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Objectives: To develop an easy, rapid and reproducible spot-inoculation method for phenotypic detection of extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases and to make the existing three-dimensional extract test more convenient for use in routine diagnostic laboratories., Methods: ESBL and AmpC producing and non-producing isolates of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, as identified by the conventional three-dimensional extract test, were used to evaluate the modified procedures. Whole bacterial cells and freeze-thaw preparations, as beta-lactamase sources, were strategically applied to culture plates near ceftazidime and cefoxitin discs on a lawn inoculum of E. coli ATCC 25922. Technical variations of the test included placing the beta-lactamase-containing inoculum into slits, wells and trenches, or onto the surface as spots at varying distances from the discs, and adding clavulanate or cloxacillin to the three-dimensional inoculum to confirm the presence of ESBLs and AmpC beta-lactamases, respectively., Results: All the methods adopted for ESBL and AmpC detection by using the whole bacterial cells gave positive results. However, the best results were given by the spot-inoculation method. In modifications using the enzymic extracts, the enhanced growth of surface organisms was better appreciated in the designed modifications compared with the conventional methods., Conclusions: The method described here is simple and cost-effective. Furthermore, up to 16 isolates may be tested on a single culture plate, thus it is a less labour-intensive and more economic technique than other reported phenotypic methods.

Objectives: Isolates previously identified as Citrobacter diversus are now known as Citrobacter koseri. We measured sequence variation at the beta-lactamase structural gene among a group of clinical isolates originally identified as C. diversus by API 20E profiling., Methods: beta-Lactamase and 16S rRNA genes were amplified by PCR and sequenced by standard methods. beta-Lactamase induction was attempted in liquid-grown cultures using cefoxitin. Nitrocefin hydrolysis assays were performed using a spectrophotometer., Results: Analysis of 16S rRNA gene sequences showed that Citrobacter spp. isolates with an inducible beta-lactamase gene, cdiA, closely related to 'C. koseri ' NF85 and ULA27 are actually Citrobacter amalonaticus. C. koseri isolates, whose identities were confirmed by 16S rRNA sequencing, produce a class A beta-lactamase, Cko, constitutively at low levels. The cko and cdiA beta-lactamase genes share <45% identity., Conclusions: We have confirmed that cko is a beta-lactamase gene carried by C. koseri, and that isolates previously identified as 'C. koseri ', but carrying the cdiA beta-lactamase gene are C. amalonaticus. Thus, beta-lactamase-gene-specific PCR may provide a valuable tool to differentiate these biochemically homogeneous Citrobacter species.

AmpC enzymes can be differentiated from other extended-spectrum beta-lactamases by their ability to hydrolyse cephamycins as well as other extended-spectrum cephalosporins. The present study was designed to determine the occurrence of AmpC enzyme-harbouring Gram-negative clinical isolates in a tertiary care hospital in Delhi, India. Among the 135 clinical isolates of Gram-negative bacilli tested, 20.7% were found to harbour AmpC enzymes using a modified three-dimensional test. Inhibition of zone distortion in the presence of cloxacillin confirmed AmpC-harbouring isolates. Maximal incidence of AmpC producers was found among Acinetobacter spp. (42.8%) followed by Klebsiella pneumoniae isolates (33.3%). No AmpC-harbouring isolates revealed decreased susceptibility to cefoxitin. Therefore, Gram-negative bacilli showing resistance to any cephalosporin or aztreonam irrespective of cefoxitin susceptibility should be screened for the AmpC enzyme. The modified three-dimensional test is easy to carry out and can be applied as a phenotypic screening method for detection of AmpC-harbouring Gram-negative organisms. This is the first study to determine the occurrence of AmpC beta-lactamases from India.

Plasma concentration of cefminox and serum bactericidal activity against four ATCC strains (Escherichia coli 25992, Klebsiella pneumoniae 13833, Serratia marcescens 8100 and Bacteroides fragilis 25285), were determined over a 24 h period after administration of cefminox 1 and 2 g to six healthy volunteers in a randomized, cross-over, single blind study. The increase observed in the area under the bactericidal curve (AUBC) with the 2 g dose was at least 3.5 times that seen with the 1 g dose for all four test strains and was larger than predicted by the corresponding increase (1.84 times) in the area under the serum concentration versus time curve (AUC); a correlation (r = 0.88, P = 0.0001) between the cefminox concentration and the serum bactericidal titres was, however, observed with all four strains tested. The MBC6h showed a better association with the serum bactericidal titre (P < 0.01) than did the MIC or MBC.

The purpose of this study was to assess the effect of different MIC statistics on the in-vitro evaluation of cefoxitin, cefotetan, ceftizoxime, cefotaxime, desacetylcefotaxime, and cefotaxime:desacetylcefotaxime (1:1 ratio) against clinical isolates of Bacteroides fragilis from the Medical University of South Carolina. MICs were determined by the agar dilution method following NCCLS guidelines. Statistical analyses included arithmetic and geometric means, median, mode, MIC50, MIC90, and range. Differences between antimicrobial agents were determined using parametric and nonparametric methods. Consistent with previous in-vitro studies on anaerobes with these agents, a wide range in the MICs was observed. The arithmetic mean MIC was lowest for cefotetan, but the geometric mean MIC was lowest for ceftizoxime. Using the MIC90, cefotetan was at least a two-fold dilution more active than the other agents. After natural logarithmic transformation of the MIC data, analysis of variance with the Scheffé post-hoc test demonstrated that the MICs of ceftizoxime were significantly lower than those of cefoxitin (P < 0.001), cefotetan (P < 0.05), cefotaxime (P < 0.05), and desacetylcefotaxime (P < 0.001). Median and mode MICs were lowest for ceftizoxime and cefotaxime:desacetylcefotaxime. Using published breakpoints, cumulative percent susceptibility was similar for all agents studied. In this analysis of the antimicrobial susceptibility of B. fragilis in our institution, the in-vitro activity of the cephalosporins and cephamycins tested appeared to be very similar when all statistical data were evaluated. Since apparent in-vitro activity may be influenced by the statistic evaluated, we suggest that all MIC data be reported to allow for a more complete analysis of microbiological potency.

Initially, cefotetan interacts with beta-lactamases to form a classical Michaelis complex characterized by a term Ki (the inhibition constant). In a second phase, this complex develops with time to form a new entity devoid of enzymic activity. This new entity may have the structure of an acyl enzyme with variable stability. In the case of TEM-1 and TEM-2 beta-lactamases, the process is progressively and totally reversible. The same phenomena were observed in varying degrees for Pitton's type 2 (PIT-2) penicillinase, OXA-1 and CARB-1. The Klebsiella oxytoca and Proteus vulgaris beta-lactamases are also inactivated by this same process. However, four other cephalosporinases appear to be unaffected by this mechanism.

Cefotetan, a novel 7 alpha-methoxy cephalosporin, was shown to be comparable to other third-generation cephalosporins when tested in vitro against 1063 Gram-negative clinical isolates of 11 species. Ninety per cent of all isolates of Escherichia coli, Klebsiella, Proteus mirabilis, Proteus vulgaris, Proteus rettgeri, Providencia stuartii, Serratia marcescens and Citrobacter freundii were inhibited by concentrations of cefotetan between 0.07 and 3.2 mg/l. Activity against Gram-positive cocci was about equal to that of moxalactam. Pseudomonas aeruginosa and Acinetobacter were resistant. Cefotetan inhibited about two-thirds of Enterobacter strains at 16 mg/l. beta-Lactamases of Enterobacter cloacae highly resistant to cefotetan inactivated this cephamycin. These strains were resistant to most other beta-lactams as well. Synergism between cefotetan and aminoglycosides was found in 8 out of 16 Gram-negatives.

The in-vitro activity of cefotetan, a recently developed cephamycin, was investigated under various experimental conditions. The compound showed moderate activity against Staphylococcus aureus and Streptococcus pyogenes, no activity against Pseudomonas aeruginosa and Streptococcus faecalis, but a high activity against Enterobacteriaceae, including beta-lactamase-producing strains. Haemophilus influenzae also was fairly susceptible. The MBC was usually equal to or two- or fourfold higher than MIC. Medium composition, pH and inoculum size had minimal influence on its activity. About 50% of recent clinical isolates of Bacteroides fragilis were susceptible to cefotetan, but some were highly resistant. Killing curves of cefotetan against different bacterial strains indicated that it was rapidly bactericidal at concentrations equal to MIC or two- to fourfold higher. However, some strains showed regrowth after initial inhibition. Combination of cefotetan with aminoglycosides, or with cefazolin, cefotaxime, moxalactam or piperacillin resulted either in synergy, addition or indifference according to the bacterial strain and the nature of the combination. Antagonism was never observed. Human serum protein binding varied from 75 to 86% according to the assay method. Binding with horse serum protein was about 28%.

The in-vitro antibacterial activity of cefotetan was assessed against recent clinical isolates of common bacteria and also against reference strains that produced known beta-lactamases. The compound was active against most staphylococci (MICs 4 to 8 mg/l) though methicillin-resistant strains were less sensitive. It was also generally active against streptococci (MICs mostly in the range 1 to 16 mg/l). However, enterococci and penicillin-resistant pneumococci were resistant. Cefotetan was highly active against enterobacteria, with 75% of isolates inhibited by 0.5 mg/l and 90% inhibited by 4 mg/l. It was also highly active against Haemophilus influenzae (MICs 0.5 to 4 mg/l) and Neisseria gonorrhoeae (MICs 0.06 to 2 mg/l) but had relatively poor activity against Acinetobacter spp. (MICs mostly 4 to 128 mg/l). Cefotetan had little useful activity against Pseudomonas aeruginosa (MICs 16 to 512 mg/l) but was more active against most other pseudomonads. Cefotetan had moderate activity against the Bacteroides fragilis group (MICs mostly less than or equal to 32 mg/l), though Bact. fragilis sensu stricto and Bact. vulgatus were more sensitive (MICs usually less than or equal to 4 mg/l). It was more active against most other anaerobes, though its activity was always exceeded by that of ampicillin. Cefotetan possessed a high degree of resistance to both plasmid-mediated and chromosomally-determined beta-lactamases.

MT-141, a new cephamycin (7 alpha-methoxy-cephalosporin) antibiotic with a D-cysteine moiety in its 7 beta-side chain, has binding affinities to penicillin-binding proteins of Escherichia coli and an inhibitory action on their transpeptidase activity similar to those of other structurally related cephamycins. Yet this antibiotic was found to exert an exceedingly strong and rapid lytic action on sensitive Gram-negative bacteria such as E. coli, Klebsiella pneumoniae, Serratia marcescens and Salmonella enteritidis. Not only rapidly growing cells, but also slowly growing dense cells of the above bacteria could be lysed by this antibiotic at low concentrations. In the presence of 20% sucrose, low concentrations of MT-141 induced smooth-surfaced single and twin bulges of the putative growth zone of the cells and irregularly orientated rough-surfaced bulges. Probably the 7 beta-side chain structure of this antibiotic is involved in its rapid and strong bacteriolytic action.

The activity of cefotetan was tested against 683 bacterial strains by measuring the minimum inhibitory concentrations. Comparisons were made with cefoxitin, cefotaxime, moxalactam, cefoperazone and piperacillin. In addition, a regression line analysis was made with cefotetan discs (30 micrograms) using the ICS technique. Cefotetan was noted to have a broader spectrum of activity than cefoxitin and its antibacterial activity was comparable to that of cefotaxime and moxalactam. According to the regression line analysis a cefotetan inhibition zone diameter of greater than or equal 19 mm is interpreted as sensitive in agar diffusion tests.

In recent years there has been a gradual increase in the number of infections caused by Klebsiella, Enterobacter and Serratia and non-fermentative bacilli such as Pseudomonas, Alcaligenes and Acinetobacter. The object of this investigation was to evaluate the antibacterial properties of the cephalosporins cefoxitin, cefoperazone, cefotaxime and of gentamicin, in comparison with cefotetan, a new cephamycin with a high degree of stability to beta-lactamases. The cefotetan MIC was not affected either by size of the bacterial inoculum, or the culture medium and the serum protein binding of cefotetan was 85%. Cefotetan showed only moderate antibacterial activity, similar to that of the other cephalosporins tested, against the Gram-positive cocci, whereas it was very active against fermentative and non-fermentative Gram-negative bacilli, except for Pseudomonas aeruginosa. The remarkable activity of cefotetan against Klebsiella, Enterobacter and Serratia is of particular interest.

Minimum inhibitory concentrations (MICs) of ampicillin, cefoxitin, cefbuperazone, latamoxef, metronidazole, clindamycin and chloramphenicol were determined for 15 different anaerobic bacteria including Bacteroides spp., anaerobic cocci and Clostridium spp., in 18 European laboratories, who used their own methodology. The degree of intra- and inter-laboratory reproducibility was surprisingly good--87% of results fell on the modal MIC or were within one dilution of it and only 4.4% of the results differed by three or more dilutions. Results for clindamycin were the least reproducible, as were those for clostridia. Of the organisms that we tested Bacteroides fragilis ATCC 25285, NCTC 9343 emerged as the most suitable for use in quality control, and Peptococcus variabilis ATCC 14956 the most appropriate if a control for more slowly-growing species is required.

The activity in vitro of the new cephamycin, cefotetan, was compared with that of cephalothin, cefoxitin, cefuroxime and cefotaxime against 226 recently isolated Gram-negative pathogens (207 Enterobacteriaceae and 19 Acinetobacter). Cefotetan was consistently more potent than cephalothin, cefoxitin and cefuroxime. Cefotaxime and cefotetan were essentially similar in activity against the Enterobacteriaceae with the singular exception of Enterobacter spp. which were markedly more susceptible to the former drug. None of the five agents was especially active against acinetobacter but cefotaxime was the best. Limited tests of bactericidal activity showed that the MBC differed from the MIC by a factor of 4 at most. Increasing the inoculum tested from 10(2) to 10(8) per ml had little adverse effect on the MIC of cefotetan. If the recommendations of the National Committee for Clinical Laboratory Standards for susceptibility to cephalosporins are applicable to cefotetan and the breakpoint criteria of less than or equal to 10 mg/l (sensitive) and greater than or equal to 32 mg/l (resistant) are acceptable, then only a few of the Enterobacteriaceae in this study were resistant. These resistant organisms were amongst Enterobacter spp. and Citrobacter spp.

Membrane preparations were made from a number of Gram-positive and Gram-negative bacterial strains. The ability of the membrane-bound penicillin-binding proteins (PBP) in these preparations to bind [14C]cefotetan has been studied. Increasing amounts of unlabelled benzylpenicillin were added and subsequently [14C]cefotetan was introduced to determine its ability to bind to additional proteins or to displace the pre-bound benzylpenicillin. In the Gram-positive series some quantitative binding differences between the two labelled compounds could be seen. However, in the majority of Gram-negative organisms tested cefotetan exhibited greatest affinity for PBP 3 and none for PBP 2. No new PBPs were detected using [14C]cefotetan, suggesting that the compound exerts its antibacterial effect by a mechanism similar to the other cephamycins. The high minimal inhibitory concentrations (MICs) of cefotetan found for the Gram-positive genera tested are reflected in only one organism by the failure to bind of some of its PBPs. In the majority of cases, permeability is assumed to play the dominant role in the relatively low sensitivity of these organisms to cefotetan.

The activity of cefotetan has been determined against strains of bacteroides, Haemophilus influenzae and klebsiella. The activity against bacteroides was lower than that of cefoxitin and moxalactam. The activity against multi-resistant klebsiella was very high, most strains requiring a concentration of 0.06 mg/l for inhibition. The stability to various beta-lactamases was demonstrated by testing the compound against beta-lactamase-producing H. influenzae and Enterobacteriaceae.

The cephamycin, cefotetan, was compared with other beta-lactam antibiotics including the monocyclic beta-lactam, azthreonam, against a total of 277 recent clinical isolates. Cefotetan had activity comparable to moxalactam against Staphylococcus aureus and against the Enterobacteriaceae, inhibiting all isolates except for Serratia marcescens and Enterobacter spp. at less than or equal to 0.5 mg/l. Cefotetan was active against beta-lactamase producing and non-beta-lactamase producing Haemophilus influenzae (MIC90 1 mg/l); was inactive against Pseudomonas aeruginosa (MIC90 greater than 128 mg/l); and was eightfold less active than cefoxitin against the Bacteroides fragilis group. Azthreonam was as active as moxalactam against the Enterobacteriaceae and was more active than moxalactam against Ps. aeruginosa, inhibiting 90% of isolates at less than or equal to 8 mg/l, but was inactive against the Bact. fragilis group (MIC90 greater than 128 mg/l) and against Staphylococcus aureus (MIC90 greater than 128 mg/l).

The antimicrobial activity of cefotetan and cefotaxime were compared in vitro against 547 strains of aerobes and 24 strains of Bacteroides fragilis. The strains were recent clinical isolates from 20 hospitals. Cefotetan was found to exhibit an activity two- to fourfold lower than that of cefotaxime against most of the enterobacteriaceae. About half the number of Enterobacter aerogenes and Enterobacter cloacae strains resistant to cefotetan were resistant also to cefotaxime. Cefotetan was more active than cefotaxime against most of the strains of Proteus vulgaris. In contrast to cefotaxime which was moderately active, cefotetan was inactive against Pseudomonas aeruginosa. Cefotetan had limited activity against Staphylococcus aureus and Streptococcus faecalis was resistant to both cefotetan and cefotaxime. Bact. fragilis was inhibited more effectively by cefotetan than by cefotaxime. Depending on the species tested, the activity of cefotetan was influenced significantly by the inoculum size in some cases. Different test media did not affect the results.

Staphylococcus aureus strains specifically resistant to cephamycin antibiotics have been found among recent clinical isolates. These strains formed penicillin-binding protein (PBP) 2' and became phenotypically resistant to methicillin after induction with cefoxitin. Other cephamycin-type antibiotics also induced methicillin-resistance, whereas non-cephamycin-type cephalosporins such as cefmenoxime and ceftizoxime did not do so. The clones that constitutively synthesized PBP 2' arose from the cephamycin-resistant strains at a frequency of 10(-5). They were indistinguishable from clinically isolated methicillin-resistant S. aureus (MRSA). Cephamycin-resistant S. aureus may be a source for emerging MRSA.

MT-141 inhibited 90% of the clinical isolates of Bacteroides fragilis at 3.13 mg/l, whereas six reference cephem antibiotics inhibited them at 12.5- greater than 100 mg/l. MT-141 was stable to the Bacteroides beta-lactamases, but the stability alone could not explain the potent activity of MT-141 against the other Bacteroides spp., which produced only small amounts of beta-lactamase, but were resistant to other cephem antibiotics. MT-141 exhibited a higher killing rate and more potent in-vivo efficacy than the reference cephem antibiotics against Bact. fragilis No. 36 combined with Escherichia coli No. 29. In conclusion, the in-vitro and in-vivo anti-bacteroides activities of MT-141 ranked first among those of the cephem antibiotics compared.

The activity of the 7 alpha-methoxycephalosporin, cefotetan was determined against 365 infecting isolates of gentamicin- and multiply-resistant Gram-negative aerobic bacilli, and compared with those of cefuroxime, cefoxitin, cephradine, cefotaxime, ceftriaxone and ceftazidime. All proteus (4), providencia (6) salmonella (3) and serratia (2) were susceptible to 8 mg/l of cefotetan, as were respectively 90 and 81% of 42 Escherichia coli and 16 citrobacter isolates. The intrinsic activity of cefotetan was high against 237 klebsiellae (38 different serotypes), only 4 being relatively insusceptible (MICs 16- greater than 32 mg/l). Activity against pseudomonas (10 isolates) acinetobacter (15 isolates) and enterobacter (30 isolates) was poor. Overall, the activities of cefotetan were similar to those of cefotaxime, ceftriaxone and ceftazidime but ceftazidime was also active against the majority of pseudomonas, acinetobacter and enterobacter. Cefotaxime was less active than cefotetan against some cefuroxime-resistant klebsiellae. Much greater numbers of isolates were insusceptible to either cefuroxime or cefoxitin. Cephradine was the least active.

In-vitro activity of cefotetan was compared to that of other cephalosporins netilmicin. Cefotetan at very low concentrations inhibited Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Serratia marcescens. Poor activity was seen for all beta-lactams against Enterobacter and Citrobacter spp. Activity of cefotetan against Staphylococcus aureus was comparable to that of ceftazidime and moxalactam. Netilmicin was the only agent equally active against all Gram-negative bacilli and Staph. aureus tested. Under conditions simulating serum levels cefotetan demonstrated rapid and long-acting bactericidal activity and bacteria exposed to these concentrations required up to 10.2 h to recover from the effect of the antibiotic. Six out of 15 strains did not recover fully.

The activity of moxalactam, a new beta-lactam antibiotic with a novel molecular structure, was examined against dense populations of a variety of Gram-negative bacilli. The lytic activity of moxalactam against ampicillin-sensitive strains was similar to ampicillin, but the activity was maintained against ampicillin-resistant strains, including Pseudomonas aeruginosa, irrespective of whether the ampicillin resistance was 'intrinsic' (non-enzymic) or due to beta-lactamase. Of the 18 strains tested only one, a Hafnia sp., failed to respond to moxalactam.