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.     

Factors determining resistance to beta-lactam combined with beta-lactamase inhibitors in Escherichia coli.

The influence of inoculum size, beta-lactamase hyperproduction (multicopy plasmid) and modifications in the outer membrane protein profile on the susceptibility of Escherichia coli to combinations of amoxycillin/clavulanate, amoxycillin/sulbactam, amoxycillin/tazobactam and piperacillin/tazobactam were studied. For all combinations the bacterial susceptibility was affected by factors determining an increase in beta-lactamase (inoculum size or hyperproduction). Clavulanic acid was the most efficient beta-lactamase inhibitor. The absence of the outer membrane proteins, OmpF and OmpC, did not significantly affect susceptibility to the combinations per se but when combined with the presence of beta-lactamase high MICs were observed. Seven out of eight amoxycillin/clavulanate resistant clinical isolates of E. coli had beta-lactamase hyperproduction and a decrease or absence of OmpF.     

Beta-lactam resistance in clinical isolates of Escherichia coli caused by elevated production of the ampC-mediated chromosomal beta-lactamase.

Among cephalothin-resistant isolates from patients with urinary tract infections, six Escherichia coli strains were found to produce elevated amounts of a beta-lactamase indistinguishable from that coded by the ampC gene of E. coli K-12. The resistance levels displayed by these isolates toward a number of beta-lactams were, for five of them, considerably higher as compared with E. coli K-12 with the same amount of beta-lactamase, implying the importance of intrinsic resistance in these isolates. Cefuroxime, and to a lesser extent cefamandole, were stable to hydrolysis by E. coli chromosomal beta-lactamase but acted as inhibitors of the enzyme. Nevertheless, increased beta-lactamase production mediated an increased resistance toward these drugs. No plasmids were found in the isolates, suggesting a chromosomal location for the respective ampC locus.     

In vitro activity of beta-lactam and non-beta-lactam antibiotics in extended-spectrum beta-lactamase-producing clinical isolates of Escherichia coli

The activity of different beta-lactam and non-beta-lactam antibiotics was assessed against extended-spectrum beta-lactamase (ESBL)-producing and non-ESBL-producing clinical isolates of Escherichia coli. A phenotypic study to discover the presence of ESBLs in 399 clinical isolates of E. coli was made by the disk diffusion method following the Clinical and Laboratory Standards Institute (formely NCCLS, 2004) guidelines. The activity of different antibiotics was subsequently studied using the automated VITEK 2 system (bioMérieux, Marcy l'Etoile, France). One hundred fifteen isolates proved to be ESBL-producing and 284 non-ESBL-producing. Among the former, percentage susceptibilities to the antibiotics assayed were meropenem and amikacin, 100%; piperacillin/tazobactam, 97.4%; cefepime, 94.8%; amoxicillin/clavulanic acid, 84.3%; tobramycin, 84.3%; gentamicin, 83.5%; cefoxitin, 83.5%; nitrofurantoin, 71.3%; cotrimoxazole, 46.1%; norfloxacin, 29.6%; ciprofloxacin, 27%; and ofloxacin, 26.1%.     

Mutations affecting the resistance of Escherichia coli K-12 to beta-lactam antibiotics

Two mutants of Escherichia coli resistant to beta-lactam antibiotics exhibited beta-lactamase activity slightly higher than the wild type strain. One showed disturbance in cephaloridine and penicillin penetration into cells. Neither differed from the wild strain in terms of the major outer membrane proteins or PBPs. It is suggested that the disturbance in penetration of beta-lactam antibiotics in one of the mutants concerns diffusion of the drug directly through the double-layer phospholipid membrane.     

In vitro susceptibilities of Capnocytophaga isolates to beta-lactam antibiotics and beta-lactamase inhibitors

The susceptibilities of 43 pharyngeal isolates of Capnocytophaga to beta-lactam antibiotics, alone or in combination with beta-lactamase inhibitors, were tested by an agar dilution method. The 34 beta-lactamase-positive strains were highly resistant to beta-lactams, but the intrinsic activities of clavulanate, tazobactam, and sulbactam against Capnocytophaga, even beta-lactamase producers, indicates that these beta-lactamase inhibitors could be used for empirical treatment of neutropenic patients with oral sources of infection.     

Effect of hyperproduction of TEM-1 beta-lactamase on in vitro susceptibility of Escherichia coli to beta-lactam antibiotics.

The susceptibility of 173 TEM-1-producing isolates of Escherichia coli was assessed by determination of MICs by the agar dilution method. MICs of amoxicillin, mezlocillin, cephaloridine, and, to a smaller extent, amoxicillin-clavulanic acid (but not cephalexin, cefuroxime, cefotaxime, ceftazidime, or imipenem) were higher for isolates that produced large amounts of beta-lactamase than for isolates that produced smaller amounts. The effect of fixed concentrations of clavulanic acid on resistance to amoxicillin was assessed for 34 selected TEM-1-producing isolates. Low concentrations of the inhibitor (0.5 to 1 microgram/ml) reduced the amoxicillin MICs substantially for almost all the isolates, although the reductions were not sufficient to render any of the isolates amoxicillin susceptible. Higher concentrations of clavulanic acid had progressively greater effects on amoxicillin MICs, but even at 8 micrograms/ml some of the isolates with high beta-lactamase activities remained resistant or only moderately susceptible to amoxicillin. All the isolates were inhibited by clavulanic acid (in the absence of amoxicillin) at concentrations of 16 to 32 micrograms/ml. TEM-1 beta-lactamase activity was inhibited in vitro by clavulanic acid, but not totally, with approximately 2% of the initial activity remaining at 2 micrograms/ml and 0.4% remaining at 8 micrograms/ml. These findings suggest that the amount of beta-lactamase activity is a major determinant of the degree of resistance to several beta-lactam antibiotics and can make the difference between susceptibility and resistance to some compounds, notably the combination of amoxicillin with clavulanic acid.     

Influence of beta-lactam antibiotics on serum resistance of K1-positive blood culture isolates of Escherichia coli.

The K1-positive strains of Escherichia coli are a group with considerable clinical importance, serum resistance being a common virulence factor of these strains. In the present paper, the influences of cephaloridine, imipenem, and ceftazidime on the serum resistance of eight serum-resistant K1-positive E. coli blood culture isolates with smooth-type lipopolysaccharide were studied. All strains were rendered more serum sensitive by treatment with subinhibitory concentrations of antibiotics. The amount of the reduction of serum resistance was dependent on the concentration of the antibiotic. Amounts of K1 produced under the influence of the antibiotics were measured and were found to be reduced for almost all strains tested. To further test the hypothesis that antibiotic-induced reduction of serum resistance is mediated by inhibition of K1 expression, isogenic mutants of one strain were produced by selection for resistance against infection with K1-specific bacteriophages. These mutants were found to be highly serum sensitive. We conclude from this study that beta-lactam antibiotics can render K1-positive serum-resistant strains of E. coli highly serum sensitive and that this effect is mediated by inhibition of K1 expression.     

Relationship between haemolysis production and resistance to fluoroquinolones among clinical isolates of Escherichia coli

The activities of ampicillin, amoxycillin-clavulanic acid, gentamicin, tetracycline, nalidixic acid, ciprofloxacin, pefloxacin and trovafloxacin against 207 consecutive clinical isolates of Escherichia coli were determined. Fifty-six (27.3%) isolates were haemolytic. The percentages of resistance to quinolones and tetracycline, but not to other agents, among haemolytic isolates were significantly lower (P < 0.05) than among non-haemolytic isolates. Ciprofloxacin-resistant mutants obtained from ciprofloxacin-susceptible haemolytic isolates still produced haemolysis. It is concluded that most quinolone-resistant clinical isolates of E. coli are non-haemolytic, although haemolysis is produced by quinolone-resistant mutants derived from haemolytic quinolone-susceptible strains.     

Emergence of clinical isolates of Escherichia coli producing TEM-1 derivatives or an OXA-1 beta-lactamase conferring resistance to beta-lactamase inhibitors.

Sixteen Escherichia coli clinical isolates which were resistant to ampicillin and amoxicillin-clavulanate but susceptible to cephalothin were studied. Eight strains showed the presence of a beta-lactamase which comigrates with reference OXA-1 enzyme. The eight other strains produced different TEM-1 derivatives which had in common a higher Km for penicillins and a higher 50% inhibitory concentration for the beta-lactamase inhibitors. By oligotyping and sequencing of PCR products, it was shown that Ser (AGC) (TEM-30; also called TRI-1) in three strains and Cys (TGC) (TEM-31; also called TRI-2) in one strain were substituted for Arg-241 (CGC), that Leu (CTG) (TEM-33) and Val (GTG) (TEM-34) in one strain each were substituted for Met-67 (ATG), and that in other mutants the two latter substitutions occurred together with the substitution of Asp (GAT) (TEM-35 and TEM-36) for Asn-272 (AAT). Therefore, different sets of amino acid substitutions of TEM-1 can be found in clinical isolates and lead to resistance to beta-lactamase inhibitors.     

大肠埃希菌临床分离株耐药性分析

目的：监测近5年来大肠埃希菌临床分离株的耐药性变迁，指导临床合理使用抗生素。方法：常规方法进行菌株分离，采用VITEK-32全自动微生物分析仪进行菌株鉴定及药敏试验，超广谱β内酰胺酶采用双纸片确认试验和仪器检测相结合的方法，对近5年来我院大肠埃希菌的耐药谱进行回顾性调查分析。结果：近5年从各种临床标本中共分离到大肠埃希菌488株，大肠埃希菌对青霉素类及第一代头孢菌素耐药率最高。2002年，对氨苄西林、替卡西林和头孢噻吩耐药率均高达98％，对亚胺培南耐药率最低，5年耐药率均为零。对培氟沙星和替卡西林耐药率上升最快，分别上升了28和26个百分点，对呋喃妥因耐药率有所下降，对庆大霉素和妥布霉素耐药率趋于稳定。产ESBLs菌株有明显上升趋势，从1998年的17％上升到2002年的36％。结论：大肠埃希菌在临床标本中的检出率、耐药性及多重耐药性均有逐年上升的趋势。     

大肠埃希菌临床分离株对喹诺酮类抗菌药的质粒介导耐药

目的 了解近年来发现的质粒介导耐药机制在大肠埃希菌临床株对喹诺酮类耐药中所起的作用。方法 78株环丙沙星耐药菌株分离自上海5所教学医院。以克隆斑点杂交及Southern杂交方法筛选质粒介导耐药基因qnr；以接合试验了解喹诺酮耐药的可转移性；对qnr基因进行序列分析，以引物步移法对qnr周边质粒DNA进行测序、分析。结果 78株大肠埃希菌中，6株（7．7％）qnr检测阳性。在6株阳性菌中，喹诺酮耐药性均可通过质粒转移，接合子对环丙沙星的MIC较受体菌上升16～250倍。qnr基因与最早报道的序列一致，qnr位于In4族的Ⅰ类整合子上，本研究中的2个新整合子命名为In36及In37。结论 与qnr相关的质粒介导喹诺酮类耐药性在大肠埃希菌临床分离株中有一定程度流行，这可能是我国细菌对喹诺酮类耐药性上升迅速的原因之一。     

AmpC beta-lactamase in an Escherichia coli clinical isolate confers resistance to expanded-spectrum cephalosporins

Cloning, sequencing, and biochemical analysis identified a novel AmpC-type beta-lactamase conferring resistance to extended-spectrum cephalosporins in an Escherichia coli clinical isolate. This enzyme, exhibiting 14 amino acid substitutions compared to a reference AmpC cephalosporinase of E. coli, hydrolyzed ceftazidime and cefepime significantly.     

Qualitative and quantitative aspects of beta-lactamase production as mechanisms of beta-lactam resistance in a survey of clinical isolates from faecal samples

A study has been conducted to identify the beta-lactamases most likely to contribute to beta-lactam resistance in clinical populations and to investigate their interactions with cefuroxime and newer cephalosporins. A total of 217 ampicillin-resistant, Gram-negative isolates from faecal samples of healthy volunteers in Germany, South America and Amman were investigated. Such strains represent the 'gene pool' from which infections might arise. Escherichia coli was the prevalent species (59.9%) followed by Klebsiella spp. (20.3%) and Enterobacter cloacae (12.0%). At least 56.7% and possibly as high as 64.5% of strains owed their principal beta-lactamase activity to enzymes mediated by R-plasmids. The most prevalent R-plasmid mediated beta-lactamase was TEM-1 which was produced by 109 strains. The beta-lactamase activity of strains producing only a chromosomal enzyme was often markedly higher than that of strains also producing an R-plasmid mediated enzyme. The qualitative and quantitative aspects of beta-lactamase production were investigated in cell free and whole cell tests and this confirmed the superior broad spectrum beta-lactamase resistance of ceftazidime over other new cephalosporins.     

Pseudomonas pseudomallei resistance to beta-lactam antibiotics due to alterations in the chromosomally encoded beta-lactamase.

Pseudomonas pseudomallei, the causative agent of melioidosis, is generally susceptible to some of the newer extended-spectrum cephalosporins or to combinations of a beta-lactam and clavulanic acid, a beta-lactamase inhibitor. Resistance to these agents may, however, emerge during treatment. We report on alterations in the chromosomal beta-lactamase associated with the development of resistance. Three resistance patterns resulted from three different mechanisms in the strains investigated. Derepression of the chromosomal enzyme resulted in a general increase in the MICs of all of the beta-lactams tested. The second mechanism observed was an insensitivity to inhibition of the beta-lactamase by clavulanic acid. In this case, the level of susceptibility to beta-lactams as independent entities remained unchanged. The final "resistance" pattern occurred in a patient treated with ceftazidime and resulted in a beta-lactamase that was capable of hydrolyzing this antibiotic at detectable levels, but with reduced efficacy against other beta-lactams. The net result was a strain that was generally susceptible to all of the beta-lactams tested except ceftazidime. In all cases, the level of susceptibility to antibiotics other than beta-lactams remained unchanged. Such variability found within one genus over a relatively short time course suggests that treatment of infections caused by this organism should be carefully monitored to detect susceptibility alterations to the chosen therapy.     

Chromosomal beta-lactamase expression and resistance to beta-lactam antibiotics in Proteus vulgaris and Morganella morganii

Indole-positive members of the Proteeae usually have inducible expression of chromosomal beta-lactamases. Mutants with stably derepressed beta-lactamase expression occur in inducible populations at frequencies in the range of 10(-6) to 10(-8). The contribution of these beta-lactamases to drug resistance was examined in Morganella morganii and Proteus vulgaris. The M. morganii enzyme was a high-molecular-weight (49,000) class I cephalosporinase with low Vmax rates for ampicillin, carbenicillin, and and broad-spectrum cephalosporins. The P. vulgaris enzyme had a lower molecular weight (32,000) and high Vmax rates for ampicillin, cephaloridine, cefotaxime, and ceftriaxone. Imipenem and cefoxitin inactivated the P. vulgaris enzyme but were low-Vmax, low-Km substrates for that of M. morganii. Despite these differences, the two beta-lactamases caused similar resistance profiles. Ampicillin and cephaloridine were strong inducers for both species, and beta-lactamase-inducible strains and their stably derepressed mutants were resistant, whereas basal mutants (those with low-level uninducible beta-lactamase) were susceptible to these two compounds. Mezlocillin, cefotaxime, ceftriaxone, and (usually) carbenicillin were almost equally active against beta-lactamase-inducible organisms and their basal mutants, but were less active against stably derepressed mutants. This behavior reflected the beta-lactamase lability of these drugs, coupled with their weak inducer activity below the MIC. Carbenicillin was a labile strong inducer for a single P. vulgaris strain, and inducible enzyme was protective against the drug in this atypical organism. Cefoxitin and imipenem, both strong inducers below the MIC, were almost equally active against beta-lactamase-inducible organisms and their basal and stably derepressed mutants.     

Prevalence of resistance to beta-lactam antibiotics in Escherichia coli isolated from blood from 1969-1991.

MICs of beta-lactam antibiotics were determined for blood culture isolates of Escherichia coli from patients in St. Thomas' Hospital, London between 1969 and 1991. There was correlation between MICs of carbenicillin and those of amoxycillin (Kendall's coefficient of rank correlation, tau = 0.67), mezlocillin (tau = 0.73), cephaloridine (tau = 0.64) and amoxycillin/clavulanic acid (tau = 0.72), but less correlation between MICs of carbenicillin and cefuroxime (tau = 0.31). During the two decades, the geometric mean MICs increased for carbenicillin (6.6-fold increase in mean MIC), mezlocillin (3.4-fold increase in mean MIC), cephaloridine (two-fold increase in mean MIC) and, to a smaller extent, amoxycillin/clavulanic acid (1.2-fold increase in mean MIC), but not for cefuroxime. These changes were the result of increases in the proportion of isolates that were resistant to carbenicillin and not of changes in the phenotype of carbenicillin-resistant isolates.     

Ampicillin-sulbactam and amoxicillin-clavulanate susceptibility testing of Escherichia coli isolates with different beta-lactam resistance phenotypes

The activities of ampicillin-sulbactam and amoxicillin-clavulanate were studied with 100 selected clinical Escherichia coli isolates with different beta-lactam susceptibility phenotypes by standard agar dilution and disk diffusion techniques and with a commercial microdilution system (PASCO). A fixed ratio (2:1) and a fixed concentration (clavulanate, 2 and 4 micrograms/ml; sulbactam, 8 micrograms/ml) were used in the agar dilution technique. The resistance frequencies for amoxicillin-clavulanate with different techniques were as follows: fixed ratio agar dilution, 12%; fixed concentration 4-micrograms/ml agar dilution, 17%; fixed ratio microdilution, 9%; and disk diffusion, 9%. Marked discrepancies were found when these results were compared with those obtained with ampicillin-sulbactam (26 to 52% resistance), showing that susceptibility to amoxicillin-clavulanic acid cannot be predicted by testing the isolate against ampicillin-sulbactam. Interestingly, the discrimination between susceptible and intermediate isolates was better achieved with 4 micrograms of clavulanate per ml than with the fixed ratio. In contrast, amoxicillin susceptibility was not sufficiently restored when 2 micrograms of clavulanate per ml was used, particularly in moderate (mean beta-lactamase activity, 50.8 mU/mg of protein) and high-level (215 mU/mg) TEM-1 beta-lactamase producer isolates. Four micrograms of clavulanate per milliliter could be a reasonable alternative to the 2:1 fixed ratio, because most high-level beta-lactamase-hyperproducing isolates would be categorized as nonsusceptible, and low- and moderate-level beta-lactamase-producing isolates would be categorized as nonresistant. This approach cannot be applied to sulbactam, either with the fixed 2:1 ratio or with the 8-micrograms/ml fixed concentration, because many low-level beta-lactamase-producing isolates would be classified in the resistant category. These findings call for a review of breakpoints for beta-lactam-beta-lactamase inhibitors combinations.