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.     

beta-Lactamases and beta-lactam resistance in Escherichia coli.

Escherichia coli strains determining 17 different plasmid-determined beta-lactamases were tested for resistance to new broad-spectrum beta-lactam antibiotics. Several beta-lactamases demonstrated enhanced resistance to cefamandole but only low-level resistance to other agents. High production of cloned E. coli chromosomal beta-lactamase, however, provided resistance to cefamandole, cefoxitin, cefotaxime, ceftazidime, and aztreonam but not to BMY-28142 or imipenem.     

Antimicrobial resistance in Escherichia coli outpatient urinary isolates from women: emerging multidrug resistance phenotypes

This study evaluated the antimicrobial resistance profile of outpatient urinary Escherichia coli isolated from women obtained throughout Hong Kong during 2004-2005. Of 1067 single patient isolates analyzed, 60.1% were resistant to ampicillin, 34% were resistant to co-trimoxazole, and 22.1% were resistant to ciprofloxacin. Thirty-four (6.6%) of 519 isolates in 2004 and 55 (10%) of 548 isolates in 2005 were extended-spectrum beta-lactamase (ESBL) producers with a CTX-M phenotype. Rates of non-beta-lactam resistance and ESBL production were strongly influenced by patient age. The age-stratified rates for dual co-trimoxazole and ciprofloxacin resistance and for ESBL production were 10.9% and 7.6% in women aged 18-35 years, 13% and 6.9% in women aged 36-50 years, 20.4% and 8.8% in women aged 51-64 years, and 23.7% and 11.8% in women aged > or =65 years, respectively. Nitrofurantoin and fosfomycin remain active against >90% of the isolates, irrespective of the resistance phenotypes for other drugs. Our results documented the emergence of problematic resistance phenotypes among community urinary E. coli and highlight the need to explore strategies for their containment.     

大肠埃希菌耐药性分析

目的：了解大肠埃希菌对常用抗生素的耐药性以及不同来源大肠埃希菌耐药性的差异，以指导临床合理用药。方法：应用WHONET5分析我院2000年1月-2002年7月临床分离的430株大肠埃希菌的药敏结果。结果：大肠埃希菌对氨苄西林、哌拉西林、阿莫西林-克拉维酸、复方磺胺甲嗯唑、环丙沙星、庆大霉素、头孢唑啉和头孢呋辛的耐药率分别为87％、78％、80％、67％、65％、53％、55％和42％；对头孢噻肟、头孢曲松、头孢他啶和氨曲南的耐药率分别为23％、18％、4％和18％；对哌拉西林-三唑巴坦、头孢吡肟、头孢西丁、亚胺培南、阿米卡星和呋喃妥因的耐药率分别为22％、15％、22％、0、15％和20％。痰液标本中的大肠埃希菌耐药率显著高于其他各类标本中分离者。结论：大肠埃希菌对青霉素类、磺胺类、喹诺酮类和庆大霉素，第一代、第二代头孢菌素等均已高度耐药，临床不宜选用；哌拉西林-三唑巴坦、第四代头孢菌素、头霉素类、碳青霉烯类、阿米卡星和呋喃类耐药率较低，临床可选用；不同来源的大肠埃希菌耐药率存在差异，痰标本中细菌耐药率最高，其余无明显差别。     

Quantitative analysis of beta-lactamase production of multiple resistance to beta-lactam antibiotics in clinical isolates of Escherichia coli

A simple test of procedure is described for the rapid evaluation of beta-lactamase substrate profiles against all the clinically important beta-lactam antibiotics. By use of this method 100 clinical isolates of Escherichia coli were classified into four beta-lactamase types: TEM-like, 35; chromosomal-like, 56; others, 3; beta-lactamase-less, 6 strains. They were tested for their susceptibilities to cefuroxime, cefoxitin, cefamandole, cephalexin, cefazolin, cephaloridine, cephalothin, ampicillin, carbenicillin and kanamycin. The isolates of each of TEM-like and chromosomal-like beta-lactamase types showed a good correlation between the beta-lactamase activity and the minimum inhibitory concentration (MIC) determined by the agar dilution method and the least square line analysis; MIC = ax + b, where x is enzyme activity, and a and b are constants. TEM-like beta-lactamase increased the resistance to ampicillin and carbenicillin markedly and that to cephalothin, cephaloridine and cefamandole moderately. In contrast, chromosomal-like beta-lactamase increased the resistance to ampicillin, cephalexin and cephalothin only slightly.     

Patterns and mechanisms of beta-lactam resistance among isolates of Escherichia coli from hospitals in the United States.

To study the national distribution of beta-lactam resistance patterns and mechanisms among Escherichia coli organisms isolated in U.S. hospitals, 652 ampicillin-resistant (Am(r)) or ampicillin-intermediate (Ami) isolates were submitted to the Centers for Disease Control from March 1983 through July 1984 by nine hospitals participating in the National Nosocomial Infections Study. Among the isolates (most of which caused urinary tract infections), 78% were Am(r) and 22% were Ami by the interpretative criteria established by the National Committee for Clinical Laboratory Standards. Resistance to carboxypenicillins ranged from 73 to 74%, and that to acylureidopenicillins ranged from 43 to 66%. A total of 26% of the isolates were resistant to cephalothin, and 4% were resistant to cefazolin. Resistance to cefoxitin was 1%, while resistances to cefuroxime and cefamandole were 2 and 7%, respectively. With the exception of cefsulodin (98% resistant) and cefoperazone (1% resistant), there was no resistance to newer cephalosporins or aztreonam. In general, only minor differences in the incidence of resistance to beta-lactam antibiotics were noted in hospital-acquired versus non-hospital-acquired isolates as well as among isolates from various regions of the United States. TEM beta-lactamases were produced by 87% of the 237 Am(r) isolates examined. By our methods, OXA and chromosomal (type I) beta-lactamases were detected in 2 and 28 isolates, respectively, and plasmid-mediated extended-spectrum cephalosporinases were detected in none of the isolates. Disk substrate and clavulanic acid inhibition assays revealed that TEM beta-lactamase conferred Am(r) and resistance to carboxypenicillins, acylureidopenicillins, cephalothin, cefamandole, cefsulodin, and cefoperazone. A total of 391 isolates were screened for plasmids, and 259 isolates were examined by DNA hybridization with a TEM probe. Among 462 plasmids probed, 129 plasmids, ranging from 4 to 140 megadaltons, harbored TEM sequences. Although beta-lactam resistance in clinical isolates of E. coli is predominantly mediated by TEM beta-lactamase, the diverse spectrum of resistance appears to be related to additional strain=dependent factors.     

Genotypic versus phenotypic characterization, with respect to beta-lactam susceptibility, of Haemophilus influenzae isolates exhibiting decreased susceptibility to beta-lactam resistance markers

Among 165 Spanish Haemophilus influenzae isolates with mutations in the ftsI gene (ftsI⁺) (2005 to 2007), 73% were β-lactamase negative and 26.7% were positive. The proportion of β-lactamase-negative isolates to β-lactamase-positive isolates was 2:1 to 4:1 in general, versus 1:3 in pediatric hospitals. Among 44 β-lactamase-positive strains, 8 strains produced ROB-1 (5 from the pediatric hospital). β-Lactamase-positive ftsI⁺ strains were phylogenetically closer than were β-lactamase-negative strains.     

Detection and reporting beta-lactam resistance phenotypes in Escherichia coli and Klebsiella pneumoniae: a multicenter proficiency study in Spain

The ability of 57 Spanish microbiology laboratories in detecting and reporting beta-lactam resistance phenotypes in Escherichia coli and Klebsiella pneumoniae was evaluated. Laboratories received 6 well-characterized isolates expressing the most widespread extended-spectrum beta-lactamases (ESBLs) in Spain (4 CTX-M type, 1 TEM type, and 1 SHV type), 3 isolates producing AmpC-type enzymes (2 plasmid mediated and 1 E. coli hyperproducing its chromosomal AmpC), and 3 quality control strains. Ninety-one percent of laboratories recognized all ESBL producers correctly, and therefore, low error rates were observed when testing cephalosporins and aztreonam. The highest error rates were observed with combinations of penicillin plus beta-lactamase inhibitor, although more than 60% of cases were due to the interpretation made by the microbiologists. Correct recognition of all AmpC beta-lactamase-producing strains occurred in only 47.4% of laboratories. These isolates were wrongly reported as ESBL producers and penicillinase hyperproducers in 7.6 % and 5.8% of cases, respectively. Detection of the AmpC-type phenotype by Spanish laboratories needs to be improved.     

Spanish multicenter study of the epidemiology and mechanisms of amoxicillin-clavulanate resistance in Escherichia coli

We conducted a prospective multicenter study in Spain to characterize the mechanisms of resistance to amoxicillin-clavulanate (AMC) in Escherichia coli. Up to 44 AMC-resistant E. coli isolates (MIC ≥ 32/16 μg/ml) were collected at each of the seven participant hospitals. Resistance mechanisms were characterized by PCR and sequencing. Molecular epidemiology was studied by pulsed-field gel electrophoresis (PFGE) and by multilocus sequence typing. Overall AMC resistance was 9.3%. The resistance mechanisms detected in the 257 AMC-resistant isolates were OXA-1 production (26.1%), hyperproduction of penicillinase (22.6%), production of plasmidic AmpC (19.5%), hyperproduction of chromosomic AmpC (18.3%), and production of inhibitor-resistant TEM (IRT) (17.5%). The IRTs identified were TEM-40 (33.3%), TEM-30 (28.9%), TEM-33 (11.1%), TEM-32 (4.4%), TEM-34 (4.4%), TEM-35 (2.2%), TEM-54 (2.2%), TEM-76 (2.2%), TEM-79 (2.2%), and the new TEM-185 (8.8%). By PFGE, a high degree of genetic diversity was observed although two well-defined clusters were detected in the OXA-1-producing isolates: the C1 cluster consisting of 19 phylogroup A/sequence type 88 [ST88] isolates and the C2 cluster consisting of 19 phylogroup B2/ST131 isolates (16 of them producing CTX-M-15). Each of the clusters was detected in six different hospitals. In total, 21.8% of the isolates were serotype O25b/phylogroup B2 (O25b/B2). AMC resistance in E. coli is widespread in Spain at the hospital and community levels. A high prevalence of OXA-1 was found. Although resistant isolates were genetically diverse, clonality was linked to OXA-1-producing isolates of the STs 88 and 131. Dissemination of IRTs was frequent, and the epidemic O25b/B2/ST131 clone carried many different mechanisms of AMC resistance.     

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.     

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。     

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.     

Chromosomal system for studying AmpC-mediated beta-lactam resistance mutation in Escherichia coli

In some enterobacterial pathogens, but not in Escherichia coli, loss-of-function mutations in the ampD gene are a common route to beta-lactam antibiotic resistance. We constructed an assay system for studying mechanism(s) of enterobacterial ampD mutation using the well-developed genetics of E. coli. We integrated the Enterobacter ampRC genes into the E. coli chromosome. These cells acquire spontaneous recombination- and SOS response-independent beta-lactam resistance mutations in ampD. This chromosomal system is useful for studying mutation mechanisms that promote antibiotic resistance.     

Tellurite susceptibility and non-plasmid-mediated resistance in Escherichia coli.

Tellurite (TeO3(2-)) is highly toxic toward Escherichia coli (MIC, approximately 1 microgram ml-1). Mutants (Tel) that were resistant to low levels of TeO3(2-) (MIC, approximately 10 micrograms ml-1) and collaterally resistant to arsenate were isolated. These Tel mutants were unable to grow on media containing low levels of Pi, which supported growth of the parent strain. When grown at much higher Pi levels they exhibited depressed levels of the outer membrane phoE protein and the periplasmic phoS protein, as well as several other proteins indicative of Pi starvation. Tel mutants were markedly defective in 32Pi transport, and TeO3(2-) was shown to be a potent competitive inhibitor of 32Pi transport in the parent strain. The Tel phenotype could be complemented by an F' plasmid harboring the phoR, phoB, and phoA loci, and curing of the F' plasmid completely restored TeO3(2-) resistance. Of a variety of well-characterized Pi transport mutants, only phoB mutants were equally resistant to TeO3(2-), and susceptibility could also be restored in strains carrying an F' plasmid for the phoB region and lost once more after F' curing. The tel and phoB loci were equally cotransducible with lac. Tel mutants still synthesized alkaline phosphatase, the phoA gene product, after Pi starvation, suggesting that the phoB locus per se was not involved because phoB is a positive regulatory gene for phoA expression. The results indicate that TeO3(2-) is transported into E. coli by a phosphate transport system and that resistance to TeO3(2-) specifically selects for as yet uncharacterized mutants in the phoB-phoA region of the chromosome.     

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。     

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.     

3-Dimensional susceptibility testing of beta-lactam antibiotics

A technique for demonstrating the substrate specificities of bacterial beta-lactamases is presented. Enzymatic degradation of beta-lactam antibiotics can be detected by cutting a wall of inoculum into the agar 3 mm from an antibiotic disc. Inactivation of the drug as it diffuses through the wall of inoculum results in distortion of the resulting inhibition zone. This procedure can be applied to the Stokes, Bauer-Kirby, International Collaborative Study (ICS) and Calibrated Dichotomous Sensitivity Test (CDS) methods so that they provide information about any detrimental effect of a pathogen on beta-lactam antibiotics, in addition to the effect the antibiotics have on the pathogen. The additional information generated by this simple procedure should increase the predictive value of in-vitro tests.