First report of New Delhi metallo-ß-lactamase-1-producing Acinetobacter soli in Japan.

New Delhi metallo-ß-lactamase (NDM)-producing gram-negative rods, including Acinetobacter species, are a global problem but have rarely been isolated in Japan. To our knowledge, this is the first study to isolate an NDM-1-producing Acinetobacter soli strain, KUH106, in Japan. We analyzed this strain using next-generation sequencing to examine the plasmid carrying NDM-1. This plasmid, named pKUH106_NDM1, is 41,135 bp in length and contains genetic contexts with the structure ISAba14-aph(3')-VI-ISAba125-blaNDM-1ble-MBL. Comparative analysis of the plasmid revealed that it resembled the plasmids of Acinetobacter detected in various countries, such as the A. soli isolate from Taiwan and the Acinetobacter baumannii isolate from a healthcare facility in Osaka Prefecture, Japan. These results suggest that blaNDM-1 may spread via this plasmid in Acinetobacter species. This phenomenon needs to be confirmed through the genetic analysis of A. baumannii and other carbapenem-resistant Acinetobacter species. In particular, blaNDM-1 and other resistance genes must be investigated, and the spread of these genes in the community must be cautioned.

Detection of Genetic Elements Carrying vanA in Vancomycin-Resistant Enterococcus saigonensis VE80T Isolated from Retail Chicken Meat.

In this study, we aimed to detect genetic elements carrying vanA in Enterococcus saigonensis VE80T isolated from retail chicken in Vietnam. The structures of vancomycin-resistance determinants and the location of vancomycin-resistance genes were detected by sequencing the vanA gene cluster, Southern hybridization analyses, and whole-genome sequence analyses. The Tn1546-related elements harboring vanA clusters, which exhibited a characteristic structure with five point mutations compared with the prototype Tn1546, were located on the 76-kb plasmid pVE80-1 of VE80T. The vanS sequence of VE80T harboring three point mutations was 100% identical to those of vancomycin-resistant enterococci isolated from poultry in Taiwan and Japan, indicating that the element may be prevalent in poultry production farms in Asia.

Foodborne Outbreak of Group G Streptococcal Pharyngitis in a School Dormitory in Osaka, Japan.

In September 2016, 140 patients with primary symptoms of sore throat and fever were identified in a school dormitory in Osaka, Japan. Epidemiological and laboratory investigations determined that these symptomatic conditions were from a foodborne outbreak of group G streptococcus (GGS), with GGS being isolated from samples from patients, cooks, and foods. The strain of GGS was identified as Streptococcus dysgalactiae subsp. equisimilis of two emm types (stG652.0 and stC36.0). The causative food, a broccoli salad, was contaminated with the two types of S. dysgalactiae subsp. equisimilis, totaling 1.3 × 104 CFU/g. Pulsed-field gel electrophoresis (PFGE) of samples from patients, cooks, and foods produced similar band patterns among samples with the same emm type. This result suggested the possibility of exposure from the contaminated food. The average onset time was 44.9 h and the prevalence rate was 62%. This is the first report to identify the causative food of a foodborne outbreak by Streptococcus dysgalactiae subsp. equisimilis.

Risk factors for fecal carriage of IMP-6-producing Enterobacteriaceae at a long-term care hospital in Japan: A follow-up report from the northern Osaka multicentre study group.

The prevalence of carbapenem-resistant Enterobacteriaceae (CRE) has been increasing at medical institutions in Japan without even noticing. Recently, we performed a point prevalence survey for CRE carriage at a medical facility in northern Osaka that demonstrated an unexpectedly high prevalence of blaIMP-6-positive CRE, particularly at long-term care hospitals (LTCH). To identify the risk factors for CRE carriage, we collected clinical data of patients at a representative LTCH. Of 140 patients who were included in this study, 27 (19.3%) were colonized with metallo-beta-lactamase (IMP-6) producers. Pulsed-field gel electrophoresis of the IMP-6 producing Enterobacteriaceae suggested a non-clonal transmission of Escherichia coli, while a clonal spread was shown for Klebsiella pneumoniae. Risk factors for CRE colonization were a longer stay at the hospital stay and a lower independence state, as measured by Norton scales. We propose that a paradigm shift in infection control, inciting a coordinated regional effort to involve LTCHs, should be discussed in the aging society of Japan.

Development of selective medium for IMP-type carbapenemase-producing Enterobacteriaceae in stool specimens.

BACKGROUND: Identification of carbapenemase-producing Enterobacteriaceae (CPE) in faecal specimens is challenging. This fact is particularly critical because low-level carbapenem-resistant organisms such as IMP-producing CPE are most prevalent in Japan. We developed a modified selective medium more suitable for IMP-type CPE. METHODS: Fifteen reference CPE strains producing different types of ß-lactamases were used to evaluate the commercially available CHROMagar KPC and chromID CARBA as well as the newly prepared MC-ECC medium (CHROMagar ECC supplemented with meropenem, cloxacillin, and ZnSO4) and M-ECC medium (CHROMagar ECC supplemented with meropenem and ZnSO4). A total of 1035 clinical samples were then examined to detect CPE using chromID CARBA and M-ECC medium. RESULTS: All tested strains producing NDM-, KPC-, and OXA-48-carbapenemases were successfully cultured in the media employed. Although most of the IMP-positive strains did not grow in CHROMagar KPC, chromID CARBA, or MC-ECC, all tested strains grew on M-ECC. When faecal samples were applied to the media, M-ECC medium allowed the best growth of IMP-type CPE with a significantly higher sensitivity (99.3%) than that of chromID CARBA (13.9%). CONCLUSIONS: M-ECC medium was determined as the most favourable selective medium for the detection of IMP-type CPE as well as other types of CPE.

Characterization of first hemin-requiring Pseudomonas aeruginosa small-colony variants from the blood of an octogenarian male-patient with double pneumonitis.

A hemin-requiring Pseudomonas aeruginosa small-colony variant (SCV) was isolated from the blood of an octogenarian male-patient with double pneumonitis. The isolate was capable of growing on both sheep blood and chocolate agars but not on MacConkey agars without blood ingredient. Furthermore, the isolate revealed to grow only around the X-factor impregnated discs when examined using the X and V disc strips. However, not only RapID-NH system but also the VITEK2 system failed to identify the isolate. The isolate was finally identified as P. aeruginosa by the sequence of the 16S rRNA genes and the MALDI-TOF MS analysis. Interestingly, the isolate represented positive reaction for δ-aminolaevulinic acid (ALA)-test despite the requirement of hemin. Detailed analysis indicated that the isolate produced protoporphyrin IX from ALA. Therefore, the reason for the hemin dependence was deduced the dysfunction of hemH-encoded ferrochelatase behaving at the end of biosynthetic pathway of heme. However, the genetic analysis of hemH gene demonstrated no variations of both the DNA and the amino-acid sequences. To the best of our knowledge, this is the first clinical isolation of a hemin-dependent P. aeruginosa SCV from blood.

Consumption of edible ice contaminated with Acinetobacter, Pseudomonas, and Stenotrophomonas is a risk factor for fecal colonization with extended-spectrum ß-lactamase-producing Escherichia coli in Vietnam.

Although Vietnamese residents frequently harbor extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-E), it is unclear which foods/beverages are risk factors for acquiring these bacteria. The aim of this study was to evaluate the frequency with which edible ice served in restaurants is contaminated with antibiotic-resistant bacteria, and thereby clarify whether this product poses a risk for ESBL-E carriage in humans. Ice from restaurants in Vietnam and Japan was screened for bacteria capable of growing on agar containing cefotaxime (BG-CTX). Of the 119 BG-CTX strains isolated in Vietnam, 40%, 39%, and 12% were identified as Pseudomonas spp., Acinetobacter spp., and Stenotrophomonas maltophilia, respectively. Meanwhile, of the six such strains isolated in Japan, five were identified as Acinetobacter spp. and one as Pseudomonas spp. More than 10% of the Acinetobacter isolates exhibited cefotaxime, ceftazidime, and sulfa/trimethoprim resistance, while 21% of Pseudomonas and 14% of S. maltophilia isolates exhibited meropenem and sulfa/trimethoprim resistance, respectively. Subsequent multiplex polymerase chain reaction (PCR) analyses detected ESBL-encoding genes in 10% of the BG-CTX. Notably, feces harvested from mice administered water contaminated with BG-CTX contained E. coli harboring the blaCTX-M-9 gene. In conclusion, our findings indicate that consumption of contaminated edible ice is a risk factor for human ESBL-E carriage.

Frequent use of colistin-based drug treatment to eliminate extended-spectrum beta-lactamase-producing Escherichia coli in backyard chicken farms in Thai Binh Province, Vietnam.

Reports of livestock infections with extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-E) are increasing. Based on interviews conducted over a 6-month period, we found that veterinarians in the Vietnamese province of Thai Binh prefer to prescribe colistin-based drugs (CBD) in chicken farms. We aimed to clarify whether CBD use selects for strains of colistin-resistant ESBL-E. With the cooperation of seven local households, we detected ESBL-E in chickens' feces after treating chickens with CBD. Phylogenetic groupings and the presence of CTX-M/AmpC genes were determined, and the multi-antibiotic susceptibility of isolates was analyzed. Our results showed that ESBL-E presented in seven chickens' feces from two households. Seventy-two percent of ESBL-E isolates harbored CTX-M9 and the phylogenetic group A; the colistin minimum inhibitory concentration (MIC) of all isolated ESBL-E ranged from 0.064 to 1 µg mL-1. Moreover, ESBL-E isolates were used to experimentally select for colistin resistance, and the effect of commercial CBD on ESBL-E was investigated. The results showed that an ESBL-E strain with a colistin MIC of 4 µg mL-1 was able to grow in media with CBD. Although CBD treatment was effective, in vitro experiments demonstrated that ESBL-E can easily acquire colistin resistance. Therefore, restrictions on colistin use are necessary to prevent the emergence of colistin-resistant bacteria.

Phylogenetic Analysis of Enteroaggregative Escherichia coli (EAEC) Isolates from Japan Reveals Emergence of CTX-M-14-Producing EAEC O25:H4 Clones Related to Sequence Type 131.

Enteroaggregative Escherichia coli (EAEC) causes acute or persistent diarrhea. The aggR gene is widely used as a marker for typical EAEC. The heterogeneity of EAEC is well known; however, there are few reports on the phylogenetic relationships of EAEC. Recently, CTX-M extended-spectrum ß-lactamase (ESBL)-producing EAEC strains have been reported worldwide. To characterize EAEC strains in Japan, we investigated the population structure of EAEC. A total of 167 aggR-positive strains isolated from stool specimens from diarrheal patients in Kagoshima (139 strains) and Osaka (28 strains), Japan, between 1992 and 2010 were examined for the prevalence of EAEC virulence markers, the blaCTX-M gene, and the capacity to form biofilms. Multilocus sequence typing was also conducted. EAEC strains were widely distributed across four major E. coli phylogroups. Strains of O111:H21/clonal group 40 (CG40) (30 strains), O126:H27/CG200 (13 strains), and O86a:H27/CG3570 (11 strains) in phylogroup B1 are the historical EAEC clones in Japan, and they exhibited strong biofilm formation. Twenty-nine strains of EAEC O25:H4/CG131 were identified in phylogroup B2, 79% of which produced CTX-M-14. This clone has emerged since 2003. The clone harbored plasmid-encoded EAEC virulence genes but not chromosomal virulence genes and had lower biofilm-forming capacity than historical EAEC strains. This clone most likely emerged from a pandemic uropathogenic O25:H4/sequence type 131 clone by acquiring an EAEC virulence plasmid from canonical EAEC. Surveillance of the horizontal transfer of both virulence and ESBL genes among E. coli strains is important for preventing a worldwide increase in antimicrobial drug resistance.

Enterococcus saigonensis sp. nov., isolated from retail chicken meat and liver.

Two Gram-stain-positive strains, VE80T and VE116, which were resistant to vancomycin, were isolated from retail chicken meat and liver in Ho Chi Minh, Vietnam, respectively. These strains were characterized by sequence analyses of 16S rRNA, RNA polymerase α-subunit (rpoA), ATP synthase α-subunit (atpA), and phenylalanyl-tRNA synthase α-subunit (pheS) genes, determination of DNA G+C content, cellular fatty acid methyl ester analysis, DNA-DNA hybridization, and conventional morphological and biochemical tests. Strains VE80T and VE116 had 99.6 % 16S rRNA gene sequence similarity with Enterococcus canintestini LMG 13590T, and 99.1 % 16S rRNA gene sequence similarity with Enterococcus dispar ATCC 51266T. However, the two isolates could be clearly differentiated from these reference strains by the low sequence similarities (86.1-86.8 %) of the atpA gene, low DNA-DNA relatedness (<22.8 %), and differences in the production of acid from melezitose and methyl α-d-glucoside. Based on the results obtained in the present study, these two isolates are considered to represent a novel species of the genus Enterococcus, for which the name Enterococcus saigonensis sp. nov., is proposed. The type strain is VE80T (=JCM 31193T=CCUG 68827T).

Current status of extended spectrum ß-lactamase-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis in Okinawa prefecture, Japan.

Enterobacteriaceae producing extended spectrum ß-lactamase (ESBL) are distributed worldwide. In this study, 114 ESBL-producing Enterobacteriaceae were isolated by analyzing 1672 clinical isolates of Enterobacteriaceae collected from an Okinawa prefectural hospital in Japan between June 2013 and July 2014. The overall prevalence of ESBL-producing Enterobacteriaceae was 6.8%; the prevalence of different bacterial species among the ESBL-producing isolates was as follows: 11.5% Escherichia coli (90 of 783 isolates), 6.2% Klebsiella pneumoniae (19 of 307 isolates), and 11.1% Proteus mirabilis (5 of 45 isolates). The ESBL types blaCTX-M-1, -3, -15, -2, -14, -27, and mutants of blaSHV-1 were detected. Among them, blaCTX-M-15 (33.3%), blaCTX-M-14 (27.8%) and blaCTX-M-27 (33.3%) were dominant in the E. coli isolates, whereas a blaSHV mutant which possessed four mutations (Tyr7Phe, Leu35Gln, Gly238Ser and Glu240Lys) in the amino acid sequence of SHV-1 dominated in the K. pneumoniae isolates (11 of 19, 57.9%). The pandemic E. coli ST131 clone was found to constitute 3.3% of the overall examined isolates and 62.2% of the ESBL-producing E. coli isolates. Our results suggest that the genetic combination of blaCTX-M, and blaSHV and antibiotics-resistant profile were different from that in other regions such as other areas of Japan, Asia, Europe, and North America, especially in the ESBL-producing K. pneumoniae isolates and in the E. coli B2-O25b-ST131 isolates possessing blaCTX-M-15 (40.7% of the E. coli B2-O25b-ST131 isolates). Taken together, our results indicate that the ESBL-producing Enterobacteriaceae in Okinawa, Japan, might be of a unique nature.

Characterization of Third-Generation-Cephalosporin-Resistant Shiga Toxin-Producing Strains of Escherichia coli O157:H7 in Japan.

We isolated Shiga toxin-producing Escherichia coli O157:H7 strains resistant to third-generation cephalosporins. The resistant strains harbored blaCMY-2, a plasmid-mediated AmpC ß-lactamase. Genotyping of isolates revealed the possible spread of this problematic bacterium. Results suggested the importance of the investigation and surveillance of enterobacteria with plasmids harboring blaCMY-2.

Evaluation of streptococcal toxic shock-like syndrome caused by group B streptococcus in adults in Japan between 2009 and 2013.

Infection with Streptococcus agalactiae has long been recognized in infants. In recent years, S. agalactiae is an important cause of morbidity and mortality among adults and among those with underlying medical condition. Several cases of GBS infection and more fulminant disease similar to streptococcal toxic shock syndrome have recently been reported. We report here that 19 S. agalactiae strains were isolated from streptococcal toxic shock-like syndrome cases involving adult patients in Japan between 2009 and 2013. The average age of the patients was 66.3 years. At least one underlying disease was present in 47.4% (9/19) of the patients. The most prevalent serotype among these strains was Ib. All serotype Ib strains belonged to clonal complex 10 and were ciprofloxacin resistant. In contrast, all strains were susceptible to penicillin G, ampicillin, cefazolin, cefotaxime, imipenem, panipenem, and linezolid. The characteristic type distributions of streptococcal toxic shock-like syndrome isolates differed between isolates obtained from vaginal swabs of women and infants with invasive infections.

Characteristics of Extended-Spectrum ß-Lactamase-Producing Escherichia coli in Retail Meats and Shrimp at a Local Market in Vietnam.

BACKGROUND: Contamination of food with multiantibiotic-resistant bacteria, particularly extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae, is considered a potential source for the wide dissemination of ESBL-producing bacteria in communities. However, little is known about the extent of contamination of food with ESBL-producing bacteria in Vietnam. OBJECTIVE: This study was conducted to assess the characteristics of ESBL-producing Escherichia coli isolated from retail meats and shrimp in Nha Trang, Vietnam. MATERIALS AND METHODS: A total of 350 food samples (poultry [n=143], pork [n=147], and shrimp [n=60]) were purchased in July and November 2013 from a local market. ESBL-producing E. coli were isolated, and ESBL genotypes, phylogenetic groups, and antibiotic resistance profiles were determined. RESULTS: The prevalence of ESBL-producing E. coli in retail foods was 40.6%. ß-Lactamase-encoding genes of the CTX-M-1 (50.7%), CTX-M-9 (41.5%), TEM (59.9%), and SHV (2.8%) groups were detected singly or in combination. The percentages of single ESBL isolates harboring CTX-M-1 or -9 plus TEM groups were 35.2% and 16.2%, respectively. B1 was the most prevalent phylogroup in ESBL isolates from pork (44.7%), poultry (55.9%), and shrimp (72.7%). B2 was the least prevalent (4.2% and 4.8% for pork and poultry isolates, respectively). The prevalence of multidrug resistance (MDR; resistance to ≥ 3 antimicrobial groups) in ESBL-producing E. coli isolated from food was 85.9%. DISCUSSION AND CONCLUSIONS: This is the first report of the characteristics of ESBL-producing E. coli in retail foods in a local city in Vietnam. Our findings indicate that retail foods are contaminated with ESBL-producing E. coli, of which many were MDR. Further monitoring and public health efforts targeting food administration are needed to control the spread of ESBL-producing bacteria in communities.

Genetic characterization of KHM-1 metallo-ß-lactamase-producing Enterobacterales isolates from inpatient sources in Osaka, Japan.

OBJECTIVES: KHM-1-metallo-ß-lactamase-producing Enterobacterales strains, of which only a few have been found, were isolated from four inpatients in Osaka, Japan during 2016 to 2020. We compared whole genomes of the four KHM-1-producing isolates, including one Enterobacter hormaechei subsp. hoffmannii, one Escherichia coli, and two Citrobacter freundii. METHODS: These isolates were characterized by whole-genome sequencing, comparative analysis of blaKHM-1-encoding plasmids with earlier reported plasmids, and antimicrobial susceptibility tests. RESULTS: Multilocus sequence typing classified the E. hormaechei subsp. hoffmannii isolate to ST78, the E. coli isolate to ST354, and the two C. freundii isolates to ST95. These isolates harboured various antimicrobial resistance genes aside from blaKHM-1 on their chromosomes and plasmids. In all four isolates, blaKHM-1 was located on 137 kbp to 213 kbp plasmids of IncC replicon type. Although there were common resistance genes such as blaKHM-1-ISEc68, class I integron cassette, and fosG, the four blaKHM-1-encoding plasmids were distinguishable into two lineages based on differences of the resistance gene components and their surrounding regions. CONCLUSION: Because no epidemiological contact was observed among the inpatients, the blaKHM-1-encoding IncC plasmids might have spread horizontally to multiple bacterial species through repeated recombination and insertion.

Detection of chromosome-mediated blaNDM-1-carrying Aeromonas spp. in the intestinal contents of fresh water river fish in Ho Chi Minh City, Vietnam.

The spread of antibiotic-resistant bacteria is a global problem that should be addressed through the perspective of the "one health" concept. The purpose of this study was to determine the contamination rate of antibiotic-resistant Aeromonas spp. in fresh water river fish purchased from a fish market in Vietnam. We then defined the pattern of antibiotic resistance to assess antibiotic-resistant contamination. Antibiotic-resistant Aeromonas spp. were detected in the intestinal contents of 32 of 80 fish. blaNDM-1 was detected in seven strains. Extended-spectrum ß-lactamase and AmpC ß-lactamase-related genes were detected in 28 strains, including blaCTX-M-55, blaCTX-M-15, blaCTX-M-1, and blaDHA,blaFOX, and blaMOX. The blaNDM-1 detected in the seven Aeromonas spp. strains were found chromosomally. This finding suggests that the blaNDM gene is stable in the natural environment and may spread widely into animals and humans via Aeromonas spp. with a transposon. Our results suggest the importance of continuing to monitor carbapenemase genes in Aeromonas spp. to evaluate the possibility that they may spread in other Enterobacterales, and to elucidate the mechanism of spread.