Identification of a Novel Plasmid Carrying mcr-4.3 in an Acinetobacter baumannii Strain in China.

Here, we identified mcr-4.3 in Acinetobacter baumannii, which had not been previously observed to carry an mcr gene. The mcr-4.3-harboring A. baumannii strain AB18PR065 was isolated from pig feces from a slaughterhouse in Guangdong Province of China. The mcr-4.3-carrying pAB18PR065 is 25,602 bp in size and could not be transferred in conjugation, transformation, and electroporation experiments, as we did not find any conjugation-related genes therein. pAB18PR065 harbors two copies of type II toxin-antitoxin systems, which are functional in plasmid stabilization and maintenance. pAB18PR065 shares similarity only with one recently identified plasmid, pAb-MCR4.3 (35,502 bp), from a clinical A. baumannii strain. It is likely that the emergence of pAb-MCR4.3 was due to the insertion of an 11,386-bp, ISAba19-based, composite transposon into pAB18PR065. These data indicate that mcr-4.3 was captured by an A. baumannii-original plasmid via horizontal gene transfer.

Complete Nucleotide Sequences of mcr-4.3-Carrying Plasmids in Acinetobacter baumannii Sequence Type 345 of Human and Food Origin from the Czech Republic, the First Case in Europe.

Here, we describe two plasmids carrying mcr-4.3 in two Acinetobacter baumannii strains isolated from imported food and a clinical sample. The comparative analysis of these plasmids, with two other plasmids reported in the NCBI database, highlighted the common origin of the plasmidic structure carrying mcr-4.3 This is the first case of the mcr-4.3 gene in a A. baumannii strain isolated from a clinical case in Europe. We hypothesize that food import is initiating the spread in Czech Republic.

Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes.

Background and aimPlasmid-mediated colistin resistance mechanisms have been identified worldwide in the past years. A multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes (mcr-1 to mcr-5, and variants) in Enterobacteriaceae was developed for surveillance or research purposes. Methods: We designed four new primer pairs to amplify mcr-1, mcr-2, mcr-3 and mcr-4 gene products and used the originally described primers for mcr-5 to obtain a stepwise separation of ca 200 bp between amplicons. The primer pairs and amplification conditions allow for single or multiple detection of all currently described mcr genes and their variants present in Enterobacteriaceae. The protocol was validated testing 49 European Escherichia coli and Salmonella isolates of animal origin. Results: Multiplex PCR results in bovine and porcine isolates from Spain, Germany, France and Italy showed full concordance with whole genome sequence data. The method was able to detect mcr-1, mcr-3 and mcr-4 as singletons or in different combinations as they were present in the test isolates. One new mcr-4 variant, mcr-4.3, was also identified. Conclusions: This method allows rapid identification of mcr-positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance. The multiplex PCR should be particularly interesting in settings or laboratories with limited resources for performing genetic analysis as it provides information on the mechanism of colistin resistance without requiring genome sequencing.

Chromosomally and Plasmid-Located mcr in Salmonella from Animals and Food Products in China.

This study aimed to investigate the prevalence and genomic characteristics of the colistin resistance gene mcr in Salmonella enterica in China. In total, 445 S. enterica isolates from animals and food products were screened through PCR and sequencing for the presence of mcr. The mcr genes were detected in nine Salmonella strains (2.02%), with complete mcr-1 in S. enterica serovar Indiana (n = 1) and an S. Typhimurium monophasic variant (S. 4,[5],12:i:-; n = 1), mcr-4.3 in S. enterica serovar London (n = 1), and an incomplete mcr-1 in S. Indiana (n = 6). They exhibited MIC values of 0.25 to 8 mg/L to colistin and showed resistance to multiple antimicrobial agents. Whole-genome sequencing was performed on mcr-positive Salmonella strains using Illumina HiSeq or PacBio single-molecule real-time sequencing. The complete mcr-1 gene was located on conjugative IncN1-IncHI2 plasmid and IncX4 plasmid, respectively, with high similarity to other mcr-1-bearing plasmids belonging to the same incompatibility type. Together with an additional 13 antimicrobial resistance genes, the incomplete mcr-1 was embedded in an 81,442-bp multiresistance region on the chromosome in S. Indiana YZ20MCS6. The Δmcr-1-pap2 segment and a set of tellurite resistance determinants (terYXWZABCDEF) in six S. Indiana strains were similar to other IncHI2 plasmid backbones. The mcr-4.3 gene was located on an untyped plasmid pYULZMPS10. Although low prevalence of mcr was observed in Salmonella, continuous surveillance of this gene in Salmonella is required. Plasmids play an important role in mcr transmission, and mcr-1, although incomplete, can be captured by chromosomes with the help of mobile elements. IMPORTANCE Colistin is a last-resort antibiotic for severe infections caused by multidrug-resistant (MDR) Gram-negative pathogens. Colistin resistance genes mcr, particularly mcr-1, have been found in Enterobacteriaceae around the world, mainly in Escherichia coli and Salmonella. Salmonella enterica is a major foodborne pathogen, with MDR Salmonella being considered a "Serious Threat Level pathogen" by the Centers for Disease Control and Prevention. Therefore, the prevalence of mcr in Salmonella strains must be monitored. In this study, a low mcr prevalence (2.02%) was observed in Salmonella strains from animals and food products, with plasmid-borne mcr-1 in S. enterica serovar Indiana and an S. Typhimurium monophasic variant (S. 4,[5],12:i:-) and chromosomally located mcr-1 in S. Indiana. The mcr-4.3 gene was first identified in S. enterica serovar London associated with an untyped plasmid. Although this study reports a low mcr prevalence in Salmonella, the transmission ability of mcr-positive Salmonella strains to humans via the food chain is a public health concern.

Distribution of mcr genes among carbapenem-resistant Enterobacterales clinical isolates: high prevalence of mcr-positive Enterobacter cloacae complex in Seoul, Republic of Korea.

Colistin is often used as a drug of last resort against infections caused by multi-drug-resistant Gram-negative bacteria, including carbapenem-resistant Enterobacterales (CRE). Recently, the acquisition of mobile colistin resistance (mcr) genes by CRE has become a cause for concern. This study investigated the prevalence of mcr genes in CRE isolates in Seoul, Republic of Korea. In total, 3675 CRE strains were collected from patients between 2018 and 2019, and initially screened for mcr genes using multiplex polymerase chain reaction assays. Upon the identification of mcr-harbouring strains, colistin susceptibility tests, identification of carbapenemase and ß-lactamase genes, and plasmid replicon typing were performed. Clonal analysis was conducted using pulsed-field gel electrophoresis. mcr genes were detected in 2.2% (80/3675) of CRE strains. There were three mcr-1 carriers, one mcr-4.3 carrier, one mcr-4.3/mcr-9 carrier, 58 mcr-9 carriers, one mcr-9/mcr-10 carrier and 16 mcr-10 carriers among various Enterobacterales species, of which 60 were Enterobacter cloacae complex (ECC) strains. The prevalence of mcr genes in ECC strains was 20.5%. Molecular detection confirmed that 21.3% and 13.8% of mcr-harbouring strains shared blaNDM-1 or blaKPC-2, respectively. In addition, an IncHI2 replicon was identified in 71.7% of mcr-9 strains. Comparative analysis revealed not only a notable diversity of mcr carriers, but also clonal spreading or nosocomial outbreaks of some ECC strains. These findings revealed a silent distribution of mcr genes in CRE strains with high genetic heterogeneity in Seoul, underscoring the urgent need for timely intervention to control and prevent mcr dissemination.

Characterisation of mcr-4.3 in a colistin-resistant Acinetobacter nosocomialis clinical isolate from Cape Town, South Africa.

OBJECTIVES: Colistin resistance in Acinetobacter spp. is increasing, resulting in potentially untreatable nosocomial infections. Plasmid-mediated colistin resistance is of particular concern due to its low fitness cost and potential transferability to other bacterial strains and species. This study investigated the colistin resistance mechanism in a clinical Acinetobacter nosocomialis isolate from Cape Town, South Africa. METHODS: A colistin-resistant A. nosocomialis isolate was identified from a blood culture in 2017. PCR and Illumina whole-genome sequencing (WGS) were performed to identify genes and mutations conferring resistance to colistin. Plasmid sequencing was performed on an Oxford Nanopore platform. mcr functionality was assessed by broth microdilution after cloning the mcr gene into pET-48b(+) and expressing it in SHuffle® T7 Escherichia coli and after curing the plasmid using 62.5 mg/L acridine orange. RESULTS: The colistin minimum inhibitory concentration (MIC) of the A. nosocomialis isolate was 16 mg/L. The mcr-4.3 gene was detected by PCR and WGS. No other previously described colistin resistance mechanism was found by WGS. The mcr-4.3 gene was identified on a 24 024-bp RepB plasmid (pCAC13a). Functionality studies showed that recombinant mcr-4.3 did not confer colistin resistance in E. coli. However, plasmid curing of pCAC13a restored colistin susceptibility in A. nosocomialis. CONCLUSION: We describe the first detection of a plasmid-mediated mcr-4.3 gene encoding colistin resistance in A. nosocomialis and the first detection of mcr-4.3 in a clinical isolate in Africa. Recombinant expression of mcr-4.3 did not confer colistin resistance in E. coli, suggesting that its functionality may be RepB plasmid-dependent or species-specific.

Genomic Characterization of VIM and MCR Co-Producers: The First Two Clinical Cases, in Italy.

BACKGROUND: the co-production of carbapenemases and mcr-genes represents a worrisome event in the treatment of Enterobacteriaceae infections. The aim of the study was to characterize the genomic features of two clinical Enterobacter cloacae complex (ECC) isolates, co-producing VIM and MCR enzymes, in Italy. METHODS: species identification and antibiotic susceptibility profiling were performed using MALDI-TOF and broth microdilution methods, respectively. Transferability of the bla VIM- and mcr- type genes was verified through conjugation experiment. Extracted DNA was sequenced using long reads sequencing technology on the Sequel I platform (PacBio). RESULTS: the first isolate showed clinical resistance against ertapenem yet was colistin susceptible (EUCAST 2020 breakpoints). The mcr-9.2 gene was harbored on a conjugative IncHI2 plasmid, while the bla VIM-1 determinant was harbored on a conjugative IncN plasmid. The second isolate, resistant to both carbapenems and colistin, harbored: mcr-9 gene and its two component regulatory genes for increased expression on the chromosome, mcr-4.3 on non-conjugative (yet co-transferable) ColE plasmid, and bla VIM-1 on a non-conjugative IncA plasmid. CONCLUSIONS: to our knowledge, this is the first report of co-production of VIM and MCR in ECC isolates in Italy.

Leclercia adecarboxylata From Human Gut Flora Carries mcr-4.3 and bla IMP-4-Bearing Plasmids.

A clinical Leclercia adecarboxylata strain harboring the mcr-4.3 and bla IMP-4 genes was isolated from active rectal screening of carbapenem-resistant Enterobacteriaceae (CRE) in a patient. The isolate was found to harbor seven plasmids, including a 94,635 bp bla IMP-4-bearing IncN plasmid and a 9,782 bp mcr-4.3-bearing ColE10-type plasmid. The isolate was susceptible to colistin despite carrying the mcr-4.3 gene, suggesting that this MCR-4 variant may not be functional. Carriage of antibiotic resistance genes in human gut L. adecarboxylata strain suggests that close surveillance of resistance strains in the human gut flora should be included as a routine clinical practice to prevent occurrence of infections, especially among immunocompromised patients.