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Antimicrobial Agents and Chemotherapy, August 2008, p. 2950-2954, Vol. 52, No. 8
0066-4804/08/$08.00+0     doi:10.1128/AAC.01672-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Spread of OXA-48-Positive Carbapenem-Resistant Klebsiella pneumoniae Isolates in Istanbul, Turkey{triangledown}

Amélie Carrër,1 Laurent Poirel,1 Haluk Eraksoy,2 A. Atahan Cagatay,2 Selim Badur,3 and Patrice Nordmann1*

Service de Bactériologie-Virologie, INSERM U914, Emerging Resistance to Antibiotics, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris Sud, Le Kremlin-Bicêtre, France,1 Department of Infectious Diseases and Clinical Microbiology,2 Department of Microbiology, Istanbul Faculty of Medicine, Capa, Istanbul, Turkey3

Received 27 December 2007/ Returned for modification 10 February 2008/ Accepted 24 May 2008


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ABSTRACT
 
The first outbreak of carbapenem-resistant Klebsiella pneumoniae isolates producing the plasmid-encoded carbapenem-hydrolyzing oxacillinase OXA-48 is reported. The 39 isolates belonged to two different clones and were collected at the University Hospital of Istanbul, Turkey, from May 2006 to February 2007, and they coproduced various β-lactamases (SHV-12, OXA-9, and TEM-1 for clone A and CTX-M-15, TEM-1, and OXA-1 for clone B).


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TEXT
 
Outbreaks of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae have been extensively reported worldwide (9, 17, 18) and carbapenems often remain the treatment of last resort. However, carbapenem resistance in K. pneumoniae has occasionally been reported and may be due to plasmid-mediated AmpC cephalosporinases associated with porin modifications (3, 14) or carbapenemases (23). Ambler class A β-lactamases of the GES type and the KPC type have been reported for K. pneumoniae isolates from several parts of the world, including for GES Greece (26) and Korea (13) and for KPC the United States (4), Israel (24), and China (6). K. pneumoniae isolates producing metallo-β-lactamase VIM-1 have been identified in Greece (11) and recently in Spain (25) and in Italy (5). The class D β-lactamase OXA-48, possessing significant carbapenemase activity, was identified from sporadic carbapenem-resistant K. pneumoniae isolates from Turkey (1, 10, 22). The blaOXA-48 gene was part of the Tn1999 composite transposon made of two copies of the insertion sequence IS1999 (2).

Here we describe a nosocomial outbreak of carbapenem-resistant K. pneumoniae strains expressing OXA-48.

Thirty-nine nonrepetitive carbapenem-resistant K. pneumoniae isolates were collected from patients hospitalized at the University Hospital of Istanbul, Turkey, from May 2006 to January 2007. Among them, 27 were isolated from clinical samples (4 endotracheal aspirate, 8 pus, 1 ascite, 7 blood culture, 2 wound, 1 urine, and 4 catheter samples). The remaining 12 isolates were grown from routine rectal screening swabs. Most of the patients were hospitalized in intensive care units (ICUs) or for emergency surgery (Table 1). Sixteen patients were infected, and 13 of them were being treated with carbapenems prior to and also after the determination of isolate susceptibility (Table 1). The three untreated patients were suffering from infections which did not require systemic administration of antibiotic. Despite treatment, 9 out of the 16 infected patients died.


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  		TABLE 1. Clinical features of the 39 isolates of carbapenem-resistant K. pneumoniae isolates

Antibiotic susceptibility was determined by the disk diffusion method according to Clinical and Laboratory Standards Institute guidelines (7). Additionally, the MICs of several antibiotics were determined by using E-test strips (AB Biodisk, Solna, Sweden). Thirty-seven isolates were resistant to all penicillins and expanded cephalosporins. They exhibited heterogeneous decreased susceptibility to carbapenems, with meropenem and ertapenem being resistant, whereas MICs of imipenem ranged from 4 to 32 µg/ml (Table 2). Isolates 11 and 21 were resistant to carbapenems but remained susceptible to cefotaxime, ceftazidime, and aztreonam. Taking resistance to β-lactam and non-β-lactam antibiotics into account, the following three distinct resistance patterns were defined: group 1 included 23 isolates susceptible to ciprofloxacin but resistant to aminoglycosides; group 2 included 14 isolates resistant to ciprofloxacin but susceptible to gentamicin; and group 3 included isolates 11 and 21, susceptible to ceftazidime and ciprofloxacin but resistant to aminoglycosides. Double-disk synergy tests were performed for ESBL detection as described previously (12) and positive results were obtained for isolates of groups 1 and 2 but not for the two isolates of group 3. These various resistance patterns may explain the treatment failure observed for most of the infected patients.


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  		TABLE 2. MICs of β-lactams and non-β-lactamsa

Specific primers were used for the detection of β-lactamase-encoding genes that had been previously identified in K. pneumoniae 11978, namely, blaTEM, blaSHV, blaOXA-1, blaOXA-9, and blaOXA-48 (22). In addition, PCR experiments were performed to identify other ESBL-encoding genes by use of specific primers for blaCTX-M, blaVEB, blaPER, and blaGES, which had been designed previously (9, 15, 19). All isolates from group 1 were positive for the blaOXA-48, blaTEM, blaSHV, and blaOXA-9 genes; all isolates from group 2 were positive for the blaOXA-48, blaTEM, blaCTX-M, and blaOXA-1 genes; and isolates from group 3 were positive for the blaOXA-48, blaOXA-9, and blaTEM genes only. Sequence analysis of the entire genes revealed perfect identity with the blaCTX-M-15, blaOXA-1,blaOXA-9, blaOXA-48, blaSHV-12, and blaTEM-1 genes.

Isoelectrofocusing analysis performed as described elsewhere (20) confirmed gene identification for each group, with pI values of 7.4, 7.2, 8.2, 8.9, and 5.4, corresponding to OXA-48, OXA-1, SHV-12, CTX-M-15, and TEM-1, respectively (data not shown). As shown for K. pneumoniae 11978, OXA-9 was not expressed, and no isoelectric focusing band at the corresponding pI was found. No additional β-lactamase signal was observed for isolates from group 3.

The genetic relationship between the different isolates studied by pulsed-field gel electrophoresis (8) revealed only two distinct profiles, defining clone A as grouping 25 isolates from groups 1 and 3 and clone B as corresponding to the 14 isolates of group 2 (Fig. 1 and data not shown). It is noteworthy that clones A and B were genetically distinct from K. pneumoniae 11978, which is known to express another ESBL clavulanic acid-inhibited determinant, SHV-2a (22) (data not shown).


Figure 1
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  		FIG. 1. Pulsed-field gel electrophoresis patterns of OXA-48-producing K. pneumoniae isolates. Lanes: 1 to 3, three isolates of clone A harboring the blaOXA-48, blaOXA-9, blaSHV-12, and blaTEM-1 genes; 4 to 6, three isolates of clone B harboring the blaOXA-48, blaOXA-1, blaCTX-M-15, and blaTEM-1 genes; M, molecular mass markers.

Conjugation experiments were performed with one isolate belonging to clone A and with one isolate belonging to clone B by use of amoxicillin, ceftazidime, or cefotaxime as the selective agent as described previously (22). Plasmid DNA extraction according to the Kieser technique (16) showed that the blaOXA-48 gene was carried by a 70-kb plasmid present in each clone (named pAb and pBb for clones A and B, respectively), while the other β-lactamases genes were carried on plasmids of more than 150 kb (pAa and pBa) and were identified by PCR (Table 2 and data not shown).

The genetic environment of the blaOXA-48 gene was determined by PCR using specific primers for the insertion sequence IS1999, located upstream and downstream of the blaOXA-48 gene in Tn1999 (2). A structure identical to Tn1999, as found in K. pneumoniae 11978, was identified in clone A isolates, whereas in clone B isolates, the IS1999 element located upstream of the blaOXA-48 gene was truncated by IS1R, which had targeted the transposase gene of IS1999 (Fig. 2). Whereas IS1999 was shown to provide –35 and –10 promoter sequences for the expression of the blaOXA-48 gene in K. pneumoniae 11978 and thus acts similarly in isolates belonging to the clone A (Fig. 3A), it is likely that the promoter driving the expression in clone B isolates was made of a –35 box located inside IS1R together with the –10 box originally located in IS1999 (Fig. 3B). This hybrid promoter possesses the features of a strong promoter for blaOXA-48 gene expression, with a more efficient –35 sequence and an optimal 17-bp spacing between the –35 and –10 boxes. Since the genetic structures identified upstream of the blaOXA-48 gene varied, their role in blaOXA-48 gene expression was studied by measuring hydrolytic activities as described previously (21). Hydrolysis of imipenem (100 µM) obtained from Escherichia coli(pAb) (23 [± 0.002] mU/mg of protein–1) was twofold lower than that obtained from E. coli(pBb) (48 [± 0.013] mU/mg of protein–1). This result indicated a higher expression of the blaOXA-48 gene in clone B.


Figure 2
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  		FIG. 2. Schematic representation of Tn1999.2 described for clone B. The coding regions are represented as boxes, with arrows indicating their transcription orientations.


Figure 3
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  		FIG. 3. Nucleotide sequence of the –35 and –10 putative promoter regions within IS1999 described for clone A (A) and clone B (B). The left inverted repeats (IRL) of IS1999 and of IS1R are shaded in gray, and the –35 and –10 putative sequences are underlined. The tnpA transposase gene is indicated.

Our study identified the first large outbreak of OXA-48-positive carbapenem-resistant K. pneumoniae isolates. Most isolates produced multiple β-lactamases, including ESBLs, narrow-spectrum oxacillinases, and penicillinases, therefore leading to resistance to all β-lactams. Several OXA-48-producing isolates also expressed ESBL SHV-12, whereas others expressed ESBL CTX-M-15. This association was not due to the dissemination of single plasmids, since the ESBL and OXA-48 genes were not located on the same plasmids. The OXA-48 producers identified here were not clonally related to the previously identified OXA-48-positive K. pneumoniae 11978 isolate from Turkey, indicating the concomitant spread of several OXA-48-producing clones in Istanbul. This identification of K. pneumoniae isolates harboring the worldwide-spread CTX-M-15 determinant but also the OXA-48 carbapenemase is worrying, since carbapenems are often the last resort for treating infections due to ESBL-producing strains. In addition, we showed here that a higher level of OXA-48 expression was likely due to a particular genetic structure providing a stronger promoter.


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ACKNOWLEDGMENTS
 
This work was funded by a grant from the Ministère de l'Education Nationale et de la Recherche (UPRES-EA3539), Université Paris XI, Paris, France, and mostly by a grant of the European community (LSHM-CT-2005-018705).


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FOOTNOTES
 
* Corresponding author. Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre cedex, France. Phone: 33-1-45-21-36-32. Fax: 33-1-45-21-63-40. E-mail: nordmann.patrice{at}bct.aphp.fr Back

{triangledown} Published ahead of print on 2 June 2008. Back


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Antimicrobial Agents and Chemotherapy, August 2008, p. 2950-2954, Vol. 52, No. 8
0066-4804/08/$08.00+0     doi:10.1128/AAC.01672-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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  • Yong, D., Toleman, M. A., Giske, C. G., Cho, H. S., Sundman, K., Lee, K., Walsh, T. R. (2009). Characterization of a New Metallo-{beta}-Lactamase Gene, blaNDM-1, and a Novel Erythromycin Esterase Gene Carried on a Unique Genetic Structure in Klebsiella pneumoniae Sequence Type 14 from India. Antimicrob. Agents Chemother. 53: 5046-5054 [Abstract] [Full Text]  
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