Sparfloxacin resistance in clinical isolates of Streptococcus pneumoniae: involvement of multiple mutations in gyrA and parC genes

Antimicrobial susceptibility testing revealed among 150 clinical isolates of Streptococcus pneumoniae 4 pneumococcal isolates with resistance to fluoroquinolones (MIC of ciprofloxacin, >/=32 microgram/ml; MIC of sparfloxacin, >/=16 microgram/ml). Gene amplification and sequencing analysis of gyrA and parC revealed nucleotide changes leading to amino acid substitutions in both GyrA and ParC of all four fluoroquinolone-resistant isolates. In the case of strains 182 and 674 for which sparfloxacin MICs were 16 and 64 microgram/ml, respectively, nucleotide changes were detected at codon 81 in gyrA and codon 79 in parC; these changes led to an Ser-->Phe substitution in GyrA and an Ser-->Phe substitution in ParC. Strains 354 and 252, for which sparfloxacin MICs were 128 microgram/ml, revealed multiple mutations in both gyrA and parC. These strains exhibited nucleotide changes at codon 85 leading to a Glu-->Lys substitution in GyrA, in addition to Ser-79-->Tyr and Lys-137-->Asn substitutions in ParC. Moreover, strain 252 showed additional nucleotide changes at codon 93, which led to a Trp-->Arg substitution in GyrA. These results suggest that sparfloxacin resistance could be due to the multiple mutations in GyrA and ParC. However, it is possible that other yet unidentified mutations may also be involved in the high-level resistance to fluoroquinolones in S. pneumoniae.     

N-1-tert-butyl-substituted quinolones: in vitro anti-Mycobacterium avium activities and structure-activity relationship studies

We determined the MICs of 63 quinolones against 14 selected reference and clinical strains of the Mycobacterium avium-Mycobacterium intracellulare complex. Sixty-one of the compounds were selected from the quinolone library at Parke-Davis, Ann Arbor, Mich., including N-1-tert-butyl-substituted agents. T 3761 and tosufloxacin were also tested. The activities of all 63 compounds were compared with those of ciprofloxacin and sparfloxacin. The results showed 45 of the quinolones to be active against the M. avium-M. intracellulare complex, with MICs at which 50% of the strains were inhibited (MIC50s) of less than 32 micrograms/ml. Twenty-four of these quinolones had activities equivalent to or greater than that of ciprofloxacin, and nine of them had activities equivalent to or greater than that of sparfloxacin. The most active compounds were the N-1-tert-butyl-substituted quinolones, PD 161315 and PD 161314, with MIC50s of 0.25 microgram/ml and MIC90s of 1 microgram/ml; comparable values for ciprofloxacin were 2 and 4 micrograms/ml, respectively, while for sparfloxacin they were 1 and 2 micrograms/ml, respectively. The next most active compounds, with MIC50s of 0.5 microgram/ml and MIC90s of 1 microgram/ml, were the N-1-cyclopropyl-substituted quinolones, PD 138926 and PD 158804. These values show that the tert-butyl substituent is at least as good as cyclopropyl in rendering high levels of antimycobacterial activity. However, none of the quinolones showed activity against ciprofloxacin-resistant laboratory-derived M. avium-M. intracellulare complex strains. A MULTICASE program-based structure-activity relationship analysis of the inhibitory activities of these 63 quinolones and 109 quinolones previously studied against the most resistant clinical strain of M. avium was also performed and led to the identification of two major biophores and two biophobes.     

Gyrase mutations in laboratory-selected, fluoroquinolone-resistant mutants of Mycobacterium tuberculosis H37Ra

To characterize mechanisms of resistance to fluoroquinolones by Mycobacterium tuberculosis, mutants of strain H37Ra were selected in vitro with ofloxacin. Their quinolone resistance-determining regions for gyrA and gyrB were amplified and sequenced to identify mutations in gyrase A or B. Three types of mutants were obtained: (i) one mutant (TKp1) had no mutations in gyrA or gyrB; (ii) mutants that had single missense mutations in gyrA, and (iii) mutants that had two missense mutations resulting in either two altered gyrase A residues or an altered residue in both gyrases A and B. The TKp1 mutant had slightly reduced levels of uptake of [14C]norfloxacin, which was associated with two- to fourfold increases in the MICs of ofloxacin, ciprofloxacin, and sparfloxacin. Gyrase mutations caused a much greater increase in the MICs of fluoroquinolones. For mutants with single gyrA mutations, the increases in the MICs were 4- to 16-fold, and for mutants with double gyrase mutations, the MICs were increased 32-fold or more compared with those for the parent. A gyrA mutation in TKp1 secondary mutants was associated with 32- to 128-fold increases in the MICs of ofloxacin and ciprofloxacin compared with the MICs for H37Ra and an eight-fold increase in the MIC of sparfloxacin. Sparfloxacin was the most active fluoroquinolone tested. No sparfloxacin-resistant single-step mutants were selected at concentrations of > 2.5 micrograms/ml, and high-level resistance (i.e., MIC, > and = 5 micrograms/ml) was associated with two gyrase mutations. Mutations in gyrB and possibly altered levels of intracellular accumulation of drug are two additional mechanisms that may be used by M. tuberculosis in the development of fluoroquinolone resistance. Because sparfloxacin is more active in vitro and selection of resistance appears to be less likely to occur, it may have important advantage over ofloxacin or ciprofloxacin for the treatment of tuberculosis.     

Resistance to fluoroquinolones in Escherichia coli isolated from poultry

Quinolone-resistant Escherichia coli strains were isolated from poultry clinical samples in Saudi Arabia. The poultry flocks had been treated with oxolinic acid or flumequine prophylaxis. The measure of the uptake of fluoroquinolones showed that none of the strains had a reduced accumulation of quinolones. The result of complementation with the wild-type E. coli gyrA gene, which restored fluoroquinolone susceptibility, and the isolation of DNA gyrase from six isolates indicated that the resistant strains had an altered DNA gyrase. The minimum effective dose of ciprofloxacin for inhibition of supercoiling catalyzed by the isolated gyrases varied from 0.085 microgram/ml for a susceptible isolate (MIC < 4 micrograms/ml) up to 96 micrograms/ml for the more resistant one (strain 215, MIC > 64 micrograms/ml). For the same two isolates, the minimum effective doses of sparfloxacin varied from 0.17 up to 380 micrograms/ml. The in vitro selection of spontaneous single-step fluoroquinolone-resistant mutants using ciprofloxacin suggested that the more resistant mutants are likely the result of several mutations. These results also show that, as in human medicine, cross-resistance between older quinolones and fluoroquinolones can exist in veterinary isolates and reiterate the need for the prudent use of these drugs.     

Development of a rapid assay for detecting gyrA mutations in Escherichia coli and determination of incidence of gyrA mutations in clinical strains isolated from patients with complicated urinary tract infections

The MICs of ofloxacin for 743 strains of Escherichia coli isolated from 1988 to 1994 were determined by testing. The strains were from patients with urinary tract infections complicated by functional or anatomical disorders of the urinary tract. Those determined to be ofloxacin resistant (MIC, > or =12.5 microg/ml) comprised 3 of 395 strains (1.3%) from the 1988 to 1990 group, 2 of 166 strains (1.2%) from the 1991 to 1992 group, and 7 of 182 strains (3.8%) from the 1993 to 1994 group. The incidence of resistant strains increased significantly during this period. The percentage of isolates with moderately decreased susceptibilities to ofloxacin (MIC, 0.39 to 3.13 microg/ml) also rose during the same period. To determine the incidence of gyrA mutations in urinary-tract-derived strains of E. coli, we developed a simple and rapid assay based on PCR amplification of the region of the gyrA gene containing the mutation sites followed by digestion of the PCR product with a restriction enzyme. Using this assay, we examined all 182 strains isolated in 1993 and 1994 for the presence of mutations at Ser-83 and Asp-87 in the gyrA gene. Of these strains, 33 (18.1%) had mutations in the gyrA gene. The incidences of mutations at Ser-83, at Asp-87, and at both codons were 10.4 (19 strains), 4.4 (8 strains), and 3.3% (6 strains), respectively. To determine the correlation of the mutations in the gyrA gene with susceptibilities to quinolones (nalidixic acid, ofloxacin, norfloxacin, and ciprofloxacin), we further examined 116 strains for which the MICs of ofloxacin were > or =0.2 microg/ml that were chosen from the isolates in the 1988 to 1992 group. The MICs of nalidixic acid for the strains without mutations at either Ser-83 or Asp-87 were < or =25 microg/ml, whereas those for the strains with single mutations or double mutations were from 50 to >800 microg/ml. For the fluoroquinolones, significant differences in the distributions of the MICs were observed among the strains without mutations, with single mutations, and with double mutations. The accumulation of mutations in the gyrA gene was associated with an increase in fluoroquinolone resistance. Ofloxacin MICs for the majority of the strains with single and double mutations were 0.39 to 3.13 and 6.25 to 100 microg/ml, respectively. This study demonstrates a chronological increase in the percentage of not only highly fluoroquinolone-resistant strains, corresponding to those with double mutations in the gyrA gene, but also strains with moderately decreased susceptibilities to fluoroquinolones, corresponding to those with single mutations. This increase in the incidence of strains with a single mutation in the gyrA gene portends a further increase in the incidence of strains with clinically significant resistance to fluoroquinolones.     

Activities of new fluoroquinolones against fluoroquinolone-resistant pathogens of the lower respiratory tract

The activities of six new fluoroquinolones (moxifloxacin, grepafloxacin, gatifloxacin, trovafloxacin, clinafloxacin, and levofloxacin) compared with those of sparfloxacin and ciprofloxacin with or without reserpine (20 microg/ml) were determined for 19 Streptococcus pneumoniae isolates, 5 Haemophilus sp. isolates, and 10 Pseudomonas aeruginosa isolates with decreased susceptibility to ciprofloxacin from patients with clinically confirmed lower respiratory tract infections. Based upon the MICs at which 50% of isolates were inhibited (MIC50s) and MIC90s, the most active agent was clinafloxacin, followed by (in order of decreasing activity) trovafloxacin, moxifloxacin, gatifloxacin, sparfloxacin, and grepafloxacin. Except for clinafloxacin (and gatifloxacin and trovafloxacin for H. influenzae), none of the new agents had improved activities compared with that of ciprofloxacin for P. aeruginosa and H. influenzae. A variable reserpine effect was observed for ciprofloxacin and S. pneumoniae; however, for 9 of 19 (47%) isolates the MIC of ciprofloxacin was decreased by at least fourfold, suggesting the presence of an efflux pump contributing to the resistance phenotype. The laboratory parC (Ser79) mutant strain of S. pneumoniae required eightfold more ciprofloxacin for inhibition than the wild-type strain, but there was no change in the MIC of sparfloxacin and only a 1-dilution increase in the MICs of the other agents. For efflux pump mutant S. pneumoniae the activities of all the newer agents, except for levofloxacin, were reduced. Except for clinafloxacin, all second-step laboratory mutants required at least 2 microg of all fluoroquinolones per ml for inhibition.     

Efflux pump-mediated quinolone resistance in Staphylococcus aureus strains wild type for gyrA, gyrB, grlA, and norA

Fluoroquinolone efflux was studied in 47 Staphylococcus aureus clinical strains with MICs of ciprofloxacin (CFX) of < or = 2 micrograms/ml. Forty-three strains were wild type for gyrA, gyrB, and grlA quinolone resistance-determining regions and for norA and its promoter region. Forty of these strains (MICs of CFX, 0.1 to 0.2 microgram/ml) did not show efflux of fluoroquinolones. Three strains (MICs of CFX, 1 to 2 micrograms/ml) showed efflux. These results suggest that efflux can appear in S. aureus clinical strains in the absence of mutations in norA and its promoter.     

Anomalous patterns of callosal connections develop in visual cortex of monocularly enucleated hamsters

Six multiply resistant isolates of Salmonella typhimurium var. copenhagen with high-level resistance to fluoroquinolones (e.g., MIC of ciprofloxacin: 32 micrograms/ml) were isolated from human patients (n = 3) and from cattle (n = 3). The isolates were examined by complementation tests using a set of broad-host-range plasmids, which carry either the gyrA+ or the gyrB+ genes or a combination of both from Escherichia coli K-12. The results indicated a combination of gyrA and gyrB mutations in all isolates. Subsequent direct sequencing of PCR-generated internal DNA fragments of gyrA revealed an identical double mutation in all six isolates (Ser-83-->Ala and Asp-87-->Asn). In addition, the results of phenotypic (i.e., phagetype, biotype, serotype) and genotypic characterization [i.e., ribotyping and polymerase chain reaction fingerprinting (PCR-fingerprinting)] were identical for all six isolates and were distinguishable from a quinolone-susceptible strain of the same serovar and an unrelated isolate of S. typhimurium. These data indicate the clonal identity of the fluoroquinolone-resistant strains of S. typhimurium isolated from men and cattle in Germany.     

Antibacterial activity of gatifloxacin (AM-1155, CG5501, BMS-206584), a newly developed fluoroquinolone, against sequentially acquired quinolone-resistant mutants and the norA transformant of Staphylococcus aureus

Alternate mutations in the grlA and gyrA genes were observed through the first- to fourth-step mutants which were obtained from four Staphylococcus aureus strains by sequential selection with several fluoroquinolones. The increases in the MICs of gatifloxacin accompanying those mutational steps suggest that primary targets of gatifloxacin in the wild type and the first-, second-, and third-step mutants are wild-type topoisomerase IV (topo IV), wild-type DNA gyrase, singly mutated topo IV, and singly mutated DNA gyrase, respectively. Gatifloxacin had activity equal to that of tosufloxacin and activity more potent than those of norfloxacin, ofloxacin, ciprofloxacin, and sparfloxacin against the second-step mutants (grlA gyrA; gatifloxacin MIC range, 1.56 to 3.13 microg/ml) and had the most potent activity against the third-step mutants (grlA gyrA grlA; gatifloxacin MIC range, 1.56 to 6.25 microg/ml), suggesting that gatifloxacin possesses the most potent inhibitory activity against singly mutated topo IV and singly mutated DNA gyrase among the quinolones tested. Moreover, gatifloxacin selected resistant mutants from wild-type and the second-step mutants at a low frequency. Gatifloxacin possessed potent activity (MIC, 0.39 microg/ml) against the NorA-overproducing strain S. aureus NY12, the norA transformant, which was slightly lower than that against the parent strain SA113. The increases in the MICs of the quinolones tested against NY12 were negatively correlated with the hydrophobicity of the quinolones (correlation coefficient, -0.93; P < 0.01). Therefore, this slight decrease in the activity of gatifloxacin is attributable to its high hydrophobicity. Those properties of gatifloxacin likely explain its good activity against quinolone-resistant clinical isolates of S. aureus harboring the grlA, gyrA, and/or norA mutations.     

Antifungal susceptibility of clinically isolated Candida albicans by broth microdilution method

In this study we investigated the antifungal susceptibility of 285 strains of Candida albicans isolates at Kinki University Hospital from March 1995 to December 1996. The antifungal agents tested were fluconazole, miconazole, intraconazole, amphotericin B and flucytosine. The susceptibility testing were performed according to the broth microdilution method standardized by National Committee for Clinical Laboratory Standards (M27-T). Most isolates of C. albicans showed relatively a low MIC value and the MIC90S were calculated at 1 microgram/ml; fluconazole, 0.125 microgram/mg; miconazole, 0.06 microgram/ml; itraconazole, 1 microgram/ml; amphotericin B, 0.25 microgram/ml; flucytosine. There was only one strain that showed high resistance against fluconazole and it showed cross-resistance against miconazole and itraconazole. There were two flucytosine resistant strains. The MICs of amphotericin B were tightly clustered and resistant strain were not observed.     

Susceptibilities of bacteria isolated from patients with lower respiratory infectious diseases to antibiotics (1996)

The bacteria isolated from the patients with lower respiratory tract infections were collected by institutions located throughout Japan, since 1981. Ikemoto et al. have been investigating susceptibilities of these isolates to various antibacterial agents and antibiotics, and characteristics of the patients and isolates from them each year. Results obtained from these investigations are discussed. In 16 institutions around the entire Japan, 557 strains of presumably etiological bacteria were isolated mainly from the sputa of 449 patients with lower respiratory tract infections during the period from October 1996 to September 1997. MICs of various antibacterial agents and antibiotics were determined against 98 strains of Staphylococcus aureus, 93 strains of Streptococcus pneumoniae, 84 strains of Haemophilus influenzae, 84 strains of Pseudomonas aeruginosa (non-mucoid strains), 17 strains of Pseudomonas aeruginosa (mucoid strains), 31 strains of Moraxella subgenus Branhamella catarrhalis, 21 strains of Klebsiella pneumoniae etc., and the drug susceptibilities of these strains were assessed except for those strains that died during transportation. 1) S. aureus S. aureus strains for which MICs of oxacillin (MPIPC) were higher than 4 micrograms/ml (methicillin-resistant S. aureus) accounted for 67.3%. The frequency of the drug resistant bacteria increased comparing to the previous year's 52.7%. Arbekacin (ABK) and vancomycin (VCM) showed the highest activities against both S. aureus and MRSA with MIC80s of 1 microgram/ml. 2) S. pneumoniae Imipenem (IPM) and panipenem (PAPM) of carbapenems showed the most potent activities with MIC80s of 0.063 microgram/ml. Faropenem (FRPM) showed the next potent activity with MIC80 of 0.125 microgram/ml. The other drugs except erythromycin (EM), clindamycin (CLDM) and tetracycline (TC) were active against S. pneumoniae tested with MIC80s of 8 micrograms/ml or below. 3) H. influenzae The activities of all drugs were potent against H. influenzae tested with MIC80s of 4 micrograms/ml or below. Cefotiam (CTM), cefmenoxime (CMX), cefditoren (CDTR) and ofloxacin (OFLX) showed the most potent activities with MIC80s of 0.063 microgram/ml. 4) P. aeruginosa (mucoid strains) Tobramycin (TOB) showed the most potent activity against P. aeruginosa (mucoid strains) with MIC80 of 1 microgram/ml. Ceftazidime (CAZ), cefsulodin (CFS), IPM, gentamicin (GM), ABK and ciprofloxacin (CPFX) showed the next potent activities, with MIC80s of 2 micrograms/ml. The MIC80s of the other drugs ranged from 4 micrograms/ml to 16 micrograms/ml. 5) P. aeruginosa (non-mucoid strains) TOB and CPFX showed the most potent activities against P. aeruginosa (non-mucoid strains) with MIC80s of 1 microgram/ml. The MIC80s of piperacillin (PIPC) and cefoperazone (CPZ) were 16 micrograms/ml in 1995, and they were 64 micrograms/ml in 1996. 6) K. pneumoniae All drugs except ampicillin (ABPC) were active against K. pneumoniae. CMX, cefpirome (CPR), cefozopran (CZOP) and carumonam (CRMN) showed the most potent activities against K. pneumoniae with MIC80s of 0.125 microgram/ml. The MIC80s of the other drugs ranged from 0.25 microgram/ml to 2 micrograms/ml. 7) M.(B) catarrhalis Against M.(B.) catarrhalis, all drugs showed good activities with MICs of 4 micrograms/ml or below. IPM and minocycline (MINO) showed the most potent activities with MICs of 0.063 microgram/ml. Also, we investigated year to year changes in the characteristics of patients, their respiratory infectious diseases, and the etiology. Patients' backgrounds were examined for 557 isolates from 449 cases. The examination of age distribution indicated that the proportion of patients with ages over 60 years was 71.0% of all the patients showing a slight increase over that in 1994. Proportions of diagnosed diseases were as follows: Bacterial pneumonia and chronic bronchitis were the most frequent with 35.9% and 30.3% respectively. They were followed by bronchiectasis with a proportion of 10.     

Skin smears in leprosy: is reduction in number of sites justified?

BACKGROUND: Infections caused by Streptococcus pneumoniae continue to be a significant cause of mortality and morbidity in humans. Diseases caused by multi-resistant pneumococci are increasing rapidly worldwide. The fluoroquinolones have been widely used clinically to treat infectious diseases. The results of a study here on the five fluoroquinolones susceptibilities of S. pneumoniae are reported from the Taichung Veterans General Hospital. METHODS: Minimum inhibitory concentrations (MICs) of five quinolones (enoxacin, norfloxacin, ofloxacin, levofloxacin and ciprofloxacin) were determined for 106 strains of S. pneumoniae. All MICs were determined by the agar dilution method utilizing Mueller-Hinton agar supplemented with 5% sheep blood. RESULTS: MIC90 of levofloxacin was 1 microgram/ ml, and was unaffected by penicillin-susceptibility. MIC90 of ofloxacin and that of ciprofloxacin were 2 and 4 micrograms/ml, respectively, with 90.6% sensitive to ofloxacin. MIC90 of enoxacin and that of norfloxacin were higher than other compounds. CONCLUSIONS: The in vitro activity of levofloxacin is twice that of ofloxacin, 4-fold of ciprofloxacin, 16-fold of norfloxacin, and 64-fold of enoxacin. MICs of these five quinolones were unaffected by penicillin-susceptibility. The antibacterial activity of levofloxacin was better than that of ofloxacin and ciprofloxacin, norfloxacin, or enoxacin against S. pneumoniae.     

Carbapenem activities against Pseudomonas aeruginosa: respective contributions of OprD and efflux systems

While meropenem MICs were strongly influenced by the presence or absence of the MexAB-OprM efflux pump in both OprD-proficient and -deficient strain backgrounds, MICs of imipenem and of ER-35786 remained unchanged, demonstrating that meropenem is a substrate of MexAB-OprM but not imipenem and ER-35786. In vitro, all three carbapenems selected loss of OprD as a first mechanism of resistance. However, in an OprD-deficient background, meropenem was able to select MexAB-OprM overproducers as a secondary resistance mechanism, while ER-35786 selected a mutant cross-resistant to sparfloxacin and cefpirome.     

In vitro antimicrobial activity of fluoroquinolones against clinical isolates obtained in 1989 and 1990

The in vitro activity of nine fluoroquinolones, enoxacin, norfloxacin, ofloxacin, ciprofloxacin, lomefloxacin, tosufloxacin, sparfloxacin, fleroxacin and levofloxacin, and two earlier quinolones, nalidixic acid and pipemidic acid, against 1,346 bacterial strains isolated clinically between 1989 and 1990, was evaluated by agar dilution method. The bacteria studied were Staphylococcus aureus (including methicillin-susceptible and -resistant strains), Staphylococcus epidermidis, Enterococcus species (including high-level gentamicin-resistant strains), Escherichia coli, Salmonella species, Proteus mirabilis, Proteus vulgaris, Morganella morganii, Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens, Citrobacter spp., Pseudomonas aeruginosa, Pseudomonas cepacia, Acinetobacter baumannii, and Bacteroides fragilis. In contrast to the moderate to poor activity of two earlier quinolones, the fluoroquinolones acted well against most Enterobacteriaceae and A. baumannii. The minimum inhibitory concentrations for 90% of the drug strains (MIC90s) were < 1 microgram/mL against most tested species. Ciprofloxacin, tosufloxacin, sparfloxacin, and levofloxacin were more effective against multi-drug-resistant nosocomial pathogens. All fluoroquinolones assayed were very active against methicillin-susceptible S. aureus, with MIC90s < or = 1 microgram/mL. For methicillin-resistant strains, on the other hand, the MIC90s were > or = 4 micrograms/mL. There was no significant difference in fluoroquinolone susceptibility between methicillin-susceptible and -resistant S. epidermidis. Sparfloxacin, tosufloxacin, ciprofloxacin and levofloxacin were more active against enterococci. Most fluoroquinolones were relatively inactive against B. fragilis, with the exception of tosufloxacin, sparfloxacin and levofloxacin. The MIC90s of most quinolones assayed against K. pneumoniae, Citrobacter spp., E. cloacae, S. aureus and S. epidermidis were at least four-fold higher in our study. Therefore, it is important for physicians to use fluoroquinolones carefully so as to prevent or delay the emergence of resistant strains.     

MIC and time-kill study of antipneumococcal activities of RPR 106972 (a new oral streptogramin), RP 59500 (quinupristin-dalfopristin), pyostacine (RP 7293), penicillin G, cefotaxime, erythromycin, and clarithromycin against 10 penicillin-susceptible and -resistant pneumococci

Broth MICs and time-kill studies were used to test the activity of RP 59500 (quinupristin-dalfopristin), RPR 106972, pyostacine (RP 7293), erythromycin, clarithromycin, and cefotaxime for four penicillin-susceptible (MICs of 0.008 to 0.03 microgram/ml), two penicillin-intermediate (MIC of 0.25 microgram/ml), and four penicillin-resistant (MIC of 2.0 to 4.0 micrograms/ml) strains of pneumococci: 6 of 10 strains were resistant to macrolides (MICs of > or = 0.5 microgram/ml). MICs of RP 59500 (0.5 to 1.0 microgram/ml), RPR 106972 (0.125 to 0.25 microgram/ml), and pyostacine (0.125 to 0.25 microgram/ml) did not alter with the strain's penicillin or macrolide susceptibility status. Three penicillin-susceptible strains and one penicillin-intermediate strain were susceptible to macrolides (MICs of < or = 0.25 microgram/ml); the macrolide MICs for the remaining strains were > or = 4.0 micrograms/ml. Cefotaxime MICs rose with those of penicillin G, but all strains were inhibited at MICs of < or = 2.0 micrograms/ml. RP 59500 was bactericidal for all strains after 24 h at 2 x MIC and yielded 90% killing of all strains at 6 h at 2 x MIC; at 8 x MIC, RP 59500 showed 90% killing of six strains within 10 min (approximately 0.2 h). In comparison, RPR 106972 was bactericidal for 9 of 10 strains at 2 x MIC after 24 h and yielded 90% killing of all strains at 2 x MIC after 6 h; 90% killing of six strains was found at 8 x MIC at 0.2 h. Results for pyostacine were similar to those of RPR 106972. Erythromycin and clarithromycin were bactericidal for three of four macrolide-susceptible strains after 24 h at 4 x MIC. Clarithromycin yielded 90% killing of three strains at 8 x MIC after 12 h. Cefotaxime was bactericidal for all strains after 24 h at 4 x MIC, yielding 90% killing of all strains after 6 h at 4 x MIC. All three streptogramins yielded rapid killing of penicillin- and erythromycin-susceptible and -resistant pneumococci and were the only compounds which killed significant numbers of strains at 0.2 h.     

Isolation and characterization of a dibenzofuran-degrading yeast: identification of oxidation and ring cleavage products

PURPOSE: To investigate the development of fluoroquinolone resistance among Neisseria gonorrhoeae isolates in Japan and the frequency and patterns of mutations involving the GyrA and ParC proteins, which confer quinolone resistance to the bacteria, in isolates. MATERIALS AND METHODS: Antimicrobial susceptibilities of 145 gonococcal isolates, including 79 isolated from February 1992 through February 1993 and 66 isolated from February 1995 through February 1996, to six fluoroquinolones and several other antibiotics were compared with those of 27 isolates obtained from 1981 through 1984. To identify mutations in gyrA and parC genes of the isolates, the quinolone resistance-determining regions of the gyrA and parC genes were PCR-amplified and the PCR products were directly sequenced. RESULTS: The minimum inhibitory concentration for 90% of strains (MIC90) values of norfloxacin for the isolates from 1992 to 93 (4 microg./ml.) and 1995 to 96 (8 microg./ml.) were 16- and 32-fold, respectively, higher than those for isolates from 1981 to 84 (0.25 microg./ml.). The MIC90 values of ciprofloxacin for isolates from 1992 to 93 (0.5 microg./ml.) and 1995 to 96 (1 microg./ml.) showed increase of 8- and 16-fold, respectively, in comparison with those from 1981 to 84 (0.063 microg./ml.). The isolates from 1992 to 93 and 1995 to 96 were also less susceptible to newer fluoroquinolones including levofloxacin, sparfloxacin, DU-6859a and AM-1155, as compared with those from 1981 to 84. In 46 (67.6%) and 16 (23.5%) of the 68 gonococcal strains sequenced, GyrA and ParC mutations were identified, respectively. No ParC substitutions were identified in any isolates without co-existence of the GyrA mutation. A Ser-91 to Phe mutation, which was detected in 30 (65.2%) of the 46 isolates with GyrA mutations, was the most common GyrA mutation. Mutants with the single Ser-91 to Phe substitution in GyrA were 12-fold and at least 13-fold, respectively, less susceptible to norfloxacin and ciprofloxacin than the wild type. CONCLUSIONS: The results obtained in this study suggest that a high prevalence of gonococcal isolates with the Ser-91 to Phe mutation in GyrA has reduced the susceptibility of this organism to fluoroquinolones in Japan.     

In vitro activity of Bay 12-8039, a new 8-methoxyquinolone, compared to the activities of 11 other oral antimicrobial agents against 390 aerobic and anaerobic bacteria isolated from human and animal bite wound skin and soft tissue infections in humans

The in vitro activity of Bay 12-8039, a new oral 8-methoxyquinolone, was compared to the activities of 11 other oral antimicrobial agents (ciprofloxacin, levofloxacin, ofloxacin, sparfloxacin, azithromycin, clarithromycin, amoxicillin clavulanate, penicillin, cefuroxime, cefpodoxime, and doxycycline) against 250 aerobic and 140 anaerobic bacteria recently isolated from animal and human bite wound infections. Bay 12-8039 was active against all aerobic isolates, both gram-positive and gram-negative isolates, at < or = 1.0 microg/ml (MICs at which 90% of isolates are inhibited [MIC90s < or = 0.25 microg/ml) and was active against most anaerobes at < or = 0.5 microg/ml; the exceptions were Fusobacterium nucleatum and other Fusobacterium species (MIC90s, > or = 4.0 microg/ml) and one strain of Prevotella loeschii (MICs, 2.0 microg/ml). In comparison, the other quinolones tested had similar in vitro activities against the aerobic strains but were less active against the anaerobes, including peptostreptococci, Porphyromonas species, and Prevotella species. The fusobacteria were relatively resistant to all the antimicrobial agents tested except penicillin G (one penicillinase-producing strain of F. nucleatum was found) and amoxicillin clavulanate.     

Susceptibilities of penicillin- and erythromycin-susceptible and -resistant pneumococci to HMR 3647 (RU 66647), a new ketolide, compared with susceptibilities to 17 other agents

Susceptibility of 230 penicillin- and erythromycin-susceptible and -resistant pneumococci to HMR 3647 (RU 66647), a new ketolide, was tested by agar dilution, and results were compared with those of erythromycin, azithromycin, clarithromycin, roxithromycin, rokitamycin, clindamycin, pristinamycin, ciprofloxacin, sparfloxacin, trimethoprim-sulfamethoxazole, doxycycline, chloramphenicol, cefuroxime, ceftriaxone, imipenem, and vancomycin. HMR 3647 was very active against all strains tested, with MICs at which 90% of the strains were inhibited (MIC90s) of 0.03 microg/ml for erythromycin-susceptible strains (MICs, < or =0.25 microg/ml) and 0.25 microg/ml for erythromycin-resistant strains (MICs, > or =1.0 microg/ml). All other macrolides yielded MIC90s of 0.03 to 0.25 and >64.0 microg/ml for erythromycin-susceptible and -resistant strains, respectively. The MICs of clindamycin for 51 of 100 (51%) erythromycin-resistant strains were < or =0.125 microg/ml. The MICs of pristinamycin for all strains were < or =1.0 microg/ml. The MIC90s of ciprofloxacin and sparfloxacin were 4.0 and 0.5 microg/ml, respectively, and were unaffected by penicillin or erythromycin susceptibility. Vancomycin and imipenem inhibited all strains at < or =1.0 microg/ml. The MICs of cefuroxime and cefotaxime rose with those of penicillin G. The MICs of trimethoprim-sulfamethoxazole, doxycycline, and chloramphenicol were variable but were generally higher in penicillin- and erythromycin-resistant strains. HMR 3647 had the best kill kinetics of all macrolides tested against 11 erythromycin-susceptible and -resistant strains, with uniform bactericidal activity (99.9% killing) after 24 h at two times the MIC and 99% killing of all strains at two times the MIC after 12 h for all strains. Pristinamycin showed more rapid killing at 2 to 6 h, with 99.9% killing of 10 of 11 strains after 24 h at two times the MIC. Other macrolides showed significant activity, relative to the MIC, against erythromycin-susceptible strains only.     

Characterisation of DNA gyrase and measurement of drug accumulation in clinical isolates of Acinetobacter baumannii resistant to fluoroquinolones

Twelve clinical isolates of Acinetobacter baumannii highly resistant to pefloxacin (MIC > or = 32 mg/L) and to ciprofloxacin (MIC > or = 16 mg/L), were studied. A susceptible isolate used as a reference (MIC of 0.032 and 0.25 mg/L for ciprofloxacin and pefloxacin, respectively) accumulated 85 mg of pefloxacin per litre of cell volume within 10 min, from a solution containing 10 mg/L of antibiotic. One resistant isolate accumulated the same amount of pefloxacin, while the 11 others accumulated between 40 and 70 mg/L of cell volume. The differences between reference and resistant isolates with respect to ciprofloxacin and sparfloxacin accumulation were less pronounced. There were no apparent differences in the outer membrane protein profiles of susceptible and resistant isolates. DNA gyrase was isolated from four A. baumannii and the minimum concentration of fluoroquinolones, required to inhibit gyrase-catalysed supercoiling of plasmid DNA was 5- to 80-fold higher for the resistant isolates than for the reference strain. Although most isolates showed some degree of reduced fluoroquinolone accumulation, a DNA gyrase mutation was more likely to be the main mechanism of the high level resistance encountered.     

Antibacterial activity of teicoplanin against Clostridium difficile

The in vitro inhibitory action of teicoplanin, vancomycin, metronidazole and clindamycin against clinical isolates of Clostridium difficile was investigated. Minimum inhibitory concentrations (MICs) were determined using E test. Teicoplanin (MIC range 0.023-0.75 microgram/ml), vancomycin (MIC range 0.5-3 micrograms/ml) and metronidazole (MIC range 0.19-1 microgram/ml) were all very active against the isolates examined. No resistant strains of C. difficile to those three antimicrobial agents were observed, whereas resistance to clindamycin was found in 39.5% of the tested strains. Teicoplanin was about 4-times more potent than vancomycin. It appears to be a more promising antimicrobial for treatment of C. difficile enteric disease.