Characterization of mcr-5-Harboring Salmonella enterica subsp. enterica Serovar Typhimurium Isolates from Animal and Food Origin in Germany.

We characterized eight mcr-5-positive Salmonella enterica subsp. enterica serovar Typhimurium sequence type 34 (ST34) isolates obtained from pigs and meat in Germany. Five plasmid types were identified harboring mcr-5 on Tn6452 or putative mobile insertion cassettes. The mobility of mcr-5 was confirmed by integration of Tn6452 into the bacterial chromosomes of two strains and the detection of conjugative mcr-5 plasmids. The association with mobile genetic elements might further enhance mcr-5 distribution.

Chromosomally Encoded mcr-5 in Colistin-Nonsusceptible Pseudomonas aeruginosa.

Whole-genome sequencing (WGS) of historical Pseudomonas aeruginosa clinical isolates identified a chromosomal copy of mcr-5 within a Tn3-like transposon in P. aeruginosa MRSN 12280. The isolate was nonsusceptible to colistin by broth microdilution, and genome analysis revealed no mutations known to confer colistin resistance. To the best of our knowledge, this is the first report of mcr in colistin-nonsusceptible P. aeruginosa.

Shotgun metagenomic analysis reveals the emergence of plasmid-encoded mcr-5.1 gene in hospital wastewater in Bangladesh.

Colistin is considered the last line therapy for treating multidrug-resistant (MDR) bacterial infections in humans. Therefore, the spread of colistin resistance poses a serious threat to human, and environmental health. Though Bangladesh is known as a hotspot of AMR, limited studies have been carried out regarding the status of colistin resistance. Information on the emerging bacterial resistance is inevitable for protecting public health. Nowadays, wastewater analysis has been prioritized for metagenomics-enabled AMR surveillance. Our study on the metagenomic analysis of untreated hospital effluents first detected the colistin resistance-conferring mcr-5.1 gene in the hospital environment of Bangladesh. Phylogenetic tree and in silico AMR analysis confirmed the detection of this mcr-5 variant, which is located in a plasmid contig. The plasmid was untypeable and belonged to the bacteria from the Enterobacteriaceae family. The mcr-5.1 operon was embedded in a Tn3 transposon, suggesting the mobility of the gene. Tnshfr1 transposon, chromate resistance protein ChrB, DNA invertase hin, and two MFS-type proteins were present in the genetic environment of mcr-5.1. Our findings provide evidence of the occurrence of mcr-5.1 in a hospital environment in Bangladesh, which calls for immediate attention and effective measures to contain the dissemination of colistin resistance in the environment.

Genomic Sequencing of Clinical Cupriavidus gilardii Isolates Revealed Their Diverse Antimicrobial Resistance Mechanisms.

Purpose: Cupriavidus gilardii is an emerging multidrug-resistant pathogen found in many environments and few clinical samples. The clinical infectiousness, pathogenicity, and resistance mechanisms of C. gilardii are still unclear due to the lack of clinical and sequencing data. We need to obtain insight into the clinical characteristics, virulence, and resistance mechanisms of C. gilardii. Patients and Methods: We isolated five C. gilardii isolates from hospitalized patients and carried out assay, culture and genome sequencing. We analyzed the genomic features of clinical C. gilardii isolates and took insight into their clinical characteristics, virulence, and resistance mechanisms. Results: These isolates were resistant to meropenem, gentamicin, and other antimicrobials due to intrinsic resistance genes. Furthermore, the sequencing results revealed the widespread presence of the MCR-5.1 gene in C. gilardii. The virulence magnitude of C. gilardii is closely correlated with the number of virulence factors they carry. Some C. gilardii strains can acquire resistance to levofloxacin through gyrA gene mutation during treatment. The diverse antimicrobial resistance mechanisms challenge the treatment of C. gilardii infections. Conclusion: We present the genomic characteristics of clinically isolated C. gilardii to improve (i) our understanding of this pathogen and (ii) treatment options.

The surveillance of colistin resistance and mobilized colistin resistance genes in multidrug-resistant Enterobacteriaceae isolated in Japan.

BACKGROUND: The plasmid-mediated bacterial colistin-resistant gene, mcr, is of global concern in clinical healthcare. However, there are few reports of surveillance for mcr in Japan. The aim of this study was to assess the prevalence of colistin resistance by identifying nine mcr genes in extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae and carbapenem-resistant Enterobacteriaceae (CRE) isolates in Japan. METHODS: A total of 273 ESBL and CRE clinical isolates were collected from patients in five tertiary hospitals from August 2016 to March 2017. Minimum inhibitory concentration (MIC) of colistin was measured using the microdilution method. Polymerase chain reaction (PCR) was performed to detect mcr-1 to mcr-9 genes in all strains. Whole-genome sequencing (WGS) analysis was conducted for any mcr-genes identified that had not been previously reported in patients from Japan. RESULTS: The rate of colistin resistance was 7.7% in all strains, with a higher rate in the CRE strains than in the ESBL-producing strains (20.4% versus 1.1%). The mcr-5 and mcr-9 gene were detected in one ESBL-producing Escherichia coli strain (1/273, 0.37%) and three CRE strains (3/273, 1.1%), respectively. As the ESBL-producing E. coli strain was the first clinical strain with mcr-5 in Japan, WGS analysis was performed for the strain. The sequence type of the mcr-5-positive strain was ST1642 and it carried two distinct plasmids, ESBL gene-carrying pN-ES-6-1, and mcr-5.1-carrying pN-ES-6-2. CONCLUSIONS: The results of this study showed that the frequency of colistin resistance and mcr-positive strains is not high in Japan. As the MIC for colistin was low in the mcr-5.1 and mcr-9 gene-positive strain, continuous monitoring of mcr genes is necessary.

MCR-5-Producing Colistin-Resistant Cupriavidus gilardii Strain from Well Water in Batna, Algeria.

This paper presents the first description of the mcr-5.1 gene in a colistin-resistant Cupriavidus gilardii isolate from well water that supplies a maternity hospital in Algeria. The whole-genome sequence of this strain showed the presence of putative ß-lactamase, aac(3)-IVa, and multidrug efflux pump-encoding genes, which could explain the observed multidrug resistance phenotype. Our findings are of great interest, as we highlight a potential contamination route for the spread of mcr genes. IMPORTANCE Colistin resistance mediated by mcr genes in Gram-negative bacteria has gained significant attention worldwide. This is due to the ability of these genes to be horizontally transferred between different bacterial genera and species. Aquatic environments have been suggested to play an important role in the emergence and spread of this resistance mechanism. Here, we describe the first report of an mcr-5-positive Cupriavidus gilardii aquatic isolate through its isolation from well water in Algeria. The significance of our study is in shedding the light on an important environmental reservoir of mcr genes.

Characterisation of mobile colistin resistance genes (mcr-3 and mcr-5) in river and storm water in regions of the Western Cape of South Africa.

BACKGROUND: Colistin is regarded as a last-resort antimicrobial against multi-drug resistant Gram-negative bacteria (GNB), therefore the dissemination of colistin resistance in the environment is of great concern. Horizontal transfer of mobile colistin resistance (mcr) genes to potential pathogens poses a serious problem. This study aimed to describe the presence of colistin resistant GNB and mcr genes in river and storm water in regions of the Western Cape. METHODS: Water samples were collected from three rivers during May 2019 and January 2020 and two storm water samples were collected in November 2019. Colistin resistant GNB were cultured on MacConkey agar containing colistin and identified by MALDI-TOF. Colistin resistance was confirmed using broth microdilution (BMD). mcr-1-5 genes were detected by PCR performed directly on the water samples and on the colistin resistant isolates. mcr functionality was assessed by BMD after cloning the mcr genes into pET-48b(+) and expression in SHuffle T7 E. coli. RESULTS: mcr-5.1 and various mcr-3 gene variants were detected in the Plankenburg-, Eerste- and Berg rivers and in storm water from Muizenberg, and only mcr-5.1 was detected in storm water from Fish Hoek. Colistin resistant GNB were isolated from all of the water sources. Aeromonas spp. were the most common colistin resistant organisms detected in the water sources; 25% (6/24) of colistin resistant Aeromonas spp. isolated from the Berg river contained novel mcr-3 variants; mcr-3.33 (n = 1), mcr-3.34 (n = 1) mcr-3.35 (n = 1) mcr-3.36 (n = 2) and mcr-3.37 (n = 1), which were confirmed to confer colistin resistance. CONCLUSIONS: The mcr-5.1 and mcr-3 colistin resistance gene variants were present in widely dispersed water sources in regions of the Western Cape. The mcr genes were only detected in water sampled downstream of and alongside communities, suggesting that their presence is driven by human influence/contamination. This is the first documentation of mcr-3 and mcr-5 gene variants in any setting in South Africa. Spill-over of these genes to communities could result in horizontal gene transfer to pathogenic bacteria, exacerbating the challenge of controlling multidrug resistant GNB infections.

Diverse and emerging molecular mechanisms award polymyxins resistance to Enterobacteriaceae clinical isolates from a tertiary hospital of Recife, Brazil.

OBJECTIVE: To describe the molecular mechanisms of polymyxins resistance in five Enterobacteriaceae clinical isolates from a tertiary hospital of Recife, Brazil. METHODS: The species identification and the susceptibility to antimicrobials were firstly performed by automatized methods and polymyxin resistance was confirmed by broth microdilution methods. The genetic basis of resistance was characterized with WGS analyses to study their resistome, plasmidome and mobilome, by BLAST searches on reference databases. RESULTS: Five (5%) Enterobacteriaceae isolates, comprising Escherichia coli (n = 2), Klebsiella pneumoniae (n = 2) and Citrobacter freundii (n = 1) species, exhibited polymyxin resistance. The mcr-1.1 gene was found in identical IncX4-plasmids harbored by both K. pneumoniae C119 (PolB MIC = 512 mg/L) and E. coli C153 (PolB MIC = 8 mg/L). The remaining E. coli strain C027 harbored the mcr-5.1 gene on an undefined Inc-plasmid (PolB MIC 256 mg/L). Some amino acid substitutions in PmrA (S29G, G144S), PmrB (S202P; D283G, W350*, Y258N) and PhoP (I44L) was detected among the E. coli clinical isolates, however they were also found in colistin-susceptible strains and predicted as neutral alterations. The mgrB of the ST54 KPC-2-producing K. pneumoniae C151 (PolB MIC = 32 g/mL) was interrupted at 69 nt by the IS903 element. The ST117 C. freundii C156 (PolB MIC = 256 mg/L) showed the A91T substitution on HAMP domain of the histidine kinase sensor CrrB, predicted as deleterious and deemed the remarkable determinant to polymyxins resistance in this strain. CONCLUSIONS: Diverse mechanisms of polymyxins resistance were identified among clinical Enterobacteriaceae from a tertiary hospital of Recife, Brazil, such as plasmid-mediated MCR-1 and MCR-5; IS903-interruption of mgrB and mutation in CrrAB regulatory system. These findings highlight the involvement of the identified plasmids on mcr dissemination among Enterobacteriaceae; warn about co-selection of the polymyxin-resistant and KPC-producer K. pneumoniae ΔmgrB lineage by carbapenems usage; and demonstrate potential role of CrrAB on emerging of polymyxin resistance among Enterobacteriaceae, besides Klebsiella species.

Isolation and plasmid characterisation of Salmonella enterica serovar Albany harbouring mcr-5 from retail chicken meat in Japan.

The emergence of plasmid-mediated colistin resistance genes (mcr), which is occurring in numerous countries, is a worldwide concern, primarily because colistin is a last-resort antibiotic. Compared to E. coli, prevalence of mcr genes in Salmonella is unclear in Japan. Here we screened for mcr-1-5 genes in our collection of Salmonella strains isolated from retail meat products collected in Japan from 2012 through 2016. We found that Salmonella Albany strain 27A-368 encodes mcr-5 and that mcr genes were undetectable among the remaining 202 isolates. The resistance plasmid p27A-368 was transferred by conjugation to S. Infantis and was stably retained as a transconjugant. Whole-genome sequencing revealed that mcr-5 resided on a 115 kb plasmid (p27A-368). The plasmid backbone of p27A-368 is more similar to that of pCOV27, an ESBL-encoding plasmid recovered from avian pathogenic E. coli, rather than pSE13-SA01718 of S. Paratyphi B that encodes mcr-5. Further, mcr-5 is located on a transposon, and its sequence is similar to that of pSE13-SA01718. A phylogenetic tree based on single nucleotide variants implies a relationship between 27A-368 and S. Albany isolated in Southeast Asian countries.

A Genomic, Evolutionary, and Mechanistic Study of MCR-5 Action Suggests Functional Unification across the MCR Family of Colistin Resistance.

A growing number of mobile colistin resistance (MCR) proteins is threatening the renewed interest of colistin as a "last-resort" defense against carbapenem-resistant pathogens. Here, the comparative genomics of a large plasmid harboring mcr-5 from Aeromonas hydrophila and the structural/functional perspectives of MCR-5 action are reported. Whole genome sequencing has identified the loss of certain parts of the Tn3-type transposon typically associated with mcr-5, providing a clue toward its mobilization. Phylogeny of MCR-5 suggests that it is distinct from the MCR-1/2 sub-lineage, but might share a common ancestor of MCR-3/4. Domain-swapping analysis of MCR-5 elucidates that its two structural motifs (transmembrane domain and catalytic domain) are incompatible with its counterparts in MCR-1/2. Like the rest of the MCR family, MCR-5 exhibits a series of conservative features, including zinc-dependent active sites, phosphatidylethanolamine-binding cavity, and the mechanism of enzymatic action. In vitro and in vivo evidence that MCR-5 catalyzes the addition of phosphoethanolamine to the suggestive 4'-phosphate of lipid A moieties is integrated, and results in the consequent polymyxin resistance. In addition, MCR-5 alleviates the colistin-induced formation of reactive oxygen species in E. coli. Taken together, the finding suggests that a growing body of MCR family resistance enzymes are functionally unified.

Microbiological surveillance of plasmid mediated colistin resistance in human Enterobacteriaceae isolates in Romagna (Northern Italy): August 2016-July 2017.

OBJECTIVES: To start a surveillance program to investigate the possible diffusion of mobilized colistin resistance genes in Enterobacteriaceae strains isolated in the Unit of Microbiology of the Great Romagna Hub Laboratory. METHODS: All the colistin resistant Enterobacteriaceae, isolated from August 1st 2016 to July 31st 2017, were prospectively evaluated for mcr-1 and mcr-2. Backdated survey of mcr-3, mcr-4 and mcr-5 was performed on the same group of isolates. Species identification was achieved by Vitek MS and the antibiotic susceptibility testing was performed both with Vitek-2 and Sensititre systems. Colistin resistant isolates were screened by PCR for the presence of the plasmid-mediated colistin resistance genes and amplicons were verified by sequencing. All mcr-1 positive isolates were subjected to MLST analysis. RESULTS: Over the total of 19053 isolates belonging to Enterobacteriaceae, 90 were colistin resistant. The presence of mcr-1 was detected in 26 Escherichia coli. The overall prevalence of mcr-1 was 0.14%. The mcr-1 positive E. coli strains were assigned to 13 distinct sequence types (STs) according to MLST. CONCLUSIONS: The prospective epidemiological survey carried out in our study gave a glimpse of the plasmid-mediated colistin resistance dissemination in Romagna. Since the prevalence rate of carbapenem resistant Enterobacteriaceae (CRE) in some hospital wards in our area is alarming, we underline the importance of a Surveillance Program to monitor the spread of the plasmid-mediated colistin resistance genes into MDR Gram-negative bacteria.