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The aim of this study was to investigate the presence of colistin- and/or tigecycline-resistant Klebsiella spp. in influents from four wastewater treatment plants (WWTPs), which partly reflect the gut microbiome of human populations. Colistin- and tigecycline-resistant Klebsiella pneumoniae isolates (K30/ST29) were detected four times from the WWTP A during a period of 3 months. Disruptions of the mgrB and ramR genes by ISEc68 and ISKpn21, respectively, were identified in those four isolates. They also shared the IncL/M 86,197-bp plasmids carrying a bla(CTX-M-3) and Tn1548-associated armA [IS26-IntI1-dfrA12-gucF-aadA2-qacE Delta 1-sul1-ISCR1-ISEc28-armA-ISEc29-msr(E)-mph(E)-IS26]. Those isolates formed a distinct cluster within wgMLST clusters of ST29 K30 public reference strains of human origin and were unique due to harboring of Tn21-like mercury resistance operon transposons in addition to silver, copper, and arsenic resistance determinants. Five K. pneumoniae strains with different STs and 1 Klebsiella quasipneumoniae strain, exhibiting colistin resistance, were detected in WWTPs B, C, and D. For these isolates, disruptions of mgrB by ISEc68 (three isolates) or ISEcl1 (one isolate), insertion of IS2 in the mgrB promoter region (one isolate), and inactivation of MgrB by a nonsense mutation (one isolate) were identified. Close monitoring of these mcr-negative colistin- and/or tigecycline-resistant bacteria in wastewater influents is imperative to avoid further limiting of treatment options., Article, Microbial Drug Resistance 27(10) : 1433-1442(2021)

Objectives: We determined the whole DNA sequences of plasmids carrying a rare extended-spectrum beta-lactamase gene (bla(TEM-61)) to precisely understand the spread of resistance among nosocomial Serratia marcescens populations. Methods: Twenty non-duplicate ceftazidime-resistant S. marcescens nosocomial isolates (ceftazidime MICs, 32 to >128 mg/L) collected over 1 year were pulsotyped and nucleotide sequences of the bla(TEM-61) gene and its promoter region were determined. Twelve representative isolates were analysed by whole-genome sequencing. Results: The 20 isolates comprised two distinct pulsotypes: I (14 isolates) and II (6 isolates). They all contained the bla(TEM-61) gene. A polymorphism in the repeat number of a 15-nucleotide sequence (5'-ATGTCATGATAATAA-3') was found in the promoter region of bla(TEM-61); two, three and four repeat units were found in 6,12 and 2 isolates, respectively. Single nucleotide polymorphism (SNP)-based phylogenetic analysis of 12 isolates revealed that 7 isolates of pulsotype I (12-44 SNP differences) and 5 isolates of pulsotype II (15-55 SNP differences) formed two distinct clusters of genotypes 1 and 2, respectively. All 12 isolates harboured a plasmid carrying the Tn1-bla(TEM-61) element, although they were slightly different in size (78 883 bp, 78 898 bp and 78 913 bp) owing to differences in the number of 15-bp repetitive sequences. A 42 542-bp broad-host-range plasmid carrying the Tn1-bla(TEM-61) element was also found in one of the isolates. Conclusions: We characterised a plasmid-encoded novel Tn1-bla(TEM-61) element and transposon-dependent mechanisms underlying the propagation of antibiotic resistance, together with repeated new polymorphic 15-bp units in the promoter of bla(TEM-61). (C) 2021 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy., Article, Journal of Global Antimicrobial Resistance 25 : 124-131(2021)

The emergence of daptomycin (DAP) resistant Enterococcus species has increased worldwide, but the mechanisms for DAP resistance are not fully understood. We report a case of DAP resistant Enterococcus faecalis, from a clinical sample of a patient with diabetic ulcers, after DAP therapy. Whole-genome sequencing analysis revealed that the isolate had a loss-of-function point mutation within liaX encoding DAP-sensing surface protein, which inhibits the LiaFSR systems and cell membrane remodeling. This is the first case report of a clinical DAP resistant E. faecalis with a mutation in liaX.

Objectives: NDM-1 is by far one of the most commonly prevalent carbapenemases in Enterobacteriaceae and Acinetobacter baumannii. This study presented an Acinetobacter pittii (A. pittii) isolate co-harboring blaNDM-1 and blaOXA-820 from a university hospital sink, where New Delhi metallo-β-lactamase (NDM) producers have not been found in either patients or their environments. Methods: Whole-genome sequencing was performed on the HiSeq 4000 platform, and the reads were de novo assembled using the A5-miSeq Assembly pipeline. Annotation of the resulting scaffolds were performed by using the DDBJ Fast Annotation and Submission Tool (DFAST). The blaNDM-1-carrying plasmid was determined. Results: The A. pittii ST220 strain SU1805 detected from a sink strainer in the treatment room was resistant to imipenem and meropenem. Antimicrobial resistance genes blaNDM-1, blaOXA-820, blaADC-43, and aphA6 were found in this strain. The blaNDM-1 was found to be located downstream of an ISAba125 element on a plasmid pSU1805NDM with a size of 41,022 bp, and GC content of 38.3% harbouring 48 protein-coding genes. The aphA6 gene was also located upstream of the ISAba125 on the same plasmid. The A. pittii intrinsic blaOXA-213-like gene blaOXA-820 was located between fxsA and yncA genes in the chromosome. The strain also harboured biofilm-associated genes such as ompA, the csu operon and their regulating genes bfmRS. Conclusion: This study described the first isolation of NDM-1-producing A. pittii in Japan, and highlighted the importance of proper implementation of measures against AMR for sink drainage systems, since NDM producers may have already been hidden in such environments in a non-endemic country of NDM., Article, Journal of Global Antimicrobial Resistance 21 : 353-356(2020)

Phylogenetic relationship of 97 I1 plasmids harboring blaCTX-M genes encoding extended-spectrum beta-lactamase (ESBL) was analyzed using the ORF-based binarized structure network analysis of plasmids (OSNAp). The majority of plasmids carrying blaCTX-M-1 or blaCTX-M-8, ESBL genes primarily associated with domestic animals, were clonal. On the other hand, plasmids carrying blaCTX-M-14 or blaCTX-M-15, identified from both humans and domestic animals, were diverse in their contents. The findings suggest that circulation of I1 plasmids among humans and animals may contribute to their diversity.

The worldwide distribution of carbapenemase-producing Enterobacterales (CPE) is a serious public health concern as they exhibit carbapenem resistance, thus limiting the choice of antimicrobials for treating CPE infections. Combination treatment with a β-lactam and one of the newly approved β-lactamase inhibitors, such as avibactam, relebactam, or vaborbactam, provides a valuable tool to cope with CPE; however, these inhibitors are active only against serine-type carbapenemases and not against metallo-β-lactamases (MβLs). Therefore, it is important to readily differentiate carbapenemases produced by CPE by using simple and reliable methods in order to choose an appropriate treatment. Here, we developed three practical agar-based disk diffusion tests (double-disk synergy test [DDST], disk potentiation test, and modified carbapenem inactivation method [mCIM]) to discriminate the production of subclass B1 MβLs, such as IMP-, NDM-, and VIM-type MβLs, from the other carbapenemases, especially serine-type carbapenemases. This was accomplished using B1 MβL-specific sulfamoyl heteroarylcarboxylic acid inhibitors, 2,5-dimethyl-4-sulfamoylfuran-3-carboxylic acid (SFC) and 2,5-diethyl-1-methyl-4-sulfamoylpyrrole-3-carboxylic acid (SPC), originally developed by us. The DDST and mCIM using SFC and SPC revealed high sensitivity (95.3%) and specificity (100%) in detecting B1 MβL-producing Enterobacterales. In the disk potentiation test, the sensitivities using SFC and SPC were 89.1% and 93.8%, respectively, whereas the specificities for both were 100%. These methods are simple and inexpensive and have a high accuracy rate. These methods would therefore be of immense assistance in the specific detection and discrimination of B1 MβL-producing Enterobacterales in clinical microbiology laboratories and would lead to better prevention against infection with such multidrug-resistant bacteria in clinical settings.

Article, Journal of Global Antimicrobial Resistance 22 : 697-699(2020)

No study has described Streptococcus dysgalactiae subsp. equisimilis (SDSE) isolates that cause repetitive infections (recurrence and reinfection). We compared the microbiological characteristics of SDSE causing repetitive infections with those causing single infections. Three patients with invasive infections were identified based on their medical records, and multiple SDSE isolates were collected at intervals over three weeks, using a laboratory repository. Isolates from 12 patients with single-episode infections served as controls. Six isolates were collected from three patients with first and second episodes of infection. All isolates causing either repetitive or single-episode infection were subjected to emm typing, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and random amplified polymorphic DNA (RAPD) analyses. Amplification of five virulence genes (sicG, prtF1, prtF2, lmb, and cbp), biofilm formation (BF), and cell invasion abilities (CIAs) were measured as virulent phenotypes. We observed close genetic similarities in the data obtained by emm typing, MLST, PFGE, and RAPD in four isolates from two patients, suggesting recurrence, whereas two isolates from one patient indicated genetic differences in these data, suggesting re-infection. The presence of the five virulence genes and the BF and CIA measurements appeared not to contribute to repetitive infections, compared with isolates causing single-episode infection. In conclusion, clinicians encountering patients with repetitive infections should be aware of both possibilities: recurrence with closely related strains and reinfection with different strains.

Over a 35-month period, group B Streptococcus isolates with reduced penicillin susceptibility (PRGBS) were detected from elderly patients at a regional hospital in Japan, accompanying population-level transition of PRGBS serotypes. The genetic relatedness of 77 non-duplicate PRGBS from 73 patients was analysed. Serotype III PRGBS predominated (16 serotype III/1 serotype Ib) in the first 9 months (period I), then 3 serotype Ib isolates appeared transiently for the next 3 months (period II), which was replaced predominantly by serotype Ia (20 serotype Ia/1 serotype III/1 non-typeable) for 9 months (period III). In the last 14 months (period IV), besides 25 serotype Ia isolates, 10 serotype III were also identified. Serotypes III and Ia isolates, belonging to ST1, shared G329V, G398A, V405A and G429D substitutions in penicillin-binding protein 2X. Of three strains subjected to whole-genome sequencing, serotype III strain SU12 (period I) had a higher degree of genomic similarity with serotype Ia strain SU97 (period III) than serotype Ib strain SU67 (period II) based on average nucleotide identity and single nucleotide polymorphisms. Analysis of the cps gene clusters and the upstream and downstream flanking sequences revealed that disruption of the hyaluronidase gene located upstream of cpsY by insertion of IS 1548 was found in strain SU12, whereas Delta ISSag8 was inserted between tRNA-Arg and rpsA genes located downstream of cpsL in strain SU97. Interestingly, most serotype III PRGBS re-emerging in period IV had this tRNA-Arg-Delta ISSag8-rpsA region. Capsular switching and nosocomial transmission may possibly contribute to population-level serotype replacement among ST1 PRGBS isolates. (c) 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved., Article, INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS.53(3):203-210(2019)

The worldwide distribution of carbapenemase-producing Enterobacterales (CPE) is a serious public health concern as they exhibit carbapenem resistance, thus limiting the choice of antimicrobials for treating CPE infections. Combination treatment with a β-lactam and one of the newly approved β-lactamase inhibitors, such as avibactam, relebactam, or vaborbactam, provides a valuable tool to cope with CPE; however, these inhibitors are active only against serine-type carbapenemases and not against metallo-β-lactamases (MβLs). Therefore, it is important to readily differentiate carbapenemases produced by CPE by using simple and reliable methods in order to choose an appropriate treatment. Here, we developed three practical agar-based disk diffusion tests (double-disk synergy test [DDST], disk potentiation test, and modified carbapenem inactivation method [mCIM]) to discriminate the production of subclass B1 MβLs, such as IMP-, NDM-, and VIM-type MβLs, from the other carbapenemases, especially serine-type carbapenemases. This was accomplished using B1 MβL-specific sulfamoyl heteroarylcarboxylic acid inhibitors, 2,5-dimethyl-4-sulfamoylfuran-3-carboxylic acid (SFC) and 2,5-diethyl-1-methyl-4-sulfamoylpyrrole-3-carboxylic acid (SPC), originally developed by us. The DDST and mCIM using SFC and SPC revealed high sensitivity (95.3%) and specificity (100%) in detecting B1 MβL-producing Enterobacterales. In the disk potentiation test, the sensitivities using SFC and SPC were 89.1% and 93.8%, respectively, whereas the specificities for both were 100%. These methods are simple and inexpensive and have a high accuracy rate. These methods would therefore be of immense assistance in the specific detection and discrimination of B1 MβL-producing Enterobacterales in clinical microbiology laboratories and would lead to better prevention against infection with such multidrug-resistant bacteria in clinical settings.

The aim of this study was to investigate the presence of colistin- and/or tigecycline-resistant

Global widespread of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, especially Escherichia coli poses a greater threat in healthcare and community settings of humans. Raw meats from food animals colonized with ESBL producers may be one of important transmission routes for those bacteria in the community. This study investigated the presence of ESBL-producing E. coli in retail raw chicken and pork meats in Japan. ESBL producers were detected from the 59 of 150 (39.3%) chicken samples, but none were from all the 50 pork samples tested. The blaCTX-M-14 (17; 24.3%) was most frequently identified, followed by blaCTX-M-2 (16; 22.9%), blaSHV-12 (11; 15.7%), and blaCTX-M-55 (10; 14.3%) among a total of 70 ESBL-producing E. coli isolates from 59 chicken samples. The isolates with blaCTX-M-14 were often combined with phylogroup B1 (9/17) mainly composed of ST162 (7/9), and phylogroup F (5/17) with diverse STs. The blaCTX-M-14 was basically associated with the common elements ISEcp1 and ΔIS903 or IS903 in all 17 isolates. In 6 isolates, comprising 5 phylogroup B1-ST162 and a nontypeable-ST162 isolates, an IS26-truncated ISEcp1 was identified upstream of the blaCTX-M-14, and a fosA3 was further located downstream of ΔIS903. Furthermore, some mobile genetic elements mediating blaCTX-M-14 unique to raw chicken meat portions were identified. The blaCTX-M-2 gene was preceded by ISEcp1 or ISCR1 in 16 isolates, whereas the presence of Δorf3 downstream of blaCTX-M-2 was limited only in 6 isolates from Brazilian samples though they exhibited diverse phylogroups and STs. The blaCTX-M-55 and blaCTX-M-1 shared classical flanking structures, ISEcp1-blaCTX-M-orf477, although the length of spacer sequences between ISEcp1 and the start codon of blaCTX-M was 45 bp and 80 bp for blaCTX-M-55 and blaCTX-M-1, respectively. Among blaSHV-12-harboring isolates, ST38 was frequently detected (6/11) though their phylogroup distribution varied. In conclusion, besides transmission of bla gene-harboring E. coli lineages which have adaptability to both human and chicken, spread of mobile genetic elements associated with bla genes from E. coli lineages adapted to chicken to those adapted to human is highly suggested. Our results provide important information to gain a better understanding of the transmission risk of bla genes from retail chicken meats to human.

Group B Streptococcus (GBS) clinical isolates with reduced penicillin susceptibility (PRGBS) have emerged through acquisition of amino acid substitutions in penicillin-binding protein 2X (PBP2X). Moreover, we also reported the emergence of penicillin-susceptible GBS clinical isolates with reduced ceftibuten susceptibility (CTB^r PSGBS) due to amino acid substitutions in PBPs. However, whether or not these amino acid substitutions are responsible for the reduced ceftibuten susceptibility (RCTBS) profile remains unclear. Furthermore, the rate of CTB^r PSGBS isolation and their multidrug resistance tendency remain uncertain. Therefore, we collected 377 clinical GBS isolates from multiple regions in Japan between August 2013 and August 2015. These isolates were characterized by determining MICs and sequencing the pbp2x gene. The isolation rate of CTB^r PSGBS was 7.2% (27/377). CTBr PSGBS isolate harbor two types of amino acid substitutions in PBP2X [(T394A type) and (I377V, G398A, Q412L, and H438H type)]. The relevance of the amino acid substitutions found to the RCTBS was confirmed with allelic exchange techniques. Allelic exchange recombinant clones acquired two types of amino acid substitutions in PBP2X showed RCTBS. Furthermore, total ratio of resistance and non-susceptibility to both macrolides and fluoroquinolones in CTB^r PSGBS was 51.9% (14/27). The isolation rate of CTB^r PSGBS is non-negligibly high and the CTB^r PSGBS tends to exhibit resistance and non-susceptible profile to both macrolides and fluoroquinolones., ファイル公開：2019/08/01

We investigated the prevalence and characteristics of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from Japanese pigs. A total of 345 pig fecal specimens were collected from 30 farms in the Aichi prefecture of Japan between June 2015 and April 2016, and 22 unique ESBL-producing E. coli were isolated from 16 samples spanning 8 farms. The ESBL types included CTX-M-15 (54.5%), CTX-M-55 (27.2%), CTX-M-3 (0.9%), and CTX-M-14 (0.9%). The predominant plasmid replicon type was IncN, and the isolates carried blaCTX-M-55. Nine sequence type (ST)s, including ST117, ST1706, ST38, and ST10, were detected in the ESBL-producers, but no B2-O25-ST131 was found. ESBL producers were highly resistant to cefotaxime, ceftiofur, and tetracycline, but were susceptible to imipenem, amikacin, and fosfomycin (FOM), although 2 ST354 isolates showed resistance to ciprofloxacin. All 11 chloramphenicol-resistant isolates, including ST117 (n = 6) and ST38 (n = 3) isolates, harbored floR, and the 2 FOM-resistant ST38 isolates harbored fosA3. Our results suggest that pigs do not act as direct reservoirs in the transmission of ESBL genes to E. coli in humans. However, ST117 E. coli carrying IncN-type plasmids mediating blaCTX-M-55 were isolated from several different farms, suggesting the potential for future spread in Japan. Therefore, plasmid sequence analyses and continuous surveillance are necessary from an epidemiological point of view and are required to better protect against ESBL-producer transmission.

Not only detection of carbapenemases but also identification of their types accurately and rapidly in Enterobacteriaceae is still a major challenge for clinical laboratories in order to prevent the intrusion and the transmission of carbapenemase-producing Enterobacteriaceae. This study aimed to evaluate the performance of the MASTDISCS® combi Carba plus disc system in the 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 exerts its discriminatory power of carbapenemase including OXA-48-types exhibiting low carbapenem MICs by targeting Enterobacteriaceae isolates with EUCAST meropenem screening cut-off of ≥0.25 mg/L.

Purpose. Thymidine-dependent small-colony variants (TD-SCVs) are difficult to detect or test for antimicrobial susceptibility. We investigated the characteristics of clonal TD-SCVs of Escherichia coli, both with and without bla CTX-M-3, isolated from a patient. Methodology. Mutation in the thyA gene was analysed by sequencing, and morphological abnormalities in the colonies and cells of the isolates were examined. Additionally, conjugational transfer experiments were performed to prove the horizontal transferability of plasmids harbouring resistance genes. Results. The TD-SCVs contained a single nucleotide substitution in the thyA gene, c.62G>A, corresponding to p.Arg21His. Morphologically, their colonies were more translucent and flattened than those of the wild-type strain. In addition, cells of the TD-SCVs were swollen and elongated, sometimes with abnormal and incomplete divisions; a large amount of cell debris was also observed. Changing c.62G>A back to the wild-type sequence reversed these abnormalities. Conjugational transfer experiments showed that the TD-SCV of E. coli with bla CTX-M-3 failed to transfer bla CTX-M-3 to E. coli CSH2. However, the TD-SCV of E. coli without bla CTX-M-3 experimentally received the plasmid encoding bla SHV-18 from Klebsiella pneumoniae ATCC 700603 and transferred it to E. coli CSH2. Conclusion. Mutation in the thyA gene causes morphological abnormalities in the colonies and cells of E. coli, as well as inducing thymidine auxotrophy. In addition, TD-SCVs horizontally transmit plasmids encoding resistance genes. It is important to detect TD-SCVs based on their characteristics because they serve as reservoirs of transferable antibiotic resistance plasmids.

A capnophilic Gram-negative rod-shaped bacterium was recovered from the urine of an octogenarian male patient with acute pyelonephritis. The isolate was found to produce CTX-M-2-type extended-spectrum β-lactamase. Interestingly, the isolate failed to grow on modified Drigalski (BTB) and MacConkey agar media, even under CO2-enriched atmosphere. Our analysis revealed that the pH-indicator dyes, bromothymol blue, and/or crystal violet that were incorporated into the agar media inhibited the growth of the isolate. Although routine identification methods using Vitek® 2 Compact systems were unsuccessful, the isolate was identified as Proteus mirabilis by 16S rRNA sequencing and MALDI-TOF MS analysis. The carbonic anhydrase (CA) region spanning approximately 2,000 bp upstream to 2,000 bp downstream, which is responsible for the CO2 requirement, was not amplified, which could be attributed to the large-scale deletion or mutation of the DNA sequences containing the CA gene region. In fact, revertants with the ability to grow without CO2 were not detected. However, a revertant that was capable of growing in both BTB and MacConkey agar was detected at frequencies less than 10-9. Therefore, the genes responsible for the highly sensitive reactions of the isolate to pH indicator dyes is not likely to be linked to the CA genes.

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 bla(CTX-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 bla(CTX-M) gene repertoires were detected. In WWTP A influents, bla(CTX-M-14) (14 isolates) was dominant, followed by bla(CTX-M-15 )(12 isolates) and bla(CTX-M-27) (10 isolates). The chimeric bla(CTX-M-)(64) and bla(CTX-M-123) genes were each identified in one of the E. coli isolates from the same WWTP A inflow port. The bla(CTX-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 bla(CTX-M-15) gene. As for the B2-025b-ST131-H30R/non-Rx lineage, two isolates with bla(CTX-M-27) were recovered from each of the WWTP B and D influents, and one isolate with bla(CTX-M-174) was also recovered from WWTP B influent. Whole-genome sequencing of chimeric bla(CTX-M) -harboring E. coli isolates revealed that the bla(CTX-M-64) gene was integrated into the chromosome of ST10 E. coli B22 via ISEcp1-mediated transposition of a 9,467-bp sequence. The bla(CTX-M-123)-carrying Incl1 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., Article, Applied and Environmental Microbiology 85(22) : e01740-19-(2019)

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.

Since around the 2000s, Escherichia coli (E. coli) resistant to both oxyimino-cephalosporins and fluoroquinolones has remarkably increased worldwide in clinical settings. The kind of E. coli is also identified in patients suffering from community-onset infectious diseases such as urinary tract infections. Moreover, recoveries of multi-drug resistant E. coli from the feces of healthy people have been increasingly documented in recent years, although the actual state remains uncertain. These E. coli isolates usually produce extended-spectrum β-lactamase (ESBL), as well as acquisition of amino acid substitutions in the quinolone-resistance determining regions (QRDRs) of GyrA and/or ParC, together with plasmid-mediated quinolone resistance determinants such as Qnr, AAC(6')-Ib-cr, and QepA. The actual state of ESBL-producing E. coli in hospitalized patients has been carefully investigated in many countries, while that in healthy people still remains uncertain, although high fecal carriage rates of ESBL producers in healthy people have been reported especially in Asian and South American countries. The issues regarding the ESBL producers have become very complicated and chaotic due to rapid increase of both ESBL variants and plasmids mediating ESBL genes, together with the emergence of various "epidemic strains" or "international clones" of E. coli and Klebsiella pneumoniae harboring transferable-plasmids carrying multiple antimicrobial resistance genes. Thus, the current state of ESBL producers outside hospital settings was overviewed together with the relation among those recovered from livestock, foods, pets, environments and wildlife from the viewpoint of molecular epidemiology. This mini review may contribute to better understanding about ESBL producers among people who are not familiar with the antimicrobial resistance (AMR) threatening rising globally.

β-lactamase genes were detected and characterized from 10 non-typhoidal Salmonella (NTS) clinical isolates resistant to third-generation cephalosporins collected between 2012 and 2014 in Japan. Five strains showed cefotaxime minimum inhibitory concentration (MIC) ≥ 64 μg/ml and positive clavulanic acid inhibition results. The blaCTX-M-2 was detected in 3 strains (serotypes Stanley and Muenchen), whereas blaTEM-52 (serotype Manhattan) and blaSHV-12 (serotype Infantis) were each found in 1 strain. blaCMY-2 was detected in the remaining 5 strains (serotypes Infantis, Rissen, Newport, and Saintpaul) with cefotaxime MICs of 4-32 μg/ml and positive cloxacillin- and 3-aminophenylboronic acid- based inhibition tests. ISEcp1 was located upstream of the blaCMY-2 in 4 strains and of the blaCTX-M-2 in 1 strain. Incompatibility (Inc)A/C, IncP, and IncI1 plasmids were present in the strains harboring blaCMY-2, which were detected predominantly in this study. Acquisition of resistance to third-generation cephalosporins by invasive NTS may limit therapeutic options for severe systemic infections and causing serious public health problems. Though such resistant clinical isolates are still rare in Salmonella species in Japan, our findings reveal the presence of cephem-resistant NTS in food handlers, thus emphasizing the necessity of more systematic nationwide investigations.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) isolates with β-hemolysis and carbohydrate groups G or C are increasingly recovered from invasive infections in Japan. The aim of this study was to determine the epidemiological characteristics of SDSE isolates circulating locally among patients with invasive and noninvasive infections. We selected groups G/C β-hemolytic streptococci from a repository at the Clinical Laboratory of Kitasato University Medical Center, from May 2014 through April 2015. Thirteen isolates were identified as SDSE based on the data from API-20 Strep and 16S rRNA sequencing. The samples were from 7 sterile specimens (blood) and 6 non-sterile specimens (pus/sputum/vaginal secretion). Information about the patients with invasive or noninvasive SDSE infections was retrieved from their medical charts. We performed emm genotyping, multilocus sequence typing, a dendrogram analysis of the samples using pulsed-field gel electrophoresis (PFGE), and amplifications of the streptococcal inhibitor of a complement-mediated cell lysis-like gene (sicG) and antimicrobial resistance determinants. We identified 8 different emm genotypes, 8 different sequence types, including 4 novel types, 9 different groups in the PFGE dendrogram, the presence or absence of sicG, and 4 different resistance genotypes. Our observations indicate genetic diversity in SDSE isolates from patients with invasive and noninvasive infections in a Japanese university hospital (2014-2015).

We evaluated the feasibility and efficacy of a commercially available methicillin-resistant Staphylococcus aureus (MRSA)-selective agar, chromID(™) MRSA, to detect group B streptococci with reduced penicillin susceptibility (PRGBS) in this study. The results showed 72.4% (21/29) sensitivity and 98.4% (60/61) specificity to detect PRGBS using this method.

Herein we report the case of a 10-year-old boy with an autosomal mosaic mutation who developed bacteremia. The causative agent was identified as Moraxella osloensis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 16S rRNA gene sequencing. In the pediatric population, there have been 13 case reports of infection attributed to M. osloensis and this is the fifth reported case of pediatric bacteremia due to M. osloensis. After Moraxella species infection was confirmed, the patient recovered with appropriate antimicrobial therapy. It is important to consider that M. osloensis can cause serious infections, such as bacteremia, in otherwise healthy children. Keywords: Moraxella osloensis, Bacteremia, MALDI-TOF MS, 16S rRNA gene sequencing

Objectives: To characterise the genotypic profiles of methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates from companion animals and to investigate their association with those from humans in Japan.Methods: Non-duplicated MRSA clinical isolates recovered between July 2016 and January 2018 were analysed. The MRSA isolates were typed by polymerase chain reaction (PCR)-based open reading frame (ORF) typing (POT) scores, SCCmec types, multilocus sequence typing, and virulence gene profiles. Phylogenetic comparison of those isolates with previously described human isolates was performed.Results: Among 56 MRSA isolates (33 cats, 20 dogs and three rabbits), 26 isolates with a POT1 score of 93, SCCmec type II mostly belonged to CC5, including ST5. Twenty-six isolates with a POT1 score of 106, SCCmec type IV showed diversity of STs: 15 isolates belonged to CC8, mainly including ST8, and 11 isolates belonged to CC1, including ST1 and newly identified STs 4768, 4775, and 4779. Two cat isolates were ST8-SCCmec type IV possessing pvl/ACME-arcA, presumed to be the hypervirulent community-associated MRSA (CA-MRSA) clone USA300. Notably, all three rabbit isolates belonged to ST4768. The POT1 score 106 CA-MRSA isolates from animals and humans were divided into two large clusters of CC1 and CC8, where host species-specific sub-clusters were not identified within each cluster. A large cluster of POT1 score 93 healthcare-associated MRSA (HA-MRSA) isolates from animals and humans consisted of sub-clusters formed exclusively by the vast majority of human isolates and those formed by animal and human isolates.Conclusion: Companion animals could be potential reservoirs and vehicles for the transmission of CA-MRSA to humans, and could transmit companion animal-adaptive HA-MRSA lineages to humans as their second reservoirs., Article, Journal of Global Antimicrobial Resistance 20 : 253-259(2020)

In this study, the selective potential of group B Streptococcus isolates with reduced penicillin susceptibility (PRGBS) in a neonate-hypervirulent sequence type (ST)17 lineage was investigated by in vitro exposure to β-lactams. After 19 passages of stepwise penicillin exposure, PRGBS with a high penicillin minimum inhibitory concentration MIC (0.5 mg/L), greatly augmented ceftibuten MIC (>512 mg/L), and acquisition of G406D predicted to provide destabilizing effect (ΔΔG 0.099 kcal/mol) on PBP2X structure were identified. In early passages of stepwise cefotaxime exposure, PRGBS possessing G398E predicted to stabilize PBP2X (ΔΔG -0.038 kcal/mol) emerged with high MICs for cefotaxime (0.5 mg/L), ceftibuten (>512 mg/L) and penicillin (0.25 mg/L). Additionally, G398E + G329V + H438Y predicted to provide more stabilizing effect (ΔΔG -0.415 kcal/mol) were detected in mutants with higher MICs to cefotaxime (1 mg/L) and penicillin (0.5 mg/L). PRGBS mutants selected by penicillin and cefotaxime had a marked growth disadvantage compared with the parent strain. After two passages of stepwise ceftibuten exposure, the mutants exhibited increased MICs toward ceftibuten and acquisition of T555S predicted to provide stabilizing effect (ΔΔG -0.111 kcal/mol) in PBP 2X. In subsequent passages, gradual increases in ceftibuten MICs from 128 mg/L to 512 mg/L were found among selected mutants with accompanying stabilizing T555S+A354V (ΔΔG -0.257 kcal/mol) followed by stabilizing T555S + A354V + A536V (ΔΔG -0.322 kcal/mol), resulting in selection of a penicillin-susceptible group B Streptococcus lineage with reduced ceftibuten susceptibility (CTBr PSGBS). Notably, growth ability of CTBr PSGBS mutants was comparable to that of the parent strain. These findings may predict future failure of treatment for neonatal invasive infections caused by the neonate-hypervirulent PRGBS ST17 lineage.

Global widespread of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, especially Escherichia coli poses a greater threat in healthcare and community settings of humans. Raw meats from food animals colonized with ESBL producers may be one of important transmission routes for those bacteria in the community. This study investigated the presence of ESBL-producing E. coli in retail raw chicken and pork meats in Japan. ESBL producers were detected from the 59 of 150 (39.3%) chicken samples, but none were from all the 50 pork samples tested. The bla

Purpose. The decline in methicillin-resistant Staphylococcus aureus (MRSA) isolation rates has become a general observation worldwide, including Japan. We hypothesized that some genetic shift in MRSA might cause this phenomenon, and therefore we investigated the genetic profiles among MRSA clinical isolates obtained from three different epidemic phases in Japan. Methodology. A total of 353 MRSA isolates were selected from 202 medical facilities in 1990 (pre-epidemic phase), 2004 (epidemic phase) and 2016 (post-epidemic phase). Molecular typing was performed by PCR detection of 22 genes using the polymerase chain reaction (PCR)-based ORF typing (POT) system, including an additional eight genes including small genomic islets and seven toxin genes. Results. Isolates with a POT1 of score 93, identified as presumed clonal complex (pCC)5-staphylococcal cassette chromosome mec (SCCmec) type II including ST5-SCCmec type II New York/Japan clone, represented the major epidemic MRSA lineage in 1990 and 2004. In 2016, however, a marked decrease in isolates with a POT1 score of 93, along with changes in the epidemiology of toxin genes carried, was noted, where the carriers of tst genes including the tst-sec combination were markedly reduced, and those possessing the seb gene alone were markedly increased. Rather, isolates with a POT1 score of 106, including pCC1 or pCC8 among the isolates with SCCmec type IV, which often links to community-associated MRSA, were predominant. Interestingly, the pCC1 and pCC8 lineages were related to sea and tst-sec carriage, respectively. Conclusions. Over time, a transition in MRSA genetic profiles from a POT1 score of 93 in 1990 and 2004 to 106 in 2014 was found in Japan.

Global spread of the plasmid-mediated colistin resistance gene, mcr-1 poses a challenge to public health because colistin is the last-line-of-defense against severe infections of multidrug-resistant Gram-negative bacteria. In Japan, a few studies have reported the prevalence of mcr-1 among food animal-derived Escherichia coli isolates, but the prevalence of mcr-1 in retail meats is not well known. We report here the first detection of mcr-1 in retail chicken meat. A total of 70 extended-spectrum beta-lactamase-producing E. coli isolates, recovered from retail chicken meats between August 2015 and June 2016, were screened for mcr-1. We found 1 CTX-M-1 beta-lactamase-producing E. coli isolate belonging to ST1684, phylogroup A. The mcr-1 gene was not located on an IncI1 plasmid encoding the blaCTX-M-1 gene. However, whole plasmid sequencing revealed that mcr-1 was located on an IncI2 plasmid. The sequences of the nikB-mcr-1-pap2-ydfA-topB region of the IncI2 plasmid in this study was almost identical to that of the previously described IncI2 plasmid, pECJS-61-63 present in E. coli isolated from pig feces in China, except for containing a synonymous mutation in the mcr-1 gene. Plasmid carrying the mcr-1 gene have not yet been identified in human isolates in Japan. Thus, strict monitoring or surveillance of colistin resistance among Gram-negative bacteria recovered from retail meat of food animals under colistin pressure, and humans, is crucial.

In recent years, besides the widespread occurrence of extended-spectrum β-lactamase (ESBL)- and/or plasmid-mediated AmpC (pAmpC)-producing Enterobacteriaceae in both healthcare and community settings of humans, the third-generation cephalosporin (3GC)-resistant microbes have also been reported from companion animals worldwide. Here, we characterized ESBL- and/or pAmpC-producing Enterobacteriaceae clinical isolates from companion animals. Among the 487 clinical isolates mainly from urine of dogs and cats between May and September 2016, 104 non-repetitive isolates were resistant to the 3GC, and they consisted of 81 of 381 (21.3%) Escherichia coli , 21 of 50 (42.0%) Klebsiella pneumoniae , and 2 of 56 (3.6%) Proteus mirabilis isolates. In the 81 E. coli , the predominant bla genes were bla CTX-M-27 and bla CMY-2 (n = 15 each), followed by bla CTX-M-15 (n = 14), bla CTX-M-14 (n = 10), and bla CTX-M-55 (n = 5). In 21 K. pneumoniae , 10 bla gene types including bla CTX-M-15 (n = 4), bla CTX-M-2 (n = 4), and bla CTX-M-14 (n = 3) were found. The bla CTX-M-2 was identified in 2 P. mirabilis . Twenty-four of the 42 E. coli belonging to phylogroup B2 were O25b-ST131 clone, mostly associated with uropathogenic E. coli pathotype, and 22 isolates of this clone were identified as specific H 30R subclone. High prevalence of the bla CTX-M-27 -harboring isolates were noted among the H 30R/non-Rx lineage (13/19, 68.4%) ( p 0.05). The genetic environment of bla CTX-M-27 of most isolates of this lineage was identical to that of human isolates, but unique flanking genetic structures were also identified. Newly emerging virulent lineage B2-non-O25b-ST1193 was also confirmed in 5 isolates. The fosA3 and/or armA genes were detected in E. coli and K. pneumoniae isolates. These data suggest that companion animals serve as a potential reservoir of antimicrobial resistant E. coli and K. pneumoniae . This also has considerable veterinary importance, since urinary tract infections are an important disease causing therapeutic challenges worldwide.

We investigated the performance of a phenotypic test, the Carbapenemase Detection Set (MAST-CDS), for the identification of carbapenemase-producing Enterobacteriaceae. Our results indicated that MAST-CDS is rapid, easily performed, simple to interpret, and highly sensitive for the identification of carbapenemase producers, particularly imipenemase producers.

We investigated the prevalence and characteristics of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from Japanese pigs. A total of 345 pig fecal specimens were collected from 30 farms in the Aichi prefecture of Japan between June 2015 and April 2016, and 22 unique ESBL-producing E. coli were isolated from 16 samples spanning 8 farms. The ESBL types included CTX-M-15 (54.5%), CTX-M-55 (27.2%), CTX-M-3 (0.9%), and CTX-M-14 (0.9%). The predominant plasmid replicon type was IncN, and the isolates carried bla

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.