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ABSTRACT: Objectives: Colistin is considered a last-resort antibiotic against carbapenem-resistant isolates. Currently, this antibiotic is facing the emergence of mobilised colistin resistance (mcr) genes, which confer colistin resistance. This study conducted genomic characterisation of an atypical multidrug-resistant Escherichia coli harbouring two mcr genes in France. Samples collected from a pig farm in Avignon (Vaucluse department) were subjected to molecular screening targeting mcr variants. Methods: Samples were cultured on selective Lucie-Bardet-Jean-Marc-Rolain medium. Growing bacteria were identified using MALDI-TOF, followed by antibiotic susceptibility testing. Whole-genome sequencing and bioinformatic genome analysis were performed. Results: Selective culture of stools revealed the presence of an E. coli strain named Q4552 harbouring mcr-1.1 and mcr-3.5 genes, which is also resistant to 14 antibiotics. Genome sequencing and assembly yielded a complete and circular chromosome and eight different plasmids. Sequence analysis demonstrated an integration of a mobile genetic element carrying mcr-1.1 in the chromosome, whereas mcr-3.5 was in the plasmid and its resistome was composed of 22 resistance genes. The Q4552 strain was identified as an ST-843 clone that belonged to the clonal complex Cplx-568 and is the only ST type of this cplx-568 that has been isolated from animals, humans, and the environment. Conclusion: We report the first co-occurrence of mcr-1 and mcr-3 genes in France from a pathogenic E. coli isolated from a pig. Because this clone (ST-843) has been reported in zoonotic transmissions, programs to monitor the bacterium are urgently required to avoid its spread and zoonotic transmission to humans.

Objectives: The increase in resistance to antibiotics has led to the revival of colistin as the last option for treatment, which automatically led to an increase of colistin-resistant, Gram-negative bacteria. In this study, we report the presence of clinical colistin-resistant Enterobacteriaceae isolated from a Lebanese hospital. Methods: From 23 rectal swabs, eight colistin-resistant clinical strains (five Escherichia coli, two Enterobacter cloacae, and one Klebsiella pneumoniae) were isolated. Antibiotic susceptibility testing was performed using the disk diffusion method and Etest. The broth microdilution method was used to determine colistin susceptibility. Reverse transcription polymerase chain reaction (RT-PCR), standard PCR and sequencing were used to investigate genes encoding for extended-spectrum β-lactamases, carbapenemases and colistin resistance. Genotyping of these isolates was conducted by multilocus sequence typing (MLST). Results: Results of antibiotic susceptibility testing revealed that all isolates were resistant to colistin. They had MICs for colistin that ranged from 8 to 32 mg/L. Real-time PCR results showed that five strains harboured blaTEM-1 and one strain harboured blaTEM-163. Moreover, four strains were positive for blaCTX-M-15, blaCTX-M-103 and blaCTX-M-189, and K. pneumoniae harboured blaSHV-1. Observed colistin resistance was linked to amino acid substitutions into protein sequences of pmrA/B, phoP/Q, and mgrB. Interestingly, we report here a mutation in the mgrB regulator and pmrA/B, phoP/Q in colistin-resistant E. cloacae and E. coli clinical isolates for the first time in Lebanon. Conclusion: This study highlights the presence of colistin-resistant Gram-negative bacteria in a Lebanese hospital, which is worrisome. An urgent strategy needs to be adopted to avoid the spread of such bacteria.

Objectives: We aimed to develop here a specific real-time PCR assay with TaqMan® probe to detect efficiently bacterial strains harboring the new plasmid mediated-colistin resistance mcr-8 gene. Methods: Specific primers and probe for mcr-8 gene were designed from sequences alignment of all mcr genes variants. Specificity of the designed primers and probe were first checked par BlastN analysis and by in silico PCR. The analytical sensitivity and specificity tests were performed in vitro on a panel of 290 genomic DNA of Gram-negative bacteria and 250 metagenomic DNA from human stool samples. Whole genome sequencing (WGS) was performed here using MiSeq technology. Results: Designed primers and probe were 100% specific tomcr-8 gene by BlastN and in silico PCR analysis. Real-time PCR screening of a collection of clinical isolates resulted to one positive Klebsiella pneumoniae isolate (KP95). WGS confirmed that this isolate harbored the mcr-8 gene and other resistance genes such as blaOXA-48, blaCTX-M-15 β-lactamases. Our real-time PCR was highly sensitive on a 10-fold dilution serie from a calibrated inoculum at 108 CFU/mL with a limit of detection at 55 CFU/mL. Conclusion: To the best of our knowledge, we propose here, the first real-time PCR assay targeting mcr-8 gene with high specificity and sensitivity, able to detect mcr-8 gene in less than 2 h from any DNA sample. This real-time PCR assay allowed the first description of a clinical K. pneumoniae strain harboring the mcr-8 gene in Algeria.