Rapid, inexpensive method for specific detection of microbial beta-lactamases by detection of fluorescent end products

A rapid method was developed for specific detection of microbial beta-lactamases which uses ampicillin and cephalexin as substrates. The end products (open beta-lactam ring forms) generated after separately incubating either substrate with beta-lactamase-producing organisms initially were separated from the unhydrolyzed substrates by high-voltage electrophoresis at pH 2.1. The end products of both antibiotics were highly fluorescent and could be analyzed visually and semiquantitatively under a long-wave UV lamp. Application of 5 microliters of the same incubation mixture onto filter paper without subsequent electrophoretic separation also resulted in development of fluorescence after brief heating at 120 degrees C for 5 min. This spot test differentiates penicillinase activity from cephalosporinase activity and distinguishes between beta-lactamase and acylase activities, since the end products of acylase [the common side chain, D(-)-alpha-aminophenylacetic acid, and the intact beta-lactam nuclei, 6-aminopenicillanic acid and 7-aminodeacetoxycephalosporanic acid] are not fluorescent. This method was relatively rapid, inexpensive, and more sensitive than the chromogenic cephalosporin (nitrocefin) method when 21 strains of 7 gram-positive species and 77 strains of 29 gram-negative species of bacteria were tested.     

The cefoperazone-sulbactam combination. In vitro qualities including beta-lactamase stability, antimicrobial activity, and interpretive criteria for disk diffusion tests

Three concentrations of the penicillanic acid sulfone, sulbactam were tested in combination with cefoperazone against 632 recent clinical bacterial isolates. Cefoperazone was effective alone (less than or equal to 16 micrograms/mL) against 95% of Enterobacteriaceae and combined with 4 micrograms/mL sulbactam inhibited 99.5% of strains. This coverage of enteric bacilli was superior to timentin (99.1%), ceftazidime (98.2%), and tobramycin (90.9%). The minimum inhibitory concentrations (MICs) of cefoperazone-susceptible strains also were markedly decreased by sulbactam (overall MIC90s, 8.0 micrograms/mL for cefoperazone and 1.0 microgram/mL for cefoperazone and 4.0 micrograms/mL for sulbactam). Sulbactam also expanded the spectrum of cefoperazone against Acinetobacter species, some rare Pseudomonas species, and Bacteroides fragilis group species. Sulbactam had direct antimicrobial activity against the acinetobacters and Pseudomonas acidovorans, but the increased activity of cefoperazone-sulbactam against some other Pseudomonas species and anaerobes was attributed to beta-lactamase inhibition. The cefoperazone MICs against beta-lactamase producing Staphylococcus species also were lowered to the level of enzyme-deficient strains. Cefoperazone bactericidal activity was improved by 4.0 micrograms/mL sulbactam, and no antagonism was observed. beta-lactamase hydrolysis studies confirmed a slow hydrolysis of cefoperazone only by TEM beta-lactamases and a high-grade resistance to enzyme breakdown by sulbactam. Differential beta-lactamase affinity studies for cefoperazone and sulbactam showed potential efficacy and applications to plasmid-mediated TEM and OXA enzymes and only marginal effective sulbactam inhibition of Pseudomonas and Klebsiella species enzymes. Disk diffusion studies on 556 strains confirmed the applicability of the cefoperazone 75-micrograms disk to testing routine isolates other than enterococci and methicillin-resistant Staphylococcus aureus. The addition of 4.0 micrograms sulbactam/mL in a fixed concentration to dilution test systems and 15 micrograms sulbactam to the 75 micrograms cefoperazone disk were recommended for in vitro tests. Susceptibility and resistant interpretive criteria for the disk and dilution tests can be applied with confidence. Only 0.4% false-susceptibility errors and a 97.5% absolute interpretive agreement were achieved using the 75 micrograms cefoperazone/15 micrograms sulbactam disk.     

Comparative activities of 15 beta-lactam antibiotics against 590 strains of Klebsiella pneumoniae according to the production of beta-lactamase

In October 1988, all non repetitive strains of K. pneumoniae isolated in 17 hospitals have been studied. Among these 590 strains: 451 (76%) only produce the specific beta-lactamase of the species SHV-1 (pI 7,7) or SHV-1 type (pI 7,1), while 74 (12.5%) produce a TEM-1 or TEM-2 type beta-lactamase, and 65 (11%) an extended broad spectrum beta-lactamase: 22 CTX-1, 5 SHV-2, 4 SHV-3, 26 SHV-4, 8 SHV-5. The minimum inhibitory concentrations of the following antibiotics were performed by a liquid micro dilution technic: amoxicillin (AMX), amoxicillin + clavulanic acid (CL), 5 mg/l, ticarcillin (TIC), piperacillin (PIP), cefazolin (CEZ), cefamandole (CFM), cefoperazone (CFP), cefotaxime (CTX), cefotaxime + clavulanic acid 5 mg/l, cefotaxime + sulbactam (SUL) 5 mg/l, cefpirome (CPI), ceftazidime (CAZ), azthreonam (AZT), latamoxef (MOX), cefoxitin (FOX), cefotetan (CTT), temocillin (TMO), imipenem (IMI). The "wild" strains with SHV-1 beta-lactamase are resistant to AMX and have a decreased susceptibility to TIC and PIP, but are susceptible to other antibiotics. The TEM producing strains are more resistant to PIP and TIC, have a decreased susceptibility to CEZ and CFM but are susceptible to other antibiotics. For the extended broad-spectrum beta-lactamase producing strains, the MIC of penicillin antibiotics (AMX, TIC, PIP) are very high and also the MIC of CEZ, CFM and CFP. The MIC of CTX are higher for CTX-1 or SHV-4 producing strains, than for SHV-2, SHV-3, or SHV-5 producing strains. The combination with CL is more efficacious than the one with SUL to reduce the MIC of CTX in susceptibility area.(ABSTRACT TRUNCATED AT 250 WORDS)     

A clinicomicrobiological study on the importance of pseudomonas in nosocomially infected ICU patients, with special reference to metallo beta1-lactamase production

The present study was conducted with a view to assess the burden of pseudomonal infection in ICU patients of a tertiary care teaching hospital in Uttaranchal. Of the 525 patients selected for the study, during a 1-year period, 60 patients developed features of nosocomial infection and among them Pseudomonas was isolated from one or more samples in 18 patients. The isolated strains were speciated and further characterized for determining their antibiogram and for production of beta-lactamase, extended spectrum beta-lactamase and metallo-beta-lactamase enzymes. Pseudomonas aeruginosa was the commonest species isolated (54.54%) and endotracheal suction material showed the highest bacterial yield. Polymyxin B was found to be the most effective antibiotic followed by imipenem and carbenicillin. Though no strain was found to be producing beta-lactamase and extended spectrum beta-lactamase enzymes, a total of 12 strains (54.54%) were metallo-beta-lactamase producers. For all the beta lactam antibiotics, excepting aztreonam, the metallo-beta-lactamase producers showed more resistance compared to the non-producers.     

Antimicrobial susceptibility of Bacteroides fragilis group isolates in Europe

Objective  To evaluate the activity of old and newer antianaerobic drugs against clinical isolates of Bacteroides fragilis group strains from different parts of Europe.
Methods  Bacteroides fragilis group isolates from 37 laboratories in 19 countries were biochemically characterized. The MICs of seven antimicrobial agents were determined by the agar dilution method as recommended by the NCCLS. Production of beta-lactamase was detected by nitrocefin.
Results  There were 1284 B. fragilis group isolates included in the study. Abdominal infections and wounds were the most common sources of isolation and B. fragilis was the dominating species. Ninety-nine percent of the strains were resistant to ampicillin (breakpoint 2 mg/L), 6% to cefoxitin (64 mg/L), 15% to clindamycin (8 mg/L) and 9% to moxifloxacin (8 mg/L). Less than 1% were resistant to imipenem (16 mg/L), piperacillin-tazobactam (128 mg/L) and metronidazole (32 mg/L). Ninety-six percent of the isolates were beta-lactamase producers.
Conclusions  Antimicrobial resistance among the B. fragilis group is increasing.     

Determination of susceptibility of anaerobic bacteria to beta-lactam antibiotics by a tablet diffusion test

A standardized tablet diffusion test and a reference agar dilution test was evaluated for susceptibility testing of anaerobic bacteria to beta-lactam antibiotics. 74 freshly isolated anaerobic bacteria and three control strains (Cl. perfringens ATCC 13124 B. fragilis ATCC 25285, B. thetaiotaomicron ATCC 29741) were tested. The in vitro activities of 7 beta-lactam antibiotics were compared with metronidazole and clindamycin. Most active were metronidazole and clindamycin. Cefoxitin had the best activity of the beta-lactam antibiotics, whereas piperacillin and carbenicillin had good activities. High resistance rates were found for penicillin, ampicillin, cefuroxime and cefotaxime. MIC on control strains fell well within range set by the National Committee for Clinical Laboratory Standards (NCCLS). Correlation between MIC and inhibition zone diameters was generally good. Tablet diffusion can be used to divide anaerobic bacteria into three susceptibility categories. In addition all bacterial strains were tested for production of beta-lactamase by a nitrocefin tube test. Beta-lactamase production by the nitrocefin test indicated reduced sensitivity to beta-lactam antibiotics.     

Detection of beta-lactamase activity among clinical isolates of Branhamella catarrhalis with six different beta-lactamase assays.

A total of 74 different clinical isolates of Branhamella catarrhalis were examined for their ability to produce beta-lactamase by six different beta-lactamase assays. These included a conventional tube and disk test, in which the chromogenic cephalosporin nitrocefin was used as a substrate; a disk procedure, in which pyridinium-2-azo-p-dimethylanaline cephalosporin was used as a substrate; broth and disk acidometric methods; and a conventional tube iodometric assay. A total of 58 of the study isolates produced beta-lactamase. In all cases, positive results were obtained with the nitrocefin tube and disk assays after 1 min. With the pyridinium-2-azo-p-dimethylanaline cephalosporin disk test, 57 of the 58 beta-lactamase-producing strains yielded a positive reaction in 1 min; the remaining strain was positive after 10 min. None of the beta-lactamase-producing strains produced positive reactions by either the broth or disk acidometric methods after 1 min. With the broth test, 10 min was required for positive test results for 42 strains; 30 min was necessary for 16 strains. By the disk acidometric procedure, all 58 strains were positive after 10 min. Of 58 beta-lactamase-producing strains, 30 were positive by the iodometric assay after 1 min, 13 strains required 10 min, and 4 strains were detected as being beta-lactamase positive only after 30 min. One beta-lactamase-producing strain remained negative by the iodometric method. Among the 16 strains of B. catarrhalis that lacked beta-lactamase that were examined in this study, no false-positive results were obtained by any of the six assays.     

Studies on beta-lactamases from Escherichia coli isolated from urinary tract infections

The beta-lactamase types present in 75 ampicillin and carbenicillin resistant E. coli were characterized using isoelectric focusing (IEF). The strains were isolated from patients with urinary tract infections from two geographically different areas of Denmark: 38 strains from Copenhagen and 37 strains from North Jutland. For 19 of the strains from Copenhagen and 18 of the strains from North Jutland, their beta-lactamase activity against nitrocefin and ampicillin, carbenicillin, benzylpenicillin, cloxacillin and cephaloridine was examined by a micro-iodometric and an UV-spectrophotometric assay. The strains from Copenhagen showed greater activity (p less than 0.001) against nitrocefin than the strains from North Jutland. The rate of hydrolysis of ampicillin was greater for the strains from Copenhagen than for the strains from North Jutland. Ninety-three per cent of the strains produced plasmid-mediated beta-lactamases, of which the most prevalent, TEM-1, was produced by 97 per cent of these strains, and OXA-1 by 3 per cent.     

Chromosomal beta-lactamase expression and antibiotic resistance in Enterobacter cloacae

The activities of beta-lactam antibiotics were compared against Enterobacter cloacae clinical isolates and mutants which had inducible, stably-derepressed, and basal expression of a pI 8.4 subtype of the Ia chromosomal beta-lactamase. These activities were correlated with the results of studies of the beta-lactamase-lability and beta-lactamase-inducer-power of the antibiotics. Cefoxitin and ampicillin were labile, and induced beta-lactamase production strongly at concentrations below their MIC values. Consequently, beta-lactamase-inducible and beta-lactamase-stably-derepressed organisms were highly resistant (MIC greater than 256 mg/L) to these antibiotics, whereas enzyme-basal strains and mutants were much more susceptible (MIC 1-16 mg/L). Imipenem also induced beta-lactamase production strongly at concentrations below its MIC, but was more stable than ampicillin and cefoxitin. It was active against enzyme-inducible and stably-derepressed organisms at 0.25-0.5 mg/L and against beta-lactamase-basal organisms at 0.06-0.25 mg/L. Thus the beta-lactamase afforded only very low-level protection against imipenem; this appeared to be by a non-hydrolytic mechanism, with the enzyme binding to the antibiotic in a relatively stable complex. This complex, which probably was an intermediate in a hydrolytic pathway, was isolated by gelfiltration chromatography and shown to have a breakdown half-life of 47 +/- 2 min. Cefotaxime, ceftriaxone and mezlocillin were labile to the pI 8.4 beta-lactamase but induced beta-lactamase production weakly at concentrations below their MIC values. Consequently, beta-lactamase-inducible and beta-lactamase-basal organisms remained equally susceptible (MIC 0.06-4 mg/L), but stably-derepressed organisms were considerably more resistant (MIC greater than 64 mg/L) to these antibiotics.     

Detection of cfxA and cfxA2, the beta-lactamase genes of Prevotella spp., in clinical samples from dentoalveolar infection by real-time PCR

While most bacteria involved in dentoalveolar infection are highly susceptible to penicillin, some Prevotella strains exhibit resistance to this agent through the production of beta-lactamase. The production of beta-lactamase by Prevotella spp. is in turn associated with the expression of the genes cfxA and cfxA2. The aim of the present study was to determine the prevalence of cfxA and cfxA2 in Prevotella strains by use of real-time PCR and to assess the performance of this molecular method for the direct detection of the genes in 87 clinical samples (pus and root canal exudates) from dentoalveolar infection. Production of beta-lactamase by each isolate was determined using a nitrocefin disk. beta-Lactamase production was seen in 31% of Prevotella isolates, while all isolates of other species were beta-lactamase negative. The penicillin resistance of isolates strongly correlated with the production of beta-lactamase. Real-time PCR was found to detect the cfxA and cfxA2 genes from at least five cells per reaction mixture (5 x 10(3) CFU/ml of pus). Using real-time PCR, the presence of cfxA and cfxA2 was evident for all 48 beta-lactamase-positive Prevotella strains. In contrast, neither beta-lactamase-negative Prevotella (n = 91) or non-Prevotella (n = 31) strains were positive for the genes. In this study, 31 of the 87 samples yielded beta-lactamase-positive Prevotella results, and cfxA and cfxA2 were detected in all 31 samples. Of the 56 culture-negative samples, 8 (14%) were positive for cfxA and cfxA2 by the real-time PCR. This sensitive and specific molecular method offers a rapid clinical test for aiding in the selection of an appropriate antibiotic for treatment of dentoalveolar infection. Although penicillin remains largely effective in the treatment of dentoalveolar infection, beta-lactamase-stable antibiotics should be considered in cases in which beta-lactamase-positive Prevotella strains are involved.     

Phenotypes of beta-lactam resistance in the genus Aeromonas

This work aimed to investigate resistance profiles towards beta-lactam antibiotics in correlation with beta-lactamases production in the genus Aeromonas. In a series of 417 wild-type strains, biochemical identification and testing with 11 beta-lactams by the disk-diffusion method revealed 5 predominant phenotypes: A. hydrophila complex/class B, C and D beta-lactamases; A. caviae complex/class C and D beta-lactamases; A. veronii complex/class B and D beta-lactamases; A. schubertii spp./class D beta-lactamase; A. trota spp./class C beta-lactamase. A subgroup of 64 representative strains was submitted to MIC determination with 8 beta-lactam compounds alone and in combination with 3 beta-lactamase inhibitors (clavulanic acid, tazobactam and BRL 42715). Visualisation of beta-lactamases and pI determination were performed in all these 64 isolates by isoelectric focusing from crude extracts. The different Aeromonas species produced 1 to 3 of the following inducible enzymes: an imipenemase with low expression, which is difficult to detect with routine phenotype studies (class B, pI 8, imipenem MIC > 2 micrograms/ml), a cephalosporinase (class C, pI > 7 +/- 0.5, cephalothin MIC > 256 micrograms/ml), and an oxacillinase widely produced in the genus Aeromonas (class D, pI > 8.5, ticarcillin MIC > 256 micrograms/ml). In Aeromonas spp. resistance profile to beta-lactam antibiotics is correlated with naturally occurring phenotypes of beta-lactamases production. As a conclusion, the characterisation of these different enzymes is of therapeutic and taxonomic interest, in species notoriously difficult to identify.     

Urease activity of Proteus penneri.

Ten strains of Proteus penneri isolated from geographically diverse laboratories were tested for urease activity. Cell lysates from urea-induced cells had a mean activity of 4.9 +/- 4.1 mumol of NH3 per min per mg of protein. On nondenaturing 6% polyacrylamide activity gels, the enzymes of P. penneri had very similar electrophoretic mobilities within species and within the Proteus genus but were distinct from the ureases of Providencia and Morganella species. On lower-percentage polyacrylamide, differences in mobilities of the ureases could be detected between the Proteus species. From representative strains, the P. penneri urease was found to be inducible by growth in urea and had an apparent molecular weight of 246,000 +/- 9,000, an isoelectric point of 5.1, and a Km for urea of 14 mM and was inhibitable by acetohydroxamic acid, hydroxyurea, and EDTA. In an in vitro model of struvite formation, a P. penneri strain produced abundant crystals on a glass rod submerged in synthetic urine in the absence but not presence of acetohydroxamic acid (500 micrograms/ml).     

In vitro activity of cefuroxime against Moraxella (Branhamella) catarrhalis

Minimal inhibitory concentrations of cefuroxime were determined by an agar dilution procedure and compared with erythromycin and four other beta-lactam antibiotics (amoxycillin, amoxycillin + clavulanate, cefadroxil, cefaclor) on 76 strains of Moraxella (Branhamella) catarrhalis. Sixty four of them produced a beta-lactamase. Results show that the beta-lactamase of Moraxella (Branhamella) catarrhalis abrogates the activity of amoxycillin (MIC 90% = 4 mg/l) meanwhile the combination of amoxycillin-clavulanate is inhibitory at low concentration (MIC 90% = 0.25 mg/l). There are no difference in MICs between strains producing or not producing beta-lactamase with erythromycin (MIC 90% = 0.25 mg/l). All the strains evaluated in this investigation, producing or not producing beta-lactamase have MIC 90% less than or equal to 2 mg/l for cefuroxime lower than those obtained for the two other cephalosporins.     

Activity of cefuroxime against bacterial strains isolated from acute otitis media

The acute otitis media is a frequent infantile disease and, in 80% of cases, a bacterial strain can be isolated from the otorrhoea. Haemophilus influenzae and Streptococcus pneumoniae are the two major species isolated from auricular exudate, and represent two thirds of all isolated strains, with the others comprising Staphylococcus aureus, Branhamella catarrhalis, Pseudomonas aeruginosa, Enterobacteriaceae and corynebacteria. The treatment of this disease is based principally on beta-lactams (aminopenicillins, cephalosporins) administered by the oral route. Cefuroxime is a cephalosporin which is absorbed via the digestive tract in the form of cefuroxim-axetil. The activity of this compound was studied against 210 strains isolated from otorrhoea, collected from children who presented an acute otitis media during the first half of 1989. These strains were: 112 strains of H. influenzae, of which 23 produced a beta-lactamase; 21 strains of Streptococcus pneumoniae; 3 strains of Streptococcus pyogenes; 10 strains of Branhamella catarrhalis of which 9 produced a beta-lactamase; 18 strains of S. aureus; 14 strains of Enterobacteriaceae, and 32 strains of corynebacteria. The minimal inhibitory concentration (MIC) of cefuroxime-axetil was measured by dilution in agar. The MICs of cefuroxime against H. influenzae were low and similar (MIC 50 = 1 mg/l; MIC 90 = 1 mg/l) regardless of whether the strain secreted a beta-lactamase. Overall, 90% and 98% of the 210 strains tested here were inhibited by 1 and 4 mg/l of cefuroxime respectively. These results show that the antibacterial spectrum of cefuroxime-axetil appears to be ideally suited to the bacterial strains isolated from acute otitis media.     

Comparative antibacterial activity of cefotaxime, ceftazidime and cefepime with regard to different strains of Enterobacter aerogenes selected for their resistance to third generation cephalosporins

After being confronted with the isolation in our laboratory of numerous antibiotic-multiresistant Enterobacter aerogenes strains, we studied the in vitro antimicrobial activity of cefotaxime, ceftazidime, and cefepime alone or in association with sulbactam. For that, we selected 67 isolates according to their low level of susceptibility to cefotaxime. First, we deduced from a synergy test in presence of clavulanic acid and cloxacillin the production of an extended spectrum beta-lactamase (ESBL) and/or an overproduction of a chromosomal cephalosporinase. Three groups of strains were thus defined: one group of ESBL strains, another group of overproducing strains of chromosomal cephalosporinase, and a last group that produced the two types of enzymes. Minimal inhibitory concentrations (MICs) of each cephalosporin alone or in presence of 8 mg/L of sulbactam, gentamicin or amikacin were measured. Our results demonstrated the best activity of cefepime: MICs were low with a value inferior to 4 mg/L independently of the type of beta-lactamase. They were lower than 0.5 mg/L in presence of sulbactam against ESBL-producing strains. The cephalosporins could be used in association with aminoglycosides according to their susceptibility.     

In vitro evaluation of pyridine-2-azo-p-dimethylaniline cephalosporin, a new diagnostic chromogenic reagent, and comparison with nitrocefin, cephacetrile, and other beta-lactam compounds.

Pyridine-2-azo-p-dimethylanaline cephalosporin (PADAC), a chromogenic reagent which is purple and changes to yellow upon cleavage of its beta-lactam ring, was evaluated in comparison with other chromogenic cephalosporins. PADAC exhibited little antimicrobial activity against gram-negative bacteria, but did have good activity (minimum inhibitory concentration, 0.12 to 0.5 microgram/ml) against Staphylococcus aureus, a quality comparable to nitrocefin. Nitrocefin, however, demonstrated an unexpected and uniquely potent activity against Streptococcus faecalis (minimum inhibitory concentration, less than or equal to 0.06 to 0.12 microgram/ml) The relative hydrolysis rate of PADAC when subjected to six different beta-lactamases was substantially greater than that of cephacetrile, but less than that of nitrocefin. The relative hydrolysis rates of PADAC and nitrocefin were comparable with type IIIa beta lactamase and the derived from Bacillus cereus. The inhibition of beta-lactamase hydrolysis of the chromogenic cephalosporin substrates by six enzyme-stable inhibitors was generally greater with PADAC than with nitrocefin. Unlike nitrocefin, PADAC mixed with 50% human serum or various broth culture media showed no evidence of color change or degradation over several hours. The subsequent enzyme hydrolysis rates of such mixtures were the same as in phosphate buffer. Beta-lactamase-containing bacterial suspensions and clinical specimens containing such bacteria produced positive visual and spectrophotometric color changes when mixed with PADAC or nitrocefin. Although color changes occurred more slowly with PADAC than with nitrocefin, PADAC was not adversely influenced (non-enzyme-related color change) by the protein content of specimens. PADAC appears to be a promising alternative for beta-lactamase diagnostic testing in the clinical and research microbiology laboratory.     

Penicillin-binding proteins, porins and outer-membrane permeability of carbenicillin-resistant and -susceptible strains of Pseudomonas aeruginosa

Reduced cell permeability and target penicillin-binding protein modification were investigated as mechanisms of intrinsic resistance in strains of Pseudomonas aeruginosa resistant to carbenicillin (MIC greater than 128 mg/L) independently of beta-lactamase production. The carbenicillin-resistant strains were also remarkably resistant to other beta-lactams, quinolones, tetracyline and chloramphenicol, whereas carbenicillin-hypersusceptible strains (MIC less than 2 mg/L) were very sensitive to these antimicrobial compounds. These observations suggested a non-specific mechanism of resistance involving reduced permeability of the outer layers of the bacterial cell. However, carbenicillin-resistant and carbenicillin-sensitive strains had identical porin levels and the target penicillin-binding proteins of carbenicillin-resistant (MIC 256-2048 mg/L), carbenicillin-sensitive (MIC 64 mg/L) and carbenicillin-hypersusceptible (MIC 0.015 mg/L) strains were equally sensitive to beta-lactams. Thus, subtle changes in porin function or additional outer-membrane barriers regulating permeability may be involved in intrinsic resistance.     

In vitro antibacterial activity of enoxacin (CI-919)

Antibacterial activity of enoxacin was evaluated against more than 3,700 clinical isolates using the agar-dilution method and an inoculum of 10(4)-10(5) cells per site. For comparison other antibiotics appropriate for each species were also included. For most enterobacteria and for Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa, the MIC90 of enoxacin was below 2 mg/l. Serratia marcescens was more resistant; the MIC90 being 4 mg/ml. Enoxacin also showed high activity against Campylobacter jejuni and Neisseria gonorrhoeae. Streptococci were comparatively resistant, 32 mg/l to 64 mg/l of the compound being required to inhibit 90% of strains.     

Antibiotic susceptibility and beta-lactamase production in clinical isolates of Enterobacter spp

The in vitro susceptibility of 237 clinical isolates of Enterobacter spp. (E. aerogenes, E. agglomerans and E. cloacae; 41, 64 and 132 respectively) to 16 different antibiotics is described. Four quinolones (ciprofloxacin, lomefloxacin, norfloxacin and ofloxacin), two new cephalosporins (cefpirome and cefepime) and imipenem, all showed high activity against the three Enterobacter species tested (MIC50 less than or equal to 0.125 mg/l, MIC90 less than or equal to 0.5 mg/l). Also the aminoglycosides gentamicin and tobramycin were highly active antibiotics (MIC50 less than or equal to 0.5 mg/l, MIC90 less than or equal to 1.0 mg/l). The susceptibility of beta-lactam-antibiotics to beta-lactamase produced by Enterobacter spp. was evaluated, and imipenem and cefepime were found to be most stable. Different methods for detection of inducible beta-lactamases were used, the agar dilution method being more sensitive than the double-disc diffusion test. Elevated beta-lactamase production was detected, via induction, in 83% of E. aerogenes strains and 70% of E. cloacae strains, with cefamandole used as the substrate and cefoxitin as the inducer. Constitutive, high level enzyme production was detected in 7 and 13% respectively of the E. aerogenes and the E. cloacae strains. In all the strains of E. agglomerans, 10% of E. aerogenes and 13% of E. cloacae, no beta-lactamases could be detected with the methods studied.     

Cefoxitin sensitivity as a marker for inducible beta-lactamases

Inducibility of beta-lactamase activity by cefoxitin was examined in 626 gram-negative clinical isolates selected for amoxycillin and cephalothin resistance. The results indicated that precise identification and cefoxitin sensitivity or resistance could be used to predict the inducibility of beta-lactamase. Of 326 organisms from species capable of beta-lactamase induction, induction was shown in 68% and was predictable from the cefoxitin-sensitivity and identification data. No induction of beta-lactamase occurred in the remaining species. A comparison of beta-lactamase activities against cefotaxime, cefoperazone and latamoxef showed that induction of enzyme activity against cefotaxime and cefoperazone occurred at similar rates. Induction of activity against latamoxef did not occur or was minimal with three bacterial species. The data show that of 119 strains of Enterobacteriaceae displaying inducible beta-lactamase, 113 would have been reported as unequivocally sensitive to cefotaxime, 109 as sensitive to cefoperazone and 116 as sensitive to latamoxef if the disk-diffusion technique alone had been used. The majority of Pseudomonas strains examined produced inducible enzyme and they were more resistant to the three cephalosporins tested than were the Enterobacteriaceae.