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Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was approved for medical use in 2011 and is currently used as a rapid, accurate and lowcost technique for bacterial identification. External quality control for medical analysis is monitored using tests of the Japanese Association of Medical Technologists and Prefectural Association of Clinical Laboratory Technologists and through user surveys of reagent and equipment manufacturers. However, external quality control of bacterial typing using MS is not performed. Therefore, we examined procedures for evaluating quality control of bacterial typing using an identification reliability index at 38 facilities.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was approved for medical use in 2011, and is currently used as a rapid, accurate and low-cost technique for bacterial identification. Microbiological testing and internal accuracy control in Japan are mainly implemented in accordance with the standards of the Clinical and Laboratory Standards Institute (CLSI). However, few facilities perform internal accuracy control of bacterial identification by MALDI-TOF MS. Therefore, we examined the procedures for internal accuracy control of bacterial identification using MALDI-TOF MS in daily work at clinical laboratories in the seven hospitals.

We herein report a case of systemic phaeohyphomycosis by Exophiala dermatitidis (E. dermatitidis) with chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). The patient had been taking oral corticosteroids for years to control the GVHD. Yeast-like fungi were identified in a blood culture, so treatment with micafungin (150 mg/day) was begun, with no improvement. The patient passed away on hospital Day 12. A sequence analysis of rRNA revealed the isolate to be E. dermatitidis. This report brings attention to an emerging mycosis of community-acquired Exophiala species infection in the very-late phase after allogenic HSCT in patients with chronic GVHD.

Background: A low body mass index (BMI) has been reported to be a poor prognostic factor for Mycobacterium avium complex pulmonary disease (MAC-PD). The purpose of this study was to clarify the clinical features of MAC-PD in cases with a low BMI. Methods: This retrospective study analyzed the data of patients diagnosed with MAC-PD at Saga University Hospital between 2008 and 2019. The analyzed patient characteristics included age, gender, BMI, symptoms, laboratory data, chest computed tomography findings, and the treatment courses. We also investigated the factors associated with successful treatment. Results: In total, 144 patients were included in this study. The low-BMI group (BMI &lt, 18.5 kg/m2) had a higher incidence of sputum, Mycobacterium intracellurare infection, and cavitary lesions, in addition to lower blood lymphocyte counts, higher neutrophil–lymphocyte ratios, and a lower prognostic nutritional index (PNI) when compared to the preserved-BMI group (BMI ≥ 18.5 kg/m2). Sixty-six of the 144 patients (45.8%) received treatment. Hemosputum, acid-fast bacillus sputum smear positivity, low lymphocyte counts, a low PNI, and unsuccessful treatment (48.5% vs. 24.2%, p &lt, 0.05) were found to be associated with a low BMI. Conclusions: A low BMI is associated with cavitary lesions, malnutrition, and unsuccessful treatment in MAC-PD.

We herein report a case of systemic phaeohyphomycosis by Exophiala dermatitidis (E. dermatitidis) with chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). The patient had been taking oral corticosteroids for years to control the GVHD. Yeast-like fungi were identified in a blood culture, so treatment with micafungin (150 mg/day) was begun, with no improvement. The patient passed away on hospital Day 12. A sequence analysis of rRNA revealed the isolate to be E. dermatitidis. This report brings attention to an emerging mycosis of community-acquired Exophiala species infection in the very-late phase after allogenic HSCT in patients with chronic GVHD.

Objective To study how and to what degree the rapid pathogen identification by MALDI-TOF MS coupled with rapid disk diffusion test improve the current clinical practice of patients with bacteremia in a tertiary teaching hospital with full-time ID consultation service. Patients and methods MALDI-TOF MS and 8H disk diffusion tests were directly applied to the positive blood cultures samples and the results were reflected on antimicrobial therapy (n = 119). The appropriateness of antimicrobial selection through these interventions was verified with conventional culture results in comparison with historical control (n = 129). The mortality of patients between the two periods was also compared. Results The appropriateness of antimicrobial selection was higher (99.2%) in the intervention than in the control group (93.8%) (p 0.024), but there was no difference in 28-day mortality between the two periods (16.8%, 14.8%) (p 0.668). The duration of presumptive antimicrobial therapy with anti-MRSA agents and carbapenem antibiotics did not differ between the two periods indicating that the intervention was not effective in decreasing the unnecessary antibiotics. On the other hand, some bacteremic patients with pathogens whose drug susceptibilities were invariably sensitive to the standard class of antibiotics definitely benefitted from the intervention. Conclusion The intervention utilizing MALDI-TOF MS and the rapid disk diffusion test may not demonstrate overall improvement in bacteremia mortality in the institution with full-time infectious disease consultants. Its utility has yet to be evaluated in different setting hospitals.

A 61-year-old man was admitted to our hospital with 2-day history of malaise and dyspnea. He had mitral prolapse and type II diabetes mellitus with neurogenic bladder, which was cared for by catheterization on his own. On arrival the patient was in septic condition with hypoxemia, and physical examination revealed systolic murmur at the apex. Transthoracic echocardiography revealed vegetation of the mitral and the aortic valve. The presence of continuous bacteremia was confirmed by multiple sets of blood culture, whereby gram-positive cocci was retrieved and identified as Staphylococcus saprophyticus (S. saprophyticus) both phenotypically and genetically. Because two major criteria of the Modified Duke Criteria were met, the patient was diagnosed with native valve endocarditis due to S. saprophyticus. The urine culture was also positive for gram-positive cocci, phenotypically identified as Staphylococcus warneri, which was subsequently identified as S. saprophyticus with the use of 16S rRNA gene sequence analysis and MALDI-TOF MS (matrix-assisted laser desorption ionization time of flight mass spectrometry), indicating strongly that the intermittent catheterization-associated urinary tract infection resulted in bacteremia that eventually lead to infective endocarditis. This patient was treated with vancomycin and clindamycin. Because of multiple cerebral infarctions, the patient underwent mitral and aortic valve replacement on hospital day 5. Blood culture turned negative at 6th hospital day. Antibiotic therapy was continued for six weeks after surgery. The patient's clinical course was uneventful thereafter, and was discharged home. This is the first case report of native valve endocarditis caused by S. saprophyticus of confirmed urinary origin.

Enterococcus consists human bowel flora, but sometimes behave as an important nosocomial pathogen. In order to identify clinical characteristics that help discriminate between ampicillin-susceptible and ampicillin-resistant enterococcal bacteremia in advance for antimicrobial susceptibility testing, a retrospective eight-year study was carried out in patients with enterococcal bacteremia experienced in Saga University Hospital, Japan. A total of 143 patients were included in the analysis: 85 (59.4%) with bacteremia caused by ampicillin-susceptible enterococci and 58 (40.6%) by ampicillin-resistant strains. Hospital-acquired bacteremia was present in 79.0% (113/143) of patients. Abdominal infections, urinary tract infections, and unknown source were predominant foci for the two groups. Patients with ampicillin-resistant enterococcal bacteremia was significantly associated with hematological cancer, immunosuppressive therapy, prior use of antibiotics, and mucositis associated with febrile neutropenia. The 28-day mortality was significantly higher in ampicillin-resistant enterococcal bacteremia. On multivariate analysis, independent risk factors for ampicillin-resistant enterococci were as follows: prior exposures to penicillins and carbapenems, and bacteremia related to mucositis with febrile neutropenia. These findings would assist physicians in deciding whether glycopeptide antibiotics should be included as an empiric antibiotic therapy in patients with suspected enterococcal infections and also those with persistent neutropenic fever refractory to fourth generation cephalosporin. A few cases of MALDI-TOF MS-identified Enterococcus faecium that turned out ampicillin-sensitive were also described to emphasize the importance of taking epidemiological aspects of patients into considerations when deciding initial antimicrobial treatment.

Background Despite treatment, pulmonary nocardiosis, which is a rare opportunistic disease caused by Nocardia species, has poor clinical outcomes including recurrence and death. Currently, the treatment regimen and duration for pulmonary nocardiosis are not fully understood. The present study aimed to clarify the factors related to the clinical outcome of pulmonary nocardiosis. Methods The medical records of 24 patients with pulmonary nocardiosis were retrospectively reviewed. The patients were divided into two groups based on the outcomes within 2 years: patients with controlled disease (n = 14) and patients who developed recurrence or died (n = 10). Results Nocardia was identified by 16S ribosomal RNA sequencing in 17 patients (70.8%) and by conventional biochemical test in five patients (20.8%). The patients' characteristics, clinical findings, radiological features, and treatment history were not different between the two groups. Compared with patients who developed recurrence or died, those with controlled disease had significantly longer total duration of treatment with antibiotics, especially trimethoprim/sulfamethoxazole (67.5 ± 111.6 days vs. 9.0 ± 6.5 days; p = 0.01). Pancytopenia was the most frequent adverse effect of trimethoprim/sulfamethoxazole. Conclusions Longer duration of trimethoprim/sulfamethoxazole treatment was significantly associated with better outcomes of pulmonary nocardiosis. In such cases, antibiotics, especially trimethoprim/sulfamethoxazole, should be administered for more than 3 months.

OBJECTIVES To investigate clinical and microbiological characteristics of community-acquired bacteremia (CAB). METHODS We retrospectively analyzed subjects with CAB hospitalized at Saga University Hospital between January 2009 and September 2011. We investigated causative organisms, primary infection sites, and subject summaries and complications, and analyzed the mortality factor. RESULTS CAB incidence was 185 cases, with 192 organisms cultured. Causative organisms were gram-positive bacteria in 81 strains (42%), 9 (11%) of which were methicillin-resistant Staphylococcus aureus (MRSA). Gram-negative bacteria were identified in 111 strains (58%), with 80% Enterobacteriaceae. Five of the 111 (5%) were caused by extended-spectrum beta-lactamase (ESBL) producing bacteria. The most frequent bacteremia portal was intraabdominal infection (29%, 54/185). During hospitalization of 1-180 days, 20 subjects eventually died. Neutropenia on admission was associated with significantly higher mortality than without (30% vs. 3%, p < 0.001). Septic shock rates were higher in non-survivors than survivors (45% vs. 14%, p = 0.002), and more complications were documented in non-survivors than survivors (50% vs. 25%, p = 0.017). No specific pathogen or primary infection site was associated with higher mortality. CONCLUSIONS Antimicrobial-resistant pathogens such as MRSA and ESBL producers should be considered even in CAB, especially in subjects with healthcare-associated infection, regardless of how small the number. The CAB treatment course should consider subjects summaries, severity, and complications.

Nocardia concava was identified as a new species in 2005; however, the clinical manifestations of Nocardia concava infection have yet to be clarified. We herein present the case of an immunosuppressed patient who developed disseminated nocardiosis caused by N. concava with multiple abscesses in the lungs, cutis, subcutaneous tissue, skeletal muscles and kidneys accompanied by central nervous system involvement, including meningitis and ventriculitis. The patient was cured with appropriate treatment including linezolid after testing for susceptibility. Linezolid should be considered as an alternative agent for treating disseminated nocardiosis because of its effective distribution to multiple sites.

Using quantitative Bayesian analysis as a clinical epidemiological approach, we developed a diagnosis for lower respiratory tract infection (LRTI) due to Methicillin-resistant Staphylococcus aureus (MRSA). We retrospectively reviewed the charts of 181 subjects--a derivation cohort-with MRSA retrieved from lower respiratory specimens June 2006 to March 2008. Dividing them into infection or colonization (no infection) groups, we compared them for the presence or absence of clinical parameters, including fever > 38 degrees C, MRSA >106 CFU (colony-forming units)/mL, phagocytosis on Gram staining, serum albumin 15,000/mL. We them determined positive and negative likelihood ratios (LR +, LR -) for these parameters to quantify MRSA-LRTI diagnostic probability based on combined likelihood ratios (Bayesian analysis). We then determined Bayesian MRSA-LRTI diagnostic probabilities (BDPs) in 40 subjects with respiratory MRSA--a validation cohort-from May 2008 to October 2008 clinically judged with either infection (n = 14) or colonization (n = 26) by infection control personnel (ICP) blinded to the test (parameter LR+ and LR -). BDPs (mean +/- SD) quantified by combining the four parameters-fever, MRSA CFU, phagocytosis, and serum albumin-were 62.3 +/- 25.4% for 14 judged with infection, and 40.2% +/- 20.4% for 26 patients judged with colonization (p = 0.005). Using a diagnostic probability of 51% as the cut off, we compared positive and negative predictive Bayesian diagnoses ICP judgment, i.c., 77% vs. 85%. The Bayesian approach proved useful in quantitatively diagnosing infectious disease such as MRSA-LRTI that lack established diagnostic, and may aid physicians in deciding the need for specific antimicrobial therapy.

Microflex LT (Bruker Daltonics) and VITEK MS (bioMerieux) are bacterial identification systems that are based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). For VITEK MS, two identification softwares, VITEK MS IVD (IVD) and SARAMIS (SARAMIS), are available. Microflex LT is equipped with MALDI Biotyper RTC software (Biotyper). Although the identification accuracy of each instrument has been compared for various bacteria, no detailed examination has been conducted for the identification accuracy of Clostridium difficile. In this report, we compared the three identification softwares for identification reproducibility in three ATCC C. difficile strains and identification accuracy in 50 clinical C. difficile isolates. The results showed 100, 91.7 and 100 % identification reproducibility accuracy of ATCC strains when examined by IVD, SARAMIS and Biotyper software, respectively. For the identification of the clinical isolates, all three softwares exhibited satisfactory identification accuracy of C. difficile. Among the 50 clinical isolates, seven showed identical toxin genotype corresponding to the exact ribotype. However, MALDI-TOF MS failed to identify them as the identical type. Based on the above results, we concluded that both types of MALDI-TOF MS reproducibly identified C. difficile; however, they are currently not suitable for typing of C. difficile clones.

Objective To investigate how patients with bacteremic sepsis are managed in a tertiary care teaching hospital. Patients and Methods Prospective observational study on patients with bacteremic sepsis. Clinical and microbio-logical characteristics of bacteremic sepsis were analyzed in relation to prognosis. Severity of the illness was quantitatively analyzed by the APACHE (Acute Physiology, Age, Chronic Health Evaluation) III scoring system. Also investigated was how closely physicians paid attention to acute physiological alterations in patients. Results The 28-day mortalities in fifty hemodynamically stable patients and in twenty-three septic shock patients were 26% and 52%, respectively (p=0.028). Gram-positive organisms accounted for 54% of all organisms, with the mortality and incidence of septic shock being the same as with Gram-negative infections. The mean APACHE III score was 42.9 in survivors, and 76.5 in non-survivors (p < 0.001). Although serum levels of C-reactive protein and acute physiology score (APS) was significantly higher in non-survivors than in survivors, the correlation with APACHE III score was more prominent in APS. The number of vital signs recorded was 1.67 in physicians and 3.6 in nurses (p < 0.001). Conclusions The present study proved that the APACHE HI score accurately discriminates between survivors and non-survivors of patients with sepsis. By addressing the need for an objective evaluation of severity of illness, it strongly recommends that physicians should be made aware of physiologically defined sepsis and that they should pay closer attention to patients' physiological alterations to identify the development of sepsis in critically ill patients.(Internal Medicine 39: 901-909, 2000)

We attempted to analyze any influences to %TMIC achievement probability due to the difference of the MIC measurement concentration range of MEPM for 613 strains of Pseudomonas aeruginosa by the Monte Carlo simulation method. As for the analysis, we calculated the achievement probability of 30% and 50% for MEPM %TMIC by the administration volume of MEPM: 250 mg, 500 mg, and 1,000 mg, the administration time: 0.5 h, and 3 h, the administration frequency: 2 times, and 3 times, and the renal excretion capability: Normal, Slight, Moderate, and High abnormal with the 3 types of MIC concentration measurement level 1)=0.06~=256 µg/ml: 13 levels, 2)=0.5~=32 µg/ml: 7 levels, and 3)=1~=16 µg/ml: 5 levels. As the result, we found the following findings; 1. In terms of the administration of normal renal excretion capability, 250 mg, in comparison with 500 mg and 1,000 mg, indicated the differential due to the difference of MIC measurement concentration range. 2. The administration volume of MEPM 500 mg which has been recommended shown the less differential of the achievement probability due to the difference of MIC measurement concentration range. As the renal excretion was shifted through Normal to Slight to Moderate to High abnormal, the differential of the achievement probability due to the difference of MIC measurement concentration range was gradually decreased. With these results, PK/PD analysis is possible for the 5 levels measurement concentration. It is significant that the facility using the automated microbiology analyzer can provide not only the MIC report, but also the information on the appropriate administration method for antibacterial drug by PK/PD analysis.

Some automated systems of the identification and susceptibility for microorganisms are used and prevail in hospital laboratories. One of the most serious problems is to perform accurate susceptibility testing for low-level resistant organisms, while antibiotic resistant microbes are increasing in medical fields. To evaluate automated machines for the susceptibility testing, several antibiotic resistant organisms were examined by plural technicians in some laboratories. Each strain of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycinintermediate S. aureus (VISA), extended-spectrum β-lactamase (ESBL) producing Escherichia coli and Klebsiella pneumoniae was tested by three automated systems of WalkAway, VITEK2/VITEK2 compact and Phoenix for susceptibility. The results for antibiotics generated by the systems were compared to those generated by reference methods according to CLSI guidelines. The results of WalkAway, VITEK2/VITEK2 compact, and Phoenix demonstrated 92%, 91%, and 96% of reproducibilities, 92%, 94%, and 91% of MIC agreements, 0.5%, 0.8%, and 0.3% of very major error (VME) and 0.3%, 1.4%, and 2.3% of major error (ME), respectively. All automated systems had a high reproducibility even under the performance of plural technicians, although the differences of VMEs and MEs were observed among the systems. From these data, the automated systems for antimicrobial susceptibility testing were more useful for the detection of antibiotic resistant organisms by understanding the characteristics of each system.

Using quantitative Bayesian analysis as a clinical epidemiological approach, we developed a diagnosis for lower respiratory tract infection (LRTI) due to Methicillin-resistant Staphylococcus aureus (MRSA). We retrospectively reviewed the charts of 181 subjects--a derivation cohort-with MRSA retrieved from lower respiratory specimens June 2006 to March 2008. Dividing them into infection or colonization (no infection) groups, we compared them for the presence or absence of clinical parameters, including fever38 degrees C, MRSA106 CFU (colony-forming units)/mL, phagocytosis on Gram staining, serum albumin3.0 g/dL, and peripheral WBC count15,000/mL. We them determined positive and negative likelihood ratios (LR +, LR -) for these parameters to quantify MRSA-LRTI diagnostic probability based on combined likelihood ratios (Bayesian analysis). We then determined Bayesian MRSA-LRTI diagnostic probabilities (BDPs) in 40 subjects with respiratory MRSA--a validation cohort-from May 2008 to October 2008 clinically judged with either infection (n = 14) or colonization (n = 26) by infection control personnel (ICP) blinded to the test (parameter LR+ and LR -). BDPs (mean +/- SD) quantified by combining the four parameters-fever, MRSA CFU, phagocytosis, and serum albumin-were 62.3 +/- 25.4% for 14 judged with infection, and 40.2% +/- 20.4% for 26 patients judged with colonization (p = 0.005). Using a diagnostic probability of 51% as the cut off, we compared positive and negative predictive Bayesian diagnoses ICP judgment, i.c., 77% vs. 85%. The Bayesian approach proved useful in quantitatively diagnosing infectious disease such as MRSA-LRTI that lack established diagnostic, and may aid physicians in deciding the need for specific antimicrobial therapy.

The serotyping of free coagulase, one of the most reliable ways to identify strains of Staphylococcus aureus, and widely employed in Japan, has been improved by adding magnetite sand to the reaction mixture. Culture medium supernatant and a type-specific antibody are mixed in a well of a microtiter plate, and plasma-enriched bovine fibrinogen is treated with magnetite sand. The use of tranexamic acid and gum arabic in the reaction mixture also increases the sensitivity of the reaction. Finally, the plate is placed on a magnetic stirrer. If the type of the coagulase corresponds to that of the antibody, no clot formation will occur, and this is easily confirmed by the movement of the sand. Although the amount of reaction mixture required is much less than that for the conventional tube method, our new method is able to detect slight increases in viscosity of the reaction mixture due to fibrin formation even before complete clotting occurs, thus providing very high sensitivity. Clot formation can also be judged by observing a turbid mass of fibrin in the well (Hwang's method), but this approach is a little slower than our method involving immobilization of magnetite sand.

We have reported in this journal in vitro susceptibilities of clinical isolates to antibiotics every year since 1992. In this paper, we report the results of an analysis of in vitro susceptibilities of 12,919 clinical isolates from 72 centers in Japan to selected antibiotics in 2007 compared with the results from previous years. The common respiratory pathogens, Streptococcus pyogenes, Streptococcus pneumoniae, Moraxella catarrhalis and Haemophilus influenzae maintained a high susceptibility to fluoroquinolones (FQs). The resistance of S. pyogenes to macrolides has been increasing every year and this was especially clear this year. Most strains of Enterobacteriaceae except for Escherichia coli showed a high susceptibility to FQs. Almost 30% of E. coli strains were resistant to FQs and the resistance increased further this year. FQs resistance of methicillin-resistant Staphylococcus aureus (MRSA) was approximately 95% with the exception of 45% for sitafloxacin (STFX). FQs resistance of methicillin-susceptible S. aureus (MSSA) was low at about 10%. FQs resistance of methicillin-resistant coagulase negative Staphylococci (MRCNS) was higher than that of methicillin-susceptible coagulase negative Staphylococci (MSCNS), but it was lower than that of MRSA. However, FQs resistance of MSCNS was higher than that of MSSA. FQs resistance of Enterococcus faecalis was 22.5% to 29.6%, while that of Enterococcusfaecium was more than 85% except for STFX (58.3%). In clinical isolates of Pseudomonas aeruginosa derived from urinary tract infections, FQs resistance was 21-27%, which was higher than that of P. aeruginosa from respiratory tract infections at 13-21%, which was the same trend as in past years. Multidrug resistant strains accounted for 5.6% in the urinary tract and 1.8% in the respiratory tract. Acinetobacter spp. showed high susceptibility to FQs. The carbapenem resistant strains, which present a problem at present, accounted for 2.7%. Neisseria gonorrhoeae showed high resistance of 86-88% to FQs. The results of the present survey indicated that although methicillin-resistant Staphylococci, Enterococci, E. coli, P. aeruginosa, and N. gonorrhoeae showed resistance tendencies, and other species maintained high susceptibility rates more than 90% against FQs, which have been used clinically for over 15 years.

In empirical antibacterial therapy, regional surveillance is expected to yield important information for the determination of the class and dosage regimen of antibacterial agents to be used when dealing with infections with organisms such as Pseudomonas aeruginosa, in which strains resistant to antibacterial agents have been increasing. The minimal inhibitory concentrations (MICs) of five carbapenem antibiotics against P. aeruginosa strains isolated in the Northern Kyushu district of Japan between 2005 and 2006 were measured, and 100 strains for which carbapenem MICs wereor =0.5-32 microg/ml were selected. In this study, MIC was measured by two methods, i.e., the common serial twofold dilution method and an integrated concentration method, in which the concentration was changed, in increments of 2 microg/ml, from 2 to 16 microg/ml. The MIC(50)/MIC(90) values for imipenem, meropenem, biapenem, doripenem, and panipenem, respectively, with the former method were 8/16, 4/16, 4/16, 2/8, and 16/16 microg/ml; and the values were 6/10, 4/12, 4/10, 2/6, and 10/16 microg/ml with the latter method. The MIC data obtained with both methods were subjected to pharmacokinetic/pharmacodynamic (PK/PD) analysis with Monte Carlo simulation to calculate the probability of achieving the target of time above MIC (TMIC) with each carbapenem. The probability of achieving 25% time above the MIC (TMIC; % of TMIC for dosing intervals) and 40% TMIC against P. aeruginosa with any dosage regimen was higher with doripenem than with any other carbapenem tested. When the two sets of MIC data were subjected to PK/PD analysis, the difference between the two methods in the probability of achieving each % TMIC was small, thus endorsing the validity of the serial twofold dilution method.

Quantitative DNA-DNA hybridization to measure the genetic distances among bacterial species is indispensable for taxonomical determination. In the current studies, we developed a method to determine bacterial DNA relatedness on a glass microarray. Reference DNAs representing a total 93 species of Enterobacteriaceae were arrayed on a glass microplate, and signal intensities were measured after 2 hr of hybridization with Cy3-labeled bacterial DNAs. All immobilized DNAs from members of the family Enterobacteriaceae were identified by this method except for DNAs from Yersinia pseudotuberculosis and Y. pestis. These results suggest that quantitative microarray hybridization could be an alternative to conventional DNA-DNA hybridization for measuring chromosome relatedness among bacterial species.

Isolation frequency and antimicrobial susceptibility of Haemophilus influenzae isolated in Saga University hospital from October 2002 to September 2003 were investigated. Out of 155 H. influenzae strains subjected 77 were isolated from pediatrics specimens. beta-Lactamase negative ampicillin (ABPC)-resistant H. influenzae (BLNAR), against which MICs of ABPC were higher than 4 microg/mL, were 32 strains (20.6%), and it became 63 strains (41.3%) when Low-BLNAR, against which MICs of ABPC were higher than 2 microg/mL, were included. beta-Lactamase positive ABPC-resistant H. influenzae (BLPAR) were 8 strains (5.2%). Although those BLNAR were also resistant to variety of beta-lactams, fluoroquinolones and other antibiotics were not affected by the level of ABPC-resistance. Resistant strains of BLPAR against SBT/ABPC, a combination of a beta-lactamase inhibitor, were detected but all of them were sensitive to TAZ/PIPC, an another combination. Those strains were able to be considered as beta-lactamase positive amoxicillin-clavulanate resistant H. influenzae (BLPACR). PIPC, TAZ/PIPC, CTRX, CDTR, MEPM, LVFX and CPFX showed good activity among tested antibiotics.

We determined the antibacterial activities of oral Cephems against isolated from the patients with the respiratory infections, the urinary tract infections, and infections in the obstetrics field of an adult and a child, during the period from 2002 to 2003; Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Branhamella catarrhalis, Klebsiella pneumoniae and Escherichia coli of 40 strains of each, and Peptostreptococcus spp. 22 strains. S. pneumoniae and H. influenzae strains that resistant is regarded were collected mainly, penicillin-intermediate S. pneumoniae (PISP), penicillin-resistant S. pneumoniae (PRSP) and beta-lactamase negative ampicillin-resistant H. influenzae (BLNAR) strains. The MICs of Cephems except cefaclor (CCL) wereor = 0.03 microgram/mL against all strains of S. pyogenes. The MICs of cefteram (CFTM) and cefditoren (CDTR) wereor = 0.0125 microgram/mL activity against 7 strains penicillin-susceptible S. pneumoniae (PSSP). However the MIC90s of cefditoren (CDTR) was 1 microgram/mL, cefteram (CFTM), and cefcapene (CFPN) were 2 micrograms/mL against PISP and PRSP, were higher than those of other drugs, but showed slightly higher than PSSP. The MIC90s of Cephems. were 0.5-4 micrograms/mL against strains of E. coli. The MIC90s of CFTM was 0.5 microgram/mL, and CDTR, CFPN were 1 microgram/mL against E. coli were higher than those of other drugs. The four strains of E. coli however were highly-resistant which MIC90s of CCL were more than 32 micrograms/mL were obtains. Furthermore it is necessary to pay much attention to the trend of resistant such as E. coli of Cephems. Although all strains showed resistant to AMPC, MIC90 of Cephems were 0.25-1 microgram/mL, good activities against K. pneumoniae. Against beta-lactamase negative ampicillin-susceptible H. influenzae (BLNAS) 23 strains the MIC90s of CCL and other Cephems were 64 micrograms/mL and 0.25-8 micrograms/mL. The MIC90s of CDTR and CFTM wereor = 1 microgram/mL of BLNAR (15 strains). However there of CFDN and CPDX were 8 micrograms/mL and CCL wereor = 16 micrograms/mL. Two strains which were produced beta-lactamase were highly--ABPC resistant. Although B. catarrhalis all strains were produced beta-lactamase and Cephems except for CCL showed better susceptibility than AMPC. The MIC90s of Cephems were 0.25-2 micrograms/mL against Peptostreptococcus spp.

Although cagD and cagE (cagDE) identified upstream of cagA have been shown to be involved in the induction of interleukin (IL)-8 expression, the relationship between cagDE status and gastroduodenal diseases still remains to be examined. Thus we investigated prevalence and genetic diversity of cagD, cagE, and vacA in Helicobacter pylori strains isolated from patients with peptic ulcer or gastritis.We analyzed 73 H. pylori strains isolated from Japanese patients (gastritis (GA), 15; gastric ulcer (GU), 28; duodenal ulcer (DU), 23; GU and DU, 7). The presence of cagDE was evaluated by polymerase chain reaction (PCR) and Southern hybridization. The vacA genotype was examined by PCR, using type-specific primers.cagDE was present in 13 (86.7%) of 15 patients with GA, 26 (92.9%) of 28 patients with GU, 21 (91.3%) of 23 patients with DU, and 6 (85.7%) of 7 patients with GU and DU (P = 0.89). vacA signal sequence type s1 was found in 14 (93.3%) of 15 patients with GA, 26 (92.9%) of 28 patients with GU, 22 (95.7%) of 23 patients with DU, and 6 (85.7%) of 7 patients with GU and DU (P = 0.84). Sequences of cagDE and vacA in our Japanese strains were highly homologous with one another, and there were no disease-specific mutations.Most of the H. pylori strains in Japan were cagDE-positive, vacA s1 type, regardless of clinical outcome. The present study also indicated that these genes were conserved well among our H. pylori isolates.

We found a Factor (factor T) in aged mixtures of tungstate and phosphate which greatly enhanced the antibacterial effects of beta-lactams upon methicillin-resistant Staphylococcus aureus (MRSA). Factor T suppressed penicillinase production and the amount of penicillin-binding protein-2' in the membrane fraction, thus sensitizing MRSA strains to beta-lactams. In addition, Factor T caused a metachromatic reaction and prolonged the blood coagulation time, indicating that it is a heparin-like polyanion. Since Factor T becomes ineffective in the presence of a polycation, a charge interaction may play an important role in the enhancing effect. One possibility is that Factor T non-specifically inhibits several anion-sensitive enzymes. Factor T inhibited several nucleotide-interacting enzymes, but not most serum enzymes.

Clinical features in Vibrio infection are generally represented by gastrointestinal involvements such as food poisoning, and its prognosis is usually good. However, Vibrio vulnificus infection not uncommonly causes serious problems including sepsis, necrotizing fasciitis of the extremities, and other conditions, sometimes resulting in fatal outcome. In the present study, we analyzed clinical microbiological aspects of five cases with V. vulnificus infection. All the strains of V. vulnificus isolated in five patients are oxidase-positive Gram negative rods presenting comma-like configuration, which were yielded on TCBS agar forming green colonies; they were grayish-white in color and viscous in texture on 5% sheep blood agar, identification of bacteria were done using VITEK AMS (BioMerieux). Piperacillin and third-generation cephalosporins were found to have bactericidal activities against these strains. All five cases we experienced have primary ailments, and three cases out of the five had taken perishable sea-food before showing disease symptoms. V. vulnificus has two infection channels; one is external wound and the other is oral intake. The latter is said that it may become serious. This has a rather short period from the starting the symptoms to death, and there is high death rate. For life-saving, it is inevitably necessary to dose an effective antibacterial medicine in the early stage. If we suspect this bacteria in the test laboratory, it is important to report this to the clinical doctor. In other words, this is one of the bacteria that needs prompt treatment and further microbiology testing.

We have recently found a factor (Factor T) in aged mixtures of tungstate and phosphate which greatly enhances the antibacterial effects of beta-lactams on methicillin-resistant strains of staphylococcal species such as methicillin-resistant Staphylococcus aureus (MRSA), but shows only weak effects on methicillin-susceptible S. aureus and bacterial strains other than staphylococci. Factor T alone did not strongly inhibit cell metabolism and bacterial growth unless an excess amount was added. When Factor T was added to the culture medium beforehand, the growth of MRSA cells was rapidly suppressed just after addition of oxacillin (MPIPC). However, the growth of the cells was inhibited gradually when these two reagents were added in reverse order. For full expression of the enhancing effect, it seemed necessary for cells of MRSA strains to be incubated with Factor T for at least 2-3 hr. When the cells were washed after being sensitized by incubating them for 5 hr with Factor T, it took approximately 1 hr for the cells to recover their resistance to MPIPC. Factor T reduced the amount of penicillin-binding protein-2' (PBP 2'), and thus sensitized the MRSA strains to beta-lactams.