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April 21, 2020  |  

Occurrence and Characterization of mcr-1-Positive Escherichia coli Isolated From Food-Producing Animals in Poland, 2011-2016.

The emergence of plasmid-mediated colistin resistance (mcr genes) threatens the effectiveness of polymyxins, which are last-resort drugs to treat infections by multidrug- and carbapenem-resistant Gram-negative bacteria. Based on the occurrence of colistin resistance the aims of the study were to determine possible resistance mechanisms and then characterize the mcr-positive Escherichia coli. The research used material from the Polish national and EU harmonized antimicrobial resistance (AMR) monitoring programs. A total of 5,878 commensal E. coli from fecal samples of turkeys, chickens, pigs, and cattle collected in 2011-2016 were screened by minimum inhibitory concentration (MIC) determination for the presence of resistance to colistin (R) defined as R > 2 mg/L. Strains with MIC = 2 mg/L isolated in 2014-2016 were also included. A total of 128 isolates were obtained, and most (66.3%) had colistin MIC of 2 mg/L. PCR revealed mcr-1 in 80 (62.5%) isolates recovered from 61 turkeys, 11 broilers, 2 laying hens, 1 pig, and 1 bovine. No other mcr-type genes (including mcr-2 to -5) were detected. Whole-genome sequencing (WGS) of the mcr-1-positive isolates showed high diversity in the multi-locus sequence types (MLST) of E. coli, plasmid replicons, and AMR and virulence genes. Generally mcr-1.1 was detected on the same contig as the IncX4 (76.3%) and IncHI2 (6.3%) replicons. One isolate harbored mcr-1.1 on the chromosome. Various extended-spectrum beta-lactamase (blaSHV-12, blaCTX-M-1, blaCTX-M-15, blaTEM-30, blaTEM-52, and blaTEM-135) and quinolone resistance genes (qnrS1, qnrB19, and chromosomal gyrA, parC, and parE mutations) were present in the mcr-1.1-positive E. coli. A total of 49 sequence types (ST) were identified, ST354, ST359, ST48, and ST617 predominating. One isolate, identified as ST189, belonged to atypical enteropathogenic E. coli. Our findings show that mcr-1.1 has spread widely among production animals in Poland, particularly in turkeys and appears to be transferable mainly by IncX4 and IncHI2 plasmids spread across diverse E. coli lineages. Interestingly, most of these mcr-1-positive E. coli would remain undetected using phenotypic methods with the current epidemiological cut-off value (ECOFF). The appearance and spread of mcr-1 among various animals, but notably in turkeys, might be considered a food chain, and public health hazard.

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April 21, 2020  |  

A Phage-Like Plasmid Carrying blaKPC-2 Gene in Carbapenem-Resistant Pseudomonas aeruginosa.

Background: Lateral gene transfer plays a central role in the dissemination of carbapenem resistance in bacterial pathogens associated with nosocomial infections, mainly Enterobacteriaceae and Pseudomonas aeruginosa. Despite their clinical significance, there is little information regarding the mobile genetic elements and mechanism of acquisition and propagation of lateral genes in P. aeruginosa, and they remain largely unknown. Objectives: The present study characterized the genetic context of blaKPC-2 in carbapenem-resistant P. aeruginosa strain BH9. Methods:Pseudomonas aeruginosa BH9 sequencing was performed using the long-read PacBio SMRT platform and the Ion Proton System. De novo assembly was carried out using the SMRT pipeline and Canu, and gene prediction and annotation were performed using Prokka and RAST. Results:Pseudomonas aeruginosa BH9 exhibited a 7.1 Mb circular chromosome. However, the blaKPC-2 gene is located in an additional contig composed by a small plasmid pBH6 from P. aeruginosa strain BH6 and several phage-related genes. Further analysis revealed that the beginning and end of the contig contain identical sequences, supporting a circular plasmid structure. This structure spans 41,087 bp, exhibiting all the Mu-like phage landmarks. In addition, 5-bp direct repeats (GGATG) flanking the pBH6 ends were found, strongly indicating integration of the Mu-like phage into the pBH6 plasmid. Mu phages are commonly found in P. aeruginosa. However, for the first time showing a potential impact in shaping the vehicles of the dissemination of antimicrobial (e.g., plasmid pBH6) resistance genes in the Pseudomonas genus. Conclusion: pBH6 captured the Mu-like Phage BH9, creating a co-integrate pBH6::Phage BH9, and this phage-plasmid complex may represent novel case of a phage-like plasmid.

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April 21, 2020  |  

Assignment of virus and antimicrobial resistance genes to microbial hosts in a complex microbial community by combined long-read assembly and proximity ligation.

We describe a method that adds long-read sequencing to a mix of technologies used to assemble a highly complex cattle rumen microbial community, and provide a comparison to short read-based methods. Long-read alignments and Hi-C linkage between contigs support the identification of 188 novel virus-host associations and the determination of phage life cycle states in the rumen microbial community. The long-read assembly also identifies 94 antimicrobial resistance genes, compared to only seven alleles in the short-read assembly. We demonstrate novel techniques that work synergistically to improve characterization of biological features in a highly complex rumen microbial community.

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April 21, 2020  |  

Characterization of an NDM-5 carbapenemase-producing Escherichia coli ST156 isolate from a poultry farm in Zhejiang, China.

The emergence of carbapenem-resistant Enterobacteriaceae strains has posed a severe threat to public health in recent years. The mobile elements carrying the New Delhi metallo-ß-lactqtamase (NDM) gene have been regarded as the major mechanism leading to the rapid increase of carbapenem-resistant Enterobacteriaceae strains isolated from clinics and animals.We describe an NDM-5-producing Escherichia coli strain, ECCRA-119 (sequence type 156 [ST156]), isolated from a poultry farm in Zhejiang, China. ECCRA-119 is a multidrug-resistant (MDR) isolate that exhibited resistance to 27 antimicrobial compounds, including imipenem and meropenem, as detected by antimicrobial susceptibility testing (AST). The complete genome sequence of the ECCRA-119 isolate was also obtained using the PacBio RS II platform. Eleven acquired resistance genes were identified in the chromosome; four were detected in plasmid pTB201, while six were detected in plasmid pTB202. Importantly, the carbapenem-resistant gene blaNDM-5 was detected in the IncX3 plasmid pTB203. In addition, seven virulence genes and one metal-resistance gene were also detected. The results of conjugation experiments and the transfer regions identification indicated that the blaNDM-5-harboring plasmid pTB203 could be transferred between E. coli strains.The results reflected the severe bacterial resistance in a poultry farm in Zhejiang province and increased our understanding of the presence and transmission of the blaNDM-5 gene.

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April 21, 2020  |  

Genome plasticity favours double chromosomal Tn4401b-blaKPC-2 transposon insertion in the Pseudomonas aeruginosa ST235 clone.

Pseudomonas aeruginosa Sequence Type 235 is a clone that possesses an extraordinary ability to acquire mobile genetic elements and has been associated with the spread of resistance genes, including genes that encode for carbapenemases. Here, we aim to characterize the genetic platforms involved in resistance dissemination in blaKPC-2-positive P. aeruginosa ST235 in Colombia.In a prospective surveillance study of infections in adult patients attended in five ICUs in five distant cities in Colombia, 58 isolates of P. aeruginosa were recovered, of which, 27 (46.6%) were resistant to carbapenems. The molecular analysis showed that 6 (22.2%) and 4 (14.8%) isolates harboured the blaVIM and blaKPC-2 genes, respectively. The four blaKPC-2-positive isolates showed a similar PFGE pulsotype and belonged to ST235. Complete genome sequencing of a representative ST235 isolate shows a unique chromosomal contig of 7097.241?bp with eight different resistance genes identified and five transposons: a Tn6162-like with ant(2?)-Ia, two Tn402-like with ant(3?)-Ia and blaOXA-2 and two Tn4401b with blaKPC-2. All transposons were inserted into the genomic islands. Interestingly, the two Tn4401b copies harbouring blaKPC-2 were adjacently inserted into a new genomic island (PAGI-17) with traces of a replicative transposition process. This double insertion was probably driven by several structural changes within the chromosomal region containing PAGI-17 in the ST235 background.This is the first report of a double Tn4401b chromosomal insertion in P. aeruginosa, just within a new genomic island (PAGI-17). This finding indicates once again the great genomic plasticity of this microorganism.

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