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An inducible cephalosporinase was purified from Pseudomonas maltophilia GN12873. The pI was 8.4, and the molecular weight was ca. 56,000 by gel filtration or 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that this enzyme had two subunits. The optimal pH and optimal temperature were 7.5 and 45 degrees C, respectively. Enzyme activity was inhibited by clavulanic acid, sulbactam, cephamycin derivatives, carbapenem antibiotics, iodine, HgCl2, and p-chloromercuribenzoate. The enzyme showed a broad substrate profile, hydrolyzing cephaloridine, cefazolin, cefsulodin, penicillin G, ceftizoxime, and ampicillin at a high rate.

Cefatrizine, a new orally administered cephalosporin, was tested against 400 clinical isolates. Cefatrizine had excellent activity against gram-positive cocci, inhibiting all except enterococci at minimal inhibitory concentrations below 1 mug/ml. Cefatrizine inhibited the majority of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Salmonella at concentrations below 12.5 mug/ml. Although cefatrizine was not hydrolyzed by many beta-lactamases, it did not inhibit a number of strains of Enterobacter, Serratia, or indole-positive Proteus. Cefatrizine was more active than cephalothin or cephalexin against E. coli, Klebsiella, Enterobacter, Citrobacter, Salmonella, and Shigella. Its overall activity was less than that of cefoxitin against strains resistant to cephalothin, but its activity against cephalothin-susceptible strains was equivalent to that of cefamandole.

We have developed a new micro-broth-dilution assay for determining the antimicrobial susceptibility of Haemophilus influenzae. This assay is based on the ability of viable H. influenzae to reduce nitrates to nitrites. Bacterial viability is detected by a positive nitrite reaction rather than visible turbidity. The nitrate reduction assay was compared with a standard microassay using 51 isolates of H. influenzae and six beta-lactam antibiotics. Although there was good agreement between the two methods, the nitrate reduction assay was more sensitive in detecting viable bacteria, and so established a more accurate estimate of the minimal inhibitory concentration. The nitrate reduction assay offered the additional advantage that it could be used to determine the minimal bactericidal concentration without having to subculture the broth. Ampicillin, penicillin, and cefamandole were equally effective in vitro against susceptible strains (minimal inhibitory concentrations, 0.125 to 0.5 mug/ml), whereas all three antibiotics were ineffective against two beta-lactamase-producing strains. Using the nitrate reduction assay, resistance to cefamandole was detectable with inoculum sizes ranging from 10(4) to 10(6) colony-forming units per ml, while the turbidity assay detected resistance only with the largest inoculum.

A survey of 21 clinical isolates of Achromobacter species demonstrated a high level of beta-lactamase activity in all strains tested. The beta-lactamases were characterized by isoelectric focusing, purification by affinity chromatography, determination of molecular weight, immunological identity, and genetic analysis. At least three distinct patterns of beta-lactamases were found in 19 strains. The kinetic values Km and Vmax measured by a microacidimetric method showed that all three types of enzymes are cephalosporinases and did not hydrolyse oxacillin, cloxacillin, and methicillin. Two of the three types of cephalosporinases studied, namely MULB 901 (isoelectric point (pI)7.4) and MULB 905(pI 9.3) are enzymes mediated by genes of chromosomal origin. The MULB 906 (pI 8.1) enzyme, however, which has been previously shown to be mediated by an 8.2 MDal nonconjugative plasmid, showed hydrolysis of cefoxitime, cefotaxin, and moxalactam by the bioassay. In all cases, beta-lactamase synthesis appeared constitutive. This study confirms that beta-lactamase activity is commonly found in Achromobacter and that these enzymes are different and of clinical interest when compared with those observed in other Gram-negative bacteria.

The in vitro activity of U-63196E, a new broad-spectrum cephalosporin antibiotic, was studied against various gram-positive and gram-negative bacteria and compared with the in vitro activities of cefotaxime, moxalactam, cefoperazone, ceftazidime, and aztreonam. Although U-63196E inhibited many ampicillin-resistant bacteria and its activity against gram-negative species was similar to cefoperazone, it was much less active than the other agents. U-63196E was less active than cefazolin against gram-positive species, and it was less active than cefoxitin or moxalactam against Bacteroides fragilis. U-63196E did not inhibit most cefoperazone- or cefsulodin-resistant Pseudomonas aeruginosa. There was a difference between minimal inhibitory concentrations and minimal bactericidal concentrations for isolates which contained beta-lactamases. Plasmid beta-lactamases of the TEM, HSV, OXA, and PSE types hydrolyzed U-63196E. But U-63196E was relatively stable against hydrolysis by the chromosomal beta-lactamases.

A survey of clinical isolates from a hospital laboratory showed that Escherichia coli could be grouped into three classes of beta-lactam-antibiotic resistance by results of routine susceptibility testing to ampicillin, cephalothin, and carbenicillin. E. coli highly resistant to ampicillin and carbenicillin but not to cephalothin (class I) were found to have one of two levels of R factor-mediated, periplasmic-beta-lactamase which resembled R(TEM) and was located behind a permeability barrier to penicillins but not to cephalosporins. This permeability barrier appeared to act synergistically with the beta-lactamase in producing high levels of resistance to penicillins. E. coli highly resistant to ampicillin and cephalothin but not carbenicillin (class II) were found to have a beta-lactamase with predominantly cephalosporinase activity which was neither transferable nor releasable by osmotic shock. E. coli moderately resistant to one or to all three of these antibiotics (class III) were found to have low levels of different beta-lactamases including a transferable beta-lactamase which resembled R(1818). Thus, different mechanisms producing resistance to beta-lactam antibiotics could be deduced from the patterns of resistance to ampicillin, cephalothin, and carbenicillin found on routine susceptibility testing. E. coli of class I were much more prevalent than the other classes and the proportion of E. coli that were class I increased with duration of patient hospitalization. The incidence of class I E. coli rose only slightly over the past 7 years and that of class II E. coli remained constant despite increased usage of both cephalothin and ampicillin. These observations emphasize that the properties of the apparently limited number of individual resistance mechanisms that exist in a bacterial flora, such as their genetic mobility and linkages and the spectrum of their antibiotic inactivating enzymes and permeability barriers, may govern the effect that usage of an antibiotic has upon the prevalence of resistance to it and to other antibiotics.

The isolation and properties of a beta-lactamase from E. coli are described, which hydrolyses the Cephalosporins of the third generation. The enzyme has been brought to high purity by precipitation with (NH4)2SO4, gel chromatography and affinity chromatography on Cephalosporin C bound to agarose. The enzyme has been characterized by evaluation of its molecular weight 39 000, isoelectric point (pH 7.2), pH-optimum (pH 8.4) and substrate profile. It accepts Ceftizoxime, Cefamandole, Cefazolin and Cefotaxime as substrates, but shows nearly no activity on Penicillin G, Cefoxitin and the Desacetylmetabolite of Cefotaxime.

Various properties (specific activity, inducibility, substrate profile, and susceptibility to inhibitors) of the beta-lactamase activity present in 39 strains of Enterobacteriaceae and Pseudomonas aeruginosa isolated during a two-month period in the bacteriology laboratory of a large general hospital were investigated. Among the 39 strains there appeared to be at least 16 distinct enzymes. Most enzymes from Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Proteus morganii were active against both penicillins and cephalosporins, whereas those from Proteus vulgaris, Serratia marcescens and Pseudomonas aeruginosa showed activity primarily against cephalosporins. Both types of enzyme were found among strains of Enterobacter. There was a general correlation between amount of enzymic activity and levels of resistance to several beta-lactam antibiotics. Discrepancies between enzymic activity and resistance to several beta-lactam antibiotics. Discrepancies between enzymic activity and resistance may be due to variations in the roles intrinsic mechanisms play in resistance to various antibiotics.

We investigated the relationship between beta-lactamases of Bacteroides fragilis organisms and their resistance to cephalosporins. Timed killing curves were used to study the in vitro activity of three cephalosporins, cephalothin, cefazolin, and cefamandole, and a semisynthetic cephamycin, cefoxitin. Measurements of residual antibiotic concentrations in culture supernatants were made, and they were compared with the beta-lactamase activity of the microorganism. A cephalosporin-susceptible strain was rapidly killed by cephalothin, cefazolin, cefamandole, and cefoxitin. Four cephalosporin-resistant strains were not killed by cephalothin, cefazolin, or cefamandole but were killed by cefoxitin. An inoculum effect was noted with cefazolin and not with cefoxitin. The resistant strains of Bacteroides inactivated the three cephalosporins, but there was no inactivation of cefoxitin. A constitutive beta-lactamase was detected in all the isolates of the B. fragilis group that were resistant to the cephalosporins. There was no distinction of the species based on isoelectric focusing of the enzyme. These data suggest that inactivation by beta-lactamase may be the mechanism for resistance of B. fragilis to the cephalosporins and would explain the enhanced in vitro activity of cefoxitin.

Resistance to cefotaxime (CTA) and ceftriaxone (CTR) in Enterobacter cloacae and Pseudomonas aeruginosa was investigated in several strains which are susceptible or resistant to these agents. All strains produced a chromosomally mediated cephalosporinase of the Richmond type 1. beta-Lactamases in susceptible strains were inducible, whereas resistant strains produced the enzymes constitutively. CTA and CTR were very poor substrates but potent inhibitors of all enzymes. Binding to, rather than hydrolysis by, beta-lactamases was assumed to be a major reason for resistance, and combination experiments supported this assumption. Dicloxacillin, which did not inhibit the growth and which was a poor inducer but a strong inhibitor of these beta-lactamases, exerted strong synergistic activity when combined with CTA or CTR in strains which produced large amounts of beta-lactamase constitutively. Cefoxitin, on the other hand, poorly active alone, but a good inducer, strongly antagonized CTA or CTR in susceptible strains producing inducible enzymes. In marked contrast to CTA and CTR were the findings with cefsulodin. Cefsulodin was active against CTA- and CTR-resistant Pseudomonas, and its activity was hardly influenced by dicloxacillin or cefoxitin. Since cefsulodin was found to have a very low affinity for all cephalosporinases, these findings corroborate the assumption that binding of nonhydrolyzable cephalosporins, rather than hydrolysis by cephalosporinases, may play an important role in resistance to these agents and other newer cephalosporins in Enterobacteriaceae, as well as in other gram-negative bacteria.

The authors review the procedure utilized for the naming of a new antibiotic. The cephamycins are taken as an example. Cefoxitin, derivative of the cephamycin C through an original chemical reaction is described. This antibiotic possesses a high degree of resistance to beta-lactamases due to the presence of a methoxy group in the 7alpha position of the beta-lactam ring. The results of extensive in-vitro trials are given, they indicate that cefoxitin has a unique antibacterial activity.

Using the agar dilution technique, 231 strains of Bacteroides fragilis, isolated during a 2-year period from human infections, were identified at subspecies level and were tested for susceptibility to 13 beta-lactam antibiotics. The penicillins were benzylpenicillin, ampicillin, carbenicillin, cloxacillin, and the recently described penicillin derivatives cyclacillin, ticarcillin, and PC-904. The following cephalosporin derivatives were tested: cephaloridine, cephalothin, cephalexin, cefamandole and cefuroxime. The cephamycin C derivative cefoxitin was also included in the study. Cefoxitin was the most effective drug tested since more than 80% of the strains were inhibited by 8 microgram/ml or less, and no strain had a minimal inhibitory concentration (MIC) of more than 64 microgram/ml. There was no marked difference in sensitivity among the subspecies with exception of subspecies vulgatus, which was slightly more sensitive to all antibiotics tested. The size of the inoculum was an important factor for obtaining reproducible results in the sensitivity tests. Increased inocula resulted in markedly higher MICs for cephaloridine and cefuroxime. Production of beta-lactamase was performed on all isolates by a chromogenic cephalosporin substrate and about 90% of the strains were found to be beta-lactamase producers.

(6,7R)-7-[(Z)-2-(2-Amino-4-thiazoyl)-2-methoxyiminoacetamido]-8-oxo-5-thia-1 -azabicyclo[4,2,0]oct-2-ene-2-carboxylate (ceftizoxime), a new parenteral cephalosporin, in more potently active in vitro and in vivo against various gram-negative bacilli including the opportunistic pathogens such as Enterobacter, Citrobacter species and Serratia marcescens than cephalosporins and cephamycins recently developed. Ceftizoxime is extremely stable to various types of beta-lactamases, and has a high ability to penetrate the outer membrane of gram-negative organisms.

The known mechanisms of beta-lactam resistance in anaerobic bacteria involve production of beta-lactamases, alteration of penicillin-binding proteins and blocked penetration of beta-lactams through the outer membranes. The most important factor in beta-lactam resistance is production of beta-lactamase. Beta-lactamases in various Bacteroides, Fusobacterium and Clostridium species have been described. Beta-lactam resistance in Bacteroides fragilis is most commonly mediated by beta-lactamase production mainly of cephalosporinase character. Recent studies have also shown that B. fragilis can produce a penicillinase which inactivates piperacillin and carbenicillin. Enzymes inactivating cefoxitin and imipenem have also been isolated from B. fragilis. The Bacteroides non-fragilis species produce beta-lactamases of mainly penicillinase character. Recently a penicillinase from Fusobacterium nucleatum has been characterized. Among the clostridia, Clostridium butyricum, C. clostridiiformis and C. ramosum have been shown to produce penicillinases.

In an experimental model of peritoneal infection by cephalosporinase- (Ia and Ic) producing bacteria in mice, the reduction of cefoxitin, cefmetazole, and cefazolin concentrations in peritoneal fluid was observed in the mice infected with the Ia enzyme producer, whereas cefbuperazone concentrations were not reduced.

The inability of sulfamethoxazole-trimethoprim (SXT) to eradicate Haemophilus influenzae nasopharyngeal carriage in all asymptomatic patients in closed populations was examined in vitro. A broth medium was adapted for susceptibility testing of H. influenzae which permitted us to determine minimum inhibitory concentrations and minimum bactericidal concentrations (MBCs). The minimum inhibitory concentrations were all low, but the MBCs were bimodally distributed. Trimethoprim alone or the combination SXT either was bactericidal for H. influenzae isolates at low concentrations (i.e., low MBCs) similar to minimum inhibitory concentrations or showed no bactericidal activity (i.e., high MBCs). If trimethoprim was bactericidal when tested alone against H. influenzae, then the combination SXT was also bactericidal. H. influenzae carriage could not be eradicated from asymptomatic patients with SXT therapy when that combination was not bactericidal for these isolates in vitro. H. influenzae carriage was eradicated from patients when the activity of SXT was bactericidal in vitro. H. influenzae strains that are not killed by trimethoprim or SXT seem to occur at random.

Cefuroxime is a new broad-spectrum cephalosporin antibiotic with increased stability to beta-lactamases. This stability, although no absolute in all cases, has the effect of widening the antibacterial spectrum of the compound so that many organisms resistant to the established cephalosporins are susceptible to cefuroxime. It is active against gram-positive organisms, including penicillinase-producing staphylococci, but it is less active against methicillin-resistant strains. In addition to its high activity against non-beta-lactamase-producing gram-negative bacteria, cefuroxime effectively inhibits the growth of many beta-lactamase-producing strains, including Enterobacter, Klebsiella, and indole-positive Proteus spp. It is highly active against Neisseria gonorrhoeae, Neisseria meningitidis, and also Haemophilus influenzae, including ampicillin-resistant strains. Cefuroxime is rapidly bactericidal and induces the formation and subsequent lysis of filamentous forms over a small concentration range.

One-hundred clinical isolates of Serratia marcescens were tested for susceptibility to cephalothin, carbenicillin, ticarcillin, ampicillin, and cefoxitin. The majority of the 100 isolates (>/=70%) were susceptible to carbenicillin, ticarcillin, and cefoxitin; less than one-half were susceptible to ampicillin; none were susceptible to cephalothin. Ten isolates from the 100 organisms tested were selectively assayed for their beta-lactamase activity. Enzyme activity was measured using either iodometric or spectrophotometric methods, and the microbiological assay technique. It was concluded that beta-lactamase production was not the sole determinant in beta-lactam antibiotic resistance. Resistance without demonstrable beta-lactamase was evident in strains for cephalothin, ampicillin, and cefoxitin. In addition, one strain which was susceptible to all antibiotics except cephalothin, elaborated considerable beta-lactamase activity.

The antimicrobial susceptibility of 106 strains of Campylobacter jejuni/coli recovered from animals and humans was studied using broth microdilution panel. Human and animal strains exhibit similar susceptibility, with the exception of the strains from pigs which are more resistant to erythromycin and clindamycin. Among the hippurate negative strains (C. coli), these strains are also recognizable from those recovered from other hosts.

The reason why the results of the clinical response to therapy do not always correspond to antibiotic laboratory testing include errors in performance or interpretation of test results, imprecise definition of resistance and host factors which influence the outcome of treatment regardless of the antibiotic being used. These factors are reviewed. Cephalosporins and penicillins pose problems because the prime effects of enzymes in influencing the outcome of treatment are not always clear in standard tests for susceptibility. Inter-laboratory differences in interpretation of tests makes comparisons of tests between centres difficult and the use of reference strains for validation of testing is proposed.

The beta-lactamase stability of cefoxitin, the second-generation cephamycin compound, was compared with that of cefamandole, cefoperazone, cefotaxime, moxalactam, carbenicillin, and piperacillin. Unlike cefamandole, cefoperazone, carbenicillin, and piperacillin, cefoxitin was not hydrolyzed by the common plasmid beta-lactamases, TEM-1, TEM-2, OXA-2, and SHV-1. Cefoxitin and moxalactam were the only agents stable to all chromosomal beta-lactamases, whereas cefamandole, cefoperazone, cefotaxime, carbenicillin, and piperacillin were destroyed by cephalosporinases of Proteus vulgaris and Bacteroides fragilis.

1. Four histidine-containing peptides have been isolated from a tryptic digest of the Zn2+-requiring beta-lactamase II from Bacillus cereus. One of these peptides probably contains two histidine residues. 2. The presence of one equivalent of Zn2+ substantially decreases the rate of exchange of the C-2 proton in at least two and probably three of the histidine residues of these peptides for solvent 3H. 3. It is concluded that peptides containing at least two of the three histidine residues acting as Zn2+ ligands at the tighter Zn2+-binding site of beta-lactamase II have been identified.

Escherichia coli UB1005 and two mutants of this strain (DC2 and DC3) have been used to assess indirectly the relative ability of various beta-lactam antibiotics to penetrate the outer layers of E. coli. Benzylpenicillin, ampicillin, methicillin, cloxacillin, cephaloridine, cephalothin, and cephalexin have been examined. The results confirm those obtained with other methods and show that, among the compounds studied here, cephalosporins seem to penetrate more readily than penicillins.

Strains of Serratia marcescens fall into one of two groups with respect to their resistance to to beta-lactum antibiotics. Most strains are highly resistant to cephalosporins but are significantly more susceptible to ampicillin and carbenicillin, whereas other strains are highly resistant to both penicillins and cephalosporins. Strains in the former category produce small amounts of an inducible cephalosporinase, which appears to be chromosomally mediated. Strains in the latter class also elaborate large amounts of a noninducible penicillinase-cephalosporinase, which is plasmidmediated. Ability to produce this type of enzyme can be transferred to Klebsiella pneumoniae or Escherichia coli and may be lost spontaneously or after exposure of S. marcescens to "curing" agents.

Although ampicillin has been suggested as a useful agent for the treatment of nocardiosis in man, little is known regarding the presence of beta-lactamase in Nocardia or its possible role in determining resistance to ampicillin and the other beta-lactam antibiotics. We have evaluated 55 isolates of Nocardia for susceptibility to five beta-lactam antibiotics and for the presence of beta-lactamase. Nocardia were resistant to penicillin G, cloxacillin, and cefazolin, but 27 and 62% were susceptible to 3.1 and 25 mug of ampicillin per ml, respectively. Almost 90% of these ampicillin-susceptible or intermediate strains were also susceptible to carbenicillin. The combination of ampicillin and cloxacillin was synergistic against many ampicillin-resistant strains. Beta-lactamase was detected in 89% of Nocardia isolates when intact cells were used and in six of six strains after cell fractionation. This beta-lactamase was most active against penicillin G and ampicillin, with lesser activity against carbenicillin and cephaloridine. These studies suggest that beta-lactamase may be present in all clinical isolates of Nocardia and that mechanisms of antimicrobial resistance other than or in addition to beta-lactamase are responsible for resistance of Nocardia to ampicillin and carbenicillin.

The inactivation kinetics for inhibition by sulbactam (CP45,899) of Citrobacter freundii GN346 cephalosporinase were studied in detail and compared with those of type Ib penicillinase or TEM-2 beta-lactamase mediated by R plasmid RGN823. The rate constant for progressive inactivation of the cephalosporinase was significantly larger than that measured with the penicillinase. The number of sulbactam molecules required to cause complete inactivation of one cephalosporinase molecule (turnover number) was 80. The turnover number for the penicillinase was 5,200. The powerful inhibition by sulbactam of this cephalosporinase is similar to clavulanic acid inhibition of the penicillinase (turnover number, 115; reported by others). The affinity of sulbactam for the cephalosporinase, expressed as Ki, was 500 microM; this value was much higher than that for the penicillinase, which was estimated to be 0.5 microM. These results indicated that sulbactam is an effective progressive inactivator but a poor competitive inhibitor for the cephalosporinase. Our study also revealed that the cephalosporinase and sulbactam formed a long-lived inhibitor-enzyme complex which we termed the pseudo-irreversible complex. The half-life of the complex was 550 min at pH 7.0 and 30 degrees C.

The interactions of E1040 with cephalosporinase from Citrobacter freundii, including affinity and hydrolysis, were studied in comparison with those of cefotaxime and ceftazidime. E1040 showed a higher stability at low drug concentrations and a much lower affinity for the enzyme than did cefotaxime or ceftazidime. These enzymological properties explain the high activity of E1040 against cephalosporinase-producing C. freundii.

In contrast to the results obtained with ampicillin, the minimum inhibitory concentrations of cefamandole against Haemophilus influenzae were within the same range (0.5 to 1.5 mug/ml) whether or not the strains were beta-lactamase producers. The minimum bactericidal concentrations were somewhat higher for beta-lactamase-positive strains (6.4 mug/ml) than for negative strains (1.2 mug/ml). In a culture with high initial microbial density, monitored by recording optical densities, the addition of 10 mug of cefamandole per ml brought about rapid lysis of a beta-lactamase-negative strain. Observation of a beta-lactamase-positive strain revealed, in the early part of the growth curve, absence of lysis and an increase of biomass similar to that observed in a drug-free control curve. In contrast to the results obtained with ampicillin, the culture consisted uniformly of spherical forms, probably in the process of division, which were capable of generating colonies. When the microbial density exposed to cefamandole was increased still further, persistent bacillary forms were observed, and after 24 h hydrolysis had eliminated every trace of microbiologically active cefamandole.

On the basis of a simple theoretical model, the ease of penetration of beta-lactam antibiotics through the outer membrane of Escherichia coli was measured. The cell envelope was found to act as a diffusion barrier to both penicillins and cephalosporins. The validity of the model and the cooperative action of cell-bound beta-lactamase and outer membrane were further verified by comparing calculated and experimentally determined velocities of beta-lactam hydrolysis by intact cells and sonically treated cell suspensions. The results showed good correspondence at five different antibiotic concentrations. Similar conclusions could be drawn from a comparison of beta-lactam concentrations on both sides of the outer membrane, calculated from enzyme kinetic measurements and minimal inhibitory concentrations for both a beta-lactamase-producing E. coli and its enzyme-negative variant. in the case of benzylpenicillin and cephalothin, however, no correspondence was found. The joint action of several parameters determining the efficacy of penicillins and cephalosporins against beta-lactamase-producing E. coli is discussed.

1. A cephalosporin-binding protein obtained from a strain of Citrobacter freundii was purified to the extent of a single band in analytical and sodium dodecyl sulfate-containing disc electrophoresis. 2. The molecular weight determined by disc electrophoresis was 53 000. 3. The binding protein did not show any beta-lactamase activity at substrate concentrations examined: 6 mM to 100 muM of penicillins and 12 mM to 100 muM of cephalosporins. 4. In gel filtration, [14C]benzylpenicillin was found not to bind to the binding protein. 5. In fluorescence titration, all cephalosporins tested quenched the fluorescence. Association constants of cephalosporins were in the range of 0.8-12-103 M-1, and one binding site was calculated for all cephalosporins tested.

FR10612, like cephalexin, is a broad-spectrum oral cephalosporin derivative. The antimicrobial activity of FR10612 against clinical isolates was similar to cephalexin; however, at a low inoculum size its activity was greater than cephalexin against Klebsiella pneumonia and Proteus mirabilis strains. Like cephalexin, the in vitro bactericidal activity of FR10612 was more influenced by the duration of contact with the test organism than by drug concentration. The bactericidal activity of FR10612 against E. coli 317 was greater than that of cephalexin in an in vitro model system which simulated the serum levels of FR10612 and cephalexin achieved in healthy volunteers after a single oral dose. The protein binding of FR10612 to human and animal serum was extremely low. FR10612 was resistant to beta-lactamases from gram-negative bacilli. It showed resistance similar to cephalexin, but was more resistant to beta-lactamases than were cephaloridine, cephalothin and cefazolin. The protective effect of FR10612 in mice infected with various pathogens was greater than cephalexin. The serum levels of FR10612 in rats were higher and more prolonged than those of cephalexin. Tissue levels of FR10612 in rats also persisted for a long time period reflecting the serum levels. In healthy volunteers, rabbits and monkeys the serum levels of FR10612 were initially lower than those of cephalexin but persisted for a longer time period. The total 24-hour urinary excretion of FR10612 in healthy volunteers after oral administration was almost the same as that of cephalexin, but the excretion rate of FR10612 was slower, and the urinary levels were more persistent than those of cephalexin.

The properties of cefazolin, cefoxitin, cefamandole, and cefuroxime have been compared with those of cephaloridine, cephalothin, and cephalexin. The properties included for examination are the susceptibility to a range of beta-lactamases commonly encountered in gram-negative species and the ability of a beta-lactam antibiotic to penetrate the outer layers of Escherichia coli. Of the antibiotics tested, cefamandole was the most active in its antibiotic activity but had the disadvantage that it was sensitive to hydrolysis by type IVc beta-lactamase. Cefoxitin and cefuroxime were slightly less active than cefamandole but were effectively resistant to all the beta-lactamases used in the test.

A total of 218 Proteus strains isolated from clinical sources were tested for their susceptibility to three penicillins and two cephalosporins. The ability to beta-lactamase production was examined in 36 of these strains. Proteus mirabilis strains were generally more susceptible to cephalosporins than to penicillins, whereas indole-positive Protei were almost uniformely resistant to cephalosporins as well as to ampicillin and benzylpenicillin but susceptible to carbenicillin. Fairly good correlation was found between the amount and hydrolytic soectryn if beta-lactamase activity and the pattern of resistance to penicillins and cephalosporins in the strains examined. Some observations indicate, however, that the resistance of Proteus bacilli to this group of antibiotics is partly related to permeability barriers in bacterial cell. About 37% of Proteus strains transferred their ampicillin resistance to E. coli K12. Beta-lactamase activities mediated by R plasmids in E. coli cultures were 1.5 to 5 times higher than in respective Proteus donors.

The rapid fixed-time assay for penicillinase was modified for measuring beta-lactamase activity with twelve substrates, i.e., benzylpenicillin, ampicillin, cloxacillin, methicillin, carbenicillin, cefazolin, cephalothin, cephaloglycin, cephalexin, cephalosporin C, 7-aminocephalosporanic acid, and cefoxitin. The method depends upon the reduction of iodine by the hydrolyzed substrate. Determined experimentally, 1 mol of hydrolyzed penicillins consumed 3.4 to 4.0 mol of iodine (I(2)). Iodine consumption of hydrolyzed cephalosporins varied widely from 1.7 for cephalothin to 3.7 for cefazolin. The method is useful for routine assay of beta-lactamase activity with various substrates.

A fluorescent spot test method for specific detection of microbial beta-lactamases as previously published (K. C. S. Chen, J. S. Knapp, and K. K. Holmes, J. Clin. Microbiol. 19:818-825, 1984) was improved by the use of a fluorescence developer solution. The fluorescence developer solution used in this study consisted of 0.78 M sodium tartrate buffer containing 12% formaldehyde at a final pH of 4.5. An addition of 1 volume of fluorescence developer solution to 5 volumes of ampicillin or cephalex substrate solution incubated with beta-lactamase-producing organisms, followed by heating the mixture at 45 degrees C for 10 min resulted in enhancement of fluorescence of the end products of beta-lactamase activity. This provides a more sensitive assay for microbial beta-lactamases and offers the potential for direct detection of beta-lactamases in clinical specimens.

The in vitro activity of cefmenoxime (SCE-1365) was evaluated in a multiphased collaborative investigation. Over 7,500 consecutive clinical isolates were tested in five laboratories, and greater than 90% of the following organisms were inhibited by cefmenoxime at the following concentrations: Enterobacteriaceae and non-enterococcal streptococci, /=22 mm, susceptible;