Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry for species identification of Acinetobacter strains isolated from blood cultures.

The clinical relevance of Acinetobacter species, other than A. baumannii, as human pathogens has not been sufficiently assessed owing to the insufficiency of simple phenotypic clinical diagnostic laboratory tests. Infections caused by these organisms have different impacts on clinical outcome and require different treatment and management approaches. It is therefore important to correctly identify Acinetobacter species. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been introduced to identify a wide range of microorganisms in clinical laboratories, but only a few studies have examined its utility for identifying Acinetobacter species, particularly those of the non-Acinetobacter baumannii complex. We therefore evaluated MALDI-TOF MS for identification of Acinetobacter species by comparing it with sequence analysis of rpoB using 123 isolates of Acinetobacter species from blood. Of the isolates examined, we identified 106/123 (86.2%) to species, and 16/123 (13.0%) could only be identified as acinetobacters. The identity of one isolate could not be established. Of the 106 species identified, 89/106 (84.0%) were confirmed by rpoB sequence analysis, and 17/106 (16.0%) were discordant. These data indicate correct identification of 89/123 (72.4%) isolates. Surprisingly, all blood culture isolates were identified as 13 species of Acinetobacter, and the incidence of Acinetobacter pittii was unexpectedly high (42/123; 34.1%) and exceeded that of A. baumannii (22/123; 17.9%). Although the present identification rate using MALDI-TOF MS is not acceptable for species-level identification of Acinetobacter, further expansion of the database should remedy this situation.

Onychomycosis caused by Fusarium proliferatum.

Fusarium infections in humans are usually opportunistic, but the fungus sometimes infects healthy persons, causing keratomycosis or onychomycosis. Onychomycosis is usually caused by F. solani or F. oxysporum. We report the first two cases of onychomycosis caused by F. proliferatum, and discuss methods of diagnosis and effective treatment. Nail samples from the two patients were examined by direct microscopy, cultured, and identified morphologically and genetically as F. proliferatum. Both patients were treated successfully with oral itraconazole, even though the minimum inhibitory concentration of itraconazole was relatively high in Patient 1. This is the first report of F. proliferatum as an agent of onychomycosis. Itraconazole may be effective in the treatment of onychomycosis caused by F. proliferatum.

In vitro antiseptic susceptibility of clinical isolates from nosocomial infections.

To evaluate the susceptibility of a large number of strains to various antiseptics, we elaborated a simple, qualitative broth turbidity method in which we could quickly judge the efficacy visually. For this method, we prepared a modified neutralizer broth, consisting of trypticase soy broth containing 15% Tween 80, 1% soybean lecithin and 0.5% sodium thiosulfate. The susceptibilities of Serratia marcescens No. 26 to 4 antiseptics obtained from the turbidity method showed a good agreement with those obtained from the colony-counting method; the 4 antiseptics tested were povidone-iodine (PVP-I), chlorhexidine gluconate (CHG), benzalkonium chloride (BAC) and alkyldiaminoethylglycine hydrochloride (AEG). Both PVP-I and BAC had complete efficacy in 0.5 min against all isolates tested [100 isolates of S. marcescens, 103 of Klebsiella pneumoniae, 99 of Pseudomonas aeruginosa, 19 of Alcaligenes faecalis and 30 of A. xylosoxidans subsp. xylosoxydans (A. xylosoxydans)]. In contrast, the effectiveness of CHG was weak compared with PVP-I, BAC and AEG. Strong resistance against AEG was noted even after 3-min exposure in 1 isolate each of A. faecalis and A. xylosoxydans. It is concluded that the turbidity test is a simple and accurate method to evaluate susceptibility to various antiseptics and that it is suitable for a screening of a large number of strains. Among the 4 antiseptics tested, PVP-I and BAC showed a consistently high activity against all isolates, confirming PVP-I and BAC to be clinically useful antiseptics.

[Proceedings of the workshop "The future aspect of clinical hematology laboratory and the role of medical technologists"].

In the workshop of the 34th in-service training course for University Hospital Medical Technologists in 2000 sponsored by the Ministry of Education, all participants discussed future aspects of the clinical hematology laboratory and the role of medical technologists. We report here a summary of the discussion.

Mycobacterium haemophilum infection in a Japanese patient with AIDS.

Mycobacterium haemophilum has been described as a pathogen that causes cutaneous lesions in immunocompromised patients. A specimen from a skin ulcer on the leg of a Japanese patient with acquired immunodeficiency syndrome yielded acid-fast bacilli on blood agar plates after 4 weeks of incubation at 30 degrees C, but the organism was not found on Ogawa egg slants. The organism was identified as M. haemophilum, on the basis of 16S rRNA gene sequence analysis. Prolonged culture in an optimal environment that includes an iron supplement, and growth temperatures at 28 degrees to 33 degrees C are necessary to grow M. haemophilum. Genotypic characterization of 16S rRNA is useful for a rapid diagnosis of this slowly growing mycobacterium.

Control of a methicillin-resistant Staphylococcus aureus outbreak in a neonatal intensive care unit by unselective use of nasal mupirocin ointment.

In September 1996, an outbreak of methicillin-resistant Staphylococcus aureus (MRSA) colonization occurred in the neonatal intensive care unit (NICU) of our hospital. After failing to control the outbreak by conventional methods we implemented an intranasal blanket use programme of mupirocin ointment from the beginning of November 1997. In the programme, patients who had been carrying MRSA received intranasal administration of the ointment three times daily for the first three days and consecutively three times weekly, while newly admitted patients and those who had not been colonized were prophylactically medicated three times weekly. This blanket administration was executed for one month. Methicillin-resistant Staphylococcus aureus colonization became undetectable in all but one intubated inpatient who had already been colonized before the start of the programme, and no new acquisitions occurred until the middle of January 1998, seven weeks after the termination of the blanket use programme. The rate of colonized patients in the unit also decreased. During and after the programme, neither an increase in minimum inhibitory concentration for the antibiotic nor apparent adverse reactions in any of the treated patients were observed. We concluded that this procedure is an effective method of controlling an MRSA outbreak in an NICU when the outbreak cannot be managed with conventional measures.

[Detection methods for drug-resistant bacteria in routine examination--MRSA].

Since methicillin-resistant Staphylococcus aureus(MRSA) is resistant not only to methicillin but also to multiple species of antibacterial drugs, there are cases where infections with MRSA are difficult to treat. For laboratory identification of MRSA, S. aureus is first identified, then an oxacillin (MPIPC) sensitivity test, PCR detection of the S. aureus mecA gene and an agglutination test using the latex sensitized with an anti-PBP-2' monoclonal antibody are usually utilized. However, the detection of MRSA does not necessarily indicate MRSA infection, and many cases only demonstrate MRSA colonization. Thus, a clinical investigation is indispensable for establishing the diagnosis of MRSA infection. In addition, recent reports also suggested a current trend toward decreasing sensitivity to Bactroban(MUP, mupirocin, an anti-intranasal MRSA antibacterial drug). Taking these into consideration, surveillance of the trend in patient's sensitive to various species of antibacterial drugs including MUP must continue further.

[Proceeding of the workshop "What an ideal clinical microbiological laboratory should be". The 33rd in-service training course for medical technologists of university hospitals in 1999].

In the workshop of the 33rd in-service training course for University Hospital Medical Technologists in 1999(sponsorship: the ministry of Education), all groups of participants were charged with discussing an ideal clinical microbiology laboratory. In conclusion, the successful operation of the ideal system of clinical microbiology should require a high level of competence in every staff member of the hospital. It must not be focused solely on the sophistication of laboratory methods. We must modify our behavior effectively and establish a good collaborative partnership with physicians and other health care professionals.

Susceptibility to vancomycin of methicillin-resistant Staphylococcus aureus isolated in a university hospital in Japan.

Intravenous vancomycin was approved in 1991 in Japan and has been widely used for treatment of infections caused by methicillin-resistant Staphylococcus aureus (MRSA). Consequently, ever since the initial discovery of vancomycin intermediate-resistant S. aureus in Japan, the vancomycin resistance of this organism has been a great concern in clinical settings. We investigated whether vancomycin resistance had emerged in MRSA isolated in our hospital since the approval of the use of intravenous vancomycin. Vancomycin susceptibility was evaluated on the basis of minimum inhibitory concentrations determined by the agar dilution method and a heterogeneous resistance examination. The median minimum inhibitory concentration of the 69 MRSA strains isolated in 1988 and the 74 isolated in 1998 was 0.75 microgram/ml and 1.0 microgram/ml, respectively (p < 0.001), however, all of the strains were classified in the susceptible group. None of them was an MRSA heterogeneously resistant to vancomycin (hetero-VRSA), which has been defined as a strain having a 1/10(6) or greater heterogeneously resistant subpopulation to vancomycin. In another set of investigations, no hetero-VRSA were found among 12 other MRSA strains isolated after intravenous administration of vancomycin for 14 or more days (range: 14 to 77 days). We conclude that while the use of intravenous vancomycin may have slightly lowered the vancomycin susceptibility of MRSA in our hospital, the decrease in so small that it may not be significant clinically. In addition, no hetero-VRSA were found in our hospial.

[Comparison of the newly developed MB redox system with mycobacteria growth indicator tube (MGIT) and 2% Ogawa egg media for recovery of mycobacteria in clinical specimens].

The rate of recovery and the mean time to detection of mycobacteria in clinical specimens were determined in a newly-developed MB Redox system based on liquid medium, and the results were compared with those of MGIT and 2% Ogawa egg media. From 587 sputum specimens processed, totally 203 mycobacterial isolates were detected, of which 177 (87.2%) with MB Redox, 185 (91.1%) with MGIT and 133 (65.6%) with 2% Ogawa medium. The difference in the percentages of positive cultures between either of the two liquid media and 2% Ogawa medium was significant (p < 0.0001). The mean time to detection of the Mycobacterium tuberculosis complex was 17.5 days with MB Redox, 18.7 days with MGIT, and 26.2 days with 2% Ogawa medium. The contamination rates were 1.5, 1.7, and 4.1% for MB Redox, MGIT, and 2% Ogawa medium, respectively. In conclusion, both MB Redox and MGIT systems, based on liquid medium, are more efficient than 2% Ogawa medium for the recovery of mycobacteria in clinical specimens.

Detection of methicillin-resistant Staphylococcus aureus in donor eye preservation media by polymerase chain reaction.

To examine contamination of donor eye preservation media with methicillin-resistant Staphylococcus aureus (MRSA), we studied the media to detect specific genes for MRSA by use of the polymerase chain reaction (PCR) method. The materials were 36 samples of donor eye preservation media (EP-II) in which donor eyes had been stored for keratoplasty. Polymerase chain reaction procedures were carried out to simultaneously detect the spa gene, which codes protein A of S. aureus, and the mecA gene, which codes penicillin-binding protein-2' contributing resistance to methicillin. Along with PCR analyses, the preservation media was examined by conventional culture methods to determine bacteriologic contamination. The PCR analyses of the 36 samples revealed that both the spa and the mecA genes were positive in five samples, only the spa gene was positive in two, and only the mecA gene was positive in two. The conventional culture of the media showed positive for MRSA in 5 samples, methicillin-susceptible S. aureus (MSSA) in 2, methicillin-susceptible coagulase-negative staphylococci (MS-CNS) in 4, and methicillin-resistant coagulase-negative staphylococci (MR-CNS) in 2 of the 36 samples. The results of PCR coincided well with those of conventional bacteriologic culture. Polymerase chain reaction analysis for spa and mecA genes is useful in detecting contamination of donor eye preservation media by MRSA, MSSA, MR-CNS, or MS-CNS in a shorter time than by conventional culture.

[The in vitro antifungal activities of fluconazole against pathogenic yeasts recently isolated from clinical specimens].

The emergence of Candida albicans resistance to azole antifungal agents have been reported in the U. S. and Europe. We examined the in vitro antifungal activities of fluconazole against clinical isolates collected by seven investigators in three years to examine if a tendency existed toward the development of azole-resistance among fungal isolates in Japan. The following results were obtained: 1. Sensitivities to fluconazole (FLCZ) were determined for yeast-like fungi, including 113 strains isolated in 1993, 149 strains isolated in 1994 and 205 strains isolated in 1995. No significant differences in sensitivities in the three years were detected. 2. Minimum inhibitory concentrations of FLCZ were 0.1-0.78 microgram/ml for C. albicans and 3.13-25 micrograms/ml for C. glabrata. Strains with 25 micrograms/ml of FLCZ's MIC were detected; two strains of C. krusei and one strain each of C. krusei, Trichospron beigelii and Hansenula anomala. No strains with higher than 50 micrograms/ml MIC of FLCZ were detected. 3. In vitro activities of FLCZ were compared between clinical strains isolated between 1993 and 1995 and clinical strains isolated before the marketing of FLCZ (up to December 1987) or clinical yeasts isolated between 1991 and 1992. No significant differences were observed, suggesting that no tendency existed toward azole resistance among fungal strains examined.

[The in vitro antibacterial activity of cefozopran against clinically isolated bacteria].

The in vitro antibacterial activity of cefozopran (CZOP) against recent clinical isolates was evaluated and compared with those of ceftazidime (CAZ), cefpirome (CPR), cefepime (CFPM), cefotaxime (CTX), sulbactam/cefoperazone (S/C), imipenem (IPM), oxacillin (MPIPC), and flomoxef (FMOX). MIC80 values of CZOP for methicillin-susceptible Staphylococcus aureus (MSSA, n = 41), methicillin-resistant Staphylococcus aureus (MRSA, n = 57), Streptococcus pneumoniae (n = 45), Enterococus faecalis (n = 49), Enterobacter cloacae (n = 50), Citrobacter freundii (n = 45), Serratia marcescens (n = 45), and Pseudomonas aeruginosa (n = 100) were 1, 32, 2, 16, 4, 1, 0.25, 8 micrograms/ml, respectively. CZOP was more active than CPR against P. aeruginosa and exhibited similar activity to CPR against other species. CZOP was especially active against S. marcescens with MIC values lower than 1 microgram/ml against all strains tested. CZOP was similarly active to or more active than CFPM against all species except for C. freundii. CZOP was not active against MRSA. Thus, we investigated the in vitro combination effects of CZOP/vancomycin (VCM) and CZOP/arbekacin (ABK) using the checkerboard method. The interaction between CZOP and VCM ranged from additive activity (0.5 < FIC index < or = 1.00, n = 37) to synergistic activity (FIC index < 0.50, n = 1), except for one strain showing indifference (1.00 < FIC index < or = 2.00). The interaction between CZOP and ABK ranged from additive activity (n = 22) to synergistic activity (n = 1). These date suggest the potential effect of combination therapy of (CZOP) and VCM or ABK against MRSA. The combined therapy is suggested to be useful to reduce side effects in patients with impared renal function, to reduce the administration dose of VCM or to treat infections of sites where achievable drug concentrations are lower than those commonly achieved in the bloodstream. We also investigated the combination effects of CZOP/AMK and CZOP/GM against CZOP-resistant P. aeruginosa (MIC > 16 micrograms/ml). The combination of CZOP/AMK showed additive activity (n = 9) to synergistic activity (n = 2). The combination of CZOP/GM showed additive activity (n = 5). These results suggest that combinations of CZOP with AMK or GM are effective in treating P. aeruginosa.

[Comparison of amplified Mycobacterium Tuberculosis Direct Test (MTD), Amplicor Mycobacteria kit (Amplicor) and PCR method for detection Mycobacterium tuberculosis in clinical specimens].

Accuracy amplified Mycobacterium Tuberculosis Direct Test (MTD), Amplicor Mycobacteria kit (Amplicor) and PCR method routinely used in the University of Tokyo Hospital (J. Clin. Microbiol. 31: 446-450 1991) were evaluated and compared with the same samples. The detection limits of MTD, Amplicor and PCR method (University of Tokyo) were 0.01-0.1 CFU/tube, 0.625 CFU/tube and 0.2 CFU/tube respectively, which were almost the same. These were shown to be at least as sensitive as the conventional culture techniques. The Tokyo Univ. method using radioisotope, takes up to 3 days, on the other hand 2 kits take 4-5 hours. These kits become useful tools for the early and rapid detection of M. tuberculosis in uncultured clinical specimens. But the risk of laboratory contamination and false-positive results remain. These must be further improved.

Nucleotide sequence comparison of the mycobacterial dnaJ gene and PCR-restriction fragment length polymorphism analysis for identification of mycobacterial species.

We recently reported a genus-specific PCR for the mycobacterial dnaJ gene. In the present study, we have determined the nucleotide sequences of the dnaJ gene from 19 mycobacterial species (Mycobacterium tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti, M. marinum, M. kansasii, M. gastri, M. simiae, M. scrofulaceum, M. szulgai, M. gordonae, M. avium, M. intracellulare, M. xenopi, M. fortuitum, M. chelonae, M. hemophilum, and M. paratuberculosis). On the basis of the amplified dnaJ gene nucleotide sequences, we constructed a phylogenetic tree of the mycobacterial species by using the neighbor-joining method and unweighted pairwise grouping method of arithmetic average. We found that the phylogenetic relationship inferred within the slowly growing species was in good agreement with the traditional classification, with three major branches corresponding to Runyon's groups I, II, and III. An exception was M. simiae, which was phylogenetically closer to the cluster including members of Runyon's group III than to that of Runyon's group I. On the other hand, the rapid growers, such as M. fortuitum and M. chelonae, did not form a coherent line corresponding to Runyon's group IV, indicating that our phylogenetic analysis based on the dnaJ gene reflects the phenotypic characteristics such as pigmentation but not the growth rate. Finally, we revealed the species-specific restriction sites within the amplified dnaJ gene to differentiate most of the mycobacterial DNA by a combination of PCR with restriction fragment length polymorphism analysis.

[Relationship between coagulase toxin-type and drug susceptibility in Staphylococcus aureus strains isolated in all the Japanese National University Hospitals].

Drug susceptibility of 430 Staphylococcus aureus strains isolated in 1991 from clinical specimens at all of the Japanese national university hospitals was evaluated in relationship with the epidemiological markers, namely, coagulase typing, and staphylococcal enterotoxins (SE) and toxic shock syndrome toxin 1 (TSST-1) production. There were five major methicillin-resistant Staphylococcus aureus (MRSA) groups in all the 252 MRSA strains: coagulase-type II-SEC + TSST-1- producing strains (II-SEC + TSST-1): 34.5%; coagulase-type II-no toxin-producing strains (II-): 15.4%; coagulase-type IV-SEA-producing strains (IV-SEA): 10.3%; coagulase-type II-SEA + SEC + TSST-1- producing strains (II-SEA + SEC + TSST-1): 8.7%; and coagulase-type III-no toxin-producing strains (III-): 7.1%. II-SEA + SEC + TSST-1 group was highly resistant to OFLX, whereas half of the other strain groups were sensitive to OFLX. Seventy-eight percent of the IV-SEA group was sensitive to FMOX, but there was no sensitive strain to FMOX in the II-SEA + SEC + TSST-1 group. More than 50% of the IV-SEA, III- and II-groups were sensitive to IPM, while the II-SEC + TSST-1 and II-SEA + SEC + TSST-1 groups were highly resistant to IPM. The III- and II-groups showed very good sensitivity to MINO, but the sensitivity to it of the II-SEA + SEC + TSST-1 group was very low. All of the strain groups were sensitive to ST except for the IV-SEA group. These results may provide useful information in the choice of antibacterial agents for MRSA infection.

Genus-specific polymerase chain reaction for the mycobacterial dnaJ gene and species-specific oligonucleotide probes.

Identification of tuberculous and nontuberculous mycobacteria by biochemical methods is a long-term process that takes up to 8 weeks for completion and requires expertise to interpret the results. In order to detect and differentiate the major pathogenic mycobacterial species, we developed genus-specific primers that amplify the dnaJ gene from the broad spectrum of mycobacterial species and determined the nucleotide sequences within the dnaJ genes from 19 mycobacterial species (Mycobacterium tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti, M. kansasii, M. marinum, M. gastri, M. simiae, M. scrofulaceum, M. szulgai, M. gordonae, M. avium, M. intracellulare, M. xenopi, M. fortuitum, M. chelonei, M. haemophilum, and M. paratuberculosis). On the basis of the dnaJ gene sequences, we developed dot blot hybridization analysis with species-specific oligonucleotide probes for the M. tuberculosis complex. M. avium, M. intracellulare, and M. kansaii, allowing a rapid identification of these species following polymerase chain reaction for the dnaJ gene. We conclude that polymerase chain reaction with the genus-specific primer that amplifies the dnaJ genes and subsequent dot blot analysis with species-specific oligonucleotide probes are most useful for differential diagnosis of tuberculosis and nontuberculous mycobacterial infections.

[Epidemiological study of Staphylococcus aureus isolated from the Japanese National University and Medical College Hospitals with coagulase typing, and production of enterotoxins and toxic shock syndrome toxin-1].

Coagulase typing, staphylococcus enterotoxins (SE) A to E or toxic shock syndrome toxin-1 (TSST = 1) production, and susceptibility to Oxacillin (MPIPC) were examined in 430 strains of S. aureus, which were isolated from clinical specimen of 43 Japanese National University or Medical College Hospitals during the one month period of August in 1990. Methicillin-resistant Staphylococcus aureus (MRSA): more than 4 mmg/ml of minimum inhibitory concentration for MPIPC in Mueller-Hinton broth containing 2% NaCl, occupied 58.6% of all the S. aureus, and more than 60% of the strains from admitted patients in all the areas of Japan except Hokkaidoh. Coagulase type II, SEC and TSST-1 producing strains were most frequently detected, 34.5% of all the MRSA. This kind of strain was distributed mainly in the eastern part of the Honshyu island, and showed high percentage especially in the Tohhoku and the Chyubu area. The second most frequent kind of MRSA was coagulase type II, no SE nor TSST-1 producing one, 15.4%, which was distributed mainly in the western part of Japan. Coagulase type IV, SEA producing MRSA strains and Coagulase type II, SEA, SEC and TSST-1 producing strains were detected in relatively high incidence, 10.3% and 8.7% respectively. Coagulase type III, no SE nor TSST-1 producing MRSAs demonstrated characteristic distribution, and were detected only in the western part of Japan, presenting the highest incidence in the Shikoku Island.