Epidemiology of beta-lactamases in Africa: correlation with resistance to beta-lactam antibiotics

In 45 centers from eight African countries, 2,888 bacterial isolates were collected from patients with community-acquired infections. Isolated pathogens included Staphylococcus aureus (29%), Escherichia coli (20%), Neisseria gonorrhoeae (6%), Proteus species (6%), Klebsiella species (6%), Klebsiella pneumoniae (4%), and Staphylococcus epidermidis (4%). An overall sensitivity of 16.2% was shown to penicillin G (number of isolates tested = 2,467), 31.8% to ampicillin (2,687), 45% to amoxicillin (1,959), and 84.9% to cefuroxime (2,888). Beta-lactamase presence was measured by a chromogenic method. Beta-lactamase was found in 75% of all pathogens tested, including 69.5% of gram-negative and 83.3% of gram-positive pathogens; 73% of E coli isolates, 76% of N gonorrhoeae, 75% of Klebsiella species, and 84% of S aureus were beta-lactamase positive. Beta-lactamase presence was associated with bacterial resistance for penicillin G, ampicillin, and amoxicillin, but not cefuroxime, whose sensitivity remained high. The higher resistance rates and beta-lactamase prevalence in Africa suggest the need for national antibiotic prescribing policies and surveillance schemes and replacement of relatively ineffective penicillins with newer agents such as cefuroxime.     

Inducible type I beta-lactamases of gram-negative bacteria and resistance to beta-lactam antibiotics

Mutants, showing either constitutive (depressed) or non-inducible expression of chromosomally-mediated Type I beta-lactamase were obtained from clinical isolates of Enterobacter cloacae, Ent. aerogenes, Citrobacter freundii, Providencia stuartii, Morganella morganii, Serratia marcescens and Pseudomonas aeruginosa. The wild-type and mutant strains were compared for susceptibility to a range of beta-lactam antibiotics. Derepression of beta-lactamase synthesis generally, but not always, resulted in a marked reduction in susceptibility to the agents tested, including the '3rd generation' cephalosporins. In many cases, the observed resistance would preclude, or severely compromise, the therapeutic efficacy of the drugs. In this context, depressed mutants of Enterobacter spp., Citro. freundii and Ps. aeruginosa could be of primary concern although those of Ser. marcescens, Prov. stuartii and Morg. morganii often exhibited equally high resistance levels to older beta-lactams. Comparison of the susceptibilities of the non-inducible mutants with that of their inducible parents suggested variation in the beta-lactamase inductive potency of different compounds in different organisms. For example, cefoxitin was a powerful inducer in Ent. cloacae, Citro. freundii and one strain of Ps. aeruginosa; similarly cefazolin and cefuroxime were good beta-lactamase inducers in Ser. marcescens and Morg. morganii. Aminothiazolyl-oxime cephalosporins and ureido-penicillins were generally poor inducers. From such comparisons, the contribution of inducible Type I beta-lactamase to resistance phenotype could be ascertained.     

Principal beta-lactamases responsible for resistance to beta-lactam antibiotics in urinary tract infections.

Two independent surveys have been conducted to determine the prevalent bacterial species and beta-lactamase types present in clinical populations of gram-negative, ampicillin-resistant isolates. A total of 208 isolates (112 from Nottingham Hospital and 96 from Charing Cross Hospital), all of which had been collected from out-patients suffering from urinary tract infections, were investigated. The incidence of ampicillin-resistant isolates (minimum inhibitory concentrations, 8 micrograms/ml) was 24.1% and 18.8% within the Nottingham and Charing Cross samples, respectively. The surveys gave similar results within the ampicillin-resistant samples. Escherichia coli was the prevalent bacterial species (52.9%), followed by Klebseilla pneumoniae (30.3%). The majority of isolates, at least 54.8% and possibly as high as 74.5%, owed their principal beta-lactamase activity to enzymes mediated by R-plasmids. The most prevalent beta-lactamases were TEM-1 (53.3%), SHV-1 (30.9%), and OXA-1 (11.5%). Positive associations were found between E. coli and TEM-1 or OXA-1 and between K. pneumoniae and SHV-1.     

Two-dimensional thin-layer chromatography for simultaneous detection of bacterial beta-lactam acylases and beta-lactamases.

A rapid and specific procedure was developed for the simultaneous detection of bacterial acylases and beta-lactamases, using ampicillin and cephalexin as substrates. Bacterial suspensions from agar plates were incubated separately with each beta-lactam substrate for 1 h at 37 degrees C. The supernatant of the reaction mixture was dansylated, and the dansyl derivatives were separated by two-dimensional thin-layer chromatography on polyamide sheets. The end products resulting from acylase hydrolysis, including the intact beta-lactam nucleus, 6-aminopenicillanic acid or 7-aminodeacetoxycephalosporanic acid, and the acyl side chain acid, D-(-)-alpha-aminophenylacetic acid, and the end product resulting from beta-lactamase hydrolysis (D-phenylglycylpenicilloic acid or D-phenylglycyldeacetoxycephalosporoic acid) were separated from each unhydrolyzed substrate and amino acids by this procedure. The presence of the intact beta-lactam nucleus in the reaction mixture is the indication of acylase activity. This method is sensitive and reproducible and has been successfully applied to screening for acylase activity in a variety of bacteria. It may be pharmaceutically useful for identifying organisms capable of removing the acyl side chain from naturally occurring beta-lactam antibiotics such as penicillin G, penicillin V, and cephalosporin C for production of the beta-lactam nuclei which serve as the starting materials for semisynthetic beta-lactam antibiotics.     

Role of beta-lactamases and outer membrane proteins in multiple beta-lactam resistance of Enterobacter cloacae.

The chromosomal beta-lactamase and outer membrane proteins of Enterobacter cloacae were examined to determine their relative contributions to multiple antibiotic resistance in this organism. Mutants altered in beta-lactamase expression, whether derived in the laboratory or recovered from patients treated with one of the new beta-lactam antibiotics, were found to have no detectable alterations in outer membrane proteins. Derepression of beta-lactamase in these mutants was associated with high-level resistance to multiple beta-lactam antibiotics, while loss of inducible beta-lactamase (i.e., production of basal enzyme levels only) was associated with acquisition of susceptibility to many beta-lactam antibiotics, including cephalothin. In contrast, alteration in outer membrane proteins was associated with only moderate-level resistance to beta-lactam antibiotics. However, this included resistance to such drugs as amdinocillin and Sch 34343, which were unaffected by derepression of beta-lactamase. Resistance to chloramphenicol and tetracycline also accompanied changes in outer membrane proteins. Although the outer membrane proteins of various strains of E. cloacae were similar, there did appear to be some major strain-to-strain variations. Thus, it appears that alterations in both beta-lactamase and outer membrane proteins can affect the susceptibility of E. cloacae to many antibiotics. However, alterations in beta-lactamase alone are sufficient to produce high-level multiple beta-lactam resistance in this organism.     

Inducible beta-lactamases are principally responsible for the naturally occurring resistance towards beta-lactam antibiotics in Proteus vulgaris

The role of inducible chromosomally mediated beta-lactamases was studied in 22 Proteus vulgaris isolates by monitoring enzyme induction in the presence of various inducers such as ampicillin, cefalothin, cefuroxime, cefsulodin, 6-aminopenicillanic acid (6-APS), and imipenem. 20 of the isolates exhibited resistance to ampicillin, cefalothin, and cefuroxime, whereas 2 isolates were susceptible to these compounds. In all resistant isolates marked inoculum effects could be observed. Enzyme production proved to be transient, i.e. maximum of enzyme production was achieved after 2 or 3 h. In both sensitive isolates enzyme production did not exceed 0.021 U beta-lactamase/mg protein of the cell-free supernatant even after induction with 6-APS or imipenem, whereas it ranged from 0.46 to 6.3 U in the resistant ones. Moreover, enzyme induction was found to be concentration-dependent, as revealed by the extensive study of one of the isolates (No. 4917). Three different enzymes could be distinguished by means of isoelectric focusing with isoelectric points at 7.4, 8.8, and approximately 9.5. In the presence of 2.5 mg/l clavulanic acid even strains known to be strong enzyme producers became as susceptible as the sensitive ones: moreover, the inocolum effect was markedly reduced. These findings make it apparent that it is above all the production of inducible enzymes that is responsible for resistance to beta-lactam antibiotics in P. vulgaris.     

Comparative potency of mecillinam and other beta-lactam antibiotics against Escherichia coli strains producing different beta-lactamases

The activity of mecillinam, a beta-lactam antibiotic with high affinity for gram-negative penicillin-binding protein 2 (PBP2), was assessed against ampicillin-resistant Escherichia coli strains producing beta-lactamases representing the three molecular classes, A (TEM-1 and -3, SHV-3 and IRT-5), C (AmpC) and D (OXA-3). The antimicrobial activity of mecillinam and other beta-lactam antibiotics was evaluated by determining their MICs on Mueller-Hinton agar. The time course of hydrolysis in crude extracts prepared from the various beta-lactamase-producing strains was also measured and was used to determine the relative rate of hydrolysis and the apparent affinity for ampicillin, cephalothin and mecillinam. When compared with the other beta-lactam antibiotics, mecillinam demonstrated significantly greater antibacterial potency and higher stability to beta-lactamase hydrolysis in TEM-, IRT- and AmpC-producing isolates. These findings confirm that the antimicrobial potency of mecillinam compares favourably with those of the other penicillins included in the present study, suggesting that mecillinam use in the treatment of infections caused by gram-negative bacteria should be re-evaluated.     

Characterization of beta-lactamases from non-Bacteroides fragilis group Bacteroides spp. belonging to seven species and their role in beta-lactam resistance

Twelve beta-lactamase-positive non-Bacteroides fragilis group Bacteroides spp. belonging to seven different species were examined by MIC determination and enzyme characterization. MICs of most beta-lactams except cefoxitin, cefotetan, imipenem, and meropenem were relatively high or very high. All enzymes hydrolyzed cephaloridine (Vmax, 100%; Km, 12 to 70 microM), cephalothin (Vmax, 25 to 826%; Km, 8 to 143 microM), cefamandole (Vmax, 13 to 158%; Km, 17 to 170 microM), and cefuroxime (hydrolysis rate, 19 to 98%), and 11 of 12 hydrolyzed cefotaxime (Vmax, 26 to 145%; Km, 13 to 127 microM); no hydrolysis of cefoxitin or moxalactam was observed. Penicillins were hydrolyzed at lower rates, with Vmax values less than or equal to 20% of that obtained with cephaloridine. Addition of clavulanate, sulbactam, or tazobactam led to a 4- to 2,048-fold lowering of MICs of penicillins as well as cephalosporins. All enzymes were inhibited by clavulanate (50% inhibitory concentration [IC50], 0.01 to 1.8 microM), sulbactam (IC50, 0.02 to 1.9 microM), tazobactam (IC50, 0.001 to 0.9 microM), cefoxitin (IC50, 0.002 to 0.35 microM), and moxalactam (IC50, 0.03 to 6.6 microM). No enzymes were inhibited by 100 microM EDTA or p-chloromercuribenzoic acid; an enzyme of one strain of B. loescheii was inhibited by 100 microM cloxacillin (IC50, 2.35 microM). Ten enzymes had optimal activity at pH 5.0 to 6.0, and two had optimal activity at pH 8.0. Isoelectric focusing revealed pIs between 4.2 and 5.6. These enzymes seem to belong to a previously unclassified group of beta-lactamases, related (but not identical) to beta-lactamases of the B. fragilis group.     

The beta-lactamases and beta-lactam antibiotic susceptibility of Yersinia enterocolitica

One hundred and forty-five isolates of Yersinia enterocolitica of different serotypes and biotypes, including atypical biotypes, collected from various parts of the world, were examined for their susceptibility to beta-lactam antibiotics and expression of intracellular beta-lactamases. The reasons for the specificity of patterns of susceptibility to beta-lactams for each biotype or subtype of Y. enterocolitica were elucidated by examining their ss-lactamase activity. Whilst the biotypes and subtypes were uniformly susceptible to the newer beta-lactam antibiotics, the susceptibility pattern observed with other beta-lactams was specific to each biotype or subtype, because of the characteristics of beta-lactamase expression by strains within these groups. The susceptibility to these beta-lactam agents depended entirely on the extent of elaboration or the absence of one of the two beta-lactamases, enzyme A and enzyme B, found in the species. Detection of enzyme B by a disc diffusion test yielded inconsistent results, but detection of enzyme A by disc diffusion was highly reliable. This test clearly distinguished strains of biotype 2, serotype O:5,27 from those of biotype 2, serotype O:9 and biotype 3, serotypes O:1, 2a-3, O:3 and O:5.     

Effects of beta-lactamases and omp mutation on susceptibility to beta-lactam antibiotics in Escherichia coli.

Four types of beta-lactamases consisting of a penicillinase type I (TEM-1), a penicillinase type II (OXA-1), a cephalosporinase of Citrobacter freundii, and a cephalosporinase of Proteus vulgaris were introduced into Escherichia coli MC4100 and its omp mutants, MH1160 (MC4100 ompR1) and MH760 (MC4100 ompR2), by transformation. Effects of the combination of the omp mutations and these beta-lactamases on the susceptibility of E. coli strains were studied with 15 beta-lactam antibiotics including cephalosporins, cephamycins, penicillins, imipenem, and aztreonam. The ompR1 mutant, MH1160, lacks OmpF and OmpC, and it showed reduced susceptibility to 11 of the 15 beta-lactam agents. The reduction in susceptibility to cefoxitin, moxalactam, and flomoxef was much greater than reduction in susceptibility to the other agents. When the ompR1 mutant produced the cephalosporinase of C. freundii, the susceptibility of the mutant to 12 of the 15 beta-lactam antibiotics decreased. The reduction in susceptibility of MH1160 to 10 of the 12 agents affected by the enzyme was two- to fourfold greater than that observed in MC4100. Such a synergistic effect was also observed with the cephalosporinase of P. vulgaris and ompR1 mutation against six cephalosporins, moxalactam, and aztreonam.     

Comparable evaluation of orally active beta-lactam compounds in ampicillin-resistant gram-positive and gram-negative rods: role of beta-lactamases on resistance

The antibacterial activity of the recently developed cephems cefixime and cefetamet-pivoxyl was evaluated in 408 gram-positive and gram-negative rods, all isolated recently from clinical specimens, and compared to that of other orally active agents such as ampicillin, amoxycillin + clavulanic acid, cefaclor, cefuroxime-axetil and to ceftriaxone. With regard to ampicillin-resistant Enterobacteriaceae ceftriaxone proved to be the most active agent, followed by cefixime and cefetamet, whereas cefuroxime was less active. Cefaclor and amoxycillin + claculanic acid were active against ampicillin-resistant Escherichia coli, Klebsiella pneumoniae, and Proteus ssp. isolates. All beta-lactam compounds exhibited poor activity against Acinetobacter anitratus isolates, but were highly active against Haemophilus influenzae with the exception of cefaclor. Both cefixime and cefetamet were poorly active against Staphylococcus aureus, but highly active against beta-hemolytic streptococci. Moreover, both compounds remained unaffected by the production of plasmid-mediated beta-lactamases such as the TEM or OXA enzymes. Resistance to both agents was observed in Enterobacteriaceae that produced large amounts of chromosomally mediated enzymes; their affinity to the class I enzyme from Enterobacter cloacae was somewhat lower than that of other third-generation cephalosporins. However, in contrast with these agents breakdown of cefixime and cefetamet by a class IIIa enzyme form Proteus vulgaris was marginal. In methicillin-resistant S. aureus isolates there was a complete cross-resistance between all beta-lactam compounds included in this study.     

Characterization and prevalence of the different mechanisms of resistance to beta-lactam antibiotics in clinical isolates of Escherichia coli

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.     

Global dissemination of beta-lactamases mediating resistance to cephalosporins and carbapenems

While the main era of beta-lactam discovery programs is over, these agents continue to be the most widely prescribed antimicrobials in both community and hospital settings. This has led to considerable beta-lactam pressure on pathogens, resulting in a literal explosion of new beta-lactamase variants of existing enzyme classes. Recent advances in the molecular tools used to detect and characterize beta-lactamases and their genes has, in part, fueled the large increase in communications identifying novel beta-lactamases, particularly in Gram-negative bacilli. It now seems clear that the beta-lactams themselves have shaped the field of new enzymes, and the evolution of key amino acid substitutions around the active sites of beta-lactamases continues to drive resistance. Over 130 variants of TEM beta-lactamase now exist, and more are reported in the scientific literature each month. The most disturbing current trend is that many bla structural genes normally limited to the chromosome are now mobilized on plasmids and integrons, broadening the spread of resistance to include carbapenems and cephamycins. Furthermore, in some Enterobacteriaceae, concomitant loss of outer membrane porins act in concert with these transmissible beta-lactamase genes to confer resistance to the most potent beta-lactams and inhibitor combinations available. Continued reviews of the literature are necessary in order to keep abreast of the ingenuity with which bacteria are changing the current genetic landscape to confer resistance to this important class of antimicrobials.     

产超广谱β内酰胺酶和高产AmpC酶革兰阴性杆菌耐药性检测

目的了解铜陵地区产ESBLs和高产AmpC酶革兰阴性杆菌的耐药性.方法2003年10月至2004年9月铜陵地区临床分离的革兰阴性杆菌270株,用Kirby-Bauer法进行药敏试验,根据NCCLS 2002年判断标准分析结果.结果270株革兰阴性杆菌中检出产ESBLs或（和）高产AmpC酶68株,检出率25.2%,其中单产ESBLs 49株,以肺炎克雷伯菌、大肠埃希菌中检出率最高,分别为31.6%和31.0%：单产AmpC酶3株,均为阴沟肠杆菌,同时产AmpC酶和ESBLs16株,其中鲍曼不动杆菌8株.产酶株对头孢菌素耐药率明显高于非产酶株,产ESBLs菌株对替卡西林-克拉维酸、头孢哌酮-舒巴坦等含酶抑制剂的抗菌药物耐药率在50%以上,对亚胺培南敏感性好.结论ESBLs和高产AmpC酶是导致革兰阴性杆菌耐药的主要两类酶,产酶株对多种抗菌药物耐药,仅对亚胺培南敏感.     

Mechanisms of resistance to beta-lactam antibiotics in Acinetobacter calcoaceticus.

Examination of 12 strains of Acinetobacter calcoaceticus revealed that the strains expressed different constitutive levels of beta-lactamase. Mutants resistant to cefoxitin, cefoperazone or ceftazidime were selected from a strain producing a low level of beta-lactamase. All the mutants showed no change in expression of beta-lactamase, but produced penicillin-binding proteins with altered expression and/or affinity for beta-lactams. In addition, the outer membrane of the mutants showed decreased permeability (40-80% that of the parent strain) towards small hydrophilic solutes, together with diminished production of a 46.5 kDa porin protein. It was concluded that the enhanced resistance to beta-lactams in the A. calcoaceticus mutants was the result of interplay between the altered penicillin binding proteins and the reduced outer membrane permeability.