Accurate and rapid detection of carbapenemases and identification of their types in Enterobacteriaceae are both still major challenges for clinical laboratories in attempting to prevent the intrusion and transmission of carbapenemase-producing Enterobacteriaceae. This study aimed to evaluate the performance of the MASTDISCS combi Carba plus disc system in identification of different carbapenemase types, including OXA-48-type carbapenemase, for which no specific enzyme inhibitors have so far been available. The simple disc system discriminates carbapenemases, including OXA-48-types exhibiting low carbapenem minimum inhibitory concentrations, by targeting Enterobacteriaceae isolates with a EUCAST meropenem screening cut-off of 0.25 mg/L.

ABSTRACT. To clarify the factors for occurrence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in broilers, two flocks (1 day of age) fed a diet with or without antibiotics were kept in a broiler house sanitized with disinfectants. ESBL-producing E. coli, however, was detected at a concentration of over 10 6 CFU/g of feces at 9 days of age to 49 days of age in both broiler flocks. Therefore, this indicated that the antibiotics other than cephalosporins used in this study had no effect due to co-selection on the numbers of ESBL-producing E. coli in broiler feces during this period. When a flock was kept with diet containing antibiotics for 49 days in a laboratory animal room, no ESBL-producing E. coli was detected in the flock. These results suggest that the occurrence of ESBL-producing E. coli may not be related to feeding with antibiotics and that the contamination of broiler houses with ESBL-producing E. coli might be an important factor. KEY WORDS: antibiotics, broiler, Escherichia coli, extended-spectrum β-lactamase-producing, occurrence. Extended-spectrum β-lactamase (ESBL)-producing bacteria have antibiotic resistance to third generation cephalosporins such as cefotaxime (CTX). Reports on Escherichia coli carrying broad-spectrum β-lactamases isolated from food-producing animals and humans have been published worldwide [8]. ESBL-producing Klebsiella pneumoniae, E. coli and Proteus mirabilis have been implicated in numerous outbreaks of nosocomial infections over the last 2 decades [3,10,13]. CTX-M-2-producing E. coli from cattle in Japan has been reported [11], and the Japanese Veterinary Antimicrobial Resistance Monitoring Program (JVARM) reported isolation of ESBL-producing E. coli from poultry in Japan [6]. In addition, a potential increase in the ESBLproducing E. coli isolated from broilers was also reported [4]. Our previous study [5] clarified the high prevalence of ESBL-producing commensal enteric bacteria in broilers in Japan. However, use of third-generation cephalosporins on broilers is not permitted in Japan. The factors of occurrence of ESBL-producing E. coli in broiler are still unclear. The aim of this study was to clarify these factors.Two flocks with 16 broilers each (Chunky, 1 day of age) were newly introduced to a hygienic broiler house sanitized with disinfectants. Although this windowless broiler house had been maintained in all-in-all-out system and sanitized with disinfectants immediately after all the broilers were marketed, ESBL-producing E. coli strains had been isolated from broiler feces collected in this broiler house in the last two examinations. A flock fed a diet with antibiotics (salinomycin 50 ppm and enramycin 7 ppm from 1 to 21 days of age; salinomycin sodium salt 50 ppm, gentamycin 5 ppm and colistin sulfate 5 ppm from 22 to 42 days of age; no antibiotics from 43 to 49 days of age), and another flock without antibiotics was raised until 49 days of age with hygienic water. The broiler house operating procedures were conventional. Harada et al. [2] repor...

Since a method of rapidly detecting extended-spectrum beta-lactamase (ESBL) production in gram-negative isolates from patients with severe infection is urgently required, the present study of a novel commercial kit was conducted. The Cica-Beta Test I (Kanto Chemical, Tokyo, Japan) is designed for the rapid detection of ESBL in gram-negative bacteria directly from isolated colonies in a 15-min protocol. In this study, a total of 304 strains of Klebsiella spp., Escherichia coli and Proteus mirabilis were tested using the novel kit and the phenotypic confirmatory disk test using cefotaxime and ceftazidime with and without clavulanate. The kit showed 95.5 and 98.1% sensitivity and specificity, respectively, as compared to the disk test, and thus proved to be an appropriate tool for the rapid detection of ESBL.

This study focused on the detection of the plasmid-mediated mcr colistin resistance gene in Escherichia coli isolates from wastewater treatment plants (WWTPs). Seven influent samples were collected from three WWTPs in Nagano Prefecture, Japan, during August and December 2018. Colistin-resistant E. coli isolates were selected on colistin-supplemented CHROMagar ECC plates. mcr-1-positive isolates were subjected to whole-genome sequencing (WGS) analysis. From six influent samples, seven mcr-1-positive but extended-spectrum β-lactamase (ESBL)-negative isolates belonging to different genetic lineages, namely, B2-O25:H4-ST131-fimH22, B2-O2:H1-ST135-fimH2, B1-O8:H9-ST764-fimH32, B1-O23:H16-ST453-fimH31, A-O81:H27-ST10-fimH54, A-O16:H5-ST871-fimH25, and F-O11:H6-ST457-fimH145, were detected. The MICs of colistin for these isolates ranged from 4 to 16 mg/liter. The mcr-1 genes were located on plasmids belonging to IncX4 and IncI2 in five and two isolates, respectively. Four IncX4 plasmids with the same size (33,309 bp) showed high sequence similarity (4 single-nucleotide variations). The remaining one IncX4 plasmid, with a size of 33,858 bp, carried the mcr-1 gene with the single synonymous nucleic substitution T27C. Two IncI2 plasmids with sizes of 60,710 bp and 60,733 bp had high sequence similarity (99.9% identity; 100% query coverage). Two of five isolates carrying IncX4 plasmids and both of the isolates carrying IncI2 plasmids harbored ColV plasmids carrying virulence-associated genes of avian pathogenic E. coli (APEC). In addition, another isolate of the B2-O25:H4-ST131-fimH22 lineage had those APEC-associated virulence genes on its chromosome. In conclusion, mcr-1-positive E. coli environmental isolates were mostly characterized as positive for APEC-associated virulence genes. The copresence of those genes may suggest the existence of a common source in animals and/or their associated environments. IMPORTANCE Colistin is considered a last-line therapeutic option in severe infections due to multidrug-resistant Gram-negative bacteria, in particular carbapenemase-producing Enterobacteriaceae and multidrug-resistant Acinetobacter baumannii. An increasing prevalence of mcr genes in diverse Enterobacteriaceae species, mainly Escherichia coli and Klebsiella pneumoniae from humans and food animals, has become a significant concern to public health all over the world. In Japan, mcr genes have so far been detected in food animals, raw meat, wastewater, and human clinical samples. This study reports the copresence of mcr-1 and avian pathogenic E. coli (APEC)-associated virulence genes in five of seven E. coli isolates recovered from aquatic environments in Japan. Our study highlights the importance and urgency of action to reduce environmental contamination by mcr genes that may likely occur due to exposure to untreated wastewater through combined sewer overflow by recent unusual weather.

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