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The ability of the AccuProbe (Kyokuto) to identify mycobacteria directly from the positive cultures in an automatic detector for mycobacteria (BACTEC MGIT960, Becton Dickinson) was evaluated. Sputum samples were collected from patients with suspected mycobacteriosis between February and April 1999 and conventionally incubated in MGIT960 (37 degrees C for 42 days). The MGIT-positive cultures were successively incubated for several days and were directly identified with the AccuProbe. Monomycobacterial strains were detected from 93 (93.9%) of the 99 sputum samples and polymycobacterial strains were detected from 6 sputum samples (6.1%). Viable cell counts in the positive cultures in MGIT960 were determined using Middlebrook 7H10 agar. The cell counts of Mycobacterium tuberculosis and Mycobacterium avium-intracellulare complex (MAC) varied greatly among the individual samples; the former, 3.8 x 10(2)-2.5 x 10(6) cfu/ml and the latter 1.5 x 10(3)-1.9 x 10(8) cfu/ml. The great differences in the cell counts were observed among these samples. Although the cultures in MGIT were estimated as positive, the early stage of bacterial growth might be used as the samples for the cell counting. By this method, 96 of the total 101 cases were successfully identified as follows: M. tuberculosis 57 cases (56.4%) and MAC 39 cases (38.6%). In conclusion, the present results indicate that the direct identification of mycobacteria from the positive cultures in MGIT960 using AccuProbe is useful for a rapid diagnosis in microbiological testing for mycobacteriosis which has tended to increase in recent years.

Agar dilution and semi-solid agar dilution were used to determine the MIC of faropenem (FRPM) against 24 H. pylori isolates. FRPM was active against clarithromycin resistance H. pylori isolates. And, the MICs obtained by both methods were in agreement. The results suggest that FRPM was not affected by pH and is a clinically useful oral antibiotic for the eradication therapy of H. pylori infections.

The clinical isolates of Pseudomonas aeruginosa can be roughly classified into long- and short-lipopolysaccharide (LPS) strains and LPS-deficient strains, based on the silver-stained patterns of their LPSs after SDS-PAGE. The ionic binding of 3H-gentamicin, a polycationic antibiotic, to the negatively charged sites on the surface structures of P. aeruginosa strains, often differing in LPS structure, was the highest in the long-LPS strains followed in descending order by the short-LPS strains and LPS-deficient strains. It was presumed that a clinical isolate of P. aeruginosa No. 45 is lacking in the O-polysaccharide chains and some structures of the core-regions consisting of its LPS-structure after SDS-PAGE. On the other hand, the binding of 3H-gentamicin to this strain was quite. high, i.e., similar to that to of the long-LPS strains. To clarify this finding, P. aeruginosa PAC1R and its LPS-deficient mutants were used as reference strains because the chemical structures of their LPSs containing the repeated units of O-polysaccharides and the neutral sugar contents in the core-regions were previously confirmed. The PAC605 strain of the LPS-mutants of the PAC series, was completely lacking in the repeated units of O-polysaccharide and also lacking in some neutral sugar residues of the core-oligosaccharide region. However, this strain was highly bound to 3H-gentamicin, suggesting that the negatively charged sites on the deep core-oligosaccharide region and/or on lipid A participated in the binding of 3H-gentamicin. This manner of binding may be also applied to P. aeruginosa No. 45. When P. aeruginosa PAC1R, PAC605 and No. 45 strains were each exposed to gentamicin (20 micrograms/ml) for 10 minutes, the viable cell counts of PAC1R decreased to about 70% of the initial count, whereas the viable cell counts of PAC605 and No. 45 strains decreased to 3.6 and 11.0% respectively, indicating the vulnerability of both types of the strains to be enhanced by the bactericidal action of gentamicin with short-term incubation.

The MICs of amoxycillin, clarithromycin and azithromycin for 26 strains of Helicobacter pylori were determined using Mueller-Hinton semi-solid agar (SSA) without CO2 incubation, as in the conventional agar dilution method. The tested H. pylori strains grew satisfactorily in the SSA method within 48 h of incubation. Reasonable values of MICs of three antibiotics for these strains were obtained by this method, avoiding the inactivation of macrolides due to acidification of the medium. By this method, the MICs of the antibiotics for the reference strains were within the acceptable NCCLS ranges for quality control.

Emergency of multi-drug resistance in Pseudomonas aeruginosa isolates after exposure to gentamicin was investigated with reference to their LPS-compositions. Three strains each of P. aeruginosa, different in LPS-compositions (long- and short-LPS strains, and LPS-deficient strain) were once or repeatedly incubated overnight at 35 degrees C in Mueller-Hinton broth including different concentrations of gentamicin, a poly-cationic antibiotic. LPS-compositions of the bacteria which survived and were grown after exposure to gentamicin were analyzed and the susceptibility to some of anti-pseudomonal drugs was determined. Of the test 9 strains after single exposure, No. 4 (long-LPS) and No. 41 (short-LPS) formed the LPS-deficient mutants. The resistance to ceftazidime and gentamicin was found in No. 4 strain, but not in No. 41 strain. Drug resistance in different patterns was also found in Nos. 43, 29, 45 and 52 strains without any change in their LPS-compositions. Contrary to these results, the susceptibility to norflxacin increased in Nos. 21 and 45 strains without any change in their LPS-compositions. After repeated exposures to gentamicin, the LPS-compositions of Nos. 4 and 14 (short-LPS) were altered and formed the LPS-deficient and the Intermediate-LPS (between short and long compositions) mutants, respectively. The LPS-alteration in both strains were accompanied by the development of resistance to ceftazidime and gentamicin, respectively. The drug resistance in different patterns were also found in Nos. 43, 156, 29, 41, 21, 45 and 52 strains, without any change in the LPS-compositions, whereas the increase in susceptibility to norfloxacin was simultaneously found in No. 45 strain. The ionic-binding of [3H]-gentamicin to the cell surfaces of two LPS-deficient mutants of P. aeruginosa decreased markedly, in comparison with that of the respective parents, and these results might lead to the drug resistance in the LPS-deficient mutants. However, the gentamicin-induced resistance of some strains of P. aeruginosa (but no change in the LPS-compositions) to the test drugs including gentamicin indicates a possibility that negative charges on the bacterial surface were decreased by the drug exposure. The drug resistance and LPS-compositions in the gentamicin-induced mutants of P. aeruginosa were stable during or after the 15th in vitro serial transfers.

PMAC agar, a novel, selective and differential medium has been developed and was subjected for evaluation of its selective and differential capability of methicillin resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa from other bacteria such as Bacillus, Micrococcus, Gram-negative bacteria and drug resistant ones. Growth of MRSA and P. aeruginosa on PMAC agar was facilitated and their colonies were easily differentiated. Colonies of MRSA after 24 approximately 48 h incubation at 35 degrees C were small (2 to 4 mm in diameter), smooth and egg-yolk reaction positive. On the other hand, P. aeruginosa with pigment production (pyocianin, fluorescin or pyomelanin) formed large (2.5 to 7.0 mm in diameter), brownish black or brown colonies with a creamy edge. PMAC agar did not allow to grow unwanted bacteria tested except certain species formerly classified to Pseudomonas such as Burkholderia and Stenotrophomonas. However multi-drug resistant strains such as Enterobacter cloacae, Serratia marcescens and Acinetobacter calcoaceticus formed extremely small colonies. PMAC agar is recommended as a novel, useful medium for isolation, differentiation and presumptive identification of MRSA and P. aeruginosa from clinical and environmental sources.

Eight multi-drug resistant mutants (4.94%) were found in 162 clinical isolates of P. aeruginosa after exposure to norfloxacin at different concentrations (1/4, 1/2, 1, 2 and 4 MICs) and were investigated for the mechanisms of drug resistance. All the mutants were eight-times more resistant to norfloxacin than the respective parents, and showed the cross-resistance to the other fluoroquinolones. However, these mutants differed in the drug-resistant patterns; the 94-74 mutant was resistant to carbenicillin, ceftazidime and chloramphenicol, TA-15 mutant was resistant to imipenem, and the 93-183 was resistant to carbenicillin, ceftazidime and gentamicin. TA-16 mutant only showed a marked decrease in the bacterial uptake of norfloxacin. Profiles of the outer membrane proteins of the mutants were analyzed by SDS-PAGE method. The six mutants, except for TA-52 and 93-183 mutants, increased the intensity of bands in the 46 KD region. Three mutants (TA-15, TA-16 and 93-183) decreased the intensity of 44 KD (OMPE) and also the former two decreased the intensity of 22 KD (OMP G). The gryA mutations associated with fluoroquinolone-resistance were investigated for the eight mutants, and the 93-183 mutant showed to have the gyrA mutation caused by the alteration in the amino acid sequence of gyrA; Thr-83 (ACC) to Ile (ATC). The result of the present study indicate that multi-drug resistance of clinical isolates of P. aeruginosa develops through different types of mechanisms, and is not easily explained fully by the present results, and suggest that many factors attributed to the development of the resistances in those mutants remains to be clarified.

In the presence of 40% human serum plus polymorphonuclear leukocytes (PMNs, 10(7) cells/ml), changes in the surface properties of clinical isolates of P. aeruginosa were investigated by determining their serotypes and pyocin types as the markers. Furthermore, three isolates were tested for their susceptibility to anti-pseudomonal drugs and profiles of outer membrane proteins by the SDS-PAGE analysis. P. aeruginosa No. 21 which did not change in serotype and pyocin type after exposure to serum plus PMNs did not alter their susceptibility to all the drugs tested or their profiles of OMPs. In the case of P. aeruginosa No. 1-S, the variants with different serotypes were formed after the exposure, and increased their susceptibilities to some beta-lactams and norfloxacin which could penetrate into the bacterial cells through the porin channels of the outer membrane. Furthermore, two of the three type variants formed decreased their susceptibilities to gentamicin and polymyxin B which penetrated into the cells by the self-promoted uptake pathway. P. aeruginosa No. 1-R formed the serotype A and G variants with different pyocin types, only when exposed to serum plus PMNs for 24 hours, and the results were accompanied by the appearance of the porin D2 which was not detected in the parent cells. A small number of P. aeruginosa formed the variants with different serotypes and pyocin types owing to the alteration of their surface structures, when the cells were exposed to serum plus PMNs. The alterations were accompanied by the changes in some outer membrane proteins and the drug susceptibility to anti-pseudomonal drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

The serotype variants were formed in some isolates of Pseudomonas aeruginosa in the presence of anti-pseudomonal drugs in vitro. Pyocin type, various biochemical properties and drug susceptibility of these variants were compared with those of the parent cells. Furthermore, the patterns of outer membrane proteins were also examined for some respective parent and variant cells by the SDS-PAGE and compared with their drug susceptibilities. In P. aeruginosa No. 1-S (serotype E) and No. 1-R (serotype C), simultaneously isolated from the identical specimen of the infectious patient, the serotype variants were formed by anti-pseudomonal drugs and accompanied by changes in pyocin types, some biochemical properties and susceptibilities to various kinds of anti-pseudomonal drugs. When the profiles of outer membrane proteins of P. aeruginosa No. 1-R parent and two variant cells were evaluated by the SDS-PAGE, the quantitative differences of the porin proteins between the parent and variant cells were well related to their drug susceptibilities. Although eight variant cells with other serotype were formed from the parent cells (serotype A) of P. aeruginosa No. 13, four variants with serotype M and one non-typable variant showed changes in drug susceptibility such as resistance to imipenem only and no changes in biochemical properties. On the other hand, three other variants showed marked changes in two kinds of biochemical properties and in susceptibilities to various kinds of anti-pseudomonal drugs. From the results of SDS-PAGE, it was found that the porin protein profiles of outer membranes of these parent and variant cells except a few cells which corresponded to their drug susceptibility.

"Crime, Restoration and Responsibility" Report on Research Project No.185