Prevalence and Characterization of Extended-Spectrum ß-Lactamase- and Carbapenemase-Producing Enterobacterales from Tunisian Seafood.

Aquaculture is a rapidly expanding sector in which it is important to monitor the occurrence of multi-drug resistant (MDR) bacteria. The presence of extended-spectrum ß-lactamase (ESBL-) or carbapenemase-producing Enterobacterales is a commonly used indicator of the resistance burden in a given sector. In this study, 641 pieces of farmed fish (sea bream and sea bass), as well as 1075 Mediterranean clams, were analyzed. All ESBL- and carbapenemase-producing Enterobacterales collected were whole-genome sequenced. The proportion of ESBL-producing Enterobacterales was 1.4% in fish and 1.6% in clams, carried by Escherichia coli (n = 23) and Klebsiella pneumoniae (n = 4). The ESBL phenotype was exclusively due to the presence of blaCTX-M genes, the most frequent one being blaCTX-M-15. The blaCTX-M-1 gene was also identified in six E. coli, among which four were carried by IncI1/pST3 plasmids, possibly betraying an animal origin. Carbapenemases were absent in fish but identified in two K. pneumoniae isolates from clams (blaNDM-1 and blaOXA-48). Several sequence types (STs) identified were associated with human MDR clones such as E. coli ST131 and ST617, or K. pneumoniae ST307 and ST147. Our results might indicate that bacteria from hospital or farm effluents can reach the open sea and contaminate seafood and fish that are living or raised nearby. Therefore, monitoring the quality of water discharged to the sea and the presence of MDR bacteria in seafood is mandatory to ensure the quality of fishery products.

Epidemiology and Whole-Genome Analysis of NDM-1-Producing Klebsiella pneumoniae KP3771 from Tunisia.

Objectives: The whole-genome sequence (WGS) of Klebsiella pneumoniae KP3771 isolate was characterized. This strain was recovered from the urine sample of an 80-year-old man hospitalized in an intensive care unit of the University Hospital Tahar Sfar in Tunisia. Materials and Methods: WGS using a MiSeq platform was used. The assembled genome was subjected to several software analyses. Results: K. pneumoniae KP3771 was resistant to all antibiotics but colistin and tigecycline. WGS analysis found 18 transmissible genes encoding resistance markers, including blaNDM-1 and blaCTX-M-15 genes, which were carried by four plasmids belonging to the Inc Ib, IIk, and R groups. Three families of genes encoding virulence factors were detected, including adhesins (fimH, fimA, fimB, fimC, mrkD, Kpn, and ycfM), siderophores (enterobactin, aerobactin, and yersiniabactin siderophores), and protectin/invasin (traT). The strain was assigned to the sequence type 147. Conclusions: This study describes the genome of a carbapenemase-producing K. pneumoniae clinical isolate recovered in Tunisia. Bacteria WGS has become the reference technology to address epidemiological issues; this high level of information is particularly well suited to enrich epidemiological workflows' output.

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 ß-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.

Extended-spectrum ß-lactamases and plasmid-mediated quinolone resistance in enterobacterial clinical isolates from neonates in Tunisia.

This study was conducted to investigate extended-spectrum-ß-lactamase (ESBL) producing Enterobacteriaceae isolates from the Center of Maternity and Neonatology of Monastir, Tunisia. Fourty-six strains out of 283 were found to produce ESBL: Klebsiella pneumoniae (n = 37), Escherichia coli (n = 6), Enterobacter cloacae (n = 2), and Citrobacter freundi (n = 1). Genotyping analysis, using ERIC2 and RAPD, showed that strains were clonally unrelated. PCR amplification followed by sequencing revealed that all strains produced CTX-M-15. This enzyme was co-produced with TEM and SHV determinants in 34 and 36 strains respectively. The blaCTXM-15 gene was bracked by ISEcp1 and/or IS26 in 42 out of the 46 ESBL positive strains. The quinolone resistance determinants were associated to the ESBL producing isolates: we identified the qnrB1 gene in six isolates and the aac(6')-Ib-cr gene in five isolates. This epidemiological study shows the widespread of CTX-M-15 and qnr determinants among enterobacterial isolates from neonates hospitalized at the center of Maternity and Neonatology of Monastir suggesting either mother portage or horizontal transmission.

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.

Emergence of ST147 Klebsiella pneumoniae Producing OXA-204 Carbapenemase in a University Hospital, Tunisia.

Molecular features of the first carbapenem-resistant Klebsiella pneumoniae isolates (KP1 and KP2) from the University Hospital Tahar Sfar, Tunisia, were investigated. Antimicrobial susceptibility testing, multilocus sequence typing, S1 nuclease pulsed-field gel electrophoresis, Southern blot, and polymerase chain reaction (PCR)-based replicon typing were performed. Extended-spectrum ß-lactamases and carbapenemase genes were detected by PCR and sequencing. Both isolates were multidrug resistant. KP1 was of sequence type (ST) ST101 and exhibited blaCTX-M-15 and blaTEM-1 on an untypeable plasmid and blaOXA-48 on an IncL/M plasmid. KP2 was genetically unrelated to KP1 (ST147) and harbored an IncA/C plasmid carrying blaCMY-4 and the blaOXA-48 derivative gene: blaOXA-204. This study reports the second case worldwide of an OXA-204-producing K. pneumoniae isolate from the same country, however, in a different genetic background.

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 ß-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 ß-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.