The comparative in vitro activity of amoxicillin alone and in combination with clavulanic acid against 15 isolates of Mycobacterium tuberculosis was evaluated by broth dilution susceptibility testing. Amoxicillin inhibited 4 of 15 isolates at 8 ,ug/ml or less but was not bactericidal against any of the isolates at that concentration. Amoxicillin in combination with clavulanic acid was bactericidal for 14 of 15 isolates tested at an amoxicillin concentration of 4 ,ug/ml or less and a clavulanic acid concentration of 2 ,ug/ml or less.Mycobacterium tuberculosis produces a constitutive, intracellular ,-lactamase which has activity as both a penicillinase and a cephalosporinase (3). This enzyme appears to have a role in determining the resistance of M. tuberculosis to P-lactamase-labile P-lactams (2 (strains 1268, 1540, 1560, 1578, 1694, 1789, and 1790).The strains of M. tuberculosis were tested for P-lactamase activity with nitrocefin (provided by Glaxo Research, Greenford, Middlesex, United Kingdom) as described in a previous paper (1) except that the culture medium was 7H10 broth. All of the strains of M. tuberculosis used in this study had P-lactamase activity.The antimicrobial agents evaluated in this study were provided as standard powders as follows: amoxicillin (potency 860 ,ug/mg) and potassium clavulanate (potency, 826 jig/mg) (Beecham Laboratories, Bristol, Tenn.). Stock solutions of each antimicrobial agent were prepared immediately before use by hydrating a known weight of drug in Middlebrook and Cohn 7H10 broth (5) with Middlebrook OADC Enrichment (Difco Laboratories, Detroit, Mich.) and 0.05% Tween 80, followed by filter sterilization through a GA-6 0.45-,um membrane filter (Gelman Sciences, Inc., Ann Arbor, Mich.). < The mycobacteria were grown in 7H10 broth at 37°C on a rotary shaker and subcultured 3 days before use. The cultures were diluted in 7H10 broth to yield 5 Klett units per ml (KlettSummerson colorimeter, Klett Manufacturing, Brooklyn, NY). These were used to inoculate 7H10 broth containing the following concentration (micrograms per milliliter) of drug(s): amoxicillin, 8; amoxicillin, 4; amoxicillin 2; clavulanic acid, 8; clavulanic acid, 4; amoxicillin, 8, and clavulanic acid, 4; amoxicillin, 4, and clavulanic acid, 2; amoxicillin, 2, and clavulanic acid, 1; amoxicillin, 1, and clavulanic acid, 0.5; and amoxicillin, 0.5, and clavulanic acid, 0.25. A control tube without any drug was run for each mycobacterial strain. The titers of the mycobacteria were determined in duplicate on 7H10 agar plates (BBL Microbiology Systems, Cockeysville, Md.). The final concentration of organisms was 0.1 Klett unit per ml, or about 5 x 104 CFU/ml (range, 1 x 104 to 7.7 x 104 CFU/ml).The optical density at 550 nm (OD550) of the above suspensions was determined with a Spectrophotometer Modernization System model 252 (Gilford Instrument Laboratories, Inc., Oberlin, Ohio) of a DU spectrophotometer (Beckman Instruments, Inc., Fullerton, Calif.). The OD550 of the inoculated tubes was not measurably different fr...

The in-vitro susceptibility of Mycobacterium tuberculosis, M. bovis, and M. kansasii to amoxycillin alone and in combination with 2 mg/l of clavulanic acid was evaluated by broth dilution. The MIC90 of amoxycillin plus clavulanic acid was 4 mg/l compared with greater than 32 mg/l for amoxycillin alone when tested against M. tuberculosis (n = 27). M. bovis (n = 8) was the most susceptible species with an MIC90 of amoxycillin 8 mg/l, compared with 0.5 mg/l for the combination. M. kansasii (n = 6), with an MIC90 of 16 mg/l for amoxycillin plus clavulanic acid was more resistant than either M. tuberculosis or M. bovis. Ticarcillin plus clavulanic acid with an MIC90 of 32 mg/l was less active against M. tuberculosis (n = 28) than amoxycillin plus clavulanic acid. The addition of clavulanic acid to amoxycillin greatly improves its in-vitro activity against M. tuberculosis and M. bovis.

The comparative in vitro activity of cefoxitin, cephalothin, amoxicillin, and clavulanic acid in combination with the latter two agents against 13 isolates of Mycobacterium fortuitum was evaluated by agar dilution susceptibility testing.Amoxicillin was more active than cephalothin but less active than cefoxitin against the strains tested. Clavulanic acid in combination with these 3-lactams usually improved the activity by one or two dilutions compared with the P-lactams alone.Cefoxitin and cefmetazole, two cephamycins, have been shown to have good in vitro activity against Mycobacterium fortuitum (3). These organisms have been shown to have a 3-lactamase which hydrolyzes benzylpenicillin, cephalothin, and cephaloridine but does not hydrolyze methicillin or oxacillin (4). Cephalosporins which are P-lactamase stable but do not have the 7-amethoxy group have been found to be relatively inactive against M. fortuitum (2). The purpose of this study was to evaluate the activity of clavulanic acid, a P-lactamase inhibitor, in combination with the P-lactamase-labile ,B-lactams amoxicillin and cephalothin against M. fortuitum to better define the role of ,-lactamase stability in the activity of the 7-a-methoxy derivatives. Temocillin (BRL 17421), a 6-a-methoxy derivative of ticarcillin with increased stability against 1B-lactamases, was included to study the effect of the 6-a-methoxy group on the penicillin nucleus (7).The isolates of M. fortuitum were those used in our previous studies (2, 3). They all had 1B-lactamase activity when tested with nitrocefin (2). Staphylococcus aureus ATCC 25923 (Difco Laboratories, Detroit, Mich.), a ,B-lactamasenegative strain, and S. aureus Sbg, a j-lactamase-positive clinical isolate, were used as controls.The antimicrobial agents evaluated in this study were provided as standard powders by the following: cefoxitin (potency, 948 ,ug/mg), Merck Institute for Research, Rahway, N.J.; amoxicillin (potency, 860 ,g/mg), potassium clavulanate (potency, 826 ,ug/mg), and temocillin, Beecham Laboratories, Bristol, Tenn.; and cephalothin (potency, 955 ,ug/mg), Lilly Research Laboratories, Eli Lilly & Co., Indianapolis, Ind. Nitrocefin was provided by Glaxo Research, Greenford, Middlesex, United Kingdom. Stock solutions of each antimicrobial agent were prepared immediately before use by hydrating a known weight of drug in 50 mM phosphate buffer (pH 6.0) and by filter sterilization through a GA-6 0.45-,um membrane filter (Gelman Sciences, Inc., Ann Arbor, Mich.). The appropriate concentration of antibiotic was added to Mueller-Hinton agar at 56°C before quadrant plates were prepared. The final concentrations of the antibiotics were as follows: cephalothin, cefoxitin, and amoxicillin, 2 to 256 jig/ml; potassium clavulanate and temocillin, 4 to 256 Lg/ml; amoxicillin, 2 ,ug/ml, combined with clavulanic acid, 1 ,ug/ml; and cephalothin, 256 ,ug/ml, combined with clavulanic acid, 128 .Lg/ml.The mycobacteria and S. aureus isolates were inoculated into tubes of Mueller-Hinton broth containing 0.05% Tw...

The comparative in vitro activity of N-formimidoyl thienamycin, cefmetazole, cefoxitin, cefotetan , and moxalactam against 13 isolates of Mycobacteriumfortuitum was evaluated by agar dilution susceptibility testing. N-Formimidoyl thienamycin inhibited 10 to 12 strains at 6.25 ,g/ml, cefmetazole inhibited 12 of 13 strains at 12.5 I±g/ml, cefoxitin inhibited 11 of 13 strains at 25 ,ug/ml, and cefotetan inhibited 11 of 13 strains at 50 ,ug/ml. Moxalactam was less active than the other compounds tested, inhibiting only 9 of 13 strains at 50 ,ug/ml.Mycobacterium fortuitum, an organism commonly encountered in the environment, has been associated with soft tissue abscesses and wound infections in humans (5). It is resistant in vitro to all the commonly used antituberculous agents (4). We recently reported (1) that cefoxitin, a 7-a-methoxy cephalosporin derivative with increased P-lactamase resistance (3), inhibited 12 of 13 isolates of M. fortuitum at 25 ,ug/ml when tested by agar dilution. The purpose of the present study was to compare the in vitro activity of N-formimidoyl thienamycin, cefmetazole, moxalactam, cefotetan , cefoxitin, and several other 7-a-methoxy derivatives ( Fig. 1

The susceptibility of N. asteroides to N -formimidoyl thienamycin, cefamandole, cefoxitin, and moxalactam was determined by agar dilution. N -Formimidoyl thienamycin was the most active, inhibiting eight of nine strains at 1.56 μg/ml.