Synergistic activity of the novel des-fluoro(6) quinolone, garenoxacin (BMS-284756), in combination with other antimicrobial agents against Pseudomonas aeruginosa and related species.

Non-fermentative Gram-negative bacteria (Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia and Acinetobacter spp.) are intrinsically less susceptible to many antimicrobial agents. Two-drug combinations have been used to treat infections caused by less susceptible pathogens. In this study, the antibacterial activity of garenoxacin (GARX) with non-quinolones was examined. The non-quinolones evaluated were cefepime (CEPI), imipenem (IMIP), aztreonam (AZTR), piperacillin-tazobactam (PIPC/TZ), amikacin (AMK), ceftazidime (CTAZ), trimethoprim-sulphamethoxazole (TMP/SMX) and ticarcillin-clavulanate (TICC/CA). Synergism was determined by time-kill analysis using GARX (at 2 x its MIC, not to exceed 4 mg/l) and the second drug (at 1 x MIC, not to exceed its susceptible MIC breakpoint), and is defined as > or = 2 log(10) enhanced killing at 24 h with the combination. Partial synergy is defined as > or = 1.5 log(10) but < 2 log(10) enhanced killing with the drug combination. Synergy/partial synergy was observed most often with GARX plus: CEPI, AZTR, PIPC/TZ, IMIP (five strains each) or AMK (four strains) vs. eight P. aeruginosa; CTAZ, AZTR (five strains each) vs. six B. cepacia; TICC/CA (six strains), CEPI, CTAZ or AMK (five strains each) vs. eight S. maltophilia; and CEPI, AMK (three strains each) or CTAZ, TICC/CA (two strains each) vs. four Acinetobacter spp. In conclusion, synergistic killing was observed frequently with GARX plus a non-quinolone bactericidal agents against non-fermentative Gram-negative bacteria, including strains intermediately susceptible/resistant to one or both agents.

Synergistic activities of gatifloxacin in combination with other antimicrobial agents against Pseudomonas aeruginosa and related species.

Drug combinations have been used to treat serious infections caused by Pseudomonas, Burkholderia, Stenotrophomonas, and Acinetobacter. In this study, the combined drug effects of gatifloxacin (GAT) and nonquinolones were determined by time-kill analysis at clinically achievable drug concentrations. Synergy (>or=2 log(10)-enhanced killing at 24 h) was observed with GAT plus amikacin or a beta-lactam against 50 to 75% of strains, including strains nonsusceptible to one or both drugs.

Correlation between genotype and phenotypic categorization of staphylococci based on methicillin susceptibility and resistance.

Positive correlation between methicillin and oxacillin susceptibility test results and the detection of the mecA gene was observed for Staphylococcus aureus, S. epidermidis, and S. haemolyticus as well as among mecA(+) strains of other species of coagulase-negative staphylococci (CNS). However, at least 50% of the mecA-negative strains of these other species of CNS were falsely classified as methicillin and oxacillin resistant.

Comparative killing kinetics of the novel des-fluoro(6) quinolone BMS-284756, fluoroquinolones, vancomycin and beta-lactams.

The primary bactericidal classes used therapeutically as single agents, are the quinolones and the cell-wall active agents. In this study, their rates of killing were compared. The des-fluoro(6) quinolone BMS-284756 (T-3811ME), fluoroquinolones (trovafloxacin, levofloxacin) and cell wall-active agents (beta-lactams, vancomycin) were evaluated against Enterobacteriaceae, Staphylococcus aureus, streptococci, and Enterococcus faecalis. Time-kill analysis was done at 10x the MIC, using Mueller-Hinton broth (supplemented with 7% lysed horse blood for Streptococcus pneumoniae and the viridans streptococci), or Brain Heart Infusion broth for beta-haemolytic streptococci. Using a 3-log(10) decrease in viable count as an index of bactericidal activity, BMS-284756 and the fluoroquinolones killed Enterobacteriaceae rapidly, requiring < 2 h versus > or =6 h for beta-lactams. The staphylococcal cell counts generally decreased more rapidly with quinolone exposure, compared with those treated with vancomycin or the beta-lactams. The antimicrobial agents killed streptococci and enterococci more slowly, requiring > 6 h to decrease the viable count by 99.9%. In summary, BMS-284756 killing rates are similar to those of recent fluoroquinolones and are bacterial group-dependent. Overall, the quinolones are more rapidly bactericidal than vancomycin and the beta-lactam antibiotics.

Activity of gatifloxacin against strains resistant to ofloxacin and ciprofloxacin and its ability to select for less susceptible bacterial variants.

Gatifloxacin is an 8-methoxy fluoroquinolone. On quinolones, this side chain imparts increased activity against Gram-positive bacteria and enhanced killing. Gatifloxacin was tested against ofloxacin non-susceptible (ofloxacin MIC>2 mg/l) strains of Streptococcus pneumoniae (gatifloxacin MIC(90), 1 mg/l) and methicillin-resistant Staphylococcus aureus (MRSA, gatifloxacin MIC(90), 4 mg/l), and to ciprofloxacin non-susceptible (ciprofloxacin MIC>1 mg/l) strains of Escherichia coli (gatifloxacin MIC(90),>16 mg/l) and ciprofloxacin non-susceptible (ciprofloxacin MIC>0.06 mg/l) Neisseria gonorrhoeae (gatifloxacin MIC(50), 0.12 mg/l and MIC(90), 0.5 mg/l). Though gatifloxacin showed some reduced susceptibility to these populations, the MIC(50) and MIC(90) values suggest that gatifloxacin may be useful against pneumococci and some gonococcal strains not susceptible to other fluoroquinolones. Gatifloxacin did not select for less susceptible variants of MRSA and pneumococci, in contrast to the 10- to 100-fold higher selection frequencies with ciprofloxacin and ofloxacin. The single-step E. coli mutants selected by gatifloxacin and the comparator quinolones had quinolone MICs within the susceptible range. These data suggest that gatifloxacin use may hinder the development of quinolone-resistance, particularly in Gram-positive bacteria.

Activity of gatifloxacin and ciprofloxacin in combination with other antimicrobial agents.

The influence of non-quinolone antimicrobial agents on the antibacterial activities of gatifloxacin and ciprofloxacin was determined using chequerboard, fractional inhibitory concentration, (FIC) and time-kill analysis methods. In the chequerboard method, the quinolones were tested in combination with ten antimicrobial agents (macrolides, aminoglycosides, beta-lactams, vancomycin, rifampicin and chloramphenicol) against five bacterial strains (one strain each of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis and Streptococcus pneumoniae). In no incidence was antagonism (FIC > or = 4) or synergy (FIC < or = 0.5) observed; all dual drug combinations involving gatifloxacin or ciprofloxacin showed additivity/indifference (FIC > 0.5, < 4). By time-kill analysis, the strains were tested at a quinolone concentration equal to 8 x MIC in combination with a second antibiotic at 0.5xits MIC. These combinations killed non-enterococcal strains at rates similar to those with quinolones alone. However, rifampicin and chloramphenicol were often antagonistic (100-fold lesser killing) to the lethal action of gatifloxacin and ciprofloxacin against E. faecalis. These findings indicate that, with the exception of E. faecalis, the antibacterial activities of quinolones are generally additive/indifferent to those of other antimicrobial agents.

Antibacterial spectrum of a novel des-fluoro(6) quinolone, BMS-284756.

The in vitro spectrum of a novel des-fluoro(6) quinolone, BMS-284756, was compared with those of five fluoroquinolones (trovafloxacin, moxifloxacin, levofloxacin, ofloxacin, and ciprofloxacin). BMS-284756 was among the most active and often was the most active quinolone against staphylococci (including methicillin-resistant strains), streptococci, pneumococci (including ciprofloxacin-nonsusceptible and penicillin-resistant strains), and Enterococcus faecalis. BMS-284756 inhibited approximately 60 to approximately 70% of the Enterococcus faecium (including vancomycin-resistant) strains and 90 to 100% of the Enterobacteriaceae strains and gastroenteric bacillary pathogens at the anticipated MIC susceptible breakpoint (

Comparative killing rates of fluoroquinolones and cell wall-active agents.

Killing rates of fluoroquinolones, beta-lactams, and vancomycin were compared against Enterobacteriaceae, Staphylococcus aureus, pneumococci, streptococci, and Enterococcus faecalis. The times required for fluoroquinolones to decrease viability by 3 log(10) were 1.5 h for Enterobacteriaceae, 4 to 6 h for staphylococci, and >/=6 h for streptococci and enterococci. Thus, the rate of killing by fluoroquinolones is organism group dependent; overall, they killed more rapidly than beta-lactams and vancomycin.

Antibacterial activity of BMS-180680, a new catechol-containing monobactam.

The in vitro activities of a new catechol-containing monobactam, BMS-180680 (SQ 84,100), were compared to those of aztreonam, ceftazidime, imipenem, piperacillin-tazobactam, ciprofloxacin, amikacin, and trimethoprim-sulfamethoxazole. BMS-180680 was often the most active compound against many species of the family Enterobacteriaceae, with MICs at which 90% of the isolates were inhibited (MIC90s) of < or = 0.5 microg/ml for Escherichia coli, Klebsiella spp., Citrobacter diversus, Enterobacter aerogenes, Serratia marcescens, Proteus spp., and Providencia spp. BMS-180680 had moderate activities (MIC90s of 2 to 8 microg/ml) against Citrobacter freundii, Morganella morganii, Shigella spp., and non-E. aerogenes Enterobacter spp. BMS-180680 was the only antibiotic evaluated that was active against >90% of the Pseudomonas aeruginosa (MIC90, 0.25 microg/ml), Burkholderia cepacia, and Stenotrophomonas maltophilia (MIC90s, 1 microg/ml) strains tested. BMS-180680 was inactive against most strains of Pseudomonas fluorescens, Pseudomonas stutzeri, Pseudomonas diminuta, and Burkholderia pickettii. BMS-180680 was moderately active (MIC90s of 4 to 8 microg/ml) against Alcaligenes spp. and Acinetobacter lwoffii and less active (MIC90, 16 microg/ml) against Acinetobacter calcoaceticus-Acinetobacter baumanii complex. BMS-180680 lacked activity against gram-positive bacteria and anaerobic bacteria. Both tonB and cir fiu double mutants of E. coli had greatly decreased susceptibility to BMS-180680. Of the TEM, PSE, and chromosomal-encoded beta-lactamases tested, only the K1 enzyme hydrolyzed BMS-180680 to any measurable extent. Like aztreonam, BMS-180680 bound preferentially to penicillin-binding protein 3. The MICs of BMS-180680 were not influenced by the presence of hematin or 5% sheep blood in the test medium or with incubation in an atmosphere containing 5% CO2. BMS-180680 MICs obtained under strict anaerobic conditions were significantly higher than those obtained in ambient air.

Differences in the resistant variants of Enterobacter cloacae selected by extended-spectrum cephalosporins.

The rates of development of resistance to ceftriaxone, ceftazidime, cefepime, and cefpirome in 10 strains of Enterobacter cloacae were determined by daily transfer for 7 days to fresh medium containing twofold serial dilutions of the antibiotics. Development of resistance to ceftriaxone was the most rapid; this was followed by ceftazidime, cefpirome, and cefepime. Resistant variants selected by ceftriaxone and ceftazidime were cross-resistant and produced very high levels of beta-lactamase. On the other hand, resistant variants selected by cefepime and cefpirome often had moderately high levels of beta-lactamase and diminished levels of the 39- to 40-kDa porin protein.

In vitro activity of BMS-181139, a new carbapenem with potent antipseudomonal activity.

The in vitro activities of the carbapenem BMS-181139 were determined in comparison with those of imipenem, meropenem, ciprofloxacin, ceftriaxone, and vancomycin. BMS-181139 was the most active against species of Pseudomonas and related genera Alteromonas and Burkholderia, with MICs for 147 of 149 isolates of < 4 micrograms/ml. Of 22 imipenem-resistant (MIC > 8 micrograms/ml) P. aeruginosa strains, only 1 required an MIC of BMS-181139 of > 4 micrograms/ml, compared with 14 requiring the same meropenem MIC. BMS-181139 was the most active carbapenem against the majority of other gram-negative species except members of the tribe Proteeae, against which meropenem was more active. Although imipenem was more active against gram-positive species, BMS-18139 MICs at which 90% of strain tested were inhibited were < 1 microgram/ml for these species. BMS-181139 was generally active against isolates resistant to ciprofloxacin or broad-spectrum cephalosporins, including those containing plasmid-encoded beta-lactamases or high levels of chromosome-encoded beta-lactamases, as well as anaerobes except Clostridium difficile. Inoculum effects were noted for all three carbapenems against Klebsiella pneumoniae, Enterobacter cloacae, and Serratia marcescens but not Escherichia coli, Pseudomonas aeruginosa, or Staphylococcus aureus. BMS-181139's inoculum effect tended to be more marked. BMS-181139 exhibited bactericidal activity at the MIC for some strains and up to four to eight times the MIC for others. The postantibiotic effect of BMS-181139 was equal to or less than that of imipenem and, like meropenem, exhibited intraspecies variability. BMS-181139 was 30-fold more stable than imipenem and 7-fold more stable than meropenem to hydrolysis by hog kidney dehydropeptidase.

Activity of carbapenem BMS-181139 against Pseudomonas aeruginosa is not dependent on porin protein D2.

The broad antipseudomonal spectrum of the carbapenem BMS-181139 includes clinical strains and laboratory mutants of Pseudomonas aeruginosa that are resistant to imipenem. Unlike other known carbapenems (meropenem, panipenem, biapenem, and BO-2727), which have reduced activity against imipenem-resistant strains of P. aeruginosa, BMS-181139 was equally active against imipenem-susceptible (D2-sufficient) and imipenem-resistant (D2-deficient) strains. Conversely, imipenem and meropenem activities were the same against the susceptible parental strains and their BMS-181139-resistant mutants. Whereas basic amino acids antagonized the antipseudomonal activities of imipenem and meropenem, they had no effect on the activity of BMS-181139. These results suggest that the uptake of BMS-181139 into pseudomonal cells occurs by a non-D2 pathway. Compared with imipenem and meropenem, BMS-181139 may have a slightly higher affinity for penicillin-binding protein 2 (PBP-2) of P. aeruginosa. The rates of resistance development to imipenem, meropenem, and BMS-181139 in P. aeruginosa strains were similar; resistance occurred at frequencies of approximately 10(-7) to 10(-8). Resistance to BMS-181139 in P. aeruginosa is presumed to be caused by its diminished permeability since no change in their penicillin-binding protein affinities or beta-lactamase levels could be detected. In summary, BMS-181139 is a new carbapenem which differs from other known carbapenems in its lack of cross-resistance with imipenem. This difference could be explained by the permeation of BMS-181139 through a non-D2 channel, compared to the preferential uptake of other carbapenems by the D2 porin.

Structure-activity relationships of carbapenems that determine their dependence on porin protein D2 for activity against Pseudomonas aeruginosa.

A number of carbapenem derivatives were examined to determine the structure-activity relationships required for dependence on porin protein D2 for activity against Pseudomonas aeruginosa. As suggested by J. Trias and H. Nikaido (Antimicrob. Agents Chemother. 34:52-57, 1990), carbapenem derivatives, such as imipenem and meropenem, containing a sole basic group at position 2 of the molecule utilize the D2 channel for permeation through the outer membrane of pseudomonads; they are more active against D2-sufficient strains of P. aeruginosa. Our results indicated that carbapenems with a basic group at position 1 or 6 of the molecule did not depend on the D2 channel for activity; i.e. they were equally active against D2-sufficient and D2-deficient pseudomonal strains. However, addition of a basic group at position 1 or 6 of a carbapenem derivative already containing a basic group at position 2 resulted in its lack of dependency on the D2 pathway. Comparison between meropenem and its 1-guanidinoethyl derivative, BMY 45047, indicated that they differed in their dependence on D2; while meropenem required the D2 channel for uptake, BMY 45047 activity was independent of D2. Meropenem and BMY 45047 had similar affinities for the penicillin-binding proteins of P. aeruginosa. However, BMY 45047 and meropenem differed in the morphological changes that they induced in pseudomonal cells. While meropenem induced filamentation, BMY 45047 induced filaments only in BMS-181139-resistant mutants and not in imipenem-resistant mutants or in carbapenem-susceptible P. aeruginosa strains. These results suggested that in Mueller-Hinton medium the uptake of BMY 45047 through the non-D2 pathway is more rapid than that of meropenem through the D2 porin. In summary, the presence of a basic group at position 2 of a carbapenem is important for its preferential uptake by the D2 channel. However the addition of a basic group at position 1 or 6 of a carbapenem already containing a basic group at position 2 dissociates its necessity for porin protein D2 for activity.

Development of resistance in Pseudomonas aeruginosa to broad-spectrum cephalosporins via step-wise mutations.

Step-wise resistance to cefepime, ceftazidime, cefotaxime, and cefpirome were determined for 16 Pseudomonas aeruginosa strains by daily transfer for 7 days to fresh media containing two-fold serial dilution of antibiotic. By the third transfer 4 of 16 strains (25%) were resistant (MIC > or = 32 mg/L) to ceftazidime compared with none, five (31%) and ten (60%) strains becoming resistant to cefepime, cefpirome and cefotaxime (MIC > or = 64 mg/L), respectively. At the end of the 7 day serial transfer, only four (25%) of the 16 strains were resistant to cefepime, in contrast to nine (56%) cefpirome resistant, 12 (75%) ceftazidime resistant and 13 (81%) cefotaxime resistant. These results are consistent with the infrequent, single-step development of resistance to cefepime, and this may also explain the frequent cefepime susceptibility of cefotaxime/ceftazidime-resistant clinical isolates of P. aeruginosa.

The synthesis, antibacterial, and beta-lactamase inhibitory activity of a novel asparenomycin analog.

An analog, 6-(2'-hydroxyethylidene)-4 beta-methyl-1-azabicyclo[3.2.0]hept-2-ene-2- carboxylate (11), of the carbapenem beta-lactamase inhibitor, asparenomycin A, was synthesized. It possessed a spectrum of antibacterial activity that was comparable to that of asparenomycin A but was less effective as a beta-lactamase inhibitor. With ampicillin, it only exhibited a moderate level of synergy against a variety of beta-lactamase-producing organisms. Although the presence of a 4 beta-methyl group in the analog brought about a significant increase in chemical stability relative to that of asparenomycin A, it did not result in an increase in stability to kidney dehydropeptidase enzyme.

Emergence of homogeneously methicillin-resistant Staphylococcus aureus.

Forty-seven clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), collected between 1986 and 1990 from 29 institutions, were analyzed for susceptibility to various antibiotics. Twenty-six strains were homogeneously methicillin resistant (i.e., greater than or equal to 10% of the cells in these strains were able to grow on Mueller-Hinton agar containing 50 micrograms of methicillin per ml). The MICs of gentamicin, clindamycin, trimethoprimsulfamethoxazole, methicillin, and imipenem for homogeneous MRSA strains were higher than those for heterogeneously resistant strains. Both types of strains were, for the most part, susceptible to vancomycin and trimethoprim-sulfamethoxazole. Ciprofloxacin-resistant MRSA strains were not isolated prior to 1988 but made up 40% of the post-1987 strains. The level of methicillin resistance correlated well with the imipenem MIC, suggesting that susceptibility to imipenem may serve as a marker to identify and monitor the prevalence of homogeneous MRSA strains.