The three-dimensional and clavulanate double-disk potentiation tests were compared as procedures for the detection of extended-spectrum 13-lactamase production in 32 strains of Escherichia coli and Klebsiella pneumoniae, 31 of which produced TEM-1, TEM-2, TEM-3, TEM-4, TEM-5, TEM-7, TEM-8, TEM-9, TEM-10, TEM-12, TEM-101, SHV-1, SHV-2, SHV-3, SHV-4, SHV-5, CAZ-2, MIR-1, or an unidentified extended-spectrum 13-lactamase with a pl of 5.95, with some strains producing multiple 13-lactamases. The three-dimensional test, which was performed in conjunction with a routine disk diffusion test, detected extended-spectrum 13-lactamase production in 26 of 28 (93%) of the strains that produced extended-spectrum 13-lactamases. The clavulanate double-disk potentiation test detected extended-spectrum 13-lactamases in only 22 of the 28 strains (79%o) when it was performed as currently recommended. The three-dimensional test, when performed in conjunction with the disk diffusion test, offered the advantages of providing simultaneous information about both antibiotic susceptibility and extended-spectrum 13-lactamase production, coupled with a greater sensitivity and earlier detection of extended-spectrum 13-lactamases.

Microbial drug resistance is an inescapable consequence of the utilization of antimicrobial agents in a given environment. Nowhere is the importance of resistance more evident than among agents of the beta-lactam family. Trends toward increased resistance can be seen among fastidious gram-negative bacteria like Haemophilus influenzae, where ampicillin resistance varies from 1% to 64% globally. For Escherichia coli, ampicillin resistance has risen to > or = 50% in high-risk populations, and resistance to third-generation cephalosporins is now being seen in certain areas. Inducible beta-lactamases have been responsible for increasing multiple beta-lactam resistance among certain Enterobacteriaceae and Pseudomonas aeruginosa, and this has been associated with increased use of newer cephalosporins. Xanthomonas maltophilia with its two inducible beta-lactamases is becoming an increasingly important nosocomial pathogen, especially in areas of heavy imipenem utilization. Only through the recognition of factors associated with increasing resistance and the mechanisms responsible can strategies be designed for minimizing beta-lactam resistance.

The ability of three quinolones, two I-lactams, and one aminoglycoside to select resistant mutants was examined in tests with 30 isolates of commonly encountered nosocomial pathogens. Ciprofloxacin and norfloxacin, two new quinolone derivatives, were no more likely to select resistant mutants than amikacin, whereas nalidixic acid, an older quinolone derivative, was the most likely of the six drugs examined to select resistant mutants. Mutational frequencies of 10-7 to 10-8 were observed in most instances. In general, the mutants were 8 to 16 times less susceptible to the drug used for selection. Although most quinolone-selected mutants were cross-resistant only to other drugs within this class, certain mutants of Klebsiella pneumoniae selected by nalidixic acid, ciprofloxacin, or norfloxacin were also less susceptible to 13-lactam antibiotics. This unusual pattern of multiple drug resistance was associated with changes in outer membrane proteins of the organism. Multiple drug resistance was also observed in P-lactam-selected mutants of Enterobacter cloacae and Pseudomonas aeruginosa (,-lactams), amikacin-selected mutants of Providencia stuartii and P. aeruginosa (aminoglycosides), and I-lactam-or amikacin-selected mutants of Serratia marcescens (B-lactams plus aminoglycosides). These results underscore the need to examine carefully the frequency with which resistance to any new antibiotic develops, as well as the patterns of multiple drug resistance which may occur simultaneously.The development of resistance can significantly shorten the useful lifetime of antimicrobial agents in prophylaxis and therapy of infectious diseases and has been a major problem with nalidixic acid, a quinolone derivative, and possibly with the new cephalosporins (23, 26). Unfortunately, potential problems with the development of resistance are often not thoroughly analyzed for most antibiotics before the time the drugs become available for clinical use. The occurrence of cross-resistance to related or unrelated antibiotics is also seldom studied in depth.The recent development of new quinolone derivatives, such as norfloxacin and ciprofloxacin, has stimulated interest in these problems of resistance. New members of this class of compounds must be analyzed closely to determine whether resistance will develop as frequently as it has with nalidixic acid. Several studies have already examined this aspect (4,5,8,13,15,17,29 MIC (usually 4,8,and 16 times the MIC). The actual inoculum utilized was determined by agar dilution plate counts. After overnight incubation at 35°C in air, the apparent mutational frequencies were calculated from results obtained with the highest drug concentration on which colonies were detected. Cells selected by this procedure were considered to be resistant mutants if the MIC for the drug utilized was increased at least eightfold. For most combinations, mutants occurring at a frequency as low as 10-9 could be detected.

Knowledge of the genus Enterobacter and its role in human disease has expanded exponentially in recent years. The incidence of infection in the hospital and the community has increased. New clinical syndromes have been recognized. Enterobacter spp. have also been implicated as causes of other syndromes that traditionally have been associated almost exclusively with more easily treatable pathogens, such as group A streptococci and staphylococci. Rapid emergence of multiple-drug resistance has been documented in individual patients during therapy and in populations and environments with strong selective pressure from antimicrobial agents, especially the cephalosporins. Therapeutic options for patients infected with multiply resistant strains have become severely limited. Carbapenems or, alternatively, fluoroquinolones are the most predictively active options, although resistance to both classes has been observed on rare occasions. Enterobacter spp. appear well adapted for survival and even proliferation as the turn of the century approaches.

The chromosomal ampC beta-lactamase in Citrobacter freundii and Enterobacter cloacae is inducible by beta-lactam antibiotics. When an inducible ampC gene is introduced on a plasmid into Escherichia coli together with its transcriptional regulator ampR, the plasmid-borne beta-lactamase is still inducible. We have isolated mutants, containing alterations in a novel E. coli gene, ampG, in which a cloned C. freundii ampC gene is unable to respond to beta-lactam inducers. The ampG gene was cloned, sequenced and mapped to minute 9.6 on the E. coli chromosome. The deduced amino acid sequence predicted AmpG to be a 53 kDa, transmembrane protein, which we propose acts as a signal transducer or permease in the beta-lactamase induction system. Immediately upstream of ampG there is another 579-base-pair-long open reading frame (ORF) encoding a putative lipoprotein shown to be non-essential for beta-lactamase induction. We have found that ampG and this ORF form an operon, whose promoter is located in front of the ORF. Located closely upstream of the putative promoter is the morphogene bolA, which is transcribed in the opposite orientation. However, using transcription fusions, we have found that the ampG transcription is not regulated by bolA. In addition, we show that transcription is probably not regulated by either the starvation specific sigma factor RpoS, which controls bolA, or by AmpD the negative regulator for ampC transcription.

Mutants of Enterobacter cloacae 55 were studied to delineate more completely the genetics of inducible expression of AmpC beta-lactamase. E. cloacae 55M-L, derived by mutagenesis from a mutant with high-level cefotaxime resistance (MIC, greater than 64 micrograms/ml), E. cloacae 55M, demonstrated a novel phenotype by producing only low levels of AmpC constitutively. Neither the parental phenotype of E. cloacae 55M nor the wild-type phenotype of E. cloacae 55 could be restored in E. cloacae 55M-L by the introduction of functional ampR, ampC, or ampD genes. Cloning each of these genes from E. cloacae 55M-L confirmed the same genotype for this mutant as for its parental strain. Mutation of E. cloacae 55M-L to the E. cloacae 55M phenotype was found to occur spontaneously at a frequency of 10(-8). All such revertants demonstrated an inducible wild-type phenotype after introduction of a functional ampD. These results suggested that the E. cloacae 55M-L phenotype was due to a mutation in an as yet unrecognized gene, designated ampG. Verification of this gene was obtained by the restoration of the E. cloacae 55M phenotype in E. cloacae 55M-L by introduction of a cloned 2.9-kilobase BamHI fragment from the E. cloacae 55 chromosome. Transformation of both ampG and ampD into E. cloacae 55M-L reconstituted the inducible wild-type phenotype. These results indicate that ampG is required for the activation of ampC by AmpR. Without ampG, neither induction nor high-level expression of AmpC is possible. It is likely that the ampG gene product and AmpD together modulate the ability of AmpR to activate ampC expression.

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