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.

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.

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.

[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.

[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.

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.

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.

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.

[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.

[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.

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.

Practical disk diffusion test for detecting group B streptococcus with reduced penicillin susceptibility.

Although group B streptococcus (GBS) has been considered to be uniformly susceptible to beta-lactams, the presence of GBS with reduced penicillin susceptibility (PRGBS) was recently confirmed genetically. We developed a feasible and reliable method for screening PRGBS in clinical microbiology laboratories using a combination of ceftibuten, oxacillin, and ceftizoxime disks.

Change in the prevalence of extended-spectrum-beta-lactamase-producing Escherichia coli in Japan by clonal spread.

INTRODUCTION: In the early 2000s, there was a rapid increase in extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in hospital settings throughout Japan. The reasons for this rapid increase are unclear. METHODS: Between 2002 and 2003, 142 clinical isolates of E. coli suspected of producing ESBL were obtained from 37 hospitals and commercial clinical laboratories in geographically distinct regions throughout Japan. They were tested for ESBL types and further subtyped for serogroups, fimH single nucleotide polymorphism, pulsed-field gel electrophoresis patterns and multilocus sequence type (MLST). Representative isolates were also subjected to plasmid analysis. RESULTS: Of 142 E. coli isolates suspected of producing ESBL, 130 were confirmed as harbouring blaCTX-M by PCR analysis and sequencing. Of these, 84 (65%) harboured CTX-M-9-group blaCTX-M. Two serogroups O25 and O86 accounted for 41% of the 130 blaCTX-M-positive E. coli. All O86 serogroup strains belonged to ST38 by MLST and they formed 18% of all the blaCTX-M-positive E. coli. Serogroup O25 strains belonged to ST131 and ST73, and formed 21% and 1% of blaCTX-M-positive E. coli, respectively. Seven characterized plasmids carrying blaCTX-M genes belonged to three distinct incompatibility groups: IncF, IncN and IncI1. CONCLUSIONS: In this study, clonally related strains of E. coli accounted for a large proportion of blaCTX-M-positive E. coli. This high proportion of clonal groups identified in different regions of Japan suggests their recent spread by mechanisms other than healthcare-associated transmission. These observations imply that restricting antimicrobial use in human clinical settings may have limited impact on the spread of ESBL-producing E. coli.

First molecular characterization of group B streptococci with reduced penicillin susceptibility.

Group B streptococci (GBS; Streptococcus agalactiae) are the leading cause of neonatal invasive diseases and are also important pathogens for adults. Penicillins are the drugs of first choice for the treatment of GBS infections, since GBS have been regarded to be uniformly susceptible to penicillins so far. Here we characterize the first strains of GBS with reduced penicillin susceptibility (PRGBS) identified in Japan. Fourteen PRGBS strains were clinically isolated from the sputa of elderly patients from 1995 to 2005; and the MICs of penicillin, oxacillin, and ceftizoxime ranged from 0.25 to 1 microg/ml, 2 to 8 microg/ml, and 4 to 128 microg/ml, respectively. Moreover, some strains were also insusceptible to ampicillin, cefazolin, cefepime, and cefotaxime. All the PRGBS isolates tested possessed a few amino acid substitutions adjacent to the conserved SSN and KSG motifs (amino acids 402 to 404 and 552 to 554, respectively) of PBP 2X, and the amino acid substitutions could be classified into two types, Q557E and V405A. Western blotting analysis of the 14 clinical isolates with anti-PBP 2X-specific serum suggested that the amount of PBP 2X among the 14 PRGBS isolates was reduced, although the 2 ATCC strains produced a significant amount of PBP 2X. The introduction of PRGBS-derived PBP 2X genes into penicillin-susceptible strains through allelic exchange elevated their penicillin insusceptibility, suggesting that these altered PBP 2X genes are responsible for the penicillin insusceptibility in PRGBS strains. In this study, we characterized for the first time PRGBS strains on a molecular basis, although several reports have so far mentioned the existence of beta-lactam-insusceptible GBS from a phenotypic standpoint.

Characterization and antimicrobial susceptibility of Dysgonomonas capnocytophagoides isolated from human blood sample.

Dysgonomonas capnocytophagoides belongs to a group of facultative anaerobic Gram-negative coccobacilli that was formerly designated CDC group DF-3. We evaluated the characteristics of this microbe and its susceptibility to antimicrobial agents. In this study, D. capnocytophagoides was isolated by anaerobic blood cultures from a 78-year-old male with pancreatic cancer, ischemic heart disease, and diabetes mellitus, who also showed symptoms of cholangitis. The isolated strain demonstrated resistance to various beta-lactams, erythromycin, aminoglycosides, and fluoroquinolones, but was susceptible to sulfamethoxazole-trimethoprim, clindamycin, minocycline, and chloramphenicol. The results of all biochemical tests and the homology of the 16S rRNA gene were consistent with previous reports of D. capnocytophagoides.

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.

Dissemination of 16S rRNA methylase-mediated highly amikacin-resistant isolates of Klebsiella pneumoniae and Acinetobacter baumannii in Korea.

Novel 16S rRNA methylase-mediated high-level resistance to amikacin and arbekacin has been reported recently in clinical isolates of Gram-negative bacilli only from several countries. We tested amikacin- or arbekacin-nonsusceptible Gram-negative bacilli isolated in 2003 and 2005 at a tertiary-care hospital in Korea by polymerase chain reaction to detect 16S rRNA methylase genes. armA alleles were detected in 14 isolates of Klebsiella pneumoniae, 10 other species of Enterobacteriaceae, and 16 Acinetobacter baumannii, whereas the rmtB allele was detected in 1 K. pneumoniae isolate. The resistance 1st detected in 2003 persisted in 2005. 16S rRNA methylase-producing isolates were highly resistant to arbekacin and amikacin, and were mostly coresistant to levofloxacin. Most K. pneumoniae isolates also produced extended-spectrum beta-lactamases and plasmid-mediated AmpC beta-lactamases, and most A. baumannii isolates were nonsusceptible to carbapenems.

Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa.

BACKGROUND: Bacteria develop resistance to aminoglycosides by producing aminoglycoside-modifying enzymes such as acetyltransferase, phosphorylase, and adenyltransferase. These enzymes, however, cannot confer consistent resistance to various aminoglycosides because of their substrate specificity. Notwithstanding, a Pseudomonas aeruginosa strain AR-2 showing high-level resistance (minimum inhibitory concentration >1024 mg/L) to various aminoglycosides was isolated clinically. We aimed to clone and characterise the genetic determinant of this resistance. METHODS: We used conventional methods for DNA manipulation, susceptibility testing, and gene analyses to clone and characterise the genetic determinant of the resistance seen. PCR detection of the gene was also done on a stock of P aeruginosa strains that were isolated clinically since 1997. FINDINGS: An aminoglycoside-resistance gene, designated rmtA, was identified in P aeruginosa AR-2. The Escherichia coli transformant and transconjugant harbouring the rmtA gene showed very high-level resistance to various aminoglycosides, including amikacin, tobramycin, isepamicin, arbekacin, kanamycin, and gentamicin. The 756-bp nucleotide rmtA gene encoded a protein, RmtA. This protein showed considerable similarity to the 16S rRNA methylases of aminoglycoside-producing actinomycetes, which protect bacterial 16S rRNA from intrinsic aminoglycosides by methylation. Incorporation of radiolabelled methyl groups into the 30S ribosome was detected in the presence of RmtA. Of 1113 clinically isolated P aeruginosa strains, nine carried the rmtA gene, as shown by PCR analyses. INTERPRETATION: Our findings strongly suggest intergeneric lateral gene transfer of 16S rRNA methylase gene from some aminoglycoside-producing microorganisms to P aeruginosa. Further dissemination of the rmtA gene in nosocomial bacteria could be a matter of concern in the future.