PCR-based ORF typing of Klebsiella pneumoniae for rapid identification of global clones and transmission events.

AIMS: Klebsiella pneumoniae is a major cause of healthcare-associated infections. In this study, we aimed to develop a rapid and simple genotyping method that can characterize strains causing nosocomial infections. METHODS AND RESULTS: The PCR-based open reading frame (ORF) typing (POT) method consists of two multiplex PCR reactions that were designed to detect 25 ORFs specific to bacterial genetic lineages, species, antimicrobial-resistant genes (blaCTX-M group-1 , blaCTX-M group-9 , blaIMP and blaKPC ), a capsular K1-specific gene and a virulence factor gene (rmpA/A2). The electrophoresis results are then digitized. A total of 192 strains (136 clinical and 8 reference strains of K. pneumoniae, 33 clinical and 1 reference strains of K. variicola and 14 clinical strains of K. quasipneumoniae) were classified into 95, 26 and 11 POT values, respectively. The distribution patterns of ORFs among K. pneumoniae correlated well with multilocus sequence typing (MLST). Furthermore, closely related species could be distinguished and key antimicrobial resistance and hypervirulence genes were identified as part of POT. CONCLUSIONS: The POT method was developed and validated for K. pneumoniae. In comparison to MLST, the POT method is a rapid and easy genotyping method for monitoring transmission events by K. pneumoniae in clinical microbiology laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: The POT method supplies clear and informative molecular typing results for K. pneumoniae. The method would facilitate molecular epidemiological analysis in infection control and hospital epidemiology investigations.

Dissecting the clonality of I1 plasmids using ORF-based binarized structure network analysis of plasmids (OSNAp).

OBJECTIVES: We aimed to elucidate the relationship among blaCTX-M-carrying plasmids and their transmission between humans and domestic animals. METHODS: Phylogenetic relationship of 90 I1 plasmids harboring blaCTX-M genes encoding extended-spectrum ß-lactamase (ESBL) was analyzed using the ORF-based binarized structure network analysis of plasmids (OSNAp). RESULTS: The majority of plasmids carrying blaCTX-M-1 or blaCTX-M-8 belonged to a single lineage, respectively, and were primarily associated with domestic animals especially chickens. On the other hand, plasmids carrying blaCTX-M-14 or blaCTX-M-15, identified from both humans and domestic animals, were distributed in two or more lineages. CONCLUSION: OSNAp has revealed the phylogenetic relationships and diversity of plasmids carrying blaCTX-M more distinctly than pMLST. The findings suggest that circulation of I1 plasmids between humans and animals may contribute to their diversity.

Practical Agar-Based Disk Diffusion Tests Using Sulfamoyl Heteroarylcarboxylic Acids for Identification of Subclass B1 Metallo-ß-Lactamase-Producing Enterobacterales.

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 ß-lactam and one of the newly approved ß-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-ß-lactamases (MßLs). 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 MßLs, such as IMP-, NDM-, and VIM-type MßLs, from the other carbapenemases, especially serine-type carbapenemases. This was accomplished using B1 MßL-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 MßL-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 MßL-producing Enterobacterales in clinical microbiology laboratories and would lead to better prevention against infection with such multidrug-resistant bacteria in clinical settings.

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.

Specific blaCTX-M-8/IncI1 Plasmid Transfer among Genetically Diverse Escherichia coli Isolates between Humans and Chickens.

We investigated the genetic backbones of 14 blaCTX-M-8-positive Escherichia coli isolates recovered from human stool samples and chicken meat. All isolates carried IncI1 plasmids with blaCTX-M-8 (blaCTX-M-8/IncI1), and most (9/14) belonged to a specific genetic lineage, namely, plasmid sequence type 113 (pST113). The genetic contexts of the nine blaCTX-M-8/IncI1 pST113 plasmids were similar, regardless of the source. These results suggest the probable local transfer of blaCTX-M-8/IncI1 between humans and chickens with genetically diverse E. coli.

Long-Term Colonization by bla(CTX-M)-Harboring Escherichia coli in Healthy Japanese People Engaged in Food Handling.

The actual state of intestinal long-term colonization by extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli in healthy Japanese people remains unclear. Therefore, a total of 4,314 fecal samples were collected from 2,563 food handlers from January 2010 to December 2011. Approximately 0.1 g of each fecal sample was inoculated onto a MacConkey agar plate containing cefotaxime (1 µg/ml). The bacterial colonies that grew on each plate were checked for ESBL production by the double-disk synergy test, as recommended by the Clinical and Laboratory Standards Institute. The bacterial serotype, antimicrobial susceptibility, pulsotype, sequence type (ST), and ESBL genotype were checked, and the replicon types of plasmids harboring the ESBL gene were also determined after conjugation experiments. ESBL producers were recovered from 70 (3.1%) of 2,230 participants who were checked only once. On the other hand, ESBL producers were isolated at least once from 52 (15.6%) of 333 participants who were checked more than twice, and 13 of the 52 participants carried ESBL producers for from more than 3 months to up to 2 years. Fluoroquinolone (FQ)-resistant E. coli strains harboring bla(CTX-M) were repeatedly recovered from 11 of the 13 carriers of bla(CTX-M)-harboring E. coli. A genetically related FQ-resistant E. coli O25b:H4-ST131 isolate harboring bla(CTX-M)-27 was recovered from 4 of the 13 carriers for more than 6 months. Three FQ-resistant E. coli O1:H6-ST648 isolates that harbored bla(CTX-M-15) or bla(CTX-M)-14 were recovered from 3 carriers. Moreover, multiple CTX-M-14- or CTX-M-15-producing E. coli isolates with different serotypes were recovered from 2 respective carriers. These findings predict a provable further spread of ESBL producers in both community and clinical settings.

Practical agar-based disk potentiation test for detection of fosfomycin-nonsusceptible Escherichia coli clinical isolates producing glutathione S-transferases.

The number of reports concerning Escherichia coli clinical isolates that produce glutathione S-transferases responsible for fosfomycin resistance (FR-GSTs) has been increasing. We have developed a disk-based potentiation test in which FR-GST producers expand the growth inhibition zone around a Kirby-Bauer disk containing fosfomycin in combination with sodium phosphonoformate (PPF). PPF, an analog of fosfomycin, is a transition-state inhibitor of FosA(PA), a type of FR-GST from Pseudomonas aeruginosa. Considering its mechanism of action, PPF was expected to inhibit a variety of FR-GSTs. In the presence of PPF, zone enlargement around the disk containing fosfomycin was observed for FosA3-, FosA4-, and FosC2-producing E. coli clinical isolates. Moreover, the growth inhibition zone was remarkably enlarged when the Mueller-Hinton (MH) agar plate contained 25 µg/ml glucose-6-phosphate (G6P). When we retrospectively tested 12 fosfomycin-resistant (MIC, ≥256 µg/ml) E. coli clinical isolates from our hospital with the potentiation test, 6 FR-GST producers were positive phenotypically by potentiation disk and were positive for FR-GST genes: 5 harbored fosA3 and 1 harbored fosA4. To identify the production of FR-GSTs, we set the provisional cutoff value, 5-mm enlargement, by adding PPF to a fosfomycin disk on the MH agar plates containing G6P. Our disk-based potentiation test reliably identifies FR-GST producers and can be performed easily; therefore, it will be advantageous in epidemiological surveys and infection control of fosfomycin-resistant bacteria in clinical settings.

Molecular epidemiology of extended-spectrum ß-lactamases and Escherichia coli isolated from retail foods including chicken meat in Japan.

Contamination of retail meat with extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli has been reported, but only limited data have been documented in Japan. One hundred fifty-three retail foods including chicken meat, beef, pork, and vegetables were purchased from 29 supermarkets between January and October in 2010. ESBL producers were recovered from each food sample using McConkey agar plate supplemented with 1 mg/L of cefotaxime. ESBL type was identified by DNA sequencing analysis after polymerase chain reaction amplification. Antibiogram, O serotype, plasmid replicon type, pulsotype, and multilocus sequence type were also determined. Fifty-two epidemiologically unrelated Escherichia coli isolates producing ESBL were recovered from 35 (22.9%) of 153 samples, all of which were chicken meat. ESBL types were mainly CTX-M-2 group followed by CTX-M-1 group and CTX-M-8 group. The numbers of bacterial isolates (8 of 21, 38.1%) harboring bla(CTX-M-8) recovered from imported meat samples were significantly larger than those of domestic ones (one of 31, 3.2%) (p<0.05). Nine O serotypes (mainly O8, O25, and O1) were found, together with O-antigen untypable (OUT). Four E. coli belonging to the O25b:H4-ST131 clone were recovered from domestic (n=1) and imported meat samples (n=3), respectively. These four isolates were susceptible to fluoroquinolones, although the E. coli O25b:H4-ST131 clone producing CTX-M-15, which is predominant in human isolates, is usually resistant to fluoroquinolones. By contrast, five CTX-M-15-producing E. coli strains were recovered only from domestic meat samples, and their serotypes were O8 or OUT instead of predominant serotype O25b. Our results showed that ESBL-producing E. coli isolates recovered from retail chicken meat samples in Japan are generally divergent in both genetic and serological aspects. Further comparative analyses of bla(CTX-M)-mediating genetic elements would be continued in the next step to characterize the ESBL producers from retail foods in Japan.

Evaluation of bactericidal effects of low-temperature nitrogen gas plasma towards application to short-time sterilization.

To develop a novel low-temperature plasma sterilizer using pure N(2) gas as a plasma source, we evaluated bactericidal ability of a prototype apparatus provided by NGK Insulators. After determination of the sterilizing conditions without the cold spots, the D value of the BI of Geobacillus stearothermophilus endospores on the filter paper was determined as 1.9 min. However, the inactivation efficiency of BI carrying the same endospores on SUS varied to some extent, suggesting that the bactericidal effect might vary by materials of sterilized instruments. Staphylococcus aureus and Escherichia coli were also exposed to the N(2) gas plasma and confirmed to be inactivated within 30 min. Through the evaluation of bactericidal efficiency in a sterilization bag, we concluded that the UV photons in the plasma and the high-voltage pulse to generate the gas plasma were not concerned with the bactericidal effect of the N(2) gas plasma. Bactericidal effect might be exhibited by activated nitrogen atoms or molecular radicals.

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.

Relationship of donor HLA antibody strength to the development of transfusion-related acute lung injury.

BACKGROUND: Antibodies against the human leukocyte antigen (HLA) in donors' blood are implicated in the development of transfusion-related acute lung injury (TRALI). Screening of female donors for HLA antibodies has been introduced to prevent TRALI; however, the relationship of HLA antibody strength in the transfused components to the development of TRALI has not been evaluated in detail. STUDY DESIGN AND METHODS: Donors involved in 1038 cases of nonhemolytic transfusion reactions (NHTRs) including 283 cases of TRALI were screened for HLA antibodies by the fluorescence beads method. HLA antibody specificity and strength were analyzed in detail. The usefulness of enzyme-linked immunosorbent assay (ELISA) for screening HLA antibodies was also evaluated. RESULT: Among 21 cases of TRALI, four cases of possible TRALI, and five cases of other NHTRs, the sum of mean fluorescence intensity (MFI) of donors' HLA antibodies to patients' cognate antigen(s) was determined in 18, four, and three cases, respectively. The sum of MFI in TRALI cases was significantly higher than that in other NHTR cases (p<0.05). When HLA antibody-positive samples were reevaluated by ELISA, the ELISA optical density ratio was significantly higher in donors' samples associated with TRALI than in those associated with other NHTRs (p<0.01) CONCLUSIONS: A correlation between the HLA antibody strength and development of TRALI was indicated. The antibody strength measured by ELISA could be used as the basis for the screening of HLA antibodies in place of the fluorescence beads method. This study provided clues to the establishment of a cutoff value for HLA antibody screening in an evidence-based manner for the prevention of TRALI.

Fatal fulminant community-acquired pneumonia caused by hypervirulent Klebsiella pneumoniae K2-ST86: Case report.

RATIONALE: Invasive community-acquired infections, including pyogenic liver abscesses, caused by hypervirulent Klebsiella pneumoniae (hvKp) strains have been well recognized worldwide. Among these, sporadic hvKp-related community-acquired pneumonia (CAP) is an acute-onset, rapidly progressing disease that can likely turn fatal, if left untreated. However, the clinical diagnosis of hvKp infection remains challenging due to its non-specific symptoms, lack of awareness regarding this disease, and no consensus definition of hvKp. PATIENT CONCERNS: A 39-year-old man presented with high-grade fever and sudden-onset chest pain. Laboratory testing revealed an elevated white blood cell count of 11,600 cells/µl and C-reactive protein level (>32 mg/dl). A chest X-ray and computed tomography revealed a focal consolidation in the left lower lung field. DIAGNOSIS: Diagnosis of fulminant CAP caused by a hvKp K2-ST86 strain was made based upon multilocus sequencing typing (MLST). INTERVENTIONS: The patient was treated with ampicillin/sulbactam. OUTCOMES: The pneumonia became fulminant. Despite intensive care and treatment, he eventually died 15.5 hours after admission. LESSONS: This is the first case of fatal fulminant CAP caused by a hvKp K2-ST86 strain reported in Japan. MLST was extremely useful for providing a definitive diagnosis for this infection. Thus, we propose that a biomarker-based approach should be considered even for an exploratory diagnosis of CAP related to hvKp infection.

Dissemination of IncF group F1:A2:B20 plasmid-harbouring multidrug-resistant Escherichia coli ST131 before the acquisition of blaCTX-M in Japan.

OBJECTIVES: The Escherichia coli O25-ST131 clone is responsible for global dissemination of the blaCTX-M gene. However, the prevalence of this clone in the digestive tract, devoid of antimicrobial selection, and its molecular epidemiology remain unclear. In this study, we examined the origin of blaCTX-M-positive E. coli O25-ST131 and its distribution. METHODS: We separately sequenced the chromosomal and plasmid genomes of 50 E. coli O25 isolates obtained from faecal samples of patients with diarrhoea in Japan. RESULTS: Although 36 (72%) of 50 E. coli O25 isolates were ST131, only 6 harboured blaCTX-M. According to the fimH and ybbW sequences and fluoroquinolone susceptibility, H30R1 isolates were dominant (27/36; 75%) and possessed IncFII-FIA-FIB with FAB formula subtype F1:A2:B20 plasmids at a high frequency (24/27; 89%). The F1:A2:B20 plasmids possessed more resistance genes such as blaTEM-1, aminoglycoside resistance genes and trimethoprim/sulfamethoxazole resistance genes compared with non-F1:A2:B20 plasmids. In contrast, only one blaCTX-M-14 gene was located on the F1:A2:B20 plasmids, whereas the other three were located on IncFII (F4:A-:B-) (n = 1) and IncZ (n = 2) plasmids. Two H30Rx-ST131 isolates harboured blaCTX-M-15: one was on the chromosome and the other on the IncFIA-R plasmid. The stability and conjugation ability of the F1:A2:B20 plasmids were compared with those of non-F1:A2:B20 plasmids, which revealed higher stability but lower conjugative ability. CONCLUSIONS: These results suggest that E. coli H30R1-ST131 is a multidrug-resistant clone containing several resistance genes in the F1:A2:B20 plasmid, which were widely distributed before the acquisition of blaCTX-M.

Spread of seb-Positive Methicillin-Resistant Staphylococcus aureus SCCmec Type II-ST764 Among Elderly Japanese in Nonacute Care Settings.

We investigated the prevalence and molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) among 356 residents of nine long-term care facilities (LTCFs) in Japan during 2015 and 2017. In total, 800 specimens were tested and 39 MRSA isolates were recovered from 31 (8.71%) residents. PCR-based open reading frame typing (POT) and pulsed-field gel electrophoresis typing were performed for the 39 MRSA isolates; five of them showing identical pulsotypes, and POT scores were excluded in further analysis. Staphylococcal cassette chromosome mec (SCCmec) typing, multilocus sequence typing, and toxin gene detection were performed for one representative MRSA isolate per resident. Among the 34 unrelated MRSA isolates, 15 (44.1%) and 19 (55.9%) were of SCCmec types II and IV, respectively, and belonged to seven sequence types (STs). Among the 15 SCCmec II isolates, 11 (73.3%), 3, and 1 belonged to ST764 (clonal complex [CC]5), ST5 (CC5), and ST630 (CC8), respectively. Among the 19 SCCmec IV isolates, 13 (68.4%), 3, 2, and 1 belonged to ST1 (CC1), ST474 (CC1), ST8 (CC8), and ST380 (CC8), respectively. Among the 14 CC5 lineage-SCCmec II isolates, one ST5 isolate and 7 of the 11 ST764 isolates (63.6%) carried seb gene, and 14 (87.5%) of 16 CC1 lineage-SCCmec IV isolates had sea gene (p < 0.05). The results indicate that the seb-positive SCCmec type II-ST764 clone has spread in Japanese LTCF environments. As LTCF residents have multiple comorbidities and increased susceptibility to infections, it is necessary to monitor MRSA colonization in LTCFs through periodic screening to prevent dissemination.

Detection of Escherichia coli Producing CTX-M-1-Group Extended-Spectrum ß-Lactamases from Pigs in Aichi Prefecture, Japan, between 2015 and 2016.

We investigated the prevalence and characteristics of extended-spectrum ß-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.

High prevalence of blaCTX-M-14 among genetically diverse Escherichia coli recovered from retail raw chicken meat portions in Japan.

Global widespread of extended-spectrum ß-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.

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.

Analysis of endogenous H2S and H2Sn in mouse brain by high-performance liquid chromatography with fluorescence and tandem mass spectrometric detection.

Previous studies indicated that bound sulfur species (BSS), including hydrogen polysulfide (H2Sn), have various physiological functions in mammalian cells. Although H2Sn molecules have been considered as secondary metabolites derived from hydrogen sulfide (H2S) based on in vitro studies or predetermined reaction formula, the physiological form of BSS and their endogenous concentration remain unclear. In the present study, we aimed to improve the usual method using monobromobimane (mBB) followed by high performance liquid chromatographic (HPLC) analysis for HS－ for simultaneous determination of H2S, H2S2, H2S3 and cysteine persulfide in biological samples. We demonstrated that mBB derivatization of H2S and H2Sn standards under alkaline conditions (pH 9.5) induced significant decreases in H2S2 and H2S3 levels and a significant increase in the H2S level in an incubation time-dependent manner. Conversely, the derivatization of mBB adducts of H2S2 and H2S3 were stable under neutral conditions (pH 7.0), which is physiologically relevant. Therefore, we re-examined the method using mBB and applied an improved method for the evaluation of H2S, H2S2, and H2S3 in mouse brain under physiological pH conditions. The concentrations of H2S and H2S2 were 0.030 ± 0.004µmol/g protein and 0.026 ± 0.002µmol/g protein, respectively. Although the level of H2S3 was below the quantification limit of this method, H2S3 was detected in mouse brain. Using the method established here, we reveal for the first time the existence of endogenous H2S2 and H2S3 in mammalian brain tissues. H2S2 and H2S3 exert anti-oxidant activity and anti-carbonyl stress effects through the regulation of redox balance in neuronal cells. Thus, our observations provide novel insights into the physiological functions of BSS in the brain and into neuronal diseases involved in redox imbalance.