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Objectives To characterize bla NDM-carrying Salmonella recovered from a pig slaughterhouse. Methods In this study, 46 environment samples were collected from a slaughterhouse in China, and screened for carbapenem-resistant Enterobacterales. WGS, antimicrobial susceptibility testing and conjugation experiments were carried out to identify the isolates' resistance phenotypes and genetic characteristics. The phylogenetic relatedness of the Salmonella isolates obtained in this study and Salmonella (ST34 and ST29) in GenBank was determined. Results Two ST34 Salmonella Typhimurium and one ST29 Salmonella Stanley, recovered from three environmental samples (6.52%), were positive for bla NDM-1 and bla NDM-5, respectively. The two ST34 S. Typhimurium strains exhibited a close relationship (10–36 SNPs) with two human-derived bla NDM-1-bearing isolates from China (Hong Kong and Guangxi Province) and two bla NDM-negative ST34 Salmonella strains from the UK. The bla NDM-1 genes were located on IncHI2/ST3 plasmids. The capture of bla NDM-1 by the IncHI2/ST3 plasmid seems to be due to homologous recombination mediated by circular structures, as the genetic arrangements of the bla NDM-1 gene contain two IS 26 elements of the same orientation. The bla NDM-5 gene was also carried by the IncHI2/ST3 plasmid, which shares highly similar structures with other bla NDM-5-bearing IncHI2/ST3 plasmids from other sources (fish, chicken, duck, human). Conclusions This is the first report of a bla NDM-5-carrying IncHI2/ST3 plasmid in Salmonella. The clonal spread of NDM-1-producing ST34 S. Typhimurium across human and animal-associated environments, and the widespread dissemination of epidemic bla NDM-5-carrying IncHI2/ST3 plasmids among Enterobacteriaceae in China indicate the potential of further dissemination of bla NDM among Salmonella , which poses a threat to public health. [ABSTRACT FROM AUTHOR]

A study published in the Journal of Antimicrobial Chemotherapy has identified the emergence of the mcr-8.1 gene in Citrobacter werkmanii, a type of bacteria commonly found in soil, water, retail meat, and the intestines of animals and humans. The mcr-8.1 gene, which is resistant to multiple antibiotics including colistin, was detected in drinking water and sewage samples from broiler farms in China. The study emphasizes the importance of proper sewage treatment and environmental sanitation in animal farms to prevent the spread and contamination of antibiotic-resistant bacteria. The research also suggests that C. werkmanii may act as a reservoir for the dissemination of antimicrobial resistance. [Extracted from the article]

Background: Fosfomycin is an important broad-spectrum bactericidal antibiotic to treat multidrug-resistant bacteria infections. It is generally accepted that heteroresistant bacteria are an intermediate stage in the formation of drug resistance, but there are few studies on the formation mechanism underlying fosfomycin heteroresistance (FHR).Objectives: To reveal the characteristics and formation mechanisms of FHR in Escherichia coli isolates obtained from chickens.Methods: We identified the FHR according to the population analysis profile (PAP) test and in vitro time-kill assay. Growth curves for FHR E. coli and their subpopulations were measured. Also, the subpopulations were repeatedly cultured in fosfomycin-free medium for 5-20 overnight incubation periods. The formation mechanisms of FHR in E. coli isolates were identified through accumulation assay, carbohydrate utilization testing, real-time relative quantitative PCR analysis, DNA sequencing, transcriptomic analysis, intracellular ATP and cAMP-level assessment.Results: Four of six E. coli strains were confirmed to show FHR, with a total of six subpopulations. The subpopulations restored phenotypic susceptibilities to fosfomycin within 5-20 overnight incubation sessions, but four of six subpopulations still maintained FHR characteristics. Differing from their parental isolates, the uptake of fosfomycin in the subpopulations through GlpT was reduced remarkably. Further studies identified that the low expression of glpT was due to the decrease of intracellular cAMP levels in the subpopulations, which was caused by the decreased ATP levels in cells.Conclusions: Our findings revealed the formation mechanism of E. coli isolates showing FHR obtained from chicken in China and characterized the dynamic change traits in vitro of the subpopulations. [ABSTRACT FROM AUTHOR]

Background: The increasing use of colistin causes a serious breach in our last line of defence against MDR Gram-negative pathogens. Our previous study showed that CpxR overexpression increases the susceptibility of acrB and cpxR double-deleted Salmonella enterica serovar Typhimurium to colistin.Objectives: To identify the mechanism of CpxAR and efflux pumps that synergistically enhance the susceptibility of S. Typhimurium to colistin.Methods: A series of cpxR- and tolC-deleted mutants and a cpxR-complemented strain from a multidrug-susceptible standard strain of S. Typhimurium (JS) were generated in our previous study. Herein, we investigated the susceptibility of these strains to colistin through the broth microdilution method, time-kill curves and survival assays. Growth curves were measured by OD600 in LB broth, tryptone-soy broth (TSB) and M9-glucose (0.2%) minimal media. Finally, molecular mechanisms underlying the mode of action were elucidated by transcriptomic analysis.Results: We found that in contrast to JS (0.8 mg/L), the MIC of colistin for JSΔtolC::kan showed a 16-fold decrease (0.05 mg/L). Notably, JSΔcpxRΔtolC and JSΔcpxRΔtolC/pcpxR were associated with a 256-fold decrease (0.0031 mg/L) compared with JS. Growth curves identified that JSΔcpxRΔtolC and JSΔcpxRΔtolC/pcpxR displayed a markedly lower growth rate and poorer adaptability. In addition, time-kill curves and survival assays showed that JSΔcpxRΔtolC and JSΔcpxRΔtolC/pcpxR were more susceptible to colistin. Lastly, double deletion of cpxR and tolC enhanced oxidative damage through promoting oxidative phosphorylation, the tricarboxylic acid (TCA) cycle and trimethylamine N-oxide (TMAO) respiration.Conclusions: Our findings revealed that double deletion of cpxR and tolC significantly increases the susceptibility of S. Typhimurium to colistin. [ABSTRACT FROM AUTHOR]

Objectives: To characterize a novel MDR efflux pump gene cluster tnfxB3-tmexCD3-toprJ1b carried by Proteus spp. and Pseudomonas aeruginosa strains from chickens.Methods: Antimicrobial susceptibility testing, conjugation and WGS were performed to characterize tnfxB3-tmexCD3-toprJ1b-positive isolates. Cloning and reverse transcription-quantitative PCR were performed to investigate the function of tnfxB3-tmexCD3-toprJ1b.Results: The WGS data revealed that a novel efflux pump gene cluster, tnfxB3-tmexCD3-toprJ1b, was identified on the chromosome of the Proteus cibarius strain SDQ8C180-2T, where an SXT/R391-family integrative and conjugative element (ICE) was found to co-carry tet(X6) and tnfxB3-tmexCD3-toprJ1b. Further retrospective analysis found two other tnfxB3-tmexCD3-toprJ1b variants in a Proteus mirabilis isolate and a P. aeruginosa isolate, respectively. tmexCD3-toprJ1b and its variants increased the MICs of tigecycline (8-fold) and other antibiotics (2-8-fold) in Escherichia coli host strains. The TNfxB3 protein down-regulated the expression of the tmexCD3-toprJ1b operon. Moreover, genetic-context analyses showed that tnfxB3-tmexCD3-toprJ1b together with adjacent integrase genes appeared to compose a transferable module 'int1-like+int2-like+hp1+hp2+ISCfr1+tnfxB3-tmexCD3-toprJ1b', which was inserted into the umuC-like gene of this ICE. Further analysis of the tnfxB3-tmexCD3-toprJ1b-harbouring sequences deposited in GenBank revealed similar transferable modules inserted into umuC-like genes in plasmids or chromosomes of Klebsiella pneumoniae, Pseudomonas spp. and Aeromonas spp., implying that these modules could be transferred across different bacterial species.Conclusions: To the best of our knowledge, this is the first identification of a novel tigecycline gene cluster, tmexCD3-toprJ1b, which co-exists with tet(X6) within an ICE. More attention should be paid to the co-transfer of these two tigecycline resistance determinants via an ICE to other Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Background: The two-component signalling systems PmrAB and PhoPQ of Salmonella have been extensively studied with regard to colistin resistance. We previously showed that overexpressed CpxR could significantly increase the colistin susceptibility (16-fold compared with the WT strain) of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) through PmrAB and PhoPQ.Objectives: To identify the potential target genes of CpxR in PmrAB- and PhoPQ-related signalling pathways.Methods: His6-CpxR was prokaryotically expressed and purified by Ni-NTA resin affinity chromatography. β-Galactosidase activity assays were conducted to investigate whether CpxR could regulate the promoters of colistin resistance-related genes (CRRGs). Electrophoretic mobility shift assays (EMSAs) were used to further detect His6-CpxR complexes with promoters of CRRGs.Results: We demonstrated for the first time (to the best of our knowledge) that CpxR and the AcrAB-TolC efflux pump have reciprocal effects on CRRG transcription. Additionally, CpxR could regulate the colistin susceptibility of Salmonella Typhimurium by binding directly to the promoters of phoPQ, pmrC, pmrH and pmrD at the CpxR box-like sequences or indirectly through other regulators including pmrAB and mgrB.Conclusions: CpxR could regulate the colistin susceptibility of Salmonella Typhimurium by a multitarget mechanism. [ABSTRACT FROM AUTHOR]

The initial report of the mcr-1 (mobile colistin resistance) gene has led to many reports of mcr-1 variants and other mcr genes from different bacterial species originating from human, animal and environmental samples in different geographical locations. Resistance gene nomenclature is complex and unfortunately problems such as different names being used for the same gene/protein or the same name being used for different genes/proteins are not uncommon. Registries exist for some families, such as bla (β-lactamase) genes, but there is as yet no agreed nomenclature scheme for mcr genes. The National Center for Biotechnology Information (NCBI) recently took over assigning bla allele numbers from the longstanding Lahey β-lactamase website and has agreed to do the same for mcr genes. Here, we propose a nomenclature scheme that we hope will be acceptable to researchers in this area and that will reduce future confusion. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the impact of plasmid-borne oqxAB genes on the development of fluoroquinolone resistance, mutations and bacterial fitness in Escherichia coli .Methods: MICs and mutation prevention concentrations were compared among E. coli strain TOP10 and two corresponding transformants harbouring the OqxAB-encoding plasmids. Mutants were selected by serial passages with the 0.5-fold MIC of ciprofloxacin, and were randomly selected to determine mutations. Bacterial fitness was evaluated by competition assays in vitro and in vivo .Results: The oqxAB -carrying plasmids contributed to a 4-8-fold increase in the ciprofloxacin MIC and increased the ciprofloxacin mutation prevention concentration by 8-16-fold. The MIC of ciprofloxacin for the two transformants increased faster than that of E. coli TOP10 by serial passaging. Novel mutations in gyrB (A468P or F458V) were first observed. Mutations in gyrA were distributed at codons 87 and 83 in the two transformants, whereas mutation A119E in gyrA dominated in the TOP10 mutants. Although the two oqxAB -bearing plasmids caused a decrease in fitness in vitro , their fitness increased when combined with more than one chromosomal mutation, and clear biological benefits were observed in vivo . The mutations in gyrB were associated with a fitness cost, which could be compensated for by additional mutations. The novel mutation gyrA ΔS83 significantly reduced biological fitness both in vitro and in vivo , and was thus quickly replaced by more beneficial mutations in the population.Conclusions: The possession of plasmid-borne oqxAB may facilitate the evolution of fluoroquinolone resistance, and the fitness cost of OqxAB-encoding plasmids could be compensated by additional chromosomal mutations. [ABSTRACT FROM AUTHOR]

The article presents a report on emergence of Escherichia coli co-producing New Delhi metallo-b-lactamase-1(NDM-1) and Klebsiella pneumoniae carbapenemase 2 (KPC-2) from a retail vegetable, China. Topics discussed include use of agar dilution method for determining of minimum inhibitory concentration of antimicrobial agents; its resistance to imipenem, ceftazidime, cefotaxime; and a table depicting characteristics of E. coli TS79e and its transconjugants.

Objectives To investigate the occurrence of plasmid-mediated fosfomycin resistance genes among Escherichia coli from food animals in China. Methods A total of 892 E. coli isolates collected from individual pigs (n = 368), chickens (n = 196), ducks (n = 261), geese (n = 35), pigeons (n = 20) and partridges (n = 12) in Guangdong Province during 2002–08 were screened for the presence of fosA3, fosA and fosC2 by PCR amplification and sequencing. The clonal relationship of fosA3-positive isolates, plasmid content and other associated resistance genes were also characterized. Results Twelve (1.3%) E. coli isolates showed resistance to fosfomycin and 10 (1.1%) isolates (4 from pigs, 2 from chickens, 2 from ducks, 1 from a goose and 1 from a pigeon) were positive for fosA3. None of the E. coli isolates was positive for fosA or fosC2. All of the isolates carrying fosA3 were CTX-M producers, and three of them carried rmtB. Most of the fosA3-harbouring isolates were found to be clonally unrelated. The fosA3 genes were flanked by IS26. Two fosA3 genes co-localized with rmtB and blaCTX-M-65 on indistinguishable F33:A-:B- plasmids that carried three addiction systems (pemI/pemK, hok/mok/sok and srnB). Four, one and one fosA3 genes were found to be associated with IncN (ST8 type), IncI1 and F2:A-:B- plasmids, respectively. Conclusions We discovered that fosA3 is always associated with blaCTX-M, which facilitates its quick dispersal. The emergence of fosA3 in food animals could impact on human medicine by the potential transfer of resistance through the food chain. [ABSTRACT FROM AUTHOR]

Objectives To characterize a representative self-transmissible multidrug resistance plasmid pHN7A8 isolated from an Escherichia coli from a dog in China, classified as F33:A-:B- by replicon sequence typing and carrying the blaTEM-1b, blaCTX-M-65, fosA3 and rmtB genes conferring resistance to penicillins, cephalosporins, fosfomycin and aminoglycosides, respectively. Methods pHN7A8 was sequenced using a whole-genome shotgun approach and the sequence analysed by comparison with reference plasmids. Results pHN7A8 is a circular molecule of 76 878 bp. blaCTX-M-65, fosA3 and rmtB are found in known contexts, interspersed with different mobile elements including ISEcp1, IS1, Tn2, IS1294, IS903 and four copies of IS26. This multiresistance region has only a single nucleotide difference from that of pXZ, an F2:A-:B- plasmid isolated from poultry in China. The pHN7A8 backbone carries genes encoding addiction and partitioning systems that promote plasmid maintenance and has a similar organization to pXZ, as well as IncFII plasmids such as R100, pC15-1a/pEK516 and pHK23, isolated in Japan, Canada/the UK and China, respectively, but with varying levels of identity, suggesting recombination. Conclusions pHN7A8 is a chimera that may have resulted from the acquisition, by recombination in the plasmid backbone, of the multiresistance region found in pXZ. This region appears to have evolved from the resistance determinant R100 through the stepwise integration of multiple antimicrobial resistance determinants from different sources by the actions of mobile elements and recombination. The successful dissemination of this multidrug resistance plasmid presents further challenges for the prevention and treatment of Enterobacteriaceae infections. [ABSTRACT FROM PUBLISHER]

Objectives To investigate the prevalence and characterization of 16S rRNA methylase-producing bacteria in a pig farm and its environment in East China. Methods Enterobacteriaceae isolates and metagenomic DNA from 102 pig faecal samples from a pig farm and 97 soil samples taken in or around the farm were screened for the presence of 16S rRNA methylase genes. The clonal relationships of 16S rRNA methylase-positive isolates, plasmid content and other associated resistance genes were also characterized. Results Fifty-six rmtB-positive Enterobacteriaceae isolates, including 54 Escherichia coli, 1 Morganella morganii and 1 Proteus mirabilis, were recovered from 55 pig faecal samples. Nineteen rmtB-positive bacteria, including 13 E. coli, 2 M. morganii, 2 Leclercia adecarboxylata, 1 Enterobacter aerogenes and 1 Enterobacter cloacae, were recovered from 16 soil samples. Among the 75 rmtB-positive isolates, 31 and 25 also carried the qepA and blaCTX-M genes, respectively. The qepA gene co-localized with rmtB on the F2:A-:B1 plasmids and the blaCTX-M-65 gene co-localized with rmtB on the F33:A-:B- plasmids. The rmtB gene was also found to be associated with the IncN plasmids. Clonal transmission of rmtB-positive E. coli isolates was observed between different pig groups and soil samples. Conclusions Both horizontal gene transfer and clonal spread could be responsible for the dissemination of the rmtB gene in the pig farm and its environment. To our knowledge, this study is the first report of rmtB-positive bacteria from farmland soils and indicates that these antibiotic-resistant bacteria and/or resistance genes could be acquired by humans through the food chain. [ABSTRACT FROM PUBLISHER]