Christine C. Sanders

Detection of extended-spectrum beta-lactamases in members of the family Enterobacteriaceae: comparison of the double-disk and three-dimensional tests.

The three-dimensional and clavulanate double-disk potentiation tests were compared as procedures for the detection of extended-spectrum beta-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 beta-lactamase with a pI of 5.95, with some strains producing multiple beta-lactamases. The three-dimensional test, which was performed in conjunction with a routine disk diffusion test, detected extended-spectrum beta-lactamase production in 26 of 28 (93%) of the strains that produced extended-spectrum beta-lactamases. The clavulanate double-disk potentiation test detected extended-spectrum beta-lactamases in only 22 of the 28 strains (79%) 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 beta-lactamase production, coupled with a greater sensitivity and earlier detection of extended-spectrum beta-lactamases. Images

Selection of multiple antibiotic resistance by quinolones, beta-lactams, and aminoglycosides with special reference to cross-resistance between unrelated drug classes.

The ability of three quinolones, two beta-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 beta-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 beta-lactam-selected mutants of Enterobacter cloacae and Pseudomonas aeruginosa (beta-lactams), amikacin-selected mutants of Providencia stuartii and P. aeruginosa (aminoglycosides), and beta-lactam- or amikacin-selected mutants of Serratia marcescens (beta-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. Images

Antimicrobial Resistance with Focus on β-Lactam Resistance in Gram-Negative Bacilli

beta-Lactam antibiotics are the most frequently prescribed antibiotics worldwide. Therefore, it is not surprising that resistance to this very important class of agents poses an increasingly complex and perplexing problem for physicians. Among the variety of mechanisms that can provide resistance to beta-lactam antibiotics in gram-negative bacilli, the production of beta-lactamase is by far the single most important factor. With the introduction of newer beta-lactam agents observed changes in beta-lactamases include the increased prevalence of older enzymes, the appearance of new enzymes, and alteration in the level of expression of the enzymes. These changes have been responsible for resistance to newer cephalosporins, monobactams, carbapenems, and beta-lactamase inhibitor/beta-lactam drug combinations. Resistance to beta-lactam antibiotics has also emerged through alterations in the targets of the drugs, the penicillin-binding proteins, and through alterations in outer membrane permeability of the organisms to the drugs. With some beta-lactam agents, multiple mechanisms must be acquired before clinically relevant levels of resistance are attained. This is especially true for carbapenems and fourth generation cephalosporins. Nevertheless, resistance to beta-lactam antibiotics is on the rise among clinical isolates of gram-negative bacilli, and only through more judicious use of these agents can their usefulness for treatment and prevention of infections be preserved.

AmpG, a signal transducer in chromosomal β-lactamase induction

The chromosomal ampCβ‐lactamase in Citrobacter freundii and Enterobacter cloacae is inducible by β‐lactam antibiotics. When an inducible ampC gene is introduced on a plasmid into Escherichia coli together with its transcriptional regulator ampR, the plasmid‐borne β‐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 β‐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 53kDa, trans‐membrane protein, which we propose acts as a signal transducer or permease in the β‐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 β‐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.

In vitro antagonism of beta-lactam antibiotics by cefoxitin.

We assessed the extent and mechanisms of antagonism of beta-lactam antibiotics by cefoxitin. In tests with 41 gram-negative isolates, cefoxitin antagonized cephalothin, cefamandole, cefsulodin, cefotaxime, moxalactam, ampicillin, carbenicillin, piperacillin, mezlocillin, and azlocillin, but not cephalexin, mecillinam, or N-formimidoyl thienamycin. The extent of antagonism varied with the beta-lactam and genus studied. However, antagonism occurred most often with strains possessing inducible cephalosporinases. Antagonism of cephalothin and cefamandole correlated closely with the induction of beta-lactamases capable of inactivating these drugs. Although antagonism of the remaining drugs occurred more often with strains possessing inducible beta-lactamases, these enzymes did not inactivate the drugs. Morphological studies revealed that cefoxitin inhibited filamentation and lysis produced by various beta-lactam drugs. Results of this investigation suggest that cefoxitin antagonizes beta-lactams via (i) induction of drug-inactivating beta-lactamases, and (ii) the induction of beta-lactamases that cannot inactivate the drug but serve as barriers against access to target proteins. This barrier appears most efficient for drugs that bind to penicillin-binding proteins 1 and 3. Images

PLASMID-MEDIATED RESISTANCE TO EXPANDED- SPECTRUM CEPHALOSPORINS AMONG ENTEROBACTER AEROGENES STRAINS

ABSTRACT Resistance to expanded-spectrum cephalosporins commonly develops inEnterobacter aerogenes during therapy due to selection of mutants producing high levels of the chromosomal Bush group 1 β-lactamase. Recently, resistant strains producing plasmid-mediated extended-spectrum β-lactamases (ESBLs) have been isolated as well. A study was designed to investigate ESBL production among 31 clinical isolates of E. aerogenes from Richmond, Va., with decreased susceptibility to expanded-spectrum cephalosporins and a positive double-disk potentiation test. Antibiotic susceptibility was determined by standard disk diffusion and agar dilution procedures. β-Lactamases were investigated by an isoelectric focusing overlay technique which simultaneously determined isoelectric points (pIs) and substrate or inhibitor profiles. Decreased susceptibility to cefotaxime, ceftazidime, and aztreonam (MIC range, 1 to 64 μg/ml) was detected and associated with resistance to gentamicin and trimethoprim-sulfamethoxazole. All strains produced an inducible Bush group 1 β-lactamase (pI 8.3). Twenty-nine of the 31 isolates also produced an enzyme similar to SHV-4 (pI 7.8), while 1 isolate each produced an enzyme similar to SHV-3 (pI 6.9) and to SHV-5 (pI 8.2). The three different SHV-derived ESBLs were transferred by transconjugation to Escherichia coli C600N and amplified by PCR. Plasmid profiles of the clinical isolates showed a variety of different large plasmids. Because of the linkage of resistance to aminoglycosides and trimethoprim-sulfamethoxazole with ESBL production, it is possible that the usage of these drugs was responsible for selecting plasmid-mediated resistance to extended-spectrum cephalosporins in E. aerogenes. Furthermore, it is important that strains such as these be recognized, because they can be responsible for institutional spread of resistance genes.

Emergence of resistance to beta-lactam and aminoglycoside antibiotics during moxalactam therapy of Pseudomonas aeruginosa infections.

In four patients with Pseudomonas aeruginosa infections, the infecting strain developed resistance to moxalactam during therapy with this drug. In addition, P. aeruginosa isolates from two of these four patients showed increased resistance to aminoglycosides. Isolates from a third patient acquired cross-resistance to other antipseudomonal beta-lactams. In three of the cases, disk susceptibility tests failed to detect the resistance that was demonstrated in broth dilution assays. Isolate identities were confirmed by serotyping. No new plasmids were found by agarose gel electrophoresis. The mechanisms for this resistance did not involve enzymatic antibiotic degradation. These findings suggest that currently available expanded-spectrum cephalosporin derivatives should probably not be used alone for most serious infections due to P. aeruginosa. They also suggest that strains with multiple antibiotic resistance may become more prevalent in hospitals if these drugs are used extensively.

Characterization of beta-lactamases in situ on polyacrylamide gels.

An inhibitor-based characterization system which allowed the identification of beta-lactamases after isoelectric focusing on polyacrylamide gels was developed. This system, using potassium clavulanate and oxacillin, distinguished type I chromosomally mediated enzymes from other beta-lactamases of gram-negative bacteria. Images

Clinical importance of inducible beta-lactamases in gram-negative bacteria

The clinical problems caused by inducible beta-lactamases in certain gram-negative bacteria are being recognized with increasing frequency. These problems include the rapid emergence of multiple beta-lactam resistance during therapy with many of the newer beta-lactam antibiotics. Such multiply resistant organisms are now spreading within the hospital and have become important nosocomial pathogens. This has been a particularly difficult problem for intensive care units, cystic fibrosis centers and burn units where there are clusters of patients who are highly susceptible to infections with organisms likeEnterobacter spp.,Serratia spp. andPseudomonas aeruginosa, which possess inducible beta-lactamases. Only through an awareness of these problems, their cause, and restriction of the use of certain newer betalactam antibiotics can these problems be controlled.

Emergence of resistance in gram-negative bacteria during therapy with expanded-spectrum cephalosporins

To assess the clinical importance of emergence of beta-lactam resistance caused by stable derepression of chromosomal beta-lactamases, sequential cultures from patients treated with expanded-spectrum cephalosporins were monitored for the persistence of bacteria possessing these enzymes. Antibiotic susceptibilities and beta-lactamase production before and after cefoxitin induction were determined in sequential isolates of individual bacterial strains. Of 49 strains isolated from 44 patients, 25 strains (51%) were eradicated by cephalosporin therapy, 17 strains (35%) persisted with unchanged susceptibility in sequential cultures, and 7 strains (14%) from 7 patients developed multiple beta-lactam resistance during cephalosporin therapy. In 6 of the 7 strains, resistance was associated with stable derepression of beta-lactamases. In the patient group whose strains developed resistance, subsequent use of non-beta-lactam antibiotics was more frequent and mortality was higher.