JAC Advance Access originally published online on April 1, 2008
Journal of Antimicrobial Chemotherapy 2008 62(1):133-136; doi:10.1093/jac/dkn145
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Original research |
Dissemination of ESBL and Qnr determinants in Enterobacter cloacae in Algeria
1 Laboratoire de Biologie Cellulaire et Moléculaire, Faculté des Sciences Biologiques, Université des Sciences et de la Technologie Houari Boumediene, BP 32 El-Alia, Bab-Ezzouar 16111, Alger, Algérie 2 Laboratoire de Bactériologie, CHU Beni Messous, Alger, Algérie 3 Service de Microbiologie, CHU Mustapha Bacha, Alger, Algérie 4 Université Paris VI, Faculté de Médecine Pierre et Marie Curie, Laboratoire de Bactériologie, UPRES EA 2392, Paris, France
* Correspondence address. Service de Bactériologie-Hygiène, Hôpital Tenon, 4 rue de la Chine, 75970 Paris cedex 20, France. Tel: +33-1-56-01-70-18; Fax: +33-1-56-01-61-08; E-mail: guillaume.arlet{at}tnn.aphp.fr
Received 18 January 2008; returned 31 January 2008; revised 10 March 2008; accepted 10 March 2008
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Abstract |
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Objectives: The aim of this study is to evaluate the prevalence and diversity of extended-spectrum β-lactamases (ESBLs) in Enterobacter cloacae clinical isolates collected from Algerian hospitals and to verify the association with qnr genes.
Methods: MICs were determined by Etest for isolates giving positive double-disc synergy tests, and all isolates were screened by PCR and sequenced, respectively, for blaTEM, blaCTX-M, blaSHV and blaVEB genes and for qnr genes (qnrA, qnrB, qnrS), using specific primers.
Results: The prevalence of ESBLs was 25/141 (17.7%) with 11, 9, 4 and 1 isolates testing positive for genes encoding CTX-M-15, CTX-M-3, SHV-12 and VEB-1, respectively. Two SHV-12 producers and one CTX-M-15 producer expressed QnrS1, one isolate produced CTX-M-15 and QnrB1 and one SHV-12 producer co-expressed QnrS1 and QnrB4. qnrA was not detected in our collection, and qnr alleles were not detected in non-ESBL-producing isolates.
Conclusions: SHV-12, QnrS1, QnrB1 and QnrB4 were reported for the first time in Algeria. This study also described a co-expression of qnrS1 and qnrB4 by an SHV-12 producer isolate.
Keywords: E. cloacae , CTX-M , SHV-12 , QnrB , QnrS
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Introduction |
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β-Lactams and fluoroquinolones are the most commonly prescribed antibiotic classes. The emergence of extended-spectrum β-lactamases (ESBLs) and, more recently, of Qnr proteins conferring resistance to the broad-spectrum β-lactams by their hydrolysis and to the quinolones by protecting the topoisomerases constitutes a real concern for clinicians. β-Lactamase molecular class A includes the majority of ESBLs. At first, these were derived, essentially by the accumulation of point mutations, from TEM and SHV penicillinases. Recently, other β-lactamases emerged such as VEB, PER, GES and, in particular, CTX-M, which are the most widespread enzymes.
The Qnr proteins (QnrA-like, QnrB and QnrS) have been identified worldwide in various Enterobacteriaceae. They have a high prevalence among Asian isolates and have been frequently associated with ESBLs and plasmidic cephalosporinases.1,2
Enterobacter cloacae is recognized as a cause of nosocomial infections, having been associated with several outbreaks, involving mutants that overproduce their chromosomal β-lactamases or, less frequently, produce an ESBL. The aim of this study was to investigate the presence of ESBLs and their association with qnr genes among clinical isolates of E. cloacae collected from eight different hospitals in Algeria.
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Materials and methods |
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Bacterial isolates
A total of 141 non-repetitive E. cloacae isolates (one isolate per patient) were collected from the Microbiology Laboratories of eight hospitals in Algeria: five situated in the centre of Algiers (Beni Messous, Mustapha Bacha, Ain Naadja, Zemirli and Kouba); two located 100 km east of Algiers (Tizi Ouzou and Draa El Mizan); and the last (Tlemcen) situated 600 km west of Algiers. The isolates were collected from March 2003 to July 2007 from various pathological specimens and identified by the API 20E system (bioMérieux, Marcy l'Étoile, France).
Antimicrobial susceptibility and synergy testing
Antibiograms were determined on Mueller–Hinton agar by disc diffusion and interpreted according to the guidelines of the French Society of Microbiology (www.sfm.asso.fr). Escherichia coli ATCC 25922 was used as a control strain. ESBL production was screened for using the double disc synergy test (DDST)3 with cefepime.
Antibiotic discs were purchased from Bio-Rad (Marnes la Coquette, France). MICs of cefotaxime, ceftazidime, imipenem, nalidixic acid, ciprofloxacin and norfloxacin were determined by Etest for the ESBL-producing isolates. MICs of cefepime were determined for all of the isolates.
Enterobacterial repetitive consensus PCR (ERIC-PCR)
The epidemiological relationships between ESBL-producing E. cloacae isolates were analysed by ERIC-PCR using primer ERIC2.4 Cycling conditions were as follows: 3 min at 95°C; 40 cycles of 30 s at 92°C, 1 min at 40°C and 8 min at 72°C; and final extension of 16 min at 72°C. The resulting products were analysed on 1.5% agarose gels. Fingerprints were compared visually, and the patterns differing by at least one amplification band were classified as different.
Conjugation experiments and plasmid analysis
Conjugation was carried out as described previously4 with E. coli J53-2 (Rif R) as the recipient, and transconjugants were selected on Mueller–Hinton agar containing rifampicin (250 mg/L) and cefotaxime (2.5 mg/L). Plasmid DNA was extracted using the High Pure Plasmid Isolation Kit (Roche, Mannheim, Germany), and plasmid size was estimated in comparison with plasmid size standards: pIP173 (126.8 kb) and pCFF04 (85 kb).
Characterization of ESBL- and Qnr-encoding genes
DNA was extracted from isolates with ESBL phenotypes with a QIAGEN mini kit (Qiagen, Courtaboeuf, France). These were screened for blaTEM, blaSHV, blaVEB and blaCTX-M genes by PCR, as described previously.5 PCR amplification of qnrA, qnrB and qnrS and blaLAP-1 used specific primers and conditions.6,7 The ESBL and Qnr PCR products were sequenced as described previously,4 and the nucleotide and amino acid sequences were analysed and compared by use of the BLAST computer program (National Center for Biotechnology Information).
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Results and discussion |
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Among the 141 collected isolates, 25 (17.7%; Table 1) were defined as ESBL producers according to their resistance to the marker antibiotics and DDST results. MICs of cefepime ranged from 8 to >256 mg/L for ESBL producers and <0.12 mg/L for the non-ESBL isolates. Most of the ESBL-producing isolates (20/25) showed high resistance to cefotaxime (>256 mg/L). All isolates were susceptible to imipenem (MICs 0.064–0.5 mg/L). The quinolones tested remained active versus 80% (20/25) of the ESBL producers (Table 1), but most producers were resistant to aminoglycosides and sulphonamides (data not shown).
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ERIC-PCR typing was performed on the 25 ESBL producers from the eight hospitals scattered in three distant places in Algeria. The results showed 20 different genotypes with distinct ERIC-PCR patterns, showing clear heterogeneity in isolate genetic profiles.
ESBL phenotypes were transferred in vitro from 23 of 25 ESBL producers, in association with plasmids of 
85 kb (from 20 isolates) and >150 kb (from 3 isolates). Twenty ESBL producers contained blaCTX-M genes (9 encoded CTX-M-3 and 11 CTX-M-15), four isolates harboured blaSHV-12 genes and the remaining isolate (E19) was blaVEB-positive. qnr genes were detected only in five quinolone-resistant ESBL-producing isolates; four had the QnrS1 determinant (E12, E15, E17 and E18) and one each had QnrB4 (E18) and QnrB1 (E09). Isolate E18, which harboured SHV-12 β-lactamase, was positive for both QnrB4 and QnrS1 determinants.
The 25 ESBL producers were screened for blaLAP-1 and, as shown previously,7 only the four isolates harbouring QnrS1 were positive. This study confirmed the close association between blaLAP-1 and qnrS1 genes. None of the non-ESBL-producing E. cloacae isolates was positive for qnr alleles.
The incidence of ESBL-producing E. cloacae isolates varies according to country. Our rate of 17.7% is lower than that reported in Taiwan in 2007 (28%).6 The use of cefotaxime and, recently, ceftazidime in Algeria could explain in part this high percentage of ESBLs. As no clonal relationship was established between the isolates, the high prevalence of ESBLs could be due to the dissemination of plasmids. The predominance of CTX-M-3 and CTX-M-15 enzymes is in accordance with previous reports in Algeria;8 group 1 remains the only subgroup of CTX-M ESBLs found in Algeria. This is the first detection in Algeria of SHV-12, which has been described in E. cloacae in other countries.6 The emergence of SHV-12-producing E. cloacae represents a new alert and requires more intensive monitoring. VEB-1 was found in a single E. cloacae isolate and has been reported in Providencia stuartii and Proteus mirabilis in Algeria.9
Several studies have shown a close association between the CTX-M or SHV ESBLs and Qnr determinants (QnrA, QnrB and QnrS).2,6 QnrB has previously been found to be associated with CTX-M-15 in Klebsiella pneumoniae and with SHV-12 in E. cloacae.1,10 It was also observed recently in an Algerian E. cloacae isolate, but the allele was not sequenced.8 QnrB1 was observed to be co-produced with CTX-M-15 in isolates of K. pneumoniae.10 Although QnrS1 was found to be associated with SHV-12 in French isolates of E. cloacae,9 the present study constitutes the first report of QnrS1 in isolates in Algeria. The co-expression of QnrB4 and QnrS1, as observed in isolate E18, was first reported in France in an SHV-12-producing E. cloacae.1
The emergence of these combinations of resistance determinants leaves us perplexed for the future of antimicrobial therapy in Algeria. This is a public health problem, which requires careful monitoring and implementation of a policy of antibiotic use.
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Funding |
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This work was supported by a grant from the Algerian PNE programme and from University Pierre et Marie Curie.
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Transparency declarations |
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None to declare.
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Acknowledgements |
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We wish to thank all the contributing laboratories that provided isolates for this study. We are especially grateful to T. Benhassine, S. Alouache, N. Benamrouche, D. Tiouit, H. Hassaine, S. Smail, M. Tchambaz, Mahrane and Ait-Ameur for their help.
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References |
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1 Cattoir V, Poirel L, Nordmann P. Plasmid-mediated quinolone resistance determinant QnrB4 identified in France in an Enterobacter cloacae clinical isolate coexpressing a QnrS1 determinant. Antimicrob Agents Chemother (2007) 51:2652–3.
2 Cambau E, Lascols C, Sougakoff V, et al. Occurrence of qnrA-positive clinical isolates in French teaching hospitals during 2002–2005. Clin Microbiol Infect (2006) 12:1013–20.[CrossRef][Web of Science][Medline]
3 Jarlier V, Nicolas MH, Fournier G, et al. Extended-spectrum β-lactamases conferring transferable resistance to newer β-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Rev Infect Dis (1988) 10:867–78.[Web of Science][Medline]
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Decre D, Burghoffer B, Gautier V, et al. Outbreak of multi-resistant Klebsiella oxytoca involving strains with extended-spectrum β-lactamases and strains with extended-spectrum activity of the chromosomal β-lactamase. J Antimicrob Chemother (2004) 54:881–8.
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Brasme L, Nordmann P, Fidel F, et al. Incidence of class A extended-spectrum β-lactamases in Champagne-Ardennes (France): a 1 year prospective study. J Antimicrob Chemother (2008) 61:231–2.
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Wu JJ, Ko WC, Tsai SH, et al. Prevalence of plasmid-mediated quinolone resistance determinants QnrA, QnrB, and QnrS among clinical isolates of Enterobacter cloacae in a Taiwanese hospital. Antimicrob Agents Chemother (2007) 51:1223–7.
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Poirel L, Cattoir V, Soares A, et al. Novel Ambler class A β-lactamase LAP-1 and its association with the plasmid-mediated quinolone resistance determinant QnrS1. Antimicrob Agents Chemother (2007) 51:631–7.
8 Touati A, Brasme L, Benallaoua S, et al. First report of qnrB-producing Enterobacter cloacae and qnrA-producing Acinetobacter baumannii recovered from Algerian hospitals. Diagn Microbiol Infect Dis (2008) 60:287–90.[CrossRef][Web of Science][Medline]
9 Poirel L, Villa L, Bertini A, et al. Expanded-spectrum β-lactamase and plasmid-mediated quinolone resistance. Emerg Infect Dis (2007) 13:803–5.[Web of Science][Medline]
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Jacoby GA, Kelley E, Walsh L, et al. qnrB, another plasmid-mediated gene for quinolone resistance. Antimicrob Agents Chemother (2006) 50:1178–82.
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