Wejdene Mansour

Carbapenem-Resistant Acinetobacter baumannii Isolates from Tunisia Producing the OXA-58-Like Carbapenem-Hydrolyzing Oxacillinase OXA-97

ABSTRACT The basis of the β-lactam resistance of 39 multidrug-resistant Acinetobacter baumannii isolates recovered from hospitalized patients was studied. These isolates were collected from 2001 to 2005 at the Sahloul Hospital in Sousse, Tunisia. They belonged to two distinct clones. One clone that grouped 19 isolates produced a carbapenem-hydrolyzing oxacillinase, OXA-97, that differed from OXA-58 by a single amino acid substitution and conferred the same β-lactam resistance profile as OXA-58. The blaOXA-97 gene was located on plasmids that varied in size in 18 isolates and was chromosomally located in a single isolate. Cloning and sequencing identified genetic structures surrounding the blaOXA-97 gene similar to those reported to be adjacent to the blaOXA-58 gene. In addition, the novel ISAba8 element (which is of the IS21 family) was identified. This is the first report of the nosocomial spread of carbapenemase producers in A. baumannii isolates in Africa.

Impact of food animal trade on the spread of mcr-1-mediated colistin resistance, Tunisia, July 2015

We report a high prevalence of MCR-1 and CTX-M-1-producing Escherichia coli in three Tunisian chicken farms. Chickens were imported from France or derived from French imported chicks. The same IncHI2-type plasmid reported to carry those genes in cattle in France and in a food sample in Portugal was found in Tunisian chickens of French origin. This suggests a significant impact of food animal trade on the spread of mcr-1-mediated colistin resistance in Europe.

Dissemination of OXA-23–Producing and Carbapenem-Resistant Acinetobacter baumannii in a University Hospital in Tunisia

Ninety-nine carbapenem-resistant Acinetobacter baumannii isolates were obtained from patients hospitalized between October 2005 and January 2007 at the University Hospital Sahloul, Sousse, Tunisia. Thirteen of those isolates produced the carbapenem-hydrolyzing oxacillinase OXA-23. All the OXA-23-positive isolates were clonally related, and the bla(OXA-23) gene was found to be chromosomally located and associated with an upstream-located insertion sequence ISAba1. This study further highlights the worldwide emergence of OXA-23-producing A. baumannii isolates.

Metallo-β-lactamase-producing Pseudomonas aeruginosa isolates in Tunisia

This study was conducted to identify metallo-beta-lactamase (MBL) producers among a collection of 75 nonrepetitive carbapenem-resistant Pseudomonas aeruginosa isolates recovered between November 2003 and May 2007 at the University Hospital Sahloul, Sousse, Tunisia. Five isolates produced the MBL VIM-2. Those bla(VIM-2)-positive isolates were clonally related according to pulsed-field gel electrophoresis analysis. This carbapenemase was very likely chromosomally located and as a form of a gene cassette in a class 1 integron. This is the 1st report of spread of VIM-2 producers in Tunisia.

The blaCTX-M-1 IncI1/ST3 plasmid is dominant in chickens and pets in Tunisia

Sir, Extended-spectrum b-lactamases (ESBLs) of the CTX-M group have become widespread enzymes conferring resistance to broadspectrum cephalosporins in humans and animals, and plasmids play a key role in the horizontal transfer of the corresponding genes. Contrary to the situation in humans, the blaCTX-M-1 gene is abundant in animals. It is also frequently located on plasmids of specific incompatibility (Inc) groups and subtypes, such as IncI1/ST3 plasmids, which can consequently be considered as dominant plasmids. Indeed, indistinguishable blaCTX-M-1 IncI1/ ST3 plasmids have been found in Escherichia coli from cattle, goats, poultry, horses and pets and in Salmonella enterica from cattle, poultry and humans. – 5 In Tunisia, the blaCTX-M-1 gene was also reported in pets, foodstuff and food animals, particularly in chickens. – 8 Moreover, 7.3% of Tunisian healthy humans proved to carry CTX-M-1-producing E. coli, suggesting that foodstuff of poultry origin may contribute to the transfer of the blaCTX-M-1 gene from animals to humans. The aims of this study were to characterize blaCTX-M-carrying plasmids from E. coli isolates from chickens and pets in Tunisia and to compare them with previously reported blaCTX-M-carrying plasmids. Between December 2011 and April 2012, faecal swabs from 193 chickens, 41 dogs and 4 cats were collected in Tunisia and plated onto cefotaxime-supplemented (4 mg/L) MacConkey agar. One presumptive E. coli colony was selected per plate and identification was confirmed using API20E galleries. All E. coli isolates were then tested for antimicrobial susceptibility by disc diffusion according to the Antibiogram Committee of the French Society for Microbiology guidelines (www.sfm-microbiologie.fr) and ESBL production was confirmed by the double-disc synergy test. Chickens originated from 12 unrelated farms (each with 1000–5000 animals) in the Sousse (7), Mahdia (1), Kairouan (2) and Monastir (2) governorates, and were either diseased or dead (colibacillosis). Dogs and cats were sampled at a single clinic during routine examination (Sousse), and all but three animals were healthy. Seven of the 12 chicken farms (58.3%) were found positive for ESBL producers (each governorate was represented), and eight unrelated ESBL E. coli isolates (using PFGE, not shown) were recovered from the 193 samples (8/193, 4.1%). Seven additional unrelated ESBL E. coli isolates were recovered from the 45 pets (7/45, 15.6%). No isolate belonged to the pandemic human O25b-ST131 E. coli clone. All but one isolate displayed multiple co-resistances (Table 1). ESBL-carrying plasmids were transferred into electrocompetent E. coli TOP10 cells, and the blaCTX-M-1 gene was confirmed by PCR and sequencing in 13/15 (87%) transformants. The two last transformants harboured the blaCTX-M-9 and the blaCTX-M-15 genes, respectively. Southern blots on S1-PFGE gels with blaCTX-M, IncI1 and IncF probes demonstrated that all blaCTX-M-1 and the blaCTX-M-9 genes were carried on IncI1 plasmids, whereas the blaCTX-M-15 gene was located on an IncFII plasmid (not shown). The IncI1 plasmids sizes ranged from 100 to 120 kb, whereas the size of the blaCTX-M-15 IncFII plasmid was 160 kb (Table 1). In chicken isolates, the blaCTX-M-1 IncI1 plasmids belonged to the ST3 subtype (allelic profile 2/1/4/1/2) or to the closely related ST87 (8/1/4/1/2) subtype (Table 1), which only differs from ST3 by four base pairs at the end of a single locus (repA gene; http ://pubmlst.org/plasmid). The blaCTX-M-9 gene was also located on an IncI1/ST87 plasmid. In pet isolates, all plasmids but one blaCTX-M-1 IncI1 plasmid belonged to the ST3 subtype, whereas one isolate belonged to the unrelated ST25 (1/4/5/4/1) subtype (Table 1). Restriction fragment length polymorphism (RFLP) profiles of the IncI1/ST3 and IncI1/ST87 plasmids after digestion with PstI or EcoRI were either indistinguishable or highly similar, whatever the animal origin (Figure S1, available as Supplementary data at JAC Online). Hence, this study shows that the blaCTX-M-1 IncI1/ST3 plasmid is broadly disseminated in chickens and pets in Tunisia. Interestingly enough, the blaCTX-M-1 IncI1/ST3 plasmids described here were identical or highly similar to blaCTX-M-1 IncI1/ ST3 plasmids recently reported in France and Belgium in several animal species and humans (Figure S1, available as Supplementary data at JAC Online ). These plasmids were reported not only in E. coli but also in S. enterica, in particular of serovars Llandoff, London, Newport and Typhimurium of chicken and human origin. In conclusion, the blaCTX-M-1 IncI1/ST3 plasmid, which is dominant invarious animal species in Europe and has alsobeen reported in humans infected with S. enterica, appears to be dominant in foodand non-food-producing Tunisian animals. These data underline the international role of the blaCTX-M-1 IncI1/ST3 plasmid in ESBLepidemiology in animals. The risk of dissemination of the blaCTX-M-1 IncI1/ST3 plasmid from E. coli to S. enterica in Tunisia, as reported in Europe, is also a major concern. Furthermore, this study suggests that the surprisingly high prevalence of CTX-M-1 producers in the Tunisian community might result from the abundance of the blaCTX-M-1 IncI1/ST3 plasmid in animals. There is an urgent need to set up surveillance systems for antimicrobial resistance and antibiotic usage in animals (particularly in the food chain) in Tunisia.

Emergence of SHV-2a Extended-Spectrum β-Lactamases in Clinical Isolates of Pseudomonas aeruginosa in a University Hospital in Tunisia

Extended-spectrum beta-lactamases (ESBLs) in Pseudomonas aeruginosa are increasingly reported worldwide. In our study, a total of 70 clinical isolates of multidrug-resistant P. aeruginosa were studied. Isoelectric focusing electrophoresis, PCR, and PCR product sequencing were designed to characterize the contained ESBLs. The Double Disk Synergy Test in Cloxacillin (250 microg/ml)-containing Mueller-Hinton agar plates with a 20 mm distance between disks was the most reliable ESBL-screening method. Seven out of 70 multidrug-resistant P. aeruginosa clinical isolates were positive for ESBL and have the bla(SHV-2a) ESBL gene. The bla(SHV-2a)-positive isolates were clonally related according to Enterobacterial Repetetive Intergenic Consensus-PCR (ERIC-PCR) results. The bla(SHV-2a) gene was found to be chromosomally located, and the flanking IS26 sequence in the immediate upstream region of the bla(SHV-2a) gene was detected in all SHV-2a-producing isolates. This is the first report of SHV-2a-producing P. aeruginosa isolates from Tunisia.

Dissemination of multidrug-resistant blaCTX-M-15/IncFIIk plasmids in Klebsiella pneumoniae isolates from hospital- and community-acquired human infections in Tunisia

This study investigated the molecular features of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae from hospital- and community-acquired (HA/CA) infections in the region of Mahdia, Tunisia. Among 336 K. pneumoniae isolates recovered from both clinical contexts between July 2009 and December 2011, 49 and 15 were ESBL producers and originated from clinical and community sources, respectively. All isolates produced the CTX-M-15 enzyme. As shown by Southern blot on S1 nuclease treatment followed by pulsed-field gel electrophoresis (PFGE) gels, the blaCTX-M-15 gene was carried on IncFII (n=4), IncFIIk (n=25), IncL/M (n=4), IncK (n=1), or untypeable (n=15) plasmids in HA isolates. In CA isolates, the blaCTX-M-15 gene was carried on IncFIIk (n=6), IncFII (n=1), IncHI1 (n=1), or untypeable (n=7) plasmids. In all, 23 and 11 PFGE types were found among the HA and CA isolates. Multilocus sequence typing on representative isolates shows diverse sequence types (STs), such as ST307, ST101, ST39, ST4, ST140, ST15, and ST307 in HA isolates and ST101, ST664, and ST323 in CA isolates. This study is the first comprehensive report of ESBL plasmids in K. pneumoniae from HA and CA infections in Tunisia.

blaCTX-M-15-Carrying F2:A-:B- Plasmid in Escherichia coli from Cattle Milk in Tunisia

Extended-spectrum β-lactamases (ESBL) are widespread enzymes in animals, and the risk of transmission of ESBL genes to humans has become a major issue. In Tunisia, recent data showed a high prevalence of ESBL-producing Escherichia coli isolates in healthy animals, mostly in chickens. In this study, we report the first data on ESBL in diseased Tunisian animals (chickens and cattle), highlighting a major difference in ESBL prevalence in the infectious versus noninfectious E. coli flora. Interestingly, the only ESBL producer was an ST10 E. coli from a cattle, and not from chicken. Moreover, this E. coli isolate harbored the bla(CTX-M-15) gene on an F2:A-:B- plasmid, a combination frequently found in humans. This plasmid was also highly similar to a bla(CTX-M-15) F2:A-:B- plasmid recently reported in cattle in France. Altogether, this study is also the first report of the bla(CTX-M-15) gene in food animals in Tunisia, and, to our best knowledge, the first report of an ESBL producer in cattle in Africa. Since this plasmid was recognized in cattle in France and worldwide in humans, the question of its origin in Tunisian cattle is open. The detection of ESBL producers in milk in Tunisia may also constitute a risk of ESBL transmission from animals to humans through food consumption.

Emergence of OXA-204 β-lactamase in Tunisia.

A retrospective epidemiological survey was carried out to determine the prevalence of carbapenemase producers among enterobacterial clinical isolates recovered in the center of maternity and neonatology of Monastir (Tunisia). PCR screening identified 1 OXA-48 and 2 OXA-204 producers, which coexpressed the CTX-M-15 or the CMY-4 β-lactamases. PCR mapping showed that the bla(OXA-48) gene was carried by a Tn1999.2 transposon, whereas the bla(OXA-204) gene was part of the Tn2016 transposon-like structure. The OXA-48- or OXA-204-producing Klebsiella pneumoniae clinical isolates and the OXA-204-expressing Escherichia coli clinical isolate belonged to the widespread sequence types ST11, ST101, and ST617, respectively. The OXA-204 enzyme, which is a point derivative of the OXA-48 carbapenemase, had hitherto been reported in 2013 from K. pneumoniae isolate. Our study shows for the first time the dissemination of this resistance marker in E. coli strain. The coproduction of OXA-204 with CTX-M-15 and CMY-4 enzymes may potentiate the risk of multiresistance and may enhance the risk of dissemination.

Outbreak of colistin-resistant carbapenemase-producing Klebsiella pneumoniae in Tunisia

OBJECTIVES Mechanisms of colistin and carbapenem resistance among a collection of Klebsiella pneumoniae isolates recovered in a university hospital in Tunisia were studied. METHODS In vitro antimicrobial susceptibility testing, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blotting and PCR-based replicon typing (PBRT) were performed. Extended-spectrum β-lactamases (ESBLs), carbapenemases, AmpC-type enzymes and mgrB genes were detected by PCR and sequencing. Clonality of isolates was assessed by PFGE and multilocus sequence typing (MLST). RESULTS Of 940 Enterobacteriaceae isolates recovered from June 2015 to March 2016 in Tahar Sfar Hospital (Mahdia, Tunisia), 220 were identified as K. pneumoniae, among which 29 were carbapenem-resistant. Carbapenem resistance was mostly due to expression of blaOXA-48 or blaOXA-204 in combination with blaCMY-4. Seven isolates carried blaNDM-1, of which two also harboured blaOXA-48, together with blaCMY-16 in one of them. All but two isolates also harboured blaCTX-M-15. All 20 blaOXA-48 genes were part of transposon Tn1999 on an IncL plasmid, whereas blaOXA-204 was found on transposon Tn2016 on an IncA/C plasmid. Finally, all blaNDM-1 genes were located within a Tn125 transposon on an IncFIIk plasmid. Interestingly, 7 (24.1%) of 29 carbapenem-resistant isolates were resistant to colistin, of which 6 were assigned to ST101, had similar PFGE profiles and presented the same 2-kb insertion in the mgrB gene. CONCLUSIONS This study reports, for the first time in Tunisia, the full molecular characterisation of colistin resistance in K. pneumoniae. There is an urgent need for control measures and prudent use of colistin in treatment of infections with carbapenemase-producing K. pneumoniae.