A sensitive and specific phenotypic assay for detection of metallo-ß-lactamases and KPC in Klebsiella pneumoniae with the use of meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid and cloxacillin.

Enterobacteriaceae producing carbapenemases, such as KPC or metallo-ß-lactamases (MBLs), have emerged on several continents. Phenotypic tests are urgently needed for their rapid and accurate detection. A novel carbapenemase detection test, comprising a meropenem disk, and meropenem disks supplemented with 730 µg of EDTA, 1000 µg of dipicolinic acid (DPA), 600 µg of aminophenylboronic acid (APBA), or 750 µg of cloxacillin, was evaluated against Klebsiella pneumoniae isolates with KPC (n = 34), VIM (n = 21), IMP (n = 4) or OXA-48 (n = 9) carbapenemases, and carbapenem-resistant Enterobacteriaceae with porin loss in combination with an extended-spectrum ß-lactamase (ESBL) (n = 9) or AmpC hyperproduction (n = 5). Commercially available diagnostics tablets from Rosco containing meropenem and the same inhibitors as described above (except EDTA) were also evaluated. An increased meropenem inhibition zone was sought in the presence of each added ß-lactamase inhibitor. APBA had excellent sensitivity for detecting K. pneumoniae with KPC enzymes. Isolates with combined AmpC hyperproduction and porin loss were also positive in the APBA test but, unlike KPC producers, showed cloxacillin synergy. Both DPA and EDTA had excellent sensitivity for detection of MBL-producing K. pneumoniae. However, EDTA showed poor specificity, with positive results noted for 1/9 ESBL-producing isolates, for 4/34 KPC-producing isolates, and for 4/9 OXA-48-producing isolates, whereas all of these were negative when DPA was used. The in-house test distinguished accurately between several different mechanisms mediating reduced susceptibility to carbapenems in Enterobacteriaceae. The commercial combination tablets from Rosco performed similarly to the in-house test, with the exception of one false-positive MBL result and one false-positive KPC result among the OXA-48 producers.

Antimicrobial susceptibility of Mycobacterium marinum determined by E-test and agar dilution.

Mycobacterium marinum is recognized as a cutaneous pathogen requiring antibiotic treatment. We compared the E-test with a reference agar dilution method for susceptibility testing of M. marinum to amikacin, ciprofloxacin, clarithromycin, doxycycline, rifampicin, trimethoprim-sulfamethoxazole and ethambutol. MICs obtained after 6 d showed agreement between the E-test and agar dilution within +/- 2 dilutions in 95% of all cases for amikacin, ciprofloxacin, doxycycline and rifampicin. Inhibitory concentrations of trimethoprim-sulfamethoxazole were difficult to define using the E-test because of gradually decreased growth in the presence of increasing concentrations. For clarithromycin, results were generally 1-3 dilution steps lower with the E-test and for ethambutol they were often > 3 dilution steps lower. These differences always appeared in the low MIC range and did not affect the categorization of the strains as susceptible to these 2 antimicrobial agents. All strains were interpreted as susceptible to all tested antibiotics, except for doxycycline, according to recommended breakpoints. Overall, our results suggest that the E-test can be considered an alternative for susceptibility testing of certain antibacterial agents against M. marinum.

In-vitro activity of fluorinated quinolones and macrolides against drug-resistant Mycobacterium tuberculosis.

The increasing incidence of drug-resistant Mycobacterium tuberculosis necessitates therapeutic alternatives. The in-vitro activities of seven fluoroquinolone and macrolide compounds were tested against 23 clinical isolates of M. tuberculosis, including 17 multidrug-resistant strains. Sparfloxacin was the most active fluoroquinolone, with MICs of < or = 1 mg/L for all tested strains, followed by levofloxacin and ciprofloxacin. Trovafloxacin had no inhibitory activity at the concentrations tested. The MICs of the macrolides were generally higher, clarithromycin being the most active with MICs of < or = 8 mg/L for eight of the 23 strains.

Cephadroxil promotes vaginal colonization with Escherichia coli.

The aim of this study was to examine whether a vaginal Escherichia coli colonization, mimicking the one seen in UTI-prone females, could be induced by local cephadroxil administration. When five adult cynomolgus monkeys were given a vaginal flush with a P-fimbriated E. coli strain, none became persistently colonized. When such colonization attempts were preceded by cephadroxil administration a persistent colonization occurred in 9/10 experiments. Cephadroxil also promoted a spread of fecal E. coli strains to the vagina. Reduction of the anaerobic vaginal flora can explain the breakdown of the colonization resistance. Clinical observations suggest that accumulation of E. coli around the urethral orifice increases the risk of UTI. Therefore antibiotics which promote such colonization may increase the risk for UTI in susceptible patients. From this point of view antibiotics such as cephadroxil may be less suitable for treatment of UTI-susceptible patients.

Comparative in-vitro activity of tigemonam, a new monobactam.

The in-vitro activity against Gram-negative aerobic bacterial pathogens of a new oral monobactam, tigemonam, was compared with those of amoxycillin/clavulanic acid, aztreonam, cefaclor, ceftazidime, cefuroxime, ciprofloxacin, co-trimoxazole and gentamicin, by an agar-dilution method. Tigemonam inhibited 90% of strains tested of Escherichia coli, Klebsiella spp., Proteus spp., Salmonella spp., Haemophilus influenzae and Branhamella catarrhalis at concentrations of 0.25 mg/l or below. The MIC90 for Enterobacter spp. was 16 mg/l and for Citrobacter 4 mg/l. Pseudomonas aeruginosa was resistant to tigemonam but susceptible to aztreonam. In comparison with the other oral antibiotics tested, tigemonam was generally more active. Tigemonam showed minimal inoculum dependence, between 10(2) and 10(6) cfu per spot. It is concluded that tigemonam is a candidate for clinical trials in patients with infections caused by Gram-negative aerobes other than pseudomonas and acinetobacter.

In vitro activity of amoxicillin plus clavulanic acid against Haemophilus influenzae and Branhamella catarrhalis.

The in vitro activity of amoxicillin in the presence of clavulanic acid against clinical isolates of Haemophilus influenzae and Branhamella catarrhalis was assessed in comparison with ampicillin, amoxicillin, cefaclor and erythromycin. The isolates were selected so as to yield equal numbers of beta-lactamase producing and non-beta-lactamase producing strains of the two species. MICs obtained by agar dilution indicated that amoxicillin in the presence of clavulanic acid was the most active of the drugs tested. Clavulanic acid potentiated the activity of amoxicillin against beta-lactamase-producing strains of both Haemophilus influenzae and Branhamella catarrhalis. Further studies on a few strains of each species revealed that the beta-lactamase of Haemophilus influenzae (TEM-1) rapidly inactivated ampicillin and slowly inactivated cefaclor but not cefuroxime. The Branhamella catarrhalis enzyme rapidly inactivated cefaclor, ampicillin and to some extent cefuroxime. Clavulanic acid afforded protection against the beta-lactamase action of both species when beta-lactam antibiotics were added to bacterial cultures.

Synergism between aminoglycosides and cephalosporins with antipseudomonal activity: interaction index and killing curve method.

Combinations of gentamicin with cefotaxime, moxalactam, and ceftazidime were tested against 43 bacterial strains, most of them blood isolates. With an interaction index of less than or equal to 0.5 as borderline, synergism was demonstrated against 30 to 40% of the strains by the fractional inhibitory concentration index and against 50 to 70% by the fractional bactericidal concentration index. The reproducibility of the index was within +/- 0.2 for two-thirds of 40 repetitive assays and within +/- 0.4 to 0.5 for all of these assays. Similar results were obtained when netilmicin was substituted for gentamicin. The killing curve system for studying antibiotic synergism was standardized to give results comparable to those obtained with the interaction index. This was achieved when one-half of a previously determined minimum bactericidal concentration was used for single drugs and the amount of antibiotic was at least halved again when drugs were used in combination. An initial bacterial concentration of 10(5) to 10(6) colony-forming units per ml is recommended. Given these conditions, synergism could be defined as a 2-log 10 or more decrease in viable count given by both drugs together, as compared with the more active of the pair after 24 h. Prediction of killing curve results could then be obtained with the fractional bactericidal concentration index. When cephalosporins and gentamicin were combined from the start, the beta-lactam antibiotics were less susceptible to inactivation, as demonstrated in time-killing assays. If one of the antibiotics were added after 24 h, synergism was not demonstrable. The results indicate that the new cephalosporins may be advantageously combined with aminoglycosides.

Susceptibility testing to trimethoprim alone and combined with sulphonamides.

In routine sensitivity testing by the disc diffusion methods, one member for each group of related antibiotics is chosen to represent the group. Each new antibacterial drug is evaluated with respect to its previous antibiotic class disc. In this study, trimethoprim combined with sulphadiazine in the ratio 1:4 was compared when combined with sulphamethoxazole in the ratio 1:20. Trimethoprim inhibited 86% of the bacterial strains tested at smaller than or equal to microgram/ml and the sulphonamides 50% of the strains at smaller than or equal to 64 microgram/ml. Regression line analysis showed that in sensitivity testing to trimethoprim alone discs with 5 micrograms can be used. Trimethoprim combined with sulphamethoxazole or sulphadiazine inhibited 84% respectively 91% of the strains at 0.5-8 microgram/ml. Trimethoprim combined with either sulphonamide produced superimposable regression lines. Therefore, sensitivity testing to trimethoprim/sulphadiazine can be performed by using discs with trimethoprim/sulphamethoxazole and the inhibition zone greater than or equal to 15 mm denotes susceptibility in the treatment of lower urinary tract infections.