Susceptibility of Haemophilus influenzae to amoxicillin/clavulanic acid, erythromycin, cefaclor, and trimethoprim/sulfamethoxazole

A total of 126 strains of Haemophilus influenzae were examined for susceptibility to amoxicillin/clavulanic acid, trimethoprim/sulfamethoxazole, cefaclor, and erythromycin by an agar dilution procedure. Fifty strains (eight type B, 42 non-type B), all with ampicillin minimal inhibitory concentrations (MIC) of greater than or equal to 6.2 micrograms/ml, produced beta-lactamase. The remaining 76 strains (18 type B, 59 non-type B) were beta-lactamase-negative. All of these strains had ampicillin MICs of less than or equal to 0.8 micrograms/ml. The combination of amoxicillin and clavulanic acid (2:1) was highly active against all strains tested. With the exception of two strains with amoxicillin/clavulanic acid MICs of 1.6/0.8 ug/ml, all strains were inhibited by concentrations of less than or equal to 0.8/0.4 ug/ml. Trimethoprim/sulfamethoxazole was also found to be highly active (MICs uniformly less than or equal to 0.1/1.9 ug/ml). Cefaclor and erythromycin were the least active of the agents tested. Fourteen strains (10.6%) had cefaclor MICs of greater than 32 ug/ml. Forty-seven strains (35.6%) had erythromycin MICs of greater than 8 micrograms/ml. With the exception of amoxicillin/clavulanic acid beta-lactamase production did not seem to influence the activity of any of the antimicrobials tested. Minimum inhibitory concentrations of amoxicillin/clavulanic acid, although still well within achievable serum levels, were approximately one twofold dilution higher with beta-lactamase-producing H. influenzae type B strains than with beta-lactamase-negative strains.     

Effect of clavulanic acid on activity of beta-lactam antibiotics in Serratia marcescens isolates producing both a TEM beta-lactamase and a chromosomal cephalosporinase.

An isolate of Serratia marcescens that produced both an inducible chromosomal and a plasmid-mediated TEM-1 beta-lactamase was resistant to ampicillin and amoxicillin and also demonstrated decreased susceptibility to extended-spectrum beta-lactam antibiotics (ESBAs). Clavulanic acid did not lower the MICs of the ESBAs, but it decreased the MICs of the penicillins. The TEM-1-producing plasmid was transferred to a more susceptible S. marcescens strain that produced a well-characterized inducible chromosomal beta-lactamase. The MICs of the ESBAs remained at a low level for the transconjugant. Ampicillin and amoxicillin which were good substrates for the plasmid-mediated enzyme, were not well hydrolyzed by the chromosomal enzymes; the ESBAs were hydrolyzed slowly by all the enzymes. When each of the S. marcescens strains was grown with these beta-lactam antibiotics, at least modest increases in chromosomal beta-lactamase activity were observed. When organisms were grown in the presence of clavulanic acid and an ESBA, no enhanced induction was observed. The increases in the MICs of the ESBAs observed for the initial clinical isolate may have been due to a combination of low inducibility, slow hydrolysis, and differences in permeability between the S. marcescens isolates. When clavulanic acid and a penicillin were added to strains that produced both a plasmid-mediated TEM and a chromosomal beta-lactamase, much higher levels of chromosomal beta-lactamase activity were present than were observed in cultures induced by the penicillin alone. This was due to the higher levels of penicillin that were available for induction as a result of inhibition of the TEM enzyme by clavulanate.     

Incidence and mechanisms of resistance to the combination of amoxicillin and clavulanic acid in Escherichia coli.

Among Escherichia coli organisms isolated at St. Thomas's Hospital during the years 1990 to 1994, the frequency of resistance to amoxicillin-clavulanic acid (tested by disk diffusion in a ratio of 2:1) remained constant at about 5% of patient isolates (10 to 15% of the 41 to 45% that were amoxicillin resistant). Mechanisms of increased resistance were determined for 72 consecutively collected such amoxicillin-clavulanic acid-resistant isolates. MICs of the combination were 16-8 micrograms/ml for 51 (71%) of these and > or = 32-16 micrograms/ml for the remainder. The predominant mechanism was hyperproduction of enzymes isoelectrically cofocusing with TEM-1 (beta-lactamase activities, > 200 nmol of nitrocefin hydrolyzed per min per mg of protein) which was found in 44 isolates (61%); two isolates produced smaller amounts (approximately 150 nmol/min/mg) of such enzymes, and two isolates hyperproduced enzymes cofocusing with TEM-2. Eleven isolates produced enzymes cofocusing with OXA-1 beta-lactamase, which has previously been associated with resistance to amoxicillin-clavulanic acid. Ten isolates produced increased amounts of chromosomal beta-lactamase, and four of these additionally produced TEM-1 or TEM-2. Three isolates produced inhibitor-resistant TEM-group enzymes. In one of the enzymes (pI, 5.4), the amino acid sequence change was Met-67-->Val, and thus the enzyme is identical to TEM-34. Another (pI, 5.4) had the substitution Met-67-->Ile and is identical to IRT-I67, which we propose now be given the designation TEM-40. The third (pI, 5.2) had the substitution Arg-241-->Thr; this enzyme has not been reported previously and should be called TEM-41. The rarity and diversity of inhibitor-resistant TEM-group enzymes suggest that they are the result of spontaneous mutations that have not yet spread.     

Role of beta-lactamase and different testing conditions in oxacillin-borderline-susceptible staphylococci.

A group of staphylococcal isolates for which oxacillin MICs were intermediate (1 to 4 micrograms/ml) were studied to establish the role of beta-lactamase in this phenomenon. MICs and MBCs of oxacillin and penicillin with and without clavulanic acid or sulbactam (4 or 16 micrograms/ml, respectively) were determined for 11 Staphylococcus aureus and 2 coagulase-negative Staphylococcus isolates for which oxacillin MICs were 1 to 4 micrograms/ml. The susceptibility studies were done with incubation at 35 and 30 degrees C, and the MICs were read at 24 and 48 h. Of the 13 isolates, 4 became resistant when longer incubation or 30 degrees C incubation was used, and the MICs for 9 remained in the intermediate range. Only three of these strains were susceptible to penicillin, and beta-lactamase was not detected. For 6 of 10 beta-lactamase-positive strains, there was a greater-than-twofold-dilution reduction in oxacillin MICs with the addition of clavulanic acid or sulbactam. Of the four strains that became resistant with incubation at the lower temperature, a clavulanic acid effect was observed in three but only at 35 degrees C. The oxacillin MIC for one of the beta-lactamase-negative strains was also reduced with clavulanic acid; however, this strain was inhibited by 1 microgram of clavulanic acid per ml alone. Bactericidal activity was observed with two or four times the oxacillin MIC in eight strains tested at both temperatures, and the combination with clavulanic acid was bactericidal at higher than four times the MIC in five of the strains at 30 degrees C. Our results suggest that oxacillin intermediate MICs for staphylococcal isolates are due not only to beta-lactamase hyperproduction but also some other unidentified factor. The reduction in oxacillin MIC observed when clavulanic acid was added to one strain was probably due to the intrinsic inhibitory activity of clavulanic acid.     

Antistaphylococcal activity of amoxicillin and ticarcillin when combined with clavulanic acid. Evaluation of oxacillin-resistant and oxacillin-susceptible isolates

The beta-lactamase inhibitor, clavulanic acid, was combined with amoxicillin and with ticarcillin for in vitro studies with 586 staphylococci: 97 stock cultures of oxacillin-resistant strains recovered before 1982, and 489 blood or wound isolates collected from 40 separate medical centers during 1987-1988 (300 were oxacillin resistant). Over 92% of the staphylococci produced beta-lactamase enzymes and were thus resistant to both penicillins. However, with the addition of clavulanic acid, oxacillin-susceptible strains were rendered susceptible to low concentrations of amoxicillin and ticarcillin. Staphylococcus aureus strains with borderline or partial borderline resistance to penicillinase-resistant penicillins occurred infrequently (72 of 325 S. aureus isolates). Those strains were susceptible to both clavulanic acid combinations, because their methicillin resistance is thought to be due to an excess beta-lactamase production. Strains with chromosomally mediated intrinsic heteroresistance were relatively resistant to both drug combinations. Minimal inhibitory concentration (MIC) breakpoints that best separated those heteroresistant strains from oxacillin-susceptible isolates were as follows: amoxicillin/clavulanic acid, less than or equal to 2.0/1.0 micrograms/ml for susceptible; and ticarcillin/clavulanic acid, less than or equal to 4.0/2.0 micrograms/ml for susceptible. When the broth was supplemented by 2% NaCl, MICs for both drug combinations were increased by less than one doubling dilution. Although oxacillin and methicillin broth microdilution tests were more reliable when 2% NaCl was added, tests with the two drug combinations were only minimally improved by adding 2% NaCl to the broth medium.     

Properties of IRT-14 (TEM-45), a newly characterized mutant of TEM-type beta-lactamases.

IRT-14 (TEM-45) is a new mutant TEM-type beta-lactamase that was isolated from clinical Escherichia coli P37 and that confers resistance to broad-spectrum penicillins with reduced sensitivity to beta-lactamase inhibitors. The MICs of amoxicillin alone and of amoxicillin combined with 2 micrograms of clavulanic acid or 2 micrograms of tazobactam per ml were 4,096, 2,048, and 1,024 micrograms/ml, respectively. The strain was susceptible to cephalosporins, aztreonam, moxalactam, and imipenem. The enzyme was purified to homogeneity, and values of the kinetic parameters Kcat, Km, and Kcat/Km were determined for different substrates. This enzyme, with a pI of 5.2, was found to have reduced affinity for broad-spectrum penicillins and cephalosporins. The values of 50% inhibitory concentrations of clavulanic acid, sulbactam, tazobactam, and brobactam are correlated with the higher KmS for substrates. The resistance of E. coli P37 to mechanism-based inactivators results from a higher level of production of the TEM-derived enzyme due to the G-to-T substitution at position 162 (G-162-->T) in the promoter region of blaTEM and from the structural modifications resulting from the Met-69-->Leu and Arg-275-->Gln substitutions that characterize IRT-14 beta-lactamase.     

In vitro activity of orally administered antimicrobial agents against Haemophilus influenzae recovered from children monitored longitudinally in a group day-care center.

To determine whether the prevalence of resistance to commonly used oral antimicrobial agents varied over time, we compared the in vitro susceptibilities of 217 strains of Haemophilus influenzae recovered from nasopharyngeal secretions of children in a day-care center studied longitudinally between 1979 and 1987. The overall rate of beta-lactamase production in these strains was 18%, with rates of 57% in type b isolates (n = 21) and 14% in non-type b isolates (n = 196). The percentages of isolates for which MICs were less than or equal to 1.0 micrograms/ml for amoxicillin alone, amoxicillin in combination with clavulanic acid, and cefuroxime alone were 82, 92, and 93%, respectively. The percentage of strains for which cefaclor MICs were less than or equal to 1.0 micrograms/ml was only 0.5%. Isolates for which chloramphenicol MICs were greater than 2.0 micrograms/ml or for which trimethoprim-sulfamethoxazole MICs were greater than 0.5/9.5 micrograms/ml were uncommon: 1 and less than 1%, respectively. High concentrations of erythromycin alone and in combination with sulfisoxazole were required to inhibit the majority of test strains; there was no evidence of erythromycin-sulfisoxazole synergy. In vitro susceptibility to commonly used oral antimicrobial agents remained at a constant level when H. influenzae isolates collected from children in a day-care center during 1979 through 1983 were compared with strains collected during 1984 through 1987.     

In vitro activity of amoxicillin in combination with clavulanic acid against Mycobacterium tuberculosis.

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 micrograms/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 micrograms/ml or less and a clavulanic acid concentration of 2 micrograms/ml or less.     

Activity of cefoperazone and two beta-lactamase inhibitors, sulbactam and clavulanic acid, against Bacteroides spp. correlated with beta-lactamase production.

A total of 102 isolates of Bacteroides spp. were studied for beta-lactamase production and susceptibility to cefoperazone alone or in combination with either of the beta-lactamase inhibitors sulbactam and clavulanic acid. The geometric mean minimal inhibitory concentration of cefoperazone alone was 31.5 micrograms/ml and when combined with 10 micrograms of sulbactam per ml or 2 micrograms of clavulanic acid per ml was reduced to 5.4 and 9.2 micrograms/ml, respectively. When bacterial suspensions were tested for beta-lactamase production with nitrocefin, 91 (89.2%) of these isolates produced the enzyme. The geometric mean minimal inhibitory concentrations of cefoperazone rose only slightly for isolates with low or intermediate enzyme activity but rose significantly for those with high activity. The addition of EDTA to cefoperazone significantly more frequently enhanced the activity of cefoperazone against beta-lactamase-negative as opposed to beta-lactamase-positive isolates. Furthermore, EDTA resulted in synergistic activity of the cefoperazone-sulbactam combination on beta-lactamase-positive isolates for which the combination had previously not shown a synergistic effect. This study demonstrates the relationship between beta-lactamase production and the resistance of Bacteroides spp. to cefoperazone and shows that inhibition of these enzymes can reverse this resistance.     

Susceptibility of multiply resistant Haemophilus influenzae to newer antimicrobial agents

One hundred and six isolates of Haemophilus influenzae from a national antimicrobial surveillance study demonstrated resistance to two or more of 10 primary antimicrobial agents by mechanisms other than or in addition to beta-lactamase. Of particular note were strains multiply resistant to ampicillin (by beta-lactamase production), chloramphenicol, trimethoprim/sulfamethoxazole, and tetracycline in various combinations. All of the aforementioned strains were shown to be highly susceptible to amoxicillin/clavulanate, the second generation cephalosporins cefuroxime and cefonicid, and the third generation cephalosporins cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, moxalactam, and cefixime. However, 68 strains that demonstrated resistance or marginal susceptibility (MIC greater than or equal to 2 micrograms/ml) to ampicillin by mechanisms other than beta-lactamase, also demonstrated reduced susceptibility to amoxicillin/clavulanate (MICs up to 8 micrograms/ml) and the second generation cephalosporins (MICs up to 32 micrograms/ml). While the latter strains were susceptible to the third generation cephalosporins, MICs were often 10-fold higher than MICs of ampicillin susceptible isolates or of beta-lactamase producing isolates. All of the multiply antimicrobial-resistant strains were highly susceptible (MIC less than or equal to 0.25 micrograms/ml) to the two quinolones ciprofloxacin and pefloxacin.     

An in-vitro and in-vivo comparison of the activity of beta-lactamase inhibitor combinations with imipenem and cephalosporins against Escherichia coli producing TEM-1 or TEM-2 beta-lactamase.

Reference strains of Escherichia coli (ampicillin-susceptible and -resistant ATCC strains, and known TEM-1 and TEM-2 beta-lactamase producers) were tested in vitro and in the in-vivo mouse thigh infection model against four beta-lactamase inhibitor compounds (BICs: amoxycillin/clavulanic acid, ampicillin/sulbactam, ticarcillin/clavulanic acid, and piperacillin/tazobactam), selected cephalosporins, and imipenem. The ATCC strains (ampicillin-susceptible and -resistant) were susceptible to the BICs in disc and MIC tests. Three or more logs of killing were observed at the NCCLS breakpoint concentrations. However, the TEM-1 and TEM-2 producers were resistant in disc tests to ampicillin/sulbactam and amoxycillin/clavulanic acid, and showed intermediate susceptibility to ticarcillin/clavulanic acid. MICs were at or near the breakpoint, but bactericidal activity was only noted at the probable breakpoint concentration of piperacillin/tazobactam. Cefoxitin, cefotaxime, cefpirome and imipenem, but not cephalothin, showed greater bactericidal activity and lower MICs for the TEM-producing strains than the BICs. The viable count of the TEM-1 producer was not reduced in the mouse thigh model by ampicillin/sulbactam or amoxycillin/clavulanic acid, but cefpirome and cefotaxime reduced the viable count by approximately three logs. There was a 50% mortality rate in mice receiving the two BICs. The ampicillin-susceptible ATCC strain of E. coli was killed to a similar degree by all agents tested. Overall, the BICs appeared inferior, in both in-vivo and in-vitro tests to selected cephalosporins and imipenem when tested against reference strains of E. coli producing TEM-1 or TEM-2 beta-lactamase. The large inoculum effect and poor bactericidal activity observed with the BICs suggest they could be less effective in certain clinical situations.     

Comparative activities of the beta-lactamase inhibitors YTR 830, sodium clavulanate, and sulbactam combined with amoxicillin or ampicillin.

YTR 830, a new beta-lactamase inhibitor, was compared with clavulanic acid and sulbactam against aminopenicillin-resistant clinical isolates. At a concentration of 8 micrograms/ml, YTR 830 was as effective as clavulanate or sulbactam in reducing the aminopenicillin MICs. Combined with amoxicillin, YTR 830 is a potentially useful agent for therapy of many bacterial infections.     

Role of the beta-lactamase of Campylobacter jejuni in resistance to beta-lactam agents.

We studied the role of the beta-lactamase of Campylobacter jejuni in resistance to beta-lactam agents. beta-Lactamase-positive strains were more resistant than beta-lactamase-negative strains to amoxicillin, ampicillin, and ticarcillin (P less than 0.05). With penicillin G, piperacillin, imipenem, and six cephalosporins, the susceptibility levels were similar for both beta-lactamase-positive and -negative strains. By using spectrophotometric and microbiological assays, the beta-lactamase from three strains hydrolyzed ampicillin, amoxicillin, penicillin G, cloxacillin, and, partially, cephalothin. Ticarcillin and piperacillin were partially hydrolyzed in the microbiological assay. There was no activity against five other cephalosporins or imipenem. Isoelectric focusing of the enzyme showed a pI of 8.8. Tazobactam was the best inhibitor of the enzyme, followed by clavulanic acid, sulbactam, and cefoxitin, while EDTA and p-chloromercuribenzoate had no activity. All beta-lactamase-positive strains became susceptible to amoxicillin and ampicillin with 1 micrograms of clavulanic acid per ml. With the same inhibitor, there was a reduced but significant effect for ticarcillin but no effect for penicillin G or piperacillin. Sulbactam had no effect and tazobactam was effective only at 2 micrograms/ml on amoxicillin and ampicillin. The beta-lactamase of C. jejuni seems to be a penicillinase with a role in resistance for only amoxicillin, ampicillin, and ticarcillin.     

Antimicrobial susceptibility patterns of the Bacteroides fragilis group in the United States, 1989.

A national survey of Bacteroides fragilis group was continued in 1989 for the ninth consecutive year. Seven hundred thirty-nine isolates of B fragilis group from eight centers were tested for susceptibility to 14 antimicrobials. Sulbactam and clavulanic acid, beta-lactamase inhibitors, were tested at a constant concentration of 8 micrograms/ml and 2 micrograms/ml, respectively. Sulbactam was also tested in a fixed ratio of 1:2. Imipenem, ampicillin+sulbactam, and ticarcillin+clavulanic acid had resistance of less than 1% at breakpoints of 8 micrograms/ml, 16 micrograms/ml, and 64 micrograms/ml, respectively. At 32 micrograms/ml, resistance to cefoxitin, cefotetan, ceftizoxime, and ceftriaxone were 4%, 25%, 26%, and 46%, respectively. Clindamycin resistance was 10% at a breakpoint of 4 micrograms/ml. No isolates were resistant to chloramphenicol or metronidazole. Resistance for five B fragilis species to cefoxitin, ceftizoxime, and cefotetan varied greatly among both species and participating institutions. The addition of a beta-lactamase inhibitor increased the potency of the beta-lactam drugs tested as combinations. This finding suggests that beta-lactamase production is the major resistance factor in members of the B fragilis group.     

Comparative in vitro and in vivo activities of piperacillin combined with the beta-lactamase inhibitors tazobactam, clavulanic acid, and sulbactam.

Tazobactam (YTR-830H), a novel beta-lactamase inhibitor, was compared with clavulanic acid and sulbactam for enhancement of the activity of piperacillin against beta-lactamase-producing, piperacillin-resistant clinical isolates. Piperacillin MICs were determined in media containing a fixed concentration of 2 or 4 micrograms of the inhibitors per ml. The higher concentration was generally more effective. Tazobactam was superior to sulbactam in enhancing the spectrum and potency of piperacillin. Although the calvulanic acid combination was more potent, tazobactam was effective for a similar spectrum of resistant gram-negative clinical isolates containing beta-lactamase. MICs were reduced to the susceptible range for Escherichia coli, Klebsiella pneumoniae, Proteus spp., Salmonella spp., and Shigella spp. Combinations with tazobactam and sulbactam, but not clavulanic acid, were effective against Morganella spp. Some antagonism of the activity of piperacillin was observed with clavulanic acid but not with tazobactam or sulbactam. The inhibitors were similarly effective with piperacillin against beta-lactamase-positive Staphylococcus spp. and the Bacteroides fragilis group. Piperacillin-tazobactam was more effective against a broader spectrum of gram-negative enteric bacteria than ticarcillin plus clavulanic acid was. Combinations with tazobactam or clavulanic acid had a broader spectrum of activity than combinations with sulbactam against bacteria that produce characterized plasmid-mediated enzymes of clinical significance. In particular, piperacillin with tazobactam or clavulanic acid, but not with sulbactam, inhibited TEM-1, TEM-2, and SHV-1 enzymes. In vitro activity was reflected in vivo. Tazobactam and clavulanic acid were superior to sulbactam in enhancing the therapeutic efficacy of piperacillin in mice infected with beta-lactamase-positive E. coli, K. pneumoniae, Proteus mirabilis, and Staphylococcus aureus. Only combinations with tazobactam and sulbactam were effective against the Morganella infection. Tazobactam has a good potential for enhancing the clinical efficacy of piperacillin.     

Ampicillin-sulbactam and amoxicillin-clavulanate susceptibility testing of Escherichia coli isolates with different beta-lactam resistance phenotypes

The activities of ampicillin-sulbactam and amoxicillin-clavulanate were studied with 100 selected clinical Escherichia coli isolates with different beta-lactam susceptibility phenotypes by standard agar dilution and disk diffusion techniques and with a commercial microdilution system (PASCO). A fixed ratio (2:1) and a fixed concentration (clavulanate, 2 and 4 micrograms/ml; sulbactam, 8 micrograms/ml) were used in the agar dilution technique. The resistance frequencies for amoxicillin-clavulanate with different techniques were as follows: fixed ratio agar dilution, 12%; fixed concentration 4-micrograms/ml agar dilution, 17%; fixed ratio microdilution, 9%; and disk diffusion, 9%. Marked discrepancies were found when these results were compared with those obtained with ampicillin-sulbactam (26 to 52% resistance), showing that susceptibility to amoxicillin-clavulanic acid cannot be predicted by testing the isolate against ampicillin-sulbactam. Interestingly, the discrimination between susceptible and intermediate isolates was better achieved with 4 micrograms of clavulanate per ml than with the fixed ratio. In contrast, amoxicillin susceptibility was not sufficiently restored when 2 micrograms of clavulanate per ml was used, particularly in moderate (mean beta-lactamase activity, 50.8 mU/mg of protein) and high-level (215 mU/mg) TEM-1 beta-lactamase producer isolates. Four micrograms of clavulanate per milliliter could be a reasonable alternative to the 2:1 fixed ratio, because most high-level beta-lactamase-hyperproducing isolates would be categorized as nonsusceptible, and low- and moderate-level beta-lactamase-producing isolates would be categorized as nonresistant. This approach cannot be applied to sulbactam, either with the fixed 2:1 ratio or with the 8-micrograms/ml fixed concentration, because many low-level beta-lactamase-producing isolates would be classified in the resistant category. These findings call for a review of breakpoints for beta-lactam-beta-lactamase inhibitors combinations.     

Rate of bactericidal activity for Branhamella catarrhalis of a new macrolide, CP-62,993, compared with that of amoxicillin-clavulanic acid

The rate of bactericidal activity of a new macrolide, CP-62,993, was compared with that of the combination of amoxicillin and clavulanic acid (in the proportion of 4 to 1) on strains of Branhamella catarrhalis beta-lactamase producers. The antibacterial activity of CP-62,993 was bacterostatic at 0.01 micrograms/ml. After a 6-hour period of bacteriostasis a bactericidal activity (3 log10 CFU/ml) was observed for all concentrations from 0.1 to 10 micrograms/ml after 24 h. The bactericidal rate of amoxicillin-clavulanic acid combination was more rapid during the first 6 h at 1 and 10 micrograms/ml. However, the concentration required to kill 99.9% of bacteria within 24 h was 1 microgram/ml. In conclusion, CP-62,993 was a bactericidal antibiotic for B. catarrhalis at a lower concentration. This in vitro study suggests that this macrolide may be of great interest in infections due to the B. catarrhalis beta-lactamase producer.     

Activity of cefazolin and two beta-lactamase inhibitors, clavulanic acid and sulbactam, against Bacteroides fragilis.

One hundred clinical isolates of the Bacteroides fragilis group of bacteria were tested by agar dilution for susceptibility to cefazolin alone or in combination with clavulanic acid or sulbactam. For cefazolin, the MIC for 50% of the isolates (MIC50) was 32 micrograms/ml, the breakpoint for susceptibility. With the addition of 0.5 micrograms of clavulanic acid per ml, the MIC for 90% of the isolates (MIC90) was 8 micrograms/ml, well within the achievable range of concentrations in serum or tissue. Similarly, with the addition of 0.5 micrograms of sulbactam per ml, the MIC90 was 16 micrograms/ml. The addition of a higher concentration (4.0 micrograms/ml) of clavulanic acid or sulbactam resulted in MIC90S which were fourfold lower than those with 0.5 micrograms/ml. A fixed ratio of cefazolin-beta-lactamase inhibitor of 4:1 resulted in an MIC50 and MIC90 which were intermediate between the 0.5- and 4.0-micrograms/ml fixed concentration of beta-lactamase inhibitor.     

Novel plasmid-mediated beta-lactamase (TEM-10) conferring selective resistance to ceftazidime and aztreonam in clinical isolates of Klebsiella pneumoniae.

Two clinical isolates of Klebsiella pneumoniae from seriously ill patients in Chicago, Ill., have been identified as resistant to ceftazidime and aztreonam but susceptible to other cephalosporins. This unusual antibiogram was shown to be due to a novel plasmid-mediated beta-lactamase which readily hydrolyzed ceftazidime and aztreonam in addition to penicillins such as piperacillin and carbenicillin. This enzyme and its attendant resistance were transferred to Escherichia coli by conjugation on a 50-kilobase plasmid. Isoelectric focusing revealed a single beta-lactamase band with a molecular weight of 29,000 and an isoelectric point of 5.57 in the resistant isolates and transconjugants. The beta-lactamase inhibitors clavulanic acid and sulbactam restored beta-lactam susceptibility in the resistant isolates. Fifty percent inhibitory concentrations of clavulanic acid and sulbactam were 4.4 and 940 nM, respectively. DNA hybridization studies indicated that this enzyme, designated TEM-10, is related to well-established TEM-type beta-lactamases. However, the TEM-10 enzyme was inhibited by p-chloromercuribenzoate, in contrast to TEM-2 beta-lactamase. On the basis of substrate and inhibition profiles, the TEM-10 enzyme could be easily discriminated from TEM-5 and RHH-I beta-lactamases.     

Role of Bacteroides bivius beta-lactamase in beta-lactam susceptibility

The susceptibility of 46 clinical isolates of Bacteroides bivius to amoxicillin, cefotaxime, cefoxitin, ceftizoxime, cephaloridine, cephalothin, moxalactam, penicillin G, amoxicillin plus clavulanic acid in a ratio of 2:1, carbenicillin, cefamandole, and ceftazidime was determined by an agar dilution technique. For the first eight agents susceptibility testing was also done with the addition of clavulanic acid (0.75 microgram/ml). For all agents, beta-lactamase-positive strains (35, using a nitrocefin slide test) were inhibited at higher concentrations than beta-lactamase-negative strains. Clavulanic acid reduced the susceptibility of the beta-lactamase-positive strains to the level of the beta-lactamase-negative strains to all agents. We prepared crude extracts of beta-lactamase from six strains. Activity against nitrocefin was directly related to their susceptibilities. The beta-lactamase had a mixed-substrate profile, hydrolyzing both penicillins and cephalosporins. Our results suggest a slow inactivation of cefoxitin, ceftizoxime, and moxalactam by the beta-lactamase. Clavulanic acid and cefoxitin inhibited the enzyme, whereas p-hydroxymercuribenzoate and cloxacillin did not. Thus, there was a clear relationship between beta-lactamase activity and susceptibility to beta-lactams, including cefoxitin and third-generation cephalosporins. The substrate and inhibition profiles of the B. bivius beta-lactamase were different from those of enzymes found in the "B. fragilis group."