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.

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.

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.

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.

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.

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.

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.

[Current biosafety in clinical laboratories in Japan: report of questionnaires' data obtained from clinical laboratory personnel in Japan].

To determine the status of biosafety in clinical laboratories in Japan, we conducted a survey using questionnaires on the biosafety of laboratory personnel in 2004. We obtained data from 431 hospitals (response: 59.5%). Respondents were 301 institutions (70%) having biological safety cabinets (BSCs). BSCs were held in 78% of microbiological laboratories, 7.9% of genetic laboratories, 2.7% of histopathological laboratories, and 1% or less at other laboratories. A clean bench in examination rooms for acid-fast bacilli was applied at 20 hospitals. We found 28 cases of possible laboratory-associated tuberculosis infection, 25 of which were associated with lack of BSC. Other risk factors were immature skills and insufficiently skilled eguipment operation. The frequency of rupture accidents during specimen centrifugation was 67% in dealing with blood and 9.7% in collecting acid-fast bacilli. Half or more accidents were related to inadequate sample tube materials. Technologists were shown to be working on blood collection in many hospitals (75%), and 1,534 events of self-inflicted needle puncture developed in the last 5 years. These results suggest that biosafety systems are woefully lacking or inadequate in clinical laboratories in Japan and must be established at the earliest possible opportunity.

[Isolation and identification of Helicobacter cinaedi and H. cinaedi-like organisms isolated from blood culture in practical laboratory procedures].

A spiral Gram-negative bacterium, Helicobacter cinaedi, has been isolated from clinical specimens of immunodeficient patients in Japan. We determined the isolation procedure and features using practical phenotypic tests for H. cinaedi isolated from blood culture. We applied 28 clinically isolated strains from 5 different metropolitan institutions, whose species was reconfirmed in technical support at another expert veterinary medicine institute in identifying H. cinaedi. We also used 6 strains of type and reference. Positive signals from aerobic bottles in automated blood culture systems, BACTEC, were observed 4-9 days after culture was started. A thin spread colony was found 3-4 days after the start of subcultures consisting of growth on 5% sheep blood agar plates with immediate incubation under wet and microaerophilic conditions at 35 degrees C. In phenotypic tests, all specimens of H. cinaedi were shown to be positive in nitrate reduction and to be negative in both indoxyl acetate hydrolysis and urease activity, underlining the usefulness of these 3 phenotypes. Tests of alkaline phosphatase hydrolysis, growth at different temperatures, and growth with 1% glycin provided additional information when referencing standard results of identification. We took speculative profiling data of H. cinaedi identification with a simple phenotypic kit, Api campy, indicating its usefulness in testing. These observations suggest combined methods of previously available tests (nitrate reduction, indoxyl acetate hydrolysis, and urease activity) with Api campy may be useful in differentiating H. cinaedi from H. cinaedi-like microorganisms when observing the characteristic thin spread colony within subcultures under microaerophilic conditions.

[The impact of the amendment to the Infectious Diseases Control Law on the management of clinical laboratories].

In December 2006, the Japanese parliament promulgated an amendment of the Law Concerning the Prevention of Infectious Diseases and Medical Care for Patients of Infections (the Infectious Diseases Control Law). The main elements of the amendment provide a pathogen control system that will prevent biological terrorism, global and accidental spread of infectious diseases and allow for comprehensive control of infectious diseases, including tuberculosis. State intervention was substantially curtailed in the original Infectious Diseases Control Law because of the demand at the time for decentralization. However, the rising danger of bio-terrorism has established an urgent need for direct control by the state. Developing the necessary regulations for laboratory safety and pathogen collection to prevent bioterrorism is an onerous task. The rules must take account of real conditions on the ground and to be seen to work. However, whereas the new rules may be efficient in the prevention of terrorism, there is a real suspicion that they will impede and obstruct the day-to-day routine of the clinical laboratories. We report a questionnaire concerning the influence of this law on the clinical laboratory management in 84 university hospital laboratories. The imposition of well-intentioned but implausible regulations that impair or disrupt routine laboratory work encourages employees to selectively ignore regulations that they feel are impractical or irrelevant.

[Large-scale questionnaire surveillance concerning invasive infections with group C and G streptococci].

A large-scale questionnaire surveillance was conducted regarding the onset of invasive infections with beta-hemolytic group C (GCS) and group G (GGS) streptococci from clinical specimens that are normally aseptic and the backgrounds of these cases. The surveillance period of the questionnaire was 8 months from January to August 2005. Completed questionnaires were received from the clinical laboratories of 193 medical institutions. One hundred two clinical laboratories (52.8%) had isolated these beta-hemolytic streptococci. Of all the isolates, GCS and GGS accounted for 25 and 216 cases, respectively, or a ratio of almost 1:10. Isolates from blood cultures accounted for half the number of all isolates, followed by isolates from atretic pus or joint fluid. The isolates gradually became more prevalent from patients in their 40s, and peaked in patients in their 70s. The most prevalent disorder, described in 184 cases, was suppurative disease followed by (in descending order), bacteremia, sepsis, arthritis purulenta and cellulitis. A small number of patients had developed with streptococcal toxic shock syndrome, empyema or meningitis. Most of the patients had an underlying disease, such as diabetes mellitus, malignancy or cerebrovascular disease (in descending order). We conclude from the above findings that background factors in patients as well as identification of the pathogen should be made public when GCS or GGS is isolated from normally aseptic clinical specimens.

[A case of severe necrotizing cellulitis caused by group G Streptococcus dysgalactiae subsp. equisimilis].

Group G streptococcus (GGS) is infrequently associated with severe invasive soft tissue infection and toxic shock syndrome. A 74-year-old woman with a history of lymphedema of the lower extremities after surgical and radiation therapy for uterine cancer and diabetic mellitus and admitted for swelling of the right leg, fever, and dyspnea. She presented with shock and necrotizing cellulitis of the right lower extremity. Laboratory tests showed leukocytepenia, acute renal and liver dysfunction, and muscle damage. She rapidly developed multiple organ failure and necrotizing cellulitis. A swab from skin vesicle, throat, and blood culture grew Group G Streptococcus dysgalactiae subsp. equisimilis. Despite endotoxin hemoadsorption therapy, administration of antibiotics, and intravenous immunoglobulin, she died 9 days after admission due to toxic shock syndrome caused by GGS. The M-protein gene (emm) typing of GGS isolated from both blood and skin lesion showed stG 485.0. Three virulence genes, sagA, slo and skcg, were detected from GGS isolated from them.

[The efficacy of safety winged steel needles on needlestick injuries].

Safety winged steel needles were introduced at the University of Tokyo Hospital in January 2001. We studied their effect in needlestick injuries. A total of 952 'needlestick and sharp-object injuries were reported. From January 1999 to December 2004, Cases of injury with winged steel needles decreased dramatically soon after safety devices were introduced, from 19.8% in Apr.-Dec.2000 to 6.7% in 2001 and 5.5% in 2002 (p < .01). They began to increase, however, in July 2002, decreased again after medical staff members mere given lectures and notices by e-mail. Due to the introduction of safety devices, cases classified as a "while recapping a used needle" and "when puncturing rubber stoppers" decreased. Among 17 injuries with safety winged steel needles, the most common cases were "safety mechanism not activated". We estimated that 76.5% of cases with safety winged steel needles could be prevented if they were used properly. In conclusion, the introduction of safety winged steel needles effectively reduced cases of injury with such needles. It is thus important to regularly remind hospital staff of safety device techniques and information reduce the such injuries.

[Emm typing by genetic identification of Streptococcus dysgalactiae subsp. equisimilis and susceptibility to oral antibiotics].

A total of 593 beta-hemolytic streptococci belonging to Lancefield group A (GAS), group C (GCS) or group G (GGS) according to agglutination tests were collected from 11 medical institutions between September 2003 and October 2005. In total, 128 strains were identified as Streptococcus dysgalactiae subsp. equisimilis (S. equisimilis) using physiological tests. Of these strains, 5 strains were agglutinated to Lancefield group A, 17 strains to group C, and 106 strains to group G. Most of these strains were largely isolated from clinical specimens collected from young patients with respiratory infections and middle-aged patients (in their 40s); most of the strains were isolated from blood, atretic pus, or joint fluid. Genetic analysis of the emm gene encoding the M protein revealed that these strains could be classified into 27 types. Also, many emm types were found in strains isolated from normally aseptic clinical specimens. In addition, all strains tested had slo, sagA, and skcg genes, which contributed to their virulence. The susceptibility of the strains to oral penicillin and cephalosporin antibiotics was excellent, with MICs ranging from 0.016 to 0.031mg/mL. In contrast, strains carrying the macrolide resistant elements of the ermA, ermB, and mefA genes and strains showing a high resistance to levofloxacin were also confirmed in this study. These results suggest that beta-hemolytic streptococci, except for S. pyogenes and S. agalactiae, should be reconsidered as a causative pathogen in streptococcal infections.

Genetic diversity of the internal transcribed spacers (ITS) and 5.8S rRNA genes among the clinical isolates of Candida parapsilosis in Brazil and Japan.

The internal transcribed spacer (ITS) region including 5.8S rDNA sequences of 58 isolates of Candida parapsilosis in Brazil and Japan was analyzed. Although most of the C. parapsilosis strains tested were confirmed to belong to three already reported genetically distinct groups (I, II and III) based on their ITS region sequences, 5 strains of the Brazilian isolates showed different sequences from those heretofore reported and suggested a presence of new genotype. For these strains of C. parapsilosis, we proposed a new genetic group (IV). The sequence similarities of this new group of IV to I, II and III were 87.4%, 94.7% and 87.3% in the ITS1 region, respectively. Genetic diversity in ITS regions of the remaining C. parapsilosis strains in Brazil and Japan was also discussed.