Molecular epidemiology of extended-spectrum beta-lactamase-producing Escherichia coli in Tunisia and characterization of their virulence factors and plasmid addiction systems

Basma Mnif 0 1 2 4 5 Hela Harhour 1 4 5 Jihne Jdidi 3 Faouzia Mahjoubi 1 4 5 Nathalie Genel 0 2 Guillaume Arlet 0 2 Adnene Hammami 1 4 5 0 Faculte de Medecine , Site Saint-Antoine , Laboratoire de Bacteriologie, Universite Pierre et Marie Curie-Paris-6 , Paris , France 1 Laboratoire de Microbiologie, Hopital Habib Bourguiba , Sfax, Tunisie 2 Faculte de Medecine , Site Saint-Antoine , Laboratoire de Bacteriologie, Universite Pierre et Marie Curie-Paris-6 , Paris , France 3 Service de medecine preventive, Hopital Hedi Chaker , Sfax, Tunisie 4 Laboratoire de Microbiologie, Hopital Habib Bourguiba , Sfax, Tunisie 5 Laboratoire de Microbiologie- Faculte de Medecine de Sfax , Avenue Majida Boulila, 3027, Sfax, Tunisie Background: Extended-spectrum -lactamases (ESBLs), particularly CTX-M- type ESBLs, are among the most important resistance determinants spreading worldwide in Enterobacteriaceae. The aim of this study was to characterize a collection of 163 ESBL-producing Escherichia coli collected in Tunisia, their ESBL-encoding plasmids and plasmid associated addiction systems. Results: The collection comprised 163 ESBL producers collected from two university hospitals of Sfax between 1989 and 2009. 118 isolates harbored blaCTX-M gene (101 blaCTX-M-15 gene and 17 blaCTX-M-14 gene). 49 isolates carried blaSHV-12 gene, 9 blaSHV-2a gene and only 3 blaTEM-26 gene. 16 isolates produced both CTX-M and SHV-12. The 101 CTX-M-15-producing isolates were significantly associated to phylogroup B2 and exhibiting a high number of virulence factors. 24 (23.7%) of the group B2 isolates belonged to clonal complex ST131. Pulsed-field gel electrophoresis (PFGE) typing revealed a genetic diversity of the isolates. 144 ESBL determinants were transferable mostly by conjugation. The majority of plasmid carrying blaCTX-M-15 genes (72/88) were assigned to various single replicon or multireplicon IncF types and had significantly a higher frequency of addiction systems, notably the VagCD module. Conclusion: This study demonstrates that the dissemination of CTX-M-15 producing E. coli in our setting was due to the spread of various IncF-type plasmids harboring multiple addiction systems, into related clones with high frequency of virulence determinants. - Background Extended-spectrum -lactamases (ESBLs) are among the most important resistance determinants spreading worldwide in Enterobacteriaceae [1,2]. During the 1980s, ESBLs evolved from TEM and SHV broad-spectrum--lactamases, frequently associated to Klebsiella pneumoniae involved in nosocomial outbreaks. Over the last decade, CTX-M-type ESBLs have increased dramatically, and become the most prevalent ESBLs worldwide, frequently associated to Escherichia coli. Among the CTX-M-type ESBLs, CTX-M-15 is now the most widely distributed in E. coli which became a major cause of infections in both community and hospitals [1,2]. A few explanations have been proposed on what makes CTX-M-15-producing E. coli isolates so successful. First, it has been proposed that the strain virulence background could be involved in this dissemination process. In fact, many reports have shown that CTX-M-15 is closely associated with the international and pandemic uropathogenic O25:H4-ST131 clone, which have specific virulence factors [3,4]. Second, the association of CTX-M-15 with the IncF plasmids, which are well adapted to E. coli, may facilitate the spread of this determinant in E. coli population [5]. In addition to virulence background and IncF plasmids bearing CTXM-15, it was recently suggested that the association of various plasmid addiction systems may contribute to the plasmid maintenance in their host [6-8]. An addiction system or a toxin-antitoxin system helps maintain plasmids in bacteria during host replication by killing of plasmid-free cells resulting from segregation or replication defects [9]. In Tunisia, SHV-2 was the first ESBL to be detected, in 1984 from a K. pneumoniae clinical isolate [10]. Then, various other types of ESBLs, SHV-12, SHV-2a, CTXM-15, CTX-M-14, CTX-M-9, CTX-M-16, CTX-M-27 and CTX-M-28 have been reported in different Tunisian hospitals with CTX-M-15 being the most prevalent [11-15]. This study was designed to characterize the ESBL-producing E. coli collected in two university hospitals of Sfax, in the southern part of Tunisia and to investigate their virulence background, their ESBL-encoding plasmids and their plasmid addiction systems. Methods E. coli isolates 163 isolates were randomly selected from the collection of ESBL-producing E. coli isolates maintained at -80C in the Microbiology laboratory of Habib Bourguiba hospital. The 163 isolates were collected from the two university hospital of Sfax in Tunisia during the following years: 1989-1990 (6), 2000 (9), 2001 (18), 2002 (9), 2003 (30), 2004 (26), 2006 (36) and 2009 (29). These isolates were obtained mainly from urine (124), but also from blood (20), wound swabs (10), abdominal fluid (5) and sputum (4). Antibiotic susceptibility testing The susceptibility to 16 antimicrobial agents (amoxicillin, amoxicillin + clavulanic acid, ticarcillin, ticarcillin + clavulanic acid, cefalothin, cefoxitin, ceftazidime, cefotaxime, cefepime, gentamicin, amikacin, nalidixic acid, norfloxacin, sulfamethoxzole/trimethoprim and tetracycline) was determined by the disk diffusion method according to the guidelines of the CLSI [16]. All isolates were confirmed for ESBL production using the double disk synergy method. Identification of bla genes The resistance genes blaTEM, blaSHV and blaCTX-M responsible for the ESBL activity were identified by PCRsequencing [17]. PCR products were sequenced on ABI Prism 3100 automated sequencer (Applied Biosystems, Foster City, CA) and were analyzed using NCBI BLAST program. (http://www.ncbi.nlm.nih.gov/). Strain typing The phylogenetic group of the ESBL-producing E. coli was determined by a multiplex PCR assay [18]. Isolates belonging to phylogenetic group B2 were screened with a previously established PCR-based method to identify the O25b subtype [19]. Furthermore, multilocus sequence typing (MLST) using the scheme of the Institut Pasteur, Paris, France (www.pasteur.fr/mlst) was used to confirm that CTX-M-15-producing E. coli O25b belonged to the international clone ST131 [19]. Genetic relatedness of the ESBL-producing strains was studied by PFGE following extraction of genomic DNA and digestion with XbaI PFGE according to a standard protocol using a GenePath system (Bio-Rad). PFGE banding profiles were compared digitally using Fingerprint II software (Bio-Rad) and relatedness was calculated using the unweighted pair group method with arithmetic mean (UPGMA) algorithm with similarity of bands using the Dice similarity indices. Isolates were considered to belong to the same PFGE cluster if their Dice similarity index was >80% [20]. Transfer of ESBL resistance determinants and plasmid analysis Transfer of ESBL encoding genes by conjugation was performed by matting- (...truncated)