[Adverse Events after the Introduction of Quadrivalent Influenza Vaccine in Comparison with AH1pdm Vaccine (2009) in Japan].

Inactivated quadrivalent influenza vaccine (IIV4) has been used as seasonal influenza vaccine since 2016 in Japan. This study examined the safety of IIV4 in comparison with the AH1pdm monovalent vaccine used for novel influenza in 2009. Questionnaire surveillance associated with adverse events (AEs) was conducted at Chiba University Hospital, Japan. After being vaccinated, all health care workers (HCWs) were given a daily AEs check sheet on which they recorded solicited events, the same surveillance program used after AH1pdm vaccination in 2009. The frequency of injection site AEs with IIV4 was significantly higher than with the monovalent vaccine, but there was no significant difference with systemic AEs. Injection site and systemic AEs were reported as 83.7% and 25.5%, respectively, with IIV4. The grades of AE, mild, moderate and severe, were 67.2%, 16.4% and 0.1% with IIV4, respectively, indicating that almost all of the AEs reported with IIV4 were mild or moderate. Systemic AEs with IIV4 and monovalent vaccine were reported to be 25.5% and 23.1%, respectively, with the difference not being significant. The grade of AEs with IIV4, mild, moderate and severe, was 19.1%, 5.6% and 0.9%, respectively. The ratio of HCWs reporting AEs peaked at around 80% on day 1, then decreasing to less than 5% by day 7. AEs with IIV4 were reported more frequently compared with the AH1pdm monovalent vaccine. However, in consideration of the grade and duration of AEs, IIV4 was a well-tolerated, safe vaccine.

Rapid Discrimination between Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus Using MALDI-TOF Mass Spectrometry.

　Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major pathogens responsible for nosocomial infections. The presence of MRSA in a hospital is detrimental to patients and to hospital management. Thus, rapid identification of MRSA is needed. Here, we report on a prospective method to rapidly discriminate of MSSA from MRSA using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and support vector machine (SVM) analysis in 160 clinical isolates of S. aureus. The predictive model was tested using 100 S. aureus isolates (50 MSSA and 50 MRSA). The identification rates were 90.0% for MSSA and 87.5% for MRSA in a 10-fold cross-validation SVM. In blind test sets, 60 S. aureus isolates (30 MSSA and 30 MRSA) were correctly classified, with identification rates of 93.3% for MSSA and 86.7% for MRSA. The method proposed in this study using the predictive model enables detection of one colony in 5 minutes, and thus is useful at clinical sites at which rapid discrimination of MRSA from MSSA is required.

Clinical and bacteriological analyses of bacteremia due to Corynebacterium striatum.

BACKGROUND: Corynebacterium striatum was recently recognized as a potential pathogen of various infectious diseases. However, the clinical entity of this microorganism has not been clearly identified. Therefore, we analyzed C. striatum isolates from blood culture and explored their clinical determinants. METHODS: We reviewed the medical records of all patients from whom C. striatum isolates were recovered from blood culture for analysis of the patients' backgrounds and clinical course including response to antimicrobial therapy and prognosis. RESULTS: During the 5-year study period (January 2010 to December 2014), 24 C. striatum strains were isolated from blood samples, and the frequency of C. striatum bacteremia increased. The majority of the strains were multidrug resistant. All of the tested strains were susceptible to only vancomycin. The age at onset of C. striatum bacteremia encompassed all adult age groups, and at least one underlying condition was documented in all patients. Thirteen of the 24 patients were cured using appropriate antibiotics (true infection group); however, 11 of the 24 patients were cured using inappropriate antibiotic therapy or no antibiotics (contamination group). Malignancy and neutropenia significantly increased the odds of true C. striatum bloodstream infection. CONCLUSIONS: The Corynebacterium species is often considered a contaminant when isolated in culture. Instead, particularly when the strain is isolated from blood, the species should be considered clinically relevant and identified to the species level; in addition, antimicrobial susceptibility testing is recommended.

Linezolid-resistant Staphylococcus epidermidis associated with long-term, repeated linezolid use in a pediatric patient.

We report an 8-year-old patient with catheter-related bacteremia caused by linezolid-resistant Staphylococcus epidermidis that was isolated after the long-term, repeated use of linezolid. Three S. epidermidis strains isolated from this patient were bacteriologically analyzed. While the strain isolated prior to linezolid initiation was susceptible to linezolid, two strains after linezolid therapy displayed low-level linezolid susceptibility (MIC, 4 mg/L) and linezolid resistance (MIC, 16 mg/L). T2500A mutation in two copies and G2575T mutations in three copies of 23S rRNA were detected in the low-susceptible strain and the resistant strain, respectively. Linezolid-resistant S. epidermidis infection is rare, but may occur with the long-term administration of linezolid.

Identification of Nocardia species using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

BACKGROUND: The MALDI (matrix-assisted laser desorption/ionization) Biotyper system for bacterial identification has already been utilized in clinical microbiology laboratories as a successful clinical application of protoemics. However, in cases of Nocardia, mass spectra suitable for MALDI Biotyper identification are often not obtained if such specimens are processed like general bacteria. This problem is related to the insufficiencies in bacterial spectrum databases that preclude accurate specimen identification. Here, we developed a bacterial processing method to improve mass spectra from specimens of the genus Nocardia. In addition, with the new processing method, we constructed a novel in-house bacterial database that combines a commercial database and mass spectra of Nocardia strains from the Department of Clinical Laboratory at Chiba University Hospital (DCLC) and the Medical Mycology Research Center at Chiba University (MMRC). RESULTS: The newly developed method (Nocardia Extraction Method at DCLC [NECLC]) based on ethanol-formic acid extraction (EFAE) improved mass spectra obtained from Nocardia specimens. The Nocardia in-house database at Chiba University Hospital (NDCUH) was then successfully validated. In brief, prior to introduction of the NECLC and NDCUH, 10 of 64 (15.6%) clinical isolates were identified at the species level and 16 isolates (25.0%) could only be identified at the genus level. In contrast, after the introduction, 58 isolates (90.6%) were identified at the species level and 6 isolates (9.4%) were identified at the genus level. CONCLUSIONS: The results of this study suggest that MALDI-TOF (time-of-flight) Biotyper system can identify Nocardia accurately in a short time in combination with a simple processing method and an in-house database.

Molecular characterization of linezolid-resistant CoNS isolates in Japan.

OBJECTIVES: Linezolid has been reported to remain active against 98% of staphylococci with resistance identified in 0.05% of Staphylococcus aureus and 1.4% of CoNS. The objective of this study was to characterize the linezolid-resistance mechanisms in the linezolid-resistant CoNS strains isolated in Japan. METHODS: Staphylococcus capitis strains exhibiting linezolid MICs >8 mg/L isolated from inpatients between 2012 and 2014 were screened for cfr and mutations in 23S rRNA, L3 and L4 by PCR/sequencing. Isolates were also examined for mutations in the rlmN gene. RESULTS: S. capitis had six 23S rRNA alleles. Five S. capitis isolates displayed linezolid MICs of 8, 16 and 32 mg/L. G2576U mutations were detected in three, four or five copies of 23S rRNA in all isolates. In two isolates exhibiting the highest linezolid MIC (32 mg/L) there was a large deletion in a single copy of 23S rRNA. Repeated 10 bp sequences were found in both 16S and 23S rRNAs, suggesting deletion by recombination between the repeats. One isolate had the mutation Ala-142→Thr in the ribosomal protein L3. All linezolid-resistant isolates also demonstrated mutations in the gene encoding RlmN methyltransferase, leading to Thr-62→Met and Gly-148→Ser. CONCLUSIONS: Multiple mechanisms appeared to be responsible for the elevated linezolid resistance in S. capitis isolates: a G2576U mutation in different numbers of copies of 23S rRNA, loss of a single copy of 23S rRNA and a mutation in the ribosomal protein L3, suggesting the accumulation of independent mutational events.

[Nationwide surveillance of parenteral antibiotics containing meropenem activities against clinically isolated strains in 2012].

The nationwide surveillance of antibacterial susceptibility to meropenem (MEPM) and other parenteral antibiotics against clinical isolates during 2012 in Japan was conducted. A total of 2985 strains including 955 strains of Gram-positive bacteria, 1782 strains of Gram-negative bacteria, and 248 strains of anaerobic bacteria obtained from 31 medical institutions were examined. The results were as follows; 1. MEPM was more active than the other carbapenem antibiotics tested against Gram-negative bacteria, especially against enterobacteriaceae and Haemophilus influenzae. MEPM was also active against most of the species tested in Gram-positive and anaerobic bacteria, except for multi-drug resistant strains including methicillin-resistant Staphylococcus aureus (MRSA). 2. Of all species tested, there were no species, which MIC90 of MEPM was more than 4-fold higher than those in our previous studies in 2009 or 2006. Therefore, the tendency to increase in antimicrobial resistance rates was not observed. 3. MEPM resistance against Pseudomonas aeruginosa was 17.8% (56/315 strains). Compared to our previous results, it was the lowest than that in 2006 and 2009. 4. Carbapenem-resistant Klebsiella pneumoniae, and multi-drug-resistant Acinetobacter species, which emerged in worldwide, were not observed. 5. The proportion of extended-spectrum beta-lactamase (ESBL) strains was 6.2% (59/951 strains) in enterobacteriaceae, which increased compared with that of our previous studies in 2009 or before. Whereas, the proportion of metallo-beta-lactamase strains was 1.6% (5/315 strains) in P. aeruginosa, which was stable. In conclusion, the results from this surveillance suggest that MEPM retains its potent and broad antibacterial activity and therefore is a clinically useful carbapenem for serious infections treatment at present, 17 years passed after available for commercial use in Japan.

Direct application of MALDI-TOF mass spectrometry to cerebrospinal fluid for rapid pathogen identification in a patient with bacterial meningitis.

BACKGROUND: Bacterial meningitis is a neurological emergency. Early diagnosis and rapid initiation of antimicrobial therapy are vital. METHODS: Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is increasingly used as a rapid and accurate microbial diagnostic method for species identification of pathogens. Although this technology requires a growth step to obtain bacterial colonies for the acquisition of substantial spectra in most cases, it can also be used to analyze clinical specimens such as urine and cerebrospinal fluid for direct bacterial identification. There are very few reports describing the use of MALDI-TOF MS for the direct detection of microorganisms causing bacterial meningitis. RESULTS: We describe a case of bacterial meningitis caused by Klebsiella pneumoniae in which MALDI-TOF MS provided a rapid bacteriological diagnosis, thus enabling early and appropriate treatment. CONCLUSIONS: Identification of microbes based on MALDI-TOF MS is now an important technology in clinical microbiology laboratories that are required to provide a rapid diagnosis of bacterial meningitis.

Changes in nasopharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis among healthy children attending a day-care centre before and after official financial support for the 7-valent pneumococcal conjugate vaccine and H. influenzae type b vaccine in Japan.

The 7-valent pneumococcal conjugate vaccine (PCV7) and Haemophilus influenzae type b (Hib) vaccine reduce nasopharyngeal carriage of vaccine-type bacteria, which may in turn influence the presence of other nasopharyngeal bacterial pathogens. To investigate this possibility, nasopharyngeal carriage of potential pathogens was examined before and after official financial support was provided to offer the PCV7 and Hib vaccines in healthy children attending a day care centre in Japan during 2011-2012. Despite a virtual disappearance of PCV7 serotypes over time, the overall pneumococcal carriage rate remained unchanged. Although others have reported an increase in PCV13 serotypes following PCV7 vaccination, only non-PCV13 serotypes were observed to have increased in this study. The majority of H. influenzae isolates were non-typeable and Hib was not found. Our data identified an unexpected pattern of pneumococcal serotype replacement following PCV7. Continuous monitoring of pneumococcal carriage is important for decisions regarding the future of national vaccination policy in Japan.

Efficacy of Levofloxacin Based Triple and High-Dose PPI-Amoxicillin Dual Eradication Therapy for Helicobacter pylori after Failures of First- and Second-Line Therapies.

Objectives. The aim of this study was to investigate and compare the eradication rate of Helicobacter pylori as the third-line triple therapy with rabeprazole (RPZ) + amoxicillin (AMPC) + levofloxacin (LVFX) and high-dose RPZ + AMPC. Methods. 51 patients who failed Japanese first-line (proton pump inhibitor (PPI) + AMPC + clarithromycin) and second-line (PPI + AMPC + metronidazole) eradication therapy were randomly assigned at a 1 : 1 ratio to one of the following third-line eradication groups: (1) RAL group: RPZ 10 mg (b.i.d.), AMPC 750 mg (b.i.d.), and LVFX 500 mg (o.d.) for 10 days; (2) RA group: RPZ 10 mg (q.i.d.) and AMPC 500 mg (q.i.d.) for 14 days. Patients who failed to respond to third-line eradication therapy received salvage therapy. Results. The rates of eradication success, based on intention to treat (ITT) analysis, were 45.8% in the RAL group and 40.7% in the RA group. The overall eradication rates were 73.9% in the RAL group and 64.0% in the RA group. There was no significant difference between the two groups. Conclusions. The third-line triple therapy with RPZ, AMPC, and LVFX was as effective as that with high-dose RPZ and AMPC.

[Rapid identification of microorganisms using MALDI-TOF mass spectrometry].

In a clinical diagnostic microbiology laboratory, the current method of identifying bacterial isolates is based mainly on phenotypic characteristics, such as the growth pattern on different media, colony morphology, Gram stain, and various biochemical reactions. These techniques collectively allow high-level accuracy in identifying most bacterial isolates, but they are costly and time-consuming. In our clinical microbiology laboratory, we prospectively assessed the ability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify bacterial strains that were routinely isolated from clinical samples. Bacterial colonies obtained from a total of 468 strains of 92 bacterial species isolated at the Department of Clinical Laboratory at Chiba University were directly placed on target MALDI plates, followed by the addition of CHCA matrix solution. The plates were then subjected to MALDI-TOF MS measurement, and the microorganisms were identified by pattern matching by the libraries in the BioTyper 2.0 software. The identification rates at species and genus levels were 91.7% (429/468) and 97.0% (454/468), respectively. MALDI-TOF MS is a rapid, simple, and high-throughput proteomic technique for the identification of a variety of bacterial species. Since colony to colony differences and the effects of culture duration on the results are minimal, it can be implemented in a conventional laboratory setting. Although for some pathogens, the preanalytic processes should be refined and current database should be improved to obtain more accurate results, the MALDI-TOF MS-based method generally performs as well as the conventional methods and is a promising technology in clinical laboratories.

Discrimination of methicillin-resistant Staphylococcus aureus from methicillin-susceptible Staphylococcus aureus or coagulase-negative staphylococci by detection of penicillin-binding protein 2 and penicillin-binding protein 2' using a bioluminescent enzyme immunoassay.

For the discrimination of methicillin-resistant S. aureus (MRSA) from methicillin-susceptible S. aureus (MSSA) or coagulase-negative staphylococci (CNS), we developed a bioluminescent enzyme immunoassay (BLEIA) for detecting penicillin-binding protein 2 (PBP2) and penicillin-binding protein 2' (PBP2') using biotinylated firefly luciferase. The BLEIA was able to detect recombinant PBP2 at 50 pg/ml and recombinant PBP2' at 500 pg/ml. PBP2 and PBP2' present in the membranes of S. aureus were extracted by acid and detergent treatment. The method was able to detect PBP2 or PBP2' extracted from 10(6) colony forming units of S. aureus because of efficient extraction and the high sensitivity of luciferase. In a study of clinical isolates previously characterized as either MRSA or MSSA by antibiotic susceptibility testing, all 34 specimens identified as MRSA were both PBP2 and PBP2' positive. The 34 MSSA specimens were PBP2 positive and PBP2' negative. Moreover, the BLEIA could detect PBP2' extracted from four species of methicillin-resistant CNS, but not PBP2 extracted from four species of methicillin-resistant and methicillin-susceptible CNS. This result suggested that PBP2 could be a unique marker for discrimination of S. aureus from CNS. A BLEIA that is able to detect PBP2 and PBP2' may be useful in clinical diagnostics.

Immunogenicity of a monovalent pandemic influenza A H1N1 virus vaccine with or without prior seasonal influenza vaccine administration.

The immunogenicity of pandemic influenza A H1N1 virus (A/H1pdm) vaccine might be modified by prior seasonal trivalent influenza vaccine (sTIV) administration. We conducted a retrospective analysis of immunogenicity of 243 health care workers (number of sTIV-positive [sTIV(+)] subjects, 216; number of sTIV(-) subjects, 27) by hemagglutination inhibition. There was no significant difference in the ratios of antibody titers of ≥40 (41.2% versus 48.1%; P = 0.49) and fold increases in geometric mean titer (3.8 versus 4.5; P = 0.37). sTIV injected 7 to 10 days prior to A/H1pdm vaccine administration did not interfere with the immunogenicity of the latter.

Rapid identification of microorganisms by mass spectrometry: improved performance by incorporation of in-house spectral data into a commercial database.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used as a microbial diagnostic method for species identification of pathogens. However, MALDI-TOF identification of bacteria at the species level remains unsatisfactory, with the major problem being an incomplete database that still needs refinement and expansion. Augmentation of the original MALDI BioTyper 2.0 (Bruker) database by incorporating mass spectra obtained in-house from clinical isolates may increase the identification rate at the species level. We conducted a prospective study to assess whether the augmented database can improve the performance of MALDI-TOF MS for routine identification of species. Cluster analyses revealed distinct differences in MS spectral profiles of clinical isolates obtained in our hospital and those of ATCC strains in the Bruker database. In the first part of the study, which was performed over 3 weeks, 259 bacterial isolates were subjected to analysis by MALDI-TOF MS, and MS spectra of 229 successfully identified isolates (49 species) were incorporated into the original database to give the augmented Bruker-Chiba database. In a second separate analysis, the concordance of identification of 498 clinical isolates of the 49 species with conventional methods was 87.1% (434/498) with the commercial Bruker database and 98.0% (488/498) using the Bruker-Chiba database. These results indicate that refinement of a commercial database can be achieved relatively easy and effectively by incorporating MS spectra of clinical isolates obtained in a clinical laboratory.

Effectiveness and safety of pandemic influenza A (H1N1) 2009 vaccine in healthcare workers at a university hospital in Japan.

Pandemic influenza A (H1N1) virus (AH1pdm) emerged in April 2009. An inactivated, split-virus, unadjuvanted AH1pdm vaccine was manufactured in Japan, and vaccination was initiated with top priority for healthcare workers (HCWs) on October 19, 2009. A retrospective cohort study was conducted to evaluate the effectiveness of a single-dose vaccine for HCWs in a hospital in Japan. A total of 1,567 (84.5%) of 1,854 HCWs were vaccinated. Thirty-seven were infected with AH1pdm before the vaccine became available, and were excluded. The other 250 were not vaccinated for personal reasons. We analyzed the influenza infection rate with or without vaccination and related adverse events. Among the 1,817 HCWs without previous infection, 37 were infected with AH1pdm; 13 (5.2%) of 250 unvaccinated HCWs became infected, which was a significantly higher rate than the 24 (1.5%) of 1,567 vaccinated HCWs (P=0.001). Multivariate analysis revealed that age of 20-29 years was a risk factor for infection (adjusted odds ratio [aOR], 3.7; P<0.001), and that vaccination was a preventive factor (aOR, 0.20; P<0.001). Adverse events occurred in 549 of 1,060 HCWs, but most were mild. Although vaccination was carried out during AH1pdm epidemic expansion, the single-dose AH1pdm vaccine proved effective in HCWs, and severe adverse events were rare.

Use of the MALDI BioTyper system with MALDI-TOF mass spectrometry for rapid identification of microorganisms.

In a clinical diagnosis microbiology laboratory, the current method of identifying bacterial isolates is based mainly on phenotypic characteristics, for example growth pattern on different media, colony morphology, Gram stain, and various biochemical reactions. These techniques collectively enable great accuracy in identifying most bacterial isolates, but are costly and time-consuming. In our clinical microbiology laboratory, we prospectively assessed the ability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify bacterial strains that were routinely isolated from clinical samples. Bacterial colonies obtained from a total of 468 strains of 92 bacterial species isolated at the Department of Clinical Laboratory at Chiba University were directly placed on target MALDI plates followed by addition of CHCA matrix solution. The plates were then subjected to MALDI-TOF MS measurement and the microorganisms were identified by pattern matching with the libraries in the BioTyper 2.0 software. Identification success at the species and genus levels was 91.7% (429/468) and 97.0% (454/468), respectively. MALDI-TOF MS is a rapid, simple, and high-throughput proteomic technique for identification of a variety of bacterial species. Because colony-to-colony differences and effects of culture duration on the results are minimal, it can be implemented in a conventional laboratory setting. Although for some pathogens, preanalytical processes should be refined, and the current database should be improved to obtain more accurate results, the MALDI-TOF MS based method performs, in general, as well as conventional methods and is a promising technology in clinical laboratories.

Immunogenicity of a monovalent pandemic influenza A H1N1 vaccine in health-care workers of a university hospital in Japan.

A phase III observational study evaluating a single-dose of an inactivated, split-virus, unadjuvanted AH1pdm vaccine in HCW was conducted. A safe and effective vaccine was needed after the emergence of AH1pdm in April 2009. We analyzed the immunogenicity and safety of the vaccine. A total of 409 subjects were enrolled and given 15 µg hemagglutinin antigen by s.c. injection. Antibody titers were measured using hemagglutination-inhibition antibody assays before vaccination and 28 days after. The co-primary immunogenicity end-points were the proportion of subjects with antibody titers of 1:40 or more, the proportion of subjects with either seroconversion or a significant increase in antibody titer, and the factor increase in geometric mean titer. We collected 389 pair samples. Antibody titers of 1:40 or more were observed in 148 of 389 subjects (38.0%, 95% CI: 33.2-42.9). The immunogenicity was also confirmed in other end-points, but was not sufficient and was lower than in previous reports. A total of 96 of adverse events was reported: 51 local events and 57 systemic events. There were 12 subjects with both local and systemic events. Nearly all events were mild to moderate except in four subjects. A single 15-µg dose of AH1pdm vaccine did not induce sufficient immunogenicity in HCW, with mild-to-moderate vaccine-associated adverse events. We need to consider further improvement of the AH1pdm vaccine program in HCW for the prevention of nosocomial infection, as well as for the benefit of HCW.

[Appropriate use of anti-methicillin-resistant Staphylococcus aureus agents in a retrospective study].

The track records of the use of anti-methicillin-resistant Staphylococcus aureus agents (anti-MRSA agents) in a 5-year period (2001.4-2006.3) were collected, and cases in which anti-MRSA agents were used for >4 days were selected. In each case, the results of laboratory data and bacterial examination before and after administering the anti-MRSA agents were investigated retrospectively. In addition, it was also investigated in each case whether therapeutic drug monitoring (TDM) was carried out. It was observed that the number of patients treated with anti-MRSA agents and the total dose of anti-MRSA agents used tended to increase over time, except for arbekacin sulfate. It was, however, shown that treatment with anti-MRSA agents resulted in significant decreases in body temperature, C-reactive protein, and white blood cell counts. Bacterial examination was conducted in 75.6% of the patients treated with anti-MRSA agents, with MRSA being detected in 72.4% of the cases examined. On the other hand, TDM was also conducted in 60% of the cases, but this was at a lower percentage than that of the other examinations. Quantitative bacterial examination after treatment with anti-MRSA agents indicates that TDM can be considered important for the appropriate use of anti-MRSA agents.

[Usefulness of PCR in rapidly diagnosing subcutaneous abscess and costal osteomyelitis caused by Mycobacterium bovis BCG].

Conventional polymerase chain reaction (PCR) used to identify mycobacterial species does not distinguish between Mycobacterium tuberculosis and M. bovis BCG, and several weeks or months may be needed to identify individual slow-growing Mycobacterial species. We report a 4-year-old girl who had subcutaneous abscess and sternal osteomyelitis after BCG vaccination at 4 month of age. We directly identified M. bovis BCG genome in the punctured abscess within a few days using PCR and PCR-restriction fragment length polymorphism. Such PCR is useful for rapidly diagnosing and managing of appropriate therapy in patients with infection due to M. bovis BCG.

Two distinct cytotoxic activities of subtilase cytotoxin produced by shiga-toxigenic Escherichia coli.

Subtilase cytotoxin (SubAB) is a recently identified AB5 subunit toxin produced by Shiga-toxigenic Escherichia coli. The A subunit is thought to be a subtilase-like, serine protease, whereas the B subunit binds to the toxin receptor on the cell surface. We cloned the genes from a clinical isolate; the toxin was produced as His-tagged proteins. SubAB induced vacuolation at concentrations greater than 1 microg/ml after 8 h, in addition to the reported cytotoxicity induced at a ng/ml level after 48 h. Vacuolation was induced with the B, but not the A, subunit and was dependent on V-type ATPase. The cytotoxicity of SubAB at low concentrations was associated with the inhibition of protein synthesis; the 50% inhibitory dose was approximately 1 ng/ml. The A subunit, containing serine 272, which is thought to be a part of the catalytic triad of a subtilase-like serine protease, plus the B subunit was necessary for this activity, both in vivo and in vitro. SubAB did not cleave azocasein, bovine serum albumin, ovalbumin, or synthetic peptides. These data suggest that SubAB is a unique AB toxin: first, the B subunit alone can induce vacuolation; second, the A subunit containing serine 272 plus the B subunit inhibited protein synthesis, both in vivo and in vitro; and third, the A subunit proteolytic activity may have a strict range of substrate specificity.