, Caliskan A. The optimization of a rapid pulsed-field gel electrophoresis protocol for the typing of Acinetobacter baumannii, Escherichia coli and Klebsiella spp. Jpn J Infect Dis 2009;62:372-377.

11. Nordmann P, Poirel L. Emerging carbapenemases in Gram-negative aerobes. Clin Microbiol Infect. 2002;8:321-331.

12. Poirel L, Héritier C, Tolün V, Nordmann P. Emergence of oxacillinase-mediated resistance to imipenem in Klebsiella pneumoniae. Antimicrob Agents Chemother 2004;48:15-22.

13. Nazic H, Poirel L, Nordmann P. Further identification of plasmid-mediated quinolone resistance determinant in Enterobacteriaceae in Turkey. Antimicrob Agents Chemother 2005;49:2146-7214.

14. Gulmez D, Woodford N, Palepou MF, Mushtaq S, Metan G, Yakupogullari Y, Kocagoz S, Uzun O, Hascelik G, Livermore DM. Carbapenem-resistant Escherichia coli and Klebsiella pneumoniae isolates from Turkey with OXA-48-like carbapenemases and outer membrane protein loss. Int J Antimicrob Agents 2008;31:523-526.

15. Carrër A, Poirel L, Eraksoy H, Cagatay AA, Badur S, Nordmann P. Spread of OXA-48-positive carbapenem-resistant Klebsiella pneumoniae isolates in Istanbul, Turkey. Antimicrob Agents Chemother 2008;52:2950-2954.

16. Bratu S, Landman D, Haag R, Recco R, Eramo A, Alam M, Quale J. Rapid spread of carbapenem-resistant Klebsiella pneumoniae in New York City: a new threat to our antibiotic armamentarium. Arch Intern Med 2005;165:1430-1435.

17. Borer A, Saidel-Odes L, Riesenberg K, Eskira S, Peled N, Nativ R, Schlaeffer F, Sherf M. Attributable mortality rate for carbapenem-resistant Klebsiella pneumoniae bacteremia. Infect Control Hosp Epidemiol 2009;30:972-976.

18. Walther-Rasmussen J, Høiby N. OXA-type carbapenemases. J Antimicrob Chemother 2006;57:373-383.

19. Nadkarni AS, Schliep T, Khan L, Zeana CB. Cluster of bloodstream infections caused by KPC-2 carbapenemase-producing Klebsiella pneumoniae in Manhattan. Am J Infect Control 2009;37:121-126.

20. Gales AC, Reis AO, Jones RN. Contemporary assessment of antimicrobial susceptibility testing methods for polymyxin B and colistin: review of available interpretative criteria and quality control guidelines. J Clin Microbiol 2001;39:183-190.