Molecular characterization of multidrug-resistant extended-spectrum β-lactamase-producing Enterobacteriaceae isolated in Antananarivo, Madagascar

Hanitra C Rakotonirina 0 1 Benot Garin 0 Frdrique Randrianirina 0 Vincent Richard 0 Antoine Talarmin 0 Guillaume Arlet 1 0 Institut Pasteur de Madagascar , Antananarivo 101, BP 1274 , Madagascar 1 Faculte de Medecine, Laboratoire de Bacteriologie, Universite Pierre et Marie Curie , Paris 6, 27 rue Chaligny, Paris 75012 , France Background: We investigated the molecular characteristics of multidrug-resistant, extended-spectrum -lactamase (ESBL)-producing Enterobacteriaceae isolated in community settings and in hospitals in Antananarivo, Madagascar. Results: Forty-nine E. coli, K. pneumoniae, K. oxytoca and E. cloacae ESBL-producing isolates were studied. In antimicrobial susceptibility analyses, many of the isolates exhibited resistance to aminoglycosides, fluoroquinolones and trimethoprim-sulfamethoxazole. Gene amplification analysis and sequencing revealed that 75.5% (n=37) of the isolates harbored blaCTX-M-15 and 38.7% (n=19) harbored blaSHV-12. The non-ESBLs resistance genes detected were blaTEM-1, blaOXA-1, aac(60)-Ib, aac(60)-Ib-cr, tetA, sul-1, sul-2, qnrA, qnrB and catB-3. We found dfrA and aadA gene cassettes in the class 1 integron variable regions of the isolates, and the combination of dfrA17-aadA5 to be the most prevalent. All blaCTX-M-15 positive isolates also contained the ISEcp1 insertion element. Conjugation and transformation experiments indicated that 70.3% of the antibiotic resistance genes resided on plasmids. Through a PCR based replicon typing method, plasmids carrying the blaSHV-12 or blaCTX-M-15 genes were assigned to either the IncFII replicon type or, rarely, to the HI2 replicon type. All isolates were subtyped by the rep-PCR and ERIC-PCR methods. Phylogenetic grouping and virulence genotyping of the E. coli isolates revealed that most of them belonged to group A1. One isolate assigned to group B2 harbored blaCTX-M-15 and five virulence genes (traT, fyuA, iutA, iha and sfa) and was related to the O25b-ST131 clone. Conclusions: Our results highlight the dissemination of multidrug resistant Enterobacteriaceae isolates in Antananarivo. These findings underline the need for a rational use of antibiotic and for appropriate methods of screening ESBL in routine laboratories in Antananarivo. - Background Extended-spectrum -lactamase (ESBL)-producing bacteria represent a major worldwide threat among drug-resistant bacteria in both hospital and community settings [1]. ESBLs are among the Ambler classes A, confer resistance to -lactam antibiotics except cephamycins and carbapenems, and are inhibited by clavulanic acid [1]. ESBLs are often located on large plasmids that also harbor resistant genes to other antimicrobial classes with resulting multidrug-resistant isolates [2]. The first ESBLs have evolved by genetic mutation from native -lactamases TEM and SHV [3] [4]. Recently, a novel type of ESBLs, the CTX-M enzymes, emerged worldwide, mostly from Enterobacteriaceae [5,6]. CTX-M -lactamases are not closely related to TEM or SHV ESBLs but share high amino-acid identity with chromosomal -lactamases from Kluyvera spp. [7]. Now, blaCTX-M-15 is recognized as the most widely distributed CTX-M enzyme [8]. It is derived from CTX-M-3 by a substitution of Asp-240-Gly which increases its catalytic efficiency against ceftazidime [9]. blaCTX-M-15 are encoded on plasmids belonging to the incompatibility group IncF [10]. In the upstream region of CTX-M genes an insertion sequence element, ISEcp1, is commonly present and is likely responsible for the transposition process of the genes [11]. E. coli is among the most prevalent causes of hospitalacquired and community-acquired bacterial infections and their resistances to antimicrobial agents have become a serious concern for healthcare providers [5]. Phylogenetic analyses have classified E. coli into four main phylogenetic groups (A, B1, B2, and D). Commensal isolates belong mainly to A and B1 groups whereas virulent extraintestinal pathogenic E. coli (ExPEC) are essentially from the B2 and D groups [12,13]. ExPEC harbor numerous virulence factors including -hemolysin, cytotoxic necrotizing factor, adhesins and iron acquisition systems [12]. The spread of blaCTX-M-15 has been mainly associated with the dissemination of a particular clone of E. coli ST131 belonging to phylogenetic group B2 [14,15]. Recently, an E. coli clone O25 ST131, producing CTX-M -15, with high virulence potential and belonging to the B2 group, has been reported and represent a major public health problem [14,15]. Many reports have documented the emergence of ESBLproducing Enterobacteriaceae [16-18]. In Antananarivo, ESBLs were first detected in 2005 from UTI in 9.7% of isolated Enterobacteriaceae [19]. In 2006, outbreaks of CTX-M-15 and SHV-2-producing K. pneumoniae isolates have been described in two pediatric units [20]. More recently, 21.3% of clinical isolates from patients in surgery and intensive care units [21] and 21.2% of intestinal carriage isolates from children hospitalized in a pediatric department of a large teaching hospital [22] were ESBLproducers. For 49 multidrug-resistant Enterobacteriaceae isolates from Antananarivo, we characterized: i) the genes encoding the ESBLs; ii) the drug resistance genes associated with the ESBL genes; iii) gene cassettes present in the isolates; and iv) the plasmid incompatibility groups of the isolates. We also determined the phylogenetic groups and virulence factors of the E. coli isolates. Methods Ethical clearance The study protocols were approved by the National Ethics Committee of Madagascar. Written informed consents were obtained from all patients and at least one parent of each child before enrollment. Patients Between September 2006 and December 2007, a total of 909 non-duplicate bacterial isolates were obtained from 909 patients. 830 patients were recruited from several wards in four hospitals in Antananarivo, Madagascar (two national university teaching hospitals: Joseph Ravoahangy Andrianavalona Hospital and Befelatanana Hospital; a military hospital: Soavinandriana Hospital; and a pediatric hospital: Tsaralalana Hospital) and 79 patients referred to the Pasteur Institute Medical Laboratory in Antananarivo. Laboratory methods Various clinical specimens (including blood-culture, urine, pus, sputum and CSF) were collected and submitted for bacterial analysis at the Pasteur Institute Medical Laboratory in Antananarivo. Presumptive Enterobacteria isolates were identified using standard microbiological methods and the API 20E system (Bio-Mrieux SA, Marcy lEtoile, France). Antimicrobial susceptibility testing and ESBL detection Antimicrobial susceptibilities were determined by the disk diffusion method on Mueller-Hinton agar (Bio-Rad, Marne la Coquette, France) according to the guidelines of the Comit de lantibiogramme de la Socit Franaise de Microbiologie. The following antibiotics were tested: amoxicillin, amoxicillin-clavulanate, ticarcillin, cephalotin, cefamandole, cefoxitin, (...truncated)