Pathotypes and Drug Susceptibility of Escherichia coli Isolated from Companion Dogs in Japan.

Considering the possibility that Escherichia coli carried by companion dogs could infect owners and human society, we investigated their pathogenicity and drug resistance. E. coli was isolated from stool samples of companion dogs (n = 90) to examine the O-serogroup, virulence genes, and drug susceptibility. The age of dogs ranged from 4 months to 16 years, and they were mainly treated with cefalexin, enrofloxacin, or amoxicillin. A total of 69 samples were positive for E. coli (76% of examined dogs), and the most common O-serogroup was O18 (n = 13). Nine diarrheagenic E. coli, including enteropathogenic E. coli (n = 3), enteroaggregative E. coli (n = 1), and astA-carrying E. coli (n = 5), were isolated. In addition, we isolated 28 E. coli strains resistant to at least one of six antimicrobials, including cephalothin (CET), ceftazidime (CAZ), cefotaxime (CTX), chloramphenicol (CP), fosfomycin (FOM), and norfloxacin (NLFX). The resistance pattern was as follows: CET, n = 16; NLFX, n = 3; CET/CP (resistance to both CET and CP), n = 1; CET/NLFX, n = 1; CET/CAZ/CTX, n = 3; CET/CTX/NLFX, n = 2; CET/CP/NLFX, n = 1; and CET/CAZ/CTX/NLFX, n = 1. Moreover, ten E. coli isolates were found to produce extended-spectrum ß-lactamase (ESBL), including AmpC (n = 4; OUT, O18, O74, and O166), CTX-M-1 (n = 1; O25), CTX-M-9 (n = 4; OUT, O18, O18, and O125), and AmpC/CTX-M-9 (n = 1; OUT) groups. The AmpC-producing E. coli strains included enteropathogenic and astA-carrying E. coli. Our results showed that the human-infectious diarrheagenic E. coli was isolated from some dogs, and some strains exhibited ESBL. Therefore, future studies are needed to investigate the possibility of transmission of these E. coli strains to humans.

Novel Sequence Type in Bacillus cereus Strains Associated with Nosocomial Infections and Bacteremia, Japan.

Bacillus cereus is associated with foodborne illnesses characterized by vomiting and diarrhea. Although some B. cereus strains that cause severe extraintestinal infections and nosocomial infections are recognized as serious public health threats in healthcare settings, the genetic backgrounds of B. cereus strains causing such infections remain unknown. By conducting pulsed-field gel electrophoresis and multilocus sequence typing, we found that a novel sequence type (ST), newly registered as ST1420, was the dominant ST isolated from the cases of nosocomial infections that occurred in 3 locations in Japan in 2006, 2013, and 2016. Phylogenetic analysis showed that ST1420 strains belonged to the Cereus III lineage, which is much closer to the Anthracis lineage than to other Cereus lineages. Our results suggest that ST1420 is a prevalent ST in B. cereus strains that have caused recent nosocomial infections in Japan.

Corynebacterium striatum Bacteremia Associated with a Catheter-Related Blood Stream Infection.

A 49-year-old woman visited our emergency department because of exertional dyspnea due to severe left ventricular functional failure. It progressed to disseminated intravascular coagulation and disturbance of consciousness on day 67 of admission. Gram-positive bacilli were detected from two different blood culture samples on day 67 of admission. An API-Coryne test and sequencing (1~615 bp) of the 16S rRNA gene were performed, and the strain was identified as Corynebacterium striatum. The bacterium was detected from the removed central venous catheter tip too, and the patient was diagnosed with catheter-related bloodstream infection by C. striatum. However, treatment was not effective, and the patient died on day 73 of admission.

Effect of vitamin K2 on the development of stress-induced osteopenia in a growing senescence-accelerated mouse prone 6 strain.

Vitamin K2 (VK2) has been used as a therapeutic agent for osteoporosis, since it has been suggested to be able to reduce the frequency of fractures by improving bone quality; however, bone turnover is strictly regulated by various cytokines and hormones. In the present study, the effect of menaquinone-4 (MK-4) on bone turnover was investigated using the senescence-accelerated mouse prone 6 (SAMP6) strain. Since water-immersion restraint stress (WRS) causes a significant decrease in bone mineral density (BMD), WRS was used as the bone resorption model in the SAMP6 strain. Six-week-old SAMP6 male mice were divided into the following three groups: Control, WRS and WRS + MK-4. WRS was performed for 6 h per day, 5 times a week, for 4 weeks. Following WRS, MK-4 (30 mg/kg) was injected subcutaneously 3 times a week for 4 weeks. No growth retardation was observed in the WRS groups as compared with the control group. In the WRS groups, the BMD was significantly lower than that in the control group. The levels of bone formation and resorption markers were increased in the WRS groups, indicating that WRS reduced the BMD by promoting high bone turnover. A bone histomorphometrical examination showed that the trabecular (Tb) bone mass in the secondary spongiosa at the distal femur was significantly reduced in the WRS mice, and this reduction was abrogated by MK-4 treatment. Specifically, the Tb bone reduction was caused by the activation of osteoclasts (Ocs), and Oc activity was suppressed by MK-4. The number of osteoblasts and the mineral apposition rate were significantly increased in the WRS and WRS + MK-4 mice, suggesting that WRS triggered a significantly higher mineral apposition rate. These results indicate that MK-4 can induce recovery from the bone mineral loss caused by WRS treatment. Further studies are required to clarify the association between bone quality and MK-4.

Molecular epidemiological characterization of uropathogenic escherichia coli from an outpatient urology clinic in rural Japan.

In the remote Japanese community of Saku, a rural town in the Nagano Prefecture, a large proportion of outpatient urinary tract infections was caused by well-recognized globally dispersed clonal lineages of uropathogenic Escherichia coli (UPEC). However, most of these strains were drug susceptible, suggesting that factors other than selection pressure account for the clonal spread of drug-susceptible UPEC.

Distribution of carbapenem resistance determinants among epidemic and non-epidemic types of Acinetobacter species in Japan.

We performed a comparative molecular analysis on three types of clinically isolated Acinetobacter spp.: epidemic sequence types (STs) of Acinetobacter baumannii (epidemic ST-AB), non-epidemic sequence types of A. baumannii (non-epidemic ST-AB) and non-baumannii Acinetobacter spp. A total of 87 isolates - 46 A. baumannii, 25 A. pittii and 16 A. nosocomialis - from 43 hospitals were analysed. Of these, 31 A. baumannii isolates were ST1 or ST2 according to the Pasteur Institute multilocus sequence typing scheme and were defined as epidemic ST-AB. The other 15 A. baumannii isolates were defined as non-epidemic ST-AB. The epidemic ST-AB isolates harboured the blaOXA-23-like gene or had an ISAba1 element upstream of blaOXA-51-like, or both, whereas non-epidemic ST-AB and non-baumannii Acinetobacter spp. isolates harboured blaOXA-58-like or metallo-ß-lactamase genes, or both. The proportion of multidrug-resistant isolates was significantly higher in the epidemic ST-AB isolates (48 %) than that in the other types of Acinetobacter isolates (5 %) (P<0.05). In addition, epidemic ST-AB isolates exhibited a relatively higher proportion of fluoroquinolone resistance. We demonstrated that, in terms of genotypes and phenotypes of antimicrobial resistance, non-epidemic ST-AB isolates shared more similarity with non-baumannii Acinetobacter spp. isolates than with epidemic ST-AB isolates, regardless of bacterial species. In addition, this study revealed that, even in Japan, where IMP-type metallo-ß-lactamase producers are endemic, epidemic ST-AB harbouring blaIMP have not yet emerged.

Screening for group B streptococci with reduced penicillin susceptibility in clinical isolates obtained between 1977 and 2005.

Group B streptococcus (GBS; Streptococcus agalactiae) is a leading cause of neonatal invasive infections, and until recently, it was thought to be completely susceptible to penicillin. However, we recently identified several clinical GBS isolates with reduced penicillin susceptibility (PRGBS) whose minimum inhibitory concentrations of penicillin were >0.12 µg/ml, which is above the susceptibility breakpoint set by the Clinical and Laboratory Standards Institute. These PRGBS were isolated between 1995 and 2005 in Japan; whether these PRGBS existed in Japan before 1995 is unknown. In the study described here, we screened for PRGBS among 349 clinical GBS isolates obtained in Japan between 1977 and 2005 using the previously developed disk diffusion method for the detection of PRGBS. With this method, we selected 6 PRGBS candidates and confirmed that 1 isolate was PRGBS, using agar dilution method, including oxacillin, ceftizoxime, and penicillin-binding protein 2X (PBP2X) gene sequencing analysis. This isolate was obtained from sputum in 2005, and we could not detect PRGBS isolates before 1995 in this investigation.

High cephalosporin resistance due to amino acid substitutions in PBP1A and PBP2X in a clinical isolate of group B Streptococcus.

OBJECTIVES: Group B Streptococcus (GBS; Streptococcus agalactiae) has been regarded as uniformly susceptible to penicillins. However, we recently reported the existence of GBS with reduced penicillin susceptibility (PRGBS), with amino acid substitutions in penicillin-binding protein (PBP) 2X. Although most PRGBS show high MICs of ceftizoxime (4-64 mg/L) and cefotaxime (0.12-1 mg/L), those for strain B1 are exceptionally high (ceftizoxime MIC ≥256 mg/L and cefotaxime MIC 2 mg/L). We previously found an amino acid substitution (G539S) neighbouring the conserved K540TG motif in PBP1A in addition to the PRGBS-specific amino acid substitution Q557E in PBP2X of B1. The aim of this study was to reveal the effect of the amino acid substitutions in PBP1A and PBP2X of B1 on the high cephalosporin resistance. METHODS: A ceftizoxime competition assay was performed to reveal the PBPs that are the main targets of ceftizoxime. We generated two allelic exchange mutants from ß-lactam-susceptible GBS BAA-611. BAA-611 (B1PBP2X) contained the PBP2X gene derived from B1 and BAA-611 (B1PBP2X, B1PBP1A) contained both the PBP2X and the PBP1A gene derived from B1. These allelic exchange mutants and strain B1 were subjected to susceptibility testing. RESULTS: The ceftizoxime competition assay revealed that PBP1A and PBP2X were the main targets of ceftizoxime. Although the MICs of ceftizoxime and cefotaxime for BAA-611 (B1PBP2X) were 64 and 0.5 mg/L, respectively, BAA-611 (B1PBP2X, B1PBP1A) showed high cephalosporin resistance (ceftizoxime MIC ≥256 mg/L and cefotaxime MIC 2 mg/L) comparable to B1. CONCLUSIONS: The high cephalosporin resistance of GBS was caused by amino acid substitutions in PBP1A and PBP2X.

[Three months survey of multidrug-resistant Enterobacteriaceae in Japan].

In 2010, a three months survey of multidrug-resistant Enterobacteriaceae was conducted by Ministry of Health, Labour and Welfare of Japan. A total of 153 isolates were obtained through this survey and we performed PCR using the NDM-1 type, KPC type, IMP-1 type, IMP-2 type and VIM-2 type carbapenemase genes specific primers. Of 153 analyzed isolates, 72 (47.1%) were positive for IMP-1 type bla(IMP), and two isolates from two patients were positive for bla(NDM-1). None of those patients had traveled abroad. Two isolates from a single patient who had traveled and hospitalized in abroad were positive for bla(KPC). 77 (50.3%) isolates were all negative for those five carbapenemase genes. It was shown that IMP-1 type is the most predominant carbapenemase gene among Enterobacteriaceae in Japan.

[KPC carbapenemase producing Gram-negative bacteria].

Carbapenem is the last resort for infections due to Gram-negative bacteria. However, carbapenem-resistant Enterobacteriaceae such as KPC-producing bugs were reported in the United States from early this decade and now worldwide. According to the nationwide surveillance study in Japan performed between September and December, 2010, two bla(KPC)-harboring Klebsiella pneumoniae were identified among 153 multi-drug-resistant Enterobacteriaceae. Moreover, 72 out of 153 multi-drug-resistant Enterobacteriaceae harbored bla(IMP-1). The results of this surveillance study demonstrated that the most epidemic carbapenemase in Japan is not bla(KPC) but bla(LMP-1). Although the bla(KPC)-harboring strain is very rare in Japan, detection of KPC-producing Gram-negative bacteria may be difficult based on the CLSI recommended method. Development of effective screening methods is needed in the Japanese healthcare environment.

Complete sequencing of the bla(NDM-1)-positive IncA/C plasmid from Escherichia coli ST38 isolate suggests a possible origin from plant pathogens.

The complete sequence of the plasmid pNDM-1_Dok01 carrying New Delhi metallo-ß-lactamase (NDM-1) was determined by whole genome shotgun sequencing using Escherichia coli strain NDM-1_Dok01 (multilocus sequence typing type: ST38) and the transconjugant E. coli DH10B. The plasmid is an IncA/C incompatibility type composed of 225 predicted coding sequences in 195.5 kb and partially shares a sequence with bla(CMY-2)-positive IncA/C plasmids such as E. coli AR060302 pAR060302 (166.5 kb) and Salmonella enterica serovar Newport pSN254 (176.4 kb). The bla(NDM-1) gene in pNDM-1_Dok01 is terminally flanked by two IS903 elements that are distinct from those of the other characterized NDM-1 plasmids, suggesting that the bla(NDM-1) gene has been broadly transposed, together with various mobile elements, as a cassette gene. The chaperonin groES and groEL genes were identified in the bla(NDM-1)-related composite transposon, and phylogenetic analysis and guanine-cytosine content (GC) percentage showed similarities to the homologs of plant pathogens such as Pseudoxanthomonas and Xanthomonas spp., implying that plant pathogens are the potential source of the bla(NDM-1) gene. The complete sequence of pNDM-1_Dok01 suggests that the bla(NDM-1) gene was acquired by a novel composite transposon on an extensively disseminated IncA/C plasmid and transferred to the E. coli ST38 isolate.

SMB-1, a novel subclass B3 metallo-beta-lactamase, associated with ISCR1 and a class 1 integron, from a carbapenem-resistant Serratia marcescens clinical isolate.

A carbapenem-resistant Serratia marcescens strain, 10mdr148, was identified in a Japanese hospital in 2010. The carbapenem resistance of this strain was attributed to the production of a novel metallo-ß-lactamase (MBL), named SMB-1 (Serratia metallo-ß-lactamase). SMB-1 possessed a zinc binding motif, H(Q)XHXDH (residues 116 to 121), H196, and H263 and was categorized as a member of subclass B3 MBL. SMB-1 has 75% amino acid identity with the most closely related MBL, AMO1, of uncultured bacterium, recently identified through the metagenomic analysis of apple orchard soil. The introduction of bla(SMB-1) into Escherichia coli conferred resistance to a variety of ß-lactam antibiotics, penicillins, cephalosporins, and carbapenems, but not aztreonam, a resistance pattern consistent with those of other MBLs. SMB-1 demonstrated high k(cat) values of >500 s(-1) for carbapenems, resulting in the highest hydrolyzing efficiency (k(cat)/K(m)) among the agents tested. The hydrolyzing activity of SMB-1 was well inhibited by chelating agents. The bla(SMB-1) gene was located on the chromosome of S. marcescens strain 10mdr148 and at the 3' end of the ISCR1 element in complex with a typical class 1 integron carrying aac(6')-Ib and catB3 gene cassettes. Downstream of bla(SMB-1), the second copy of the 3'conserved segment and ISCR1 were found. To our knowledge, this is the first subclass B3 MBL gene associated with an ISCR1 element identified in an Enterobacteriaceae clinical isolate. A variety of antibiotic resistance genes embedded with ISCR1 have been widely spread among Enterobacteriaceae clinical isolates, thus the further dissemination of bla(SMB-1) mediated by ISCR1 transposition activity may become a future concern.

RmtC introduces G1405 methylation in 16S rRNA and confers high-level aminoglycoside resistance on Gram-positive microorganisms.

Seven plasmid-mediated 16S rRNA methyltransferases (MTases), RmtA, RmtB, RmtC, RmtD, RmtE, ArmA, and NpmA, conferring aminoglycoside resistance have so far been found in Gram-negative pathogenic microorganisms. In the present study, by performing an RNase protection assay, primer extension, and HPLC, we confirmed that RmtC indeed methylates at the N7 position of nucleotide G1405 in 16S rRNA as found in ArmA and RmtB. RmtC has an MTase activity specific for the bacterial 30S ribosomal subunit consisting of 16S rRNA and several ribosomal proteins, but not for the naked 16S rRNA, as seen in ArmA, RmtB, and NpmA. All seven 16S rRNA MTases have been found exclusively in Gram-negative bacilli to date, and no plasmid-mediated 16S rRNA MTase has been reported in Gram-positive pathogenic microorganisms. Thus, we checked whether or not the RmtC could function in Gram-positive bacilli, and found that RmtC could indeed confer high-level resistance to gentamicin and kanamycin in Bacillus subtilis and Staphylococcus aureus. 16S rRNA MTases seemed to be functional to some extent in any bacterial species, regardless of the provenance of the 16S rRNA MTase gene responsible for aminoglycoside resistance.

Profile of Expression of Helicobacter pylori gamma-glutamyltranspeptidase.

BACKGROUND: Helicobacter pylori produces gamma-glutamyltranspeptidase (GGT), a potential virulence factor involved in induction of host cell apoptosis. Regulation of the production of this protein is not known. METHODS: The transcription start sites were determined by primer extension analysis. Transcription level of the GGT gene was examined by measuring the mRNA by RT-PCR and expression level of GGT protein was examined by Western blot analysis under different conditions. RESULTS: Two transcription start sites were identified; thymine at 78-bp upstream and adenine at 79-bp upstream from the ATG codon of the GGT gene. There was a possible -10 consensus promoter sequence (ATTAAT), but no apparent -35 consensus sequence was found. The transcription of the mRNA and the expression of the protein were at almost constant level during the course of culture. The mRNA level increased by exposure to low pH; however, the actual protein expression level remained almost constant. Addition of glutamine or glutamate did not affect the mRNA level and the protein expression level to a remarkable degree, nor did co-culture with AGS cells affect the GGT activity level. CONCLUSION: It was suggested that H. pylori GGT is constitutively expressed under various conditions.

Prevalence of fosfomycin resistance among CTX-M-producing Escherichia coli clinical isolates in Japan and identification of novel plasmid-mediated fosfomycin-modifying enzymes.

We evaluated the in vitro activity of fosfomycin against a total of 192 CTX-M beta-lactamase-producing Escherichia coli strains isolated in 70 Japanese clinical settings. Most of the isolates (96.4%) were found to be susceptible to fosfomycin. On the other hand, some of the resistant isolates were confirmed to harbor the novel transferable fosfomycin resistance determinants named FosA3 and FosC2, which efficaciously inactivate fosfomycin through glutathione S-transferase activity.

An outbreak of histoplasmosis among healthy young Japanese women after traveling to Southeast Asia.

Histoplasmosis, caused by Histoplasma capsulatum, is an endemic mycosis in many countries of the world except for Japan. Outbreaks of histoplasmosis among Japanese people are very rare and are mainly imported by travelers. We report an outbreak of histoplasmosis among healthy Japanese people who traveled to a resort area in Southeast Asia. Three young Japanese women traveled to Langkawi island, Malaysia and stayed on the island for five days without visiting caves, a known reservoir of H. capsulatum. All three individuals developed flu-like symptoms with multiple nodule shadows on chest X rays or chest CT scans at around ten days after their return to Japan. Serum samples obtained from the three subjects were positive for anti-Histoplasma antibody and specific PCR for H. capsulatum on lung biopsy specimens and the serum from one patient was positive. The clinical course of all three patients improved without the use of anti-fungal agents and no recurrence has been confirmed. Clinical attendants should consider histoplasmosis when they see patients with flu-like symptoms with abnormal chest X-rays after visiting H. capsulatum endemic areas, especially Southeast Asia.

[Recent trend and research issues related to antimicrobial-resistant bacteria].

The discovery of penicillin in 1928 was followed by the discovery and synthesis of various kinds of antimicrobial agents such as quinolone, aminogycoside, macrolide, tetracyclone, and oxazolidinone. These discoveries dramatically decreased the mortality rate due to infectious diseases. However, bacteria have also acquired antimicrobial-resistance genes or changed their own genes to oppose these antimicrobial agents, and now drug-resistant bacteria are becoming a serious clinical concern. Today, contagious diseases must be treated with the limited number of effective antimicrobial agents available. Infection control measures are required to prevent the spread of resistant bacteria in the clinical environment, and we must also increase our understanding of the drug-resistant mechanisms of bacteria. In this issue we wish to introduce the recent worldwide trend in antimicrobial-resistant bacteria, especially multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, and multidrug-resistant Pseudomonas aeruginosa, along with recently-discovered antimicrobial-resistant systems.

[First outbreak report of VIM-1 metallo-beta-lactamase producing Pseudomonas aeruginosa in Japan].

VIM-1 metallo-beta-lactamase (MBL) producing Pseudomonas aeruginosa was isolated from 35 Kobe City Medical Center General Hospital patients from September 2007 to July 2008. All but one were highly resistant to all beta-lactams, aminoglycoside, and fluoroquinolone, and one susceptible to amikacin. Strains negative to a disk diffusion screening test using sodium mercaptoacetate for detecting MBL numbered 35. PCR for MBL indicated all strains were positive for bla(VIWM-1). These strains were indistinguishable by pulsed-field gel electrophoresis, indicating an outbreak of infections caused by VIM-1 MBL producing Pseudomonas aeruginosa. After intervention to control contact, the outbreak was controlled.