Virulence-associated Genome Sequences of Pasteurella canis and Unique Toxin Gene Prevalence of P. canis and Pasteurella multocida Isolated from Humans and Companion Animals.

Background: Comparative analysis of virulence factors (VFs) between Pasteurella canis and Pasteurella multocida are lacking, although both cause zoonotic infections. We determined the virulence-associated genome sequence characteristics of P. canis and assessed the toxin gene prevalence unique to P. canis among clinical isolates of P. canis and P. multocida. Methods: We selected 10 P. canis and 16 P. multocida whole-genome sequences (WGSs) from the National Center for Biotechnology database. The VFanalyzer tool was used to estimate P. canis-characteristic VFs. Amino acid sequences of VFs were compared with multiple-aligned sequences. The genome structure containing P. canis-characteristic and adjacent loci was compared to the corresponding P. multocida genome structure. After designing primer sequences and assessing their accuracy, we examined the gene prevalence of the P. canis-characteristic VFs using PCR among clinical isolates of P. multocida and P. canis. Results: Using VFanalyzer, we found virulence-associated cytolethal distending toxin (cdt)A-cdtB-cdtC loci common to all P. canis WGSs that were not found in P. multocida WGSs. Similarities in the multiple alignments of CdtA-CdtB-CdtC amino acid sequences were found among the 10 P. canis WGSs. Shared or similar loci around cdtA-cdtB-cdtC were identified between the P. canis and P. multocida genome structures. The PCR-based cdtA-cdtB-cdtC prevalence differed for P. canis and P. multocida clinical isolates. Conclusions: P. canis-specific cdtA-cdtB-cdtC prevalence was identified among clinical isolates. These three loci may be unique toxin genes and promising targets for the rapid identification of P. canis in clinical settings.

Genetic shifts in methicillin-resistant Staphylococcus aureus epidemic clones and toxin gene profiles in Japan: comparative analysis among pre-epidemic, epidemic and post-epidemic phases.

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

The first cases of human bacteremia caused by Acinetobacter seifertii in Japan.

Acinetobacter seifertii, a novel species of Acinetobacter, was first reported in 2015. A. seifertii strains were isolated from human clinical specimens (blood, respiratory tract, and ulcer) and hospital environments. Here, we report the first cases of bacteremia caused by A. seifertii in patients with catheter-related bloodstream infection in Japan. The patients favorably recovered, without any complications, after removal of the peripheral intravenous catheters and administration of antibiotics. The pathogens were initially identified as Acinetobacter baumannii, using phenotypic methods and the MicroScan Walkaway System; however, rpoB gene sequence analysis indicated 99.54% similarity to A. seifertii. Moreover, antimicrobial susceptibility testing revealed that one of the strains was not susceptible to gentamicin and ceftazidime. Our report shows that Acinetobacter species other than A. baumannii can also cause nosocomial infections and that accurate methods for the identification of causative agents should be developed.

A novel cluster of Mycobacterium abscessus complex revealed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS).

Mycobacterium abscessus complex is a rapidly growing mycobacterium consisting of 3 subspecies, M. abscessus, Mycobacterium massiliense, and Mycobacterium bolletii. However, rapid and accurate species identification is difficult. We first evaluated a suitable protocol of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for distinguishing these subspecies. Then, we studied spectral signals by MALDI-TOF MS in 59 M. abscessus, 42 M. massiliense, and 2 M. bolletii. Among several specific spectral signals, 4 signals clearly differentiate M. massiliense from the other 2 subspecies, M. abscessus and M. bolletii. Moreover, 6 of the 42 M. massiliense isolates showed a spectral pattern similar to M. abscessus. These isolates correspond to the distinctive class of M. massiliense (cluster D) which is closer to M. abscessus by the previous variable number tandem repeat analysis. These results indicate that MALDI-TOF MS is not only useful for the identification of 3 subspecies of M. abscessus complex but also capable of distinguishing clusters of M. massiliense.

Desulfovibrio desulfuricans bacteremia in a patient hospitalized with acute cerebral infarction: case report and review.

Desulfovibrio spp. can be found in soil, water, and sewage, as well as in the digestive tracts of animals and humans. We report a case of Desulfovibrio desulfuricans bacteremia during hospitalization with acute cerebral infarction following aspiration bronchopneumonia and severe diarrhea, and the case strongly suggests that Desulfovibrio spp. bacteremia can occur as an infection due to disturbance of endogenous gut flora including antibiotic administration. Because Desulfovibrio spp. is difficult to detect in short-time incubation, its bacteremia is possibly overlooked in hospitalized patients. A few clinical cases of D. desulfuricans bacteremia have been reported in Japan, and they are reviewed briefly in this article.

Antimicrobial susceptibility profile of Acinetobacter species isolated from blood cultures in two Japanese university hospitals.

Carbapenem-resistant Acinetobacter baumannii has rapidly spread worldwide. This study investigated antibiotic susceptibility and genotypic resistance of 123 consecutive blood culture isolates of Acinetobacter species collected between 2003 and 2011 in two Japanese hospitals. The isolates were assigned to 13 species. Carbapenem resistance was detected in four isolates. Only one A. baumannii isolate had blaOXA-23 together with ISAba1; the remaining three isolates had IMP-1 metallo-ß-lactamase. Quinolone resistance was detected in five isolates that had point mutations in the quinolone resistance-determining region. The predominance of various non-A. baumannii species and low prevalence of carbapenem resistance among blood culture isolates of Acinetobacter species in two Japanese hospitals were confirmed.

Analysis of the influence of drug resistance factors on the efficacy of combinations of antibiotics for multidrug-resistant Pseudomonas aeruginosa isolated from hospitals located in the suburbs of Kanto area, Japan.

Infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa are very difficult to treat. The aim of this study was to develop more effective treatments by investigating in vitro the effects of combinations of antibiotics against 47 MDR P. aeruginosa isolates harbouring various resistance factors. The isolates included 41 (87%) metallo-ß-lactamase (MBL)-positive strains, 37 (79%) strains with mutations in OprD and 46 (98%) strains carrying the genes encoding aminoglycoside-modifying enzymes (AMEs). The quinolone resistance-determining region was mutated in all of the strains. These strains were classified into 16 groups according to amplified fragment length polymorphism and resistance factors. The effects of combinations of antibiotics on 16 representative strains were determined using a 'Break-point Checkerboard Plate' assay. Combinations of amikacin+aztreonam (coverage rate, 81.3%) and arbekacin+aztreonam (93.8%) inhibited growth. In contrast, combinations of ciprofloxacin+meropenem (6.3%) and ciprofloxacin+ceftazidime (12.5%) were much less effective. Aztreonam and arbekacin (or amikacin) are not substrates for MBLs and AMEs, respectively. We conclude that the combined effects of these drugs were possibly because of resistance factors.

In vitro combination effects of aztreonam and aminoglycoside against multidrug-resistant Pseudomonas aeruginosa in Japan.

The aim of this study was to evaluate the in vitro combination effects of aztreonam (AZT) and aminoglycosides against multidrug-resistant (MDR) Pseudomonas aeruginosa strains in Japan. We investigated 47 MDR P. aeruginosa strains collected from 8 facilities. We selected the aminoglycosides amikacin (AMK), gentamicin (GM), and arbekacin (ABK) to examine their effects when combined with AZT using the checkerboard method. Of the 47 MDR P. aeruginosa strains, 41 tested positive for metallo-ß-lactamase (MBL). In all combinations, aminoglycosides decreased the minimum inhibitory concentrations of AZT in a dose-dependent manner, and there was no apparent antagonism. The combination effects were scored on a scale of 0 to 4, and statistical analysis was performed using the Wilcoxon signed-rank test. In all 47 strains, AZT + ABK (mean score, 2.02) had the highest score, followed by AZT + AMK (1.68) and AZT + GM (1.38) (ABK versus GM, P < 0.0001). In 41 MBL-positive strains, AZT + ABK (mean score, 2.05) had the highest score, followed by AZT + AMK (1.56) and AZT + GM (1.37) (ABK versus AMK, P = 0.02, and ABK versus GM, P < 0.0001). AZT + ABK was the most promising combination regimen against MDR P. aeruginosa strains; the other promising combinations were AZT + AMK and AZT + GM.

Heterogeneously vancomycin-intermediate Staphylococcus aureus (hVISA) emerged before the clinical introduction of vancomycin in Japan: a retrospective study.

Vancomycin-intermediate Staphylococcus aureus (VISA) and its precursor, heterogeneous VISA (hVISA), are increasingly the cause of vancomycin treatment failure. Prolonged glycopeptide treatment causes the emergence of these pathogens. However, we recently reported that hVISA can be generated by methicillin-resistant S. aureus (MRSA) exposure to imipenem (Katayama et al., Antimicrob Agents Chemother. 53:3190-6). We report here a retrospective prevalence study of VISA and hVISA on 750 MRSA clinical strains isolated from 31 Japanese national university hospitals in 1990, the year before the introduction of injectable vancomycin into clinical use in Japan in 1991. No VISA strain was identified, but population analysis identified 38 hVISA strains (5.1%) from 19 hospitals. We also determined the nucleotide sequences of vraSR, walRK, clpP, and rpoB genes whose mutations are known to be associated with vancomycin resistance. When compared with vancomycin-susceptible MRSA strain N315, six of the 38 hVISA strains possessed nonsynonymous mutations in vraSR, seven in walRK, and two in rpoB genes, Thirteen of 38 (34.2%) hVISA strains possessed at least one of these mutations. Results were consistent with our hypothesis that hVISA was present in Japanese hospitals before the clinical introduction of vancomycin.

[Antimicrobial susceptibility and serotype distribution in perinatal group B Streptococcus isolates--a 1999-2009 multicenter study].

We determined temporary changes in group B Streptococcus antimicrobial susceptibility and serotype distribution from perinatal strains. We examined invasive microbiological isolates from neonates with early-onset group B streptococcal disease (n = 14), and colonized isolates from those born uneventfully (n = 55) and from the genital tracts of pregnant and puerperal women (n = 198), collected between 1999 and 2009. All isolates were susceptible to penicillin. No significant differences were seen in susceptibility of 12 antimicrobial agents examined between invasive and colonized isolates. MIC50, MIC90, and resistance did not differ between stage I (1999-2005) and II (2006-2009) isolates. Serotype distribution significantly differed, however, serotypes III and Ia predominated among invasive isolates, while serotypes Ib and VI were common among their colonized counterparts. These findings suggest that to date, penicillin remains effective in intrapartum prophylactic use in colonized pregnant women.

Molecular emm genotyping and antibiotic susceptibility of Streptococcus dysgalactiae subsp. equisimilis isolated from invasive and non-invasive infections.

To analyse the characteristics of infections caused by Streptococcus dysgalactiae subsp. equisimilis, clinical isolates (n=145) were collected at 11 medical institutions between September 2003 and October 2005. These isolates belonged to Lancefield group A (n=5), group C (n=18) or group G (n=122). Among all isolates, 42 strains were isolated from sterile samples such as blood, synovial fluid and tissue specimens from patients who were mostly over 50 years with invasive infections, and included seven cases of streptococcal toxic shock syndrome and necrotizing fasciitis. In contrast, the remaining 103 were isolated mainly from patients of all age groups with non-invasive infections such as pharyngotonsillitis. These isolates were classified into 25 types based on emm genotyping. A significant difference in emm types was observed between isolates from invasive and non-invasive infections (P<0.001): stG485, stG6792 and stG2078 predominated among isolates from invasive infections. A phylogenetic tree of complete open reading frames of emm genes in this organism showed high homology with those of Streptococcus pyogenes, but not with those of other streptococci. The presence of five different clones was estimated based on DNA profiles of isolates from invasive infections obtained by PFGE. Genes for resistance to macrolides [erm(A), three isolates; erm(B), five isolates; mef(A), seven isolates] and levofloxacin (mutations in gyrA and parC, four isolates) were identified in this organism. These results suggest the need for further nationwide surveillance of invasive infections caused by S. dysgalactiae subsp. equisimilis.

Fluoroquinolone enhances the mutation frequency for meropenem-selected carbapenem resistance in Pseudomonas aeruginosa, but use of the high-potency drug doripenem inhibits mutant formation.

The mutation frequency for carbapenem resistance in Pseudomonas aeruginosa strains that were selected with carbapenems was enhanced in the presence of subinhibitory concentrations of fluoroquinolones. The mutants showed either a loss of OprD activity or increased mexAB-oprM expression. The highest mutant isolation frequency was obtained by selection with meropenem, while doripenem inhibited mutant growth.

Recurrence of heterogeneous methicillin-resistant Staphylococcus aureus (MRSA) among the MRSA clinical isolates in a Japanese university hospital.

OBJECTIVES: In the early 1980s, heterogeneous methicillin-resistant Staphylococcus aureus (hetero-MRSA) strains were predominant in the University of Tokyo Hospital. But, in the 1990s, they were completely substituted by homogeneously highly methicillin-resistant S. aureus (homo-MRSA) strains. Since 2000, however, we started observing an increase in MRSA strains with low cefazolin MICs (MRCLSA). This study was performed to understand the phenomenon by characterization of the 'cefazolin-susceptible' MRSA strains. METHODS: A total of 39 MRCLSA strains were collected between July 2000 and June 2002 and compared with 10 homo-MRSA strains isolated during the same period for their antibiograms and genotypes. The strains were also compared with the hetero-MRSA strains isolated in the same hospital in the early 1980s. RESULTS: In contrast to the homogeneous genotype [multilocus sequence type 5 and SCCmec type II.1 (IIa)] and multiresistant nature of the homo-MRSA strains, the MRCLSA strains were composed of various genotypes as revealed by multilocus sequence typing and SCCmec typing and had resistance only to a limited number of antibiotics. Most of the MRCLSA strains were also genetically differentiated from the hetero-MRSA strains of the 1980s. However, population analysis revealed that all of the MRCLSA strains were classified as hetero-MRSA strains. CONCLUSIONS: A new group of hetero-MRSA strains genetically distinct from those dominant in the same hospital in the early 1980s might have emerged in the community and started invading the university hospital. This phenomenon may be caused by the change in the pattern of antibiotic use.

[Precautions regarding prevent acute urethritis caused by Neisseria meningitidis in Japan].

In Japan, Neisseria meningitidis is not sufficiently recognized as the primary causative bacteria of sexually transmitted diseases (STDs) as the number of reported cases is small. Here, we summarize reports from 3 medical institutions, present clinical courses for each case, as well recommending precautions to prevent infection with this bacterium. Fourteen cases of N. meningitidis urethritis (MU) were admitted between April 2001 and June 2006. All patients were male, consulted a doctor after experiencing subjective symptoms, such as micturition pain and pus discharge, and were diagnosed as having urethritis using isolation culture methods. In 8 of the 14 cases, history of sexual contact in the preclinical stage was confirmed, and contact was with a commercial sex worker (CSW) in 6 of these cases. Many of these patients recalled oral contact. All strains indicated susceptibility to many drugs, and there were no problems with treatment. With regard to serotype, there were 10 cases of type Y, 1 case of type B, and 3 cases that were not classifiable or unidentified. In addition, among the 9 strains that were subjected to genotype identification, 7 strains were ST-23. The recent increase in availability of nucleic acid amplification methods has facilitated simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis. However, we fear that MU will become latent. For screening of urethritis, Gram staining and culture of urethral material must be performed to detect this disease. The relationship of the detected strain and its role in the pathogenesis of meningitis are uncertain, but its serotype and genotype are common in cases of meningitis. Thus, precautions are required to prevent spread of this bacterium.