Conjugative transfer of multi-drug resistance IncN plasmids from environmental waterborne bacteria to Escherichia coli.

Watersheds contaminated with municipal, hospital, and agricultural residues are recognized as reservoirs for bacteria carrying antibiotic resistance genes (ARGs). The objective of this study was to determine the potential of environmental bacterial communities from the highly contaminated La Paz River basin in Bolivia to transfer ARGs to an Escherichia coli lab strain used as the recipient. Additionally, we tested ZnSO4 and CuSO4 at sub-inhibitory concentrations as stressors and analyzed transfer frequencies (TFs), diversity, richness, and acquired resistance profiles. The bacterial communities were collected from surface water in an urban site close to a hospital and near an agricultural area. High transfer potentials of a large set of resistance factors to E. coli were observed at both sites. Whole-genome sequencing revealed that putative plasmids belonging to the incompatibility group N (IncN, IncN2, and IncN3) were predominant among the transconjugants. All IncN variants were verified to be mobile by a second conjugation step. The plasmid backbones were similar to other IncN plasmids isolated worldwide and carried a wide range of ARGs extensively corroborated by phenotypic resistance patterns. Interestingly, all transconjugants also acquired the class 1 integron intl1, which is commonly known as a proxy for anthropogenic pollution. The addition of ZnSO4 and CuSO4 at sub-inhibitory concentrations did not affect the transfer rate. Metal resistance genes were absent from most transconjugants, suggesting a minor role, if any, of metals in the spread of multidrug-resistant plasmids at the investigated sites.

Dynamics of blaKPC-2 Dissemination from Non-CG258 Klebsiella pneumoniae to Other Enterobacterales via IncN Plasmids in an Area of High Endemicity.

Carbapenem-resistant Enterobacterales (CRE) pose a significant threat to global public health. The most important mechanism for carbapenem resistance is the production of carbapenemases. Klebsiella pneumoniae carbapenemase (KPC) represents one of the main carbapenemases worldwide. Complex mechanisms of blaKPC dissemination have been reported in Colombia, a country with a high endemicity of carbapenem resistance. Here, we characterized the dynamics of dissemination of blaKPC gene among CRE infecting and colonizing patients in three hospitals localized in a highly endemic area of Colombia (2013 and 2015). We identified the genomic characteristics of KPC-producing Enterobacterales recovered from patients infected/colonized and reconstructed the dynamics of dissemination of blaKPC-2 using both short and long read sequencing. We found that spread of blaKPC-2 among Enterobacterales in the participating hospitals was due to intra- and interspecies horizontal gene transfer (HGT) mediated by promiscuous plasmids associated with transposable elements that was originated from a multispecies outbreak of KPC-producing Enterobacterales in a neonatal intensive care unit. The plasmids were detected in isolates recovered in other units within the same hospital and nearby hospitals. The gene "epidemic" was driven by IncN-pST15-type plasmids carrying a novel Tn4401b structure and non-Tn4401 elements (NTEKPC) in Klebsiella spp., Escherichia coli, Enterobacter spp., and Citrobacter spp. Of note, mcr-9 was found to coexist with blaKPC-2 in species of the Enterobacter cloacae complex. Our findings suggest that the main mechanism for dissemination of blaKPC-2 is HGT mediated by highly transferable plasmids among species of Enterobacterales in infected/colonized patients, presenting a major challenge for public health interventions in developing countries such as Colombia.

In vitro and in vivo persistence of IncN plasmids carrying qnr genes in uropathogenic Escherichia coli isolates.

OBJECTIVES: This study aimed to evaluate the persistence of the plasmid-mediated quinolone resistance (PMQR) among uropathogenic Escherichia coli strains grown under or without exposure to subinhibitory concentrations of ciprofloxacin. Based on that, we evaluated the possible spontaneous loss or maintenance of PMQR and the possible appearance of compensatory mutations in gyrA and parC genes. METHODS: Three uropathogenic E. coli strains harbouring chromosomal mutations in the gyrA and/or parC genes coupled with qnrS1 or qnrB2 determinants carried by distinct plasmid sizes and incompatibility N groups (IncN/ST1, IncN/ST5) were evaluated using in vitro and in vivo assays. RESULTS: PMQRs remained stable in all strains throughout the generations evaluated, independently of exposure to ciprofloxacin in both in vivo and in vitro assays. Analysis of gyrA and parC genes after in vivo and in vitro assays revealed that no changes occurred in quinolone-resistance determining regions (QRDR). CONCLUSION: We demonstrated that IncN plasmids were persistent over 14 days in E. coli clinical strains independently of exposure to ciprofloxacin, as well as previous mutations in QRDR.