Molecular Epidemiology of Enterobacter cloacae Complex Isolates with Reduced Carbapenem Susceptibility Recovered by Blood Culture.

The Enterobacter cloacae complex (ECC) is one of the most common causes of bacteremia and leads to poor clinical outcomes. The aim of this study was to clarify the antimicrobial susceptibility profiles and genetic backgrounds of non-carbapenemase-producing reduced-carbapenem-susceptible (RCS) ECC blood isolates in Japan using agar dilution antimicrobial susceptibility testing, whole-genome sequencing, and quantitative polymerase chain reaction for ampC, ompC, and ompF transcripts. Forty-two ECC blood isolates were categorized into RCS and carbapenem-susceptible groups based on the minimum inhibitory concentration of imipenem. The RCS ECC blood isolates belonged to distinct species and sequence types and produced varying class C ß-lactamases. The E. roggenkampii, E. asburiae, and E. bugandensis isolates belonged only to the RCS group. Some E. hormaechei ssp. steigerwaltii isolates from the RCS group exhibited AmpC overexpression caused by amino acid substitutions in AmpD and AmpR along with ompF downregulation. These findings suggest that non-carbapenemase-producing RCS ECC blood isolates are genetically diverse.

Characterization of blaCTX-M-27/F1:A2:B20 Plasmids Harbored by Escherichia coli Sequence Type 131 Sublineage C1/H30R Isolates Spreading among Elderly Japanese in Nonacute-Care Settings.

We characterized 29 blaCTX-M-27-harboring plasmids of Escherichia coli sequence type 131 (ST131) sublineage C1/H30R isolates from healthy individuals and long-term-care facility (LTCF) residents. Most (27/29) plasmids were of the FIA, FIB, and FII multireplicon type with the same plasmid multilocus sequence typing (pMLST). Several plasmids (7/23) from LTCF residents harbored only blaCTX-M-27 as the resistance gene; however, their fundamental structures were very similar to those of previously isolated blaCTX-M-27/F1:A2:B20 plasmids, suggesting their prevalence as a newly arising public health concern.

Wastewater as a Probable Environmental Reservoir of Extended-Spectrum-ß-Lactamase Genes: Detection of Chimeric ß-Lactamases CTX-M-64 and CTX-M-123.

The presence of antimicrobial-resistant bacteria and resistance genes in aquatic environments is a serious public health concern. This study focused on Escherichia coli possessing blaCTX-M genes in wastewater inflows. Twelve crude inflow water samples from wastewater treatment plant (WWTP) A and two samples each from three other WWTPs were collected in 2017 and 2018. A total of 73 E. coli isolates with 31 different sequence types (STs) harboring distinctive blaCTX-M gene repertoires were detected. In WWTP A influents, blaCTX-M-14 (14 isolates) was dominant, followed by blaCTX-M-15 (12 isolates) and blaCTX-M-27 (10 isolates). The chimeric blaCTX-M-64 and blaCTX-M-123 genes were each identified in one of the E. coli isolates from the same WWTP A inflow port. The blaCTX-M-27 gene was associated with five of seven B2-ST131 isolates, including three isolates of the B2-O25b-ST131-H30R/non-Rx lineage. One of the remaining two isolates belonged to the B2-O25b-ST131-H30R/Rx lineage harboring the blaCTX-M-15 gene. As for the B2-O25b-ST131-H30R/non-Rx lineage, two isolates with blaCTX-M-27 were recovered from each of the WWTP B and D influents, and one isolate with blaCTX-M-174 was also recovered from WWTP B influent. Whole-genome sequencing of chimeric blaCTX-M-harboring E. coli isolates revealed that the blaCTX-M-64 gene was integrated into the chromosome of ST10 E. coli B22 via ISEcp1-mediated transposition of a 9,467-bp sequence. The blaCTX-M-123-carrying IncI1 plasmid pB64 was 109,169 bp in length with pST108. The overall findings suggest that wastewater may act as a probable reservoir of clinically significant clonal lineages mediating antimicrobial resistance genes and chimeric genes that have not yet been identified from human isolates of domestic origin in Japan.IMPORTANCE Global spread of CTX-M-type extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae is a critical concern in both clinical and community settings. This dominance of CTX-M-type ESBL producers may be largely due to the successful international spread of epidemic clones, as represented by the extraintestinal pathogenic Escherichia coli (ExPEC) ST131. Our findings highlight the worrisome presence of diverse E. coli clones associated with humans, including ExPEC lineages harboring the most common blaCTX-M variants in untreated wastewater samples. Moreover, the chimeric genes blaCTX-M-64 and blaCTX-M-123, which have not yet been identified from human isolates of domestic origin in Japan, were identified. Exposure to untreated wastewater through combined sewer overflow caused by heavy rains derived from abnormal weather change could pose a risk for human health due to ingesting those antimicrobial-resistant bacteria.

Acquisition of mcr-1 and Cocarriage of Virulence Genes in Avian Pathogenic Escherichia coli Isolates from Municipal Wastewater Influents in Japan.

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 ß-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.

Prevalence of CTX-M-Type Extended-Spectrum ß-Lactamase-Producing Escherichia coli B2-O25-ST131 H30R Among Residents in Nonacute Care Facilities in Japan.

We investigated the prevalence and characteristics of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli among 258 residents of long-term care facilities (LTCFs) in Japan. Out of 258 fecal samples collected from nine LTCFs between November 2015 and March 2017, we recovered 59 ESBL-producing E. coli isolates. All isolates carried blaCTX-M genes, mainly blaCTX-M-27 (42.4%), blaCTX-M-14 (23.7%), and blaCTX-M-15 (18.6%). The isolates showed 7 serotypes (STs), including ST131 (n = 49, 83.1%) and ST38 (n = 4, 6.8%), and 47 (79.7%) out of 49 isolates belonging to ST131 were identified as H30R. The 59 ESBL producers were divided into four groups, B2 (86.4%), D (8.5%), A (3.4%), and C (1.7%); 44 (74.6%) were epidemic clone B2-O25-ST131 H30R, of which 21, 11, and 6 harbored blaCTX-M-27, blaCTX-M-15, and blaCTX-M-14, respectively. Most plasmids were of IncF replicon types (n = 33), and 22 blaCTX-M-27-carrying plasmids showed multiple replicon types, including IncFII, FIA, and FIB. The ESBL producers were susceptible to imipenem, amikacin, and fosfomycin, but resistant to ceftazidime (49.2%), and ciprofloxacin (88.1%); in particular, the isolates harboring the blaCTX-M-15 gene showed significantly high resistance rate to ceftazidime (p < 0.01). Our findings indicate that a considerable proportion of the examined LTCF residents carried ESBL-producing E. coli isolates in feces and had high prevalence of epidemic clone B2-O25-ST131. Furthermore, continuous investigations would be very necessary to monitor actual carriage states of ESBL-producers among the LTCF residents from the viewpoint of both public health and healthcare viewpoints.

Spread of CTX-Type Extended-Spectrum ß-Lactamase-Producing Escherichia coli Isolates of Epidemic Clone B2-O25-ST131 Among Dogs and Cats in Japan.

This study was performed to investigate the carriage rates of CTX-M-type extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli among ill companion animals in Japan. Among the 178 nonrepetitive E. coli isolates, including 131 from dogs and 47 from cats, collected between September and November 2015, 42 (23.6%) isolates from 29 dogs and 13 cats were identified as ESBL producers. The antimicrobial susceptibility, O serotype, phylogenetic group, ß-lactamase genotype, plasmid replicon type, and sequence type (ST) of each isolate were analyzed. The major ESBL types were CTX-M-14 (26.8%), CTX-M-15 (24.4%), CTX-M-27 (19.5%), and CTX-M-55 (19.5%); predominant replicon types of blaCTX-M-carrying plasmid were IncF group and IncI1-Iγ. The most prevalent STs were ST131 (n = 15, 35.7%), followed by ST38, ST10, and ST410. The 15 isolates of ST131 belonged to B2-O25. E. coli B2-O25-ST131 isolates harboring blaCTX-M-15 or blaCTX-M-27 were resistant to ceftazidime and ciprofloxacin. In particular, CTX-M-15 producers showed multidrug resistance. Our results demonstrated that the CTX-M-producing pandemic E. coli clone B2-O25-ST131 has already spread in Japanese companion animals as well. Moreover, the similarity of genotypes, serotypes, phylogenetic groups, and STs of the isolates from companion animals to those from humans suggested probable transmission of resistant bacteria between pets and humans.