A simple and reliable method using a polymerase chain reaction (PCR) was devised to identify methicillinresistant staphylococci. By using lysates of the strain to be tested as templates and 22-mer oligonucleotides as primers, a 533-bp region of mecA, the structural gene of a low-affinity penicillin-binding protein (PBP 2'), was amplified by PCR and detected by agarose gel electrophoresis. Results obtained by this method were compared with those obtained by broth microdilution MIC determination for 210 and 100 clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci, respectively. Of 99 mecA-negative S. aureus isolates, 100% of the strains were methicillin susceptible and 98% of the strains were oxacillin susceptible. Three strains (3%) of 111 mecA-positive S. aureus isolates exhibited almost the same susceptibility to ,(-lactams as the mecA-negative ones and did not produce detectable amounts of PBP 2' despite the presence of the mecA gene. One of them yielded typically methicillin-resistant variants at a low frequency with concomitant recovery of PBP 2' production. The mecA gene was also found in coagulase-negative Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus sciuri, Staphylococcus saprophyticus, and Staphylococcus caprae and conferred resistance on most of the bacteria. * Corresponding author. species were identified by Staphyogram (Terumo, Tokyo, Japan), an identification kit, and the coagulase test. S. aureus SR3626, SR3633, SR3636, SR3639, SR3665, SR3681, and SR3716 were used as mecA-positive control organisms (12), and S. aureus ATCC 25923, 209P JC-1, and Smith were used as mecA-negative control organisms.

We recently cloned a gene encoding a new mitogenic factor (MF) from Streptococcus pyogenes NY-5. In the present study, we determined the distribution of this MF gene (ml) by PCR based upon its sequence. Of 371 streptococcal group A strains isolated from clinical specimens, 370 (99.7%) were positive for mf. The strain that was negative for the MF gene was also negative for the streptolysin 0 gene (slo). Some streptococcal strains belonging to groups C and G were negative for mf but positive for slo. Group B strains were negative for both. Furthermore, we examined the presence of mf in 54 strains belonging to 28 families and found mfonly in group A streptococci. These results indicate that mf is distributed specifically in group A streptococci and the presence of mf in clinical samples strongly suggests infection with group A streptococci.

On the basis of the known streptolysin O (SLO) genomic sequence of Streptococcus pyogenes group A, we identified the SLO genes in some strains of group C and group G streptococci by the polymerase chain reaction procedure (PCR). The entire open reading frame region of these genes was cloned and analyzed. Their nucleotide sequence data showed that the defined SLO genes in group C and group G are almost identical to that of group A.

A protocol based on the polymerase chain reaction (PCR) is the most sensitive method for detecting mycobacteria in clinical samples. However, few studies have assessed the usefulness of this method in the diagnosis of tuberculous effusion. We developed a highly sensitive and specific nested PCR method, that amplifies the bovine tuberculous MPB70 gene and the mycobacterial 16S rRNA gene for use in detecting Mycobacterium tuberculosis (M. tuberculosis) and mycobacteria, respectively, in clinical samples. We determined the sensitivity of this method for detecting mycobacteria in samples containing known amounts of mycobacterial DNA and in DNA extracted from pleural effusions obtained from 10 patients with pulmonary tuberculosis in whom standard microbiological techniques had detected mycobacteria in sputum but not in pleural effusion. The nested PCR method for the bovine tuberculous MPB70 gene and the mycobacterial 16S RNA gene was able to detect M. tuberculosis and mycobacterial genomes only if there were at least 2 copies per sample. Positive results for M. tuberculosis and the mycobacterial genomes were obtained by nested PCR in 2 of 10 and in 3 of 10 samples of pleural fluid, respectively but no mycobacteria were detected in malignant effusions obtained from 9 patients with lung cancer. The nested PCR method represents a rapid means for detecting mycobacteria in some pleural effusions previously found to be negative by culture. We speculate that the reaction of the host against mycobacteria is more important than the mycobacteria themselves in the pathogenesis of pleural effusion in which mycobacteria are not detected.

The species, mecA gene, beta-lactamase activity, the ability of slime formation and drug susceptibilities of 386 strains of staphylococci which were isolated from blood in our laboratory were studied. The coagulase typing of each strains identified as S. aureus was also determined. These 386 strains consisted of various species, e.g., S. aureus, S. epidermidis, S. capitis, S. caprae, S. hominis, S. simulans, S. haemolyticus, and S. lugdunensis. mecA (methicillin-resistant gene) was detected in 84 (67.7%) of 124 S. aureus and 195 (75.3%) of 259 CNS, but there was no statistical difference. However mecA positive rate was higher in S. epidermidis and S. caprae, lower in S. hominis compared with S. aureus, S. lugdunensis having mecA has not been reported, but one of two our S. lugdunensis strains had mecA. The positive rate (77.4%: 65/84) of beta-lactamase of methicillin-resistant Staphylococcus aureus (MRSA) was lower than that (95.5%: 187/195) of methicillin-resistant coagulase-negative staphylococci (MRCNS). Concerning the ability of slime formation, CNS had higher positive rate, especially in case of MRCNS (46.2%: 84/182) than S. aureus. On the other hand, MRCNS showed a tendency to be less resistant to some antimicrobials than MRSA. Especially against cephalothin (CET), the resistant rate of MRSA and MRCNS were 86.9% and 5.6%, respectively. Among the main species of MRCNS, S. capitis and S. caprae were more resistant than S. epidermidis and S. simulans to cefaclor (CCL), cefmetazole (CMZ), flomoxef (FMOX) and fosfomycin (FOM). Hereafter, one should be careful about infectious disease caused by CNS especially in immuno-compromised host, because many species of CNS have higher positive rate of beta-lactamase and slime formation than S. aureus beside mecA.

The combination of a culture method using gastric juice and a serum H. pylori immunoglobulin G assay was not invasive and clinically useful for evaluating H. pylori infections among handicapped patients.

We developed two PCR methods, which amplify bovine tuberculous MPB70 gene and mycobacterial 16S rRNA gene, for detection of tubercle bacilli and mycobacteria in sputum, respectively. Among 27 Mycobacterium species and 57 species of 30 genuses other than Mycobacterium, only M. tuberculosis (TB) complex, i.e., M. tuberculosis, M. bovis, M. africanum, M. microti showed DNA amplification by PCR for MPB70, and amplification of 16S rRNA gene were observed specific in Mycobacterium species. A combination of these PCR abilities were available to differentiate the TB complex and nontuberculous mycobacteria (NTM). We investigated the correlation between these methods and conventional methods with 311 sputa that were suspected mycobacteriosis. The PCR method could detect 12 cases of TB complex and 4 cases of NTM in 17 specimens, which were positive by conventional methods, but could not for one specimen. Among 294 specimens that were negative with conventional methods, the PCR method detected 13 and 8 cases of TB complex and NTM, respectively. These results were confirmed by commercial tuberculous specific DNA probe or investigation of the clinical background of the patients. On the other hand, 273 specimens showed negative result either PCR nor conventional methods. The PCR method did not detect tuberculous DNA in normal 197 sputa, which were not suspected mycobacteriosis. These results indicate that each one of these PCR methods is highly specific to TB complex or Mycobacterium species. We concluded that these PCR methods are useful and advanced methods for rapid and direct detection of tuberculosis and mycobacteriosis.

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