Ambler class A extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella spp. in Canadian hospitals

This report describes a study carried out to gain baseline information on the molecular characteristics of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella spp. in Canada. A total of 29,323 E. coli and 5,156 Klebsiella sp. isolates were screened at 12 participating sites. Of these, 505 clinically significant, nonrepeat isolates displaying reduced susceptibility to the NCCLS-recommended beta-lactams were submitted to a central laboratory over a 1-year period ending on 30 September 2000. A total of 116 isolates were confirmed to be ESBL producers. PCR and sequence analysis revealed the presence of TEM-11 (n = 1), TEM-12 (n = 1), TEM-29 (n = 1), TEM-52 (n = 4), CTX-M-13 (n = 1), CTX-M-14 (n = 15), CTX-M-15 (n = 11), SHV-2 (n = 2), SHV-2a (n = 12), SHV-5 (n = 6), SHV-12 (n = 45), and SHV-30 (n = 2). Five novel beta-lactamases were identified and designated TEM-115 (n = 2), TEM-120 (n = 1), SHV-40 (n = 2), SHV-41 (n = 4), and SHV-42 (n = 1). In addition, no molecular mechanism was identified for five isolates displaying an ESBL phenotype. Macrorestriction analysis of all ESBL isolates was conducted, as was restriction fragment length polymorphism analysis of plasmids harboring ESBLs. Although a "clonal" distribution of isolates was observed at some individual sites, there was very little evidence suggesting intrahospital spread. In addition, examples of identical or closely related plasmids that were identified at geographically distinct sites across Canada are given. However, there was considerable diversity with respect to plasmid types observed.     

Nationwide study of Escherichia coli and Klebsiella pneumoniae producing extended-spectrum beta-lactamases in Spain

Clonal dissemination of extended-spectrum beta-lactamases (ESBL) in 170 Escherichia coli isolates and 70 Klebsiella pneumoniae isolates from a nationwide study of 40 Spanish centers in 2000 was not observed in most centers. The most prevalent ESBL were CTX-M-9 (27.3%), SHV-12 (23.9%), and CTX-M-14 (20.5%) for E. coli and TEM-3 (16.7%) and TEM-4 (25%) for K. pneumoniae. A new ESBL, TEM-133, with mutations L21F, E104K, and R164S, was identified.     

Dissemination of SHV-12 and CTX-M-type extended-spectrum beta-lactamases among clinical isolates of Escherichia coli and Klebsiella pneumoniae and emergence of GES-3 in Korea

OBJECTIVES: To assess the prevalence and genotypes of Ambler class A extended-spectrum beta-lactamases (ESBLs) in Korea. METHODS: Clinical isolates of Escherichia coli and Klebsiella pneumoniae collected from 12 Korean hospitals during February-July 2003 were evaluated. Antimicrobial susceptibilities were determined by disc diffusion and agar dilution methods, and the putative ESBL-producing strains were tested by the double-disc synergy method. Detection of genes encoding class A beta-lactamases was performed by PCR amplification, and the PCR products were subjected to direct sequencing. RESULTS: The double-disc synergy test showed positive results in 9.3% (23/246) of E. coli and 23.0% (55/239) of K. pneumoniae isolates. The most prevalent types of Ambler class A ESBLs in E. coli isolates were CTX-M-15 (n = 4) and CTX-M-3 (n = 3), and those in K. pneumoniae isolates were SHV-12 (n = 30) and CTX-M-3 (n = 13). Two isolates produced both SHV-12 and GES-3, simultaneously. CONCLUSIONS: CTX-M-type and/or SHV-12 ESBL-producing E. coli and K. pneumoniae isolates are spreading, and a GES-type ESBL has emerged in Korea.     

CTX-M-2 and a new CTX-M-39 enzyme are the major extended-spectrum beta-lactamases in multiple Escherichia coli clones isolated in Tel Aviv, Israel

The rate of occurrence of the extended-spectrum beta-lactamase (ESBL)-producing phenotype among Escherichia coli isolates in Tel Aviv is 12% (22). The aim of this study was to understand the molecular epidemiology of E. coli ESBL producers and to identify the ESBL genes carried by them. We studied 20 single-patient ESBL-producing E. coli clinical isolates. They comprised 11 distinct nonrelated pulsed-field gel electrophoresis (PFGE) genotypes: six isolates belonged to the same PFGE clone, four other clones included two isolates each, and six unrelated clones included only one isolate. All isolates produced various beta-lactamases with pIs ranging from 5.2 to 8.2, varying within similar PFGE clones. The most prevalent ESBL gene was bla(CTX-M); 16 isolates carried bla(CTX-M-2) and three carried a new ESBL gene designated bla(CTX-M-39). Three strains carried bla(SHV) (two bla(SHV-12) and one bla(SHV-5)), and two strains carried inhibitor-resistant ESBL genes, bla(TEM-33) and bla(TEM-30); 18 strains carried bla(TEM-1) and eight strains carried bla(OXA-2). Plasmid mapping and Southern blot analysis with a CTX-M-2 probe demonstrated that bla(CTX-M-2) is plasmid borne. The wide dissemination of ESBLs among E. coli isolates in our institution is partly related to clonal spread, but more notably to various plasmid-associated ESBL genes, occurring in multiple clones, wherein the CTX-M gene family appears almost uniformly. We report here a new CTX-M gene, designated bla(CTX-M-39), which revealed 99% homology with bla(CTX-M-26), with a substitution of arginine for glutamine at position 225.     

产超广谱β-内酰胺酶的肺炎克雷伯菌相关耐药基因研究

目的了解常州地区肺炎克雷伯菌耐药状况及β-内酰胺类耐药基因。方法用琼脂稀释法检测氨苄西林、哌拉西林、哌拉西林-他唑巴坦、头孢噻肟、头孢他啶、头孢吡肟、氨曲南和亚胺培南对肺炎克雷伯菌的最低抑菌浓度（MIC）。采用聚合酶链反应（PCR）检测产超广谱β-内酰胺酶（ESBLs）肺炎克雷伯菌16种相关耐药基因,并用DNA测序仪测序。结果120株肺炎克雷伯菌ESBLs阳性率为44．2％（53株）。从53株肺炎克雷伯菌中检出TEM、SHV、CTX—M-1群、CTX—M-9群、OXA-1群、LEN、OKP和DHA8种β-内酰胺酶基因,阳性率分别为75．5％、22．6％、18．9％、7．5％、11．3％、5．7％、3．8％和41．5％,其中2株菌同时检出6种B-内酰胺酶基因。基因测序证实为TEM-1、TEM-11、SHV-13、SHV．28、CTX—M-22、CTX—M-55、OXA-1和OKP-6等。结论本地区肺炎克雷伯菌已携带多种β-内酰胺酶耐药基因,是其对β-内酰胺类抗菌药物耐药的重要原因之一。     

Characterization of extended-spectrum beta-lactamases in Salmonella spp. at a tertiary hospital in Durban, South Africa

Extended-spectrum beta-lactamases (ESBLs) were characterized in 41 Salmonella spp. isolates from patients admitted to a pediatric ward of a tertiary hospital in Durban, South Africa. The most common (17/ 41) serotype was Salmonella enterica serotype Typhimurium, followed by S. enterica serotype Isangi (16/41), S. enterica serotype Saint-paul (2/41), S. enterica serotype Kissi (2/41), S. enterica serotype Kivu (2/41), and S. enterica serotype Reading (1/41). All isolates were resistant to ampicillin, amoxicillin-clavulanate, piperacillin, ceftazidime, and aztreonam but susceptible to meropenem. SHV-12 found in 39% of the isolates was the most common ESBL. TEM-63 was produced in 29% and TEM-116 in 10% of the isolates, and TEM-131 was found in 1 isolate. Other ESBLs that were identified included SHV-2 (n = 2), CTX-M-3 (n = 1), CTX-M-15 (n = 2), and CTX-M-37 (n = 5). In addition, CMY-2 (n = 3) and the OXA-1(n = 1) beta-lactamase were also detected. The diversity of ESBLs suggests that its incidence in Salmonellae needs to be monitored.     

Extended-spectrum beta-lactamases in Klebsiella pneumoniae bloodstream isolates from seven countries: dominance and widespread prevalence of SHV- and CTX-M-type beta-lactamases

A huge variety of extended-spectrum beta-lactamases (ESBLs) have been detected during the last 20 years. The majority of these have been of the TEM or SHV lineage. We have assessed ESBLs occurring among a collection of 455 bloodstream isolates of Klebsiella pneumoniae, collected from 12 hospitals in seven countries. Multiple beta-lactamases were produced by isolates with phenotypic evidence of ESBL production (mean of 2.7 beta-lactamases per isolate; range, 1 to 5). SHV-type ESBLs were the most common ESBL, occurring in 67.1% (49 of 73) of isolates with phenotypic evidence of ESBL production. In contrast, TEM-type ESBLs (TEM-10 type, -12 type, -26 type, and -63 type) were found in just 16.4% (12 of 73) of isolates. The finding of TEM-10 type and TEM-12 type represents the first detection of a TEM-type ESBL in South America. PER (for Pseudomonas extended resistance)-type beta-lactamases were detected in five of the nine isolates from Turkey and were found with SHV-2-type and SHV-5-type ESBLs in two of the isolates. CTX-M-type ESBLs (bla(CTX-M-2) type and bla(CTX-M-3) type) were found in 23.3% (17 of 73) of isolates and were found in all study countries except for the United States. We also detected CTX-M-type ESBLs in four countries where they have previously not been described-Australia, Belgium, Turkey, and South Africa. The widespread emergence and proliferation of CTX-M-type ESBLs is particularly noteworthy and may have important implications for clinical microbiology laboratories and for physicians treating patients with serious K. pneumoniae infections.     

Frequency and diversity of Class A extended-spectrum beta-lactamases in hospitals of the Auvergne, France: a 2 year prospective study

OBJECTIVES: To evaluate the frequency and diversity of extended-spectrum beta-lactamases (ESBLs) produced by Enterobacteriaceae and Pseudomonas aeruginosa in one French region. METHODS: During 2001-2002, all the non-duplicate isolates of P. aeruginosa resistant to ceftazidime and of Enterobacteriaceae intermediate or resistant to ceftazidime and/or cefotaxime and/or aminoglycosides with an AAC(6') I phenotype were collected in nine hospitals of the area. ESBL isoelectric points were determined, bla genes were amplified and sequenced and epidemic isolates were genotyped with ERIC2-PCR. RESULTS: ESBLs were observed in 297 Enterobacteriaceae (0.8%). The most frequent were TEM-3 like (n=152; 51.2%) and TEM-24 (n=115; 38.7%). Four new enzymes were observed, TEM-112 (pI 5.4), TEM-113 (pI 6.3), TEM-114 (pI 5.9) and TEM-126 (pI 5.4). Other TEMs were TEM-8, TEM-12, TEM-16, TEM-19, TEM-20, TEM-21, TEM-29 and TEM-71. The other ESBLs were SHV-4, SHV-5 and SHV-12, CTX-M-1, CTX-M-3, CTX-M-14 and CTX-M-15. In 37 P. aeruginosa (0.7%) only one ESBL was observed, PER-1. Five epidemic strains were detected, Serratia marcescens TEM-3 and four observed in several hospitals, Enterobacter aerogenes TEM-24, Citrobacter koseri TEM-3, Proteus mirabilis TEM-3 and P. aeruginosa PER-1. CONCLUSION: ESBL frequency was lower than in 1998, and CTX-M-type frequency higher (2.1% of ESBLs in 2001, 4.9% in 2002). This long-term survey detected new sporadic enzymes (TEM-112, TEM-113, TEM-114 and TEM-126) and interhospital epidemic strains while avoiding any overestimation of ESBL frequency that may otherwise have occurred because of acute epidemics.     

Molecular survey of beta-lactamases conferring resistance to newer beta-lactams in Enterobacteriaceae isolates from Polish hospitals

The first national survey of resistance to newer beta-lactams in nosocomial populations of Enterobacteriaceae in Poland was performed. The study covered all nonrepetitive enterobacterial isolates cultured from specimens from inpatients in 13 regional secondary-care hospitals from November 2003 to January 2004. Among 2,388 isolates, the predominant species was Escherichia coli (59.6%), followed by Proteus mirabilis (14.5%) and Klebsiella spp. (8.5%). The frequency of extended-spectrum beta-lactamases (ESBLs) was very high, with ESBLs present in 11.1% of all isolates and 40.4% of Klebsiella pneumoniae isolates, the latter value greatly exceeding that for E. coli (2.5%). The contribution of outbreak isolates was significant, resulting, for example, in a particularly high rate of ESBL producers among Serratia marcescens isolates (70.8%). The pool of ESBL types was overwhelmingly dominated (81.7%) by CTX-M-like beta-lactamases CTX-M-3 (80.6%) and CTX-M-15, with SHV types (17.5%; SHV-2, SHV-5, and SHV-12) and sporadic TEM-like enzymes (0.7%; TEM-19 and TEM-48) being the next most frequent. Acquired AmpC-type cephalosporinases were observed exclusively in P. mirabilis, in 20.5% of the isolates of this species (compared with the frequency of ESBL producers of 11.5% of P. mirabilis isolates). All these cephalosporinases (CMY-12, CMY-15, and a novel variant, CMY-38) originated from Citrobacter freundii. Four isolates of E. coli (two isolates), K. pneumoniae (one isolate), and P. mirabilis (one isolate) produced class A inhibitor-resistant beta-lactamases (TEM-30, TEM-32, TEM-37, and SHV-49), being the first of such producers identified in Poland. The survey documented both specific and more global characteristics of the epidemiology of the beta-lactamase-mediated resistance in enterobacteria from Polish hospitals and demonstrated that the ESBL frequency has reached an alarming level.     

Prevalence of clinical isolates of Escherichia coli and Klebsiella spp. producing multiple extended-spectrum beta-lactamases

Eleven thousand two hundred seventy-two Escherichia coli, 1109 Klebsiella pneumoniae, 1124 Salmonella enterica, and 602 Klebsiella oxytoca unrelated clinical isolates were obtained between 2001 and 2004 in a university hospital in Salamanca, Spain. One hundred thirteen E. coli (1%), 32 K. pneumoniae (2.9%), 4 K. oxytoca (0.66%), and 5 S. enterica (0.44%) isolates produced extended-spectrum beta-lactamases (ESBLs). We obtained 42.2% of the ESBL-producing isolates from outpatients and 57.8% from inpatients. The most commonly detected ESBLs were CTX-M 14 (43.5% of ESBL-producing isolates), TEM-116 (22.1%), and SHV-2 (15.6%). A CTX-M 27-producing E. coli is 1st reported in Spain in this study. Two (20 isolates, 13%) or 3 (7 isolates, 4.5%) ESBLs were produced by 17.5% of ESBL-producing isolates (27 isolates). The most frequent combinations were CTX-M 14 + TEM-116 (5.7%), SHV-12 + TEM-116 (2.6%), and SHV-2 + CTX-M 14 + TEM-116 (2.6%). Clonal diversity was high even between isolates producing the same combinations of 2 or 3 beta-lactamases.     

Distribution of extended-spectrum beta-lactamases in clinical isolates of Enterobacteriaceae in Vietnam

Among 730 Escherichia coli, 438 Klebsiella pneumoniae, and 141 Proteus mirabilis isolates obtained between September 2000 and September 2001 in seven hospitals in Ho Chi Minh City, Vietnam, 26.6% were resistant to ceftazidime, 30% were resistant to cefotaxime, 31.5% were resistant to ceftriaxone, 15.9% were resistant to cefoperazone, and 6% were resistant to cefepime. Resistance to imipenem was found in 5.6% of the isolates. In 55 strains producing extended-spectrum beta-lactamases (32 E. coli isolates, 13 K. pneumoniae isolates, and 10 P. mirabilis isolates), structural genes for VEB-1 (25.5%), CTX-M (25.5%), SHV (38.1%), and TEM (76.3%) enzymes were detected alone or in combination. Sequencing of the PCR products obtained from the K. pneumoniae isolates revealed the presence of bla(VEB-1), bla(CTX-M-14), bla(CTX-M-17), bla(SHV-2), and bla(TEM-1). Molecular typing of the strains with a similar resistance phenotype to broad-spectrum cephalosporins indicated polyclonal spread. ISEcp1 was presumably responsible for dissemination of the bla(CTX-M-like) gene.     

High diversity of extended-spectrum beta-lactamases among clinical isolates of Enterobacteriaceae from Portugal

OBJECTIVES: To investigate the occurrence and the diversity of Ambler class A ESBLs among Enterobacteriaceae from different Portuguese clinical settings over a 2 year period (2002-04). METHODS: One hundred and nine extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae isolates from five geographically distant health institutions in Portugal were studied. ESBLs were characterized by isoelectric focussing, PCR and further sequencing. Antibiotic susceptibility testing, transfer of resistance genes and clonal diversity were determined by standard procedures. Plasmid relatedness was established by comparison of random amplified polymorphic DNA (RAPD) patterns. RESULTS: ESBLs were identified as TEM (46%), SHV (30%), CTX-M (22%) and GES (2%) types; TEM-24, TEM-52, SHV-12 and CTX-M-15 enzymes being the most frequently found. Inter-hospital dissemination of epidemic strains harbouring the most prevalent ESBLs was detected, including the TEM-24-producing Enterobacter aerogenes European epidemic clone. Conjugative transfer of ESBLs was achieved for 67% of isolates and epidemic plasmids containing specific bla genes were detected (bla(CTX-M-15) and bla(TEM-24)). We describe two new ESBLs, SHV-90 (A187T, G238S and E240K) and SHV-91 (P20S and E240K), and a new TEM-type enzyme conferring a phenotype resembling that of a complex mutant TEM beta-lactamase, designated as TEM-154 (M69L and R164S). The broad-spectrum beta-lactamases SHV-26, SHV-36 and TEM-110 were first observed in our country. CONCLUSIONS: We describe a complex ESBL epidemiology in Portugal, including widespread dissemination of known strains and plasmids coding for TEM-24 and CTX-M-15 enzymes as observed in other European countries.     

Mechanisms of resistance to expanded-spectrum cephalosporins in Escherichia coli isolates recovered in a Spanish hospital

OBJECTIVES: To characterize the beta-lactamase genes of the expanded-spectrum cephalosporin-resistant Escherichia coli isolates recovered in a Spanish hospital during the March 2002-March 2003 period. METHODS: Thirty-four of the 1700 E. coli isolates recovered from unrelated patients in a Spanish hospital showed expanded-spectrum cephalosporin resistance. The presence of genes encoding TEM, SHV, CTX-M, CMY-2-type or FOX beta-lactamases as well as the existence of mutations in the regulatory region of the chromosomal ampC gene were studied by PCR and sequencing in these 34 E. coli isolates. RESULTS: The following extended-spectrum beta-lactamases (ESBLs) or plasmidic class C beta-lactamase genes were detected (number of isolates): bla(CTX-M-14) (14), bla(CTX-M-9) (4), bla(CTX-M-32) (1), bla(TEM-52) (2), bla(SHV-12) (3) and bla(CMY-2) (2). The remaining eight isolates showed a mutation in the promoter/attenuator region of the ampC chromosomal gene at position -42, in combination with mutations at positions -18, -1 and +58. The bla(TEM-1) gene was also detected in 12 of the ESBL-producing isolates, in both CMY-2-producing isolates and in four of the eight isolates that showed a mutation at position -42 of the ampC promoter. Other mutations in the promoter/attenuator region were detected in association with ESBL or CMY-2 genes, such as the combination -18, -1 and +58, -28 and +58, or +22, +26, +27 and +32. No clonal relationship was found among the CTX-M-producing E. coli isolates by PFGE with XbaI enzyme. CONCLUSIONS: Approximately 1.5% of the E. coli isolates of our hospital harboured ESBL genes, those of the CTX-M-9 group being the most common ones.     

Extended-spectrum beta-lactamase-producing Enterobacteriaceae in community and private health care centers

In 1999, 39 of 2,599 isolates of the family Enterobacteriaceae (1.5%) collected by eight private laboratories in the Aquitaine region in France produced an extended-spectrum beta-lactamase (ESBL). Among these were 19 Enterobacter aerogenes isolates; 8 Klebsiella pneumoniae isolates; 6 Escherichia coli isolates; 3 Proteus mirabilis isolates; and 1 isolate each of Serratia marcescens, Morganella morganii, and Providencia stuartii. ESBL producers were isolated from 38 patients, including 33 residents of 11 clinics or nursing homes and 5 ambulatory patients. Seven different ESBLs were characterized. These mainly consisted of TEM-24 (25 isolates) and TEM-21 (9 isolates), but TEM-15 (2 isolates) and TEM-3, TEM-19, SHV-4, and CTX-M-1 (1 isolate each) were also characterized. Seven strains showed the coexistence of different TEM- and/or SHV-encoding genes, including a new SHV-1 variant, SHV-44, defined by the substitution R205L previously reported for SHV-3 in association with S238G. The epidemiology of the ESBL producers was investigated by random amplification of polymorphic DNA, typing by enterobacterial repetitive intergenic consensus PCR, analysis of resistance cotransferred with the ESBL, and analysis of the restriction profiles of the ESBL-encoding plasmids. Of the TEM-24-expressing strains, 18 were E. aerogenes isolates, including 9 from the same clinic, that were representatives of the epidemic clone disseminating in France. Of the TEM-21-producing strains that belonged to different species of the family Enterobacteriaceae (E. coli, K. pneumoniae, and P. mirabilis), 8 were isolated in the same nursing home. Outbreaks due to strain and/or plasmid dissemination in these clinic and nursing home were demonstrated. The presence of ESBL producers in five ambulatory patients probably resulted from nosocomial acquisition. Our data highlight the serious need to monitor patients for ESBL-producing Enterobacteriaceae in general practice.     

Extended-spectrum beta-lactamases among Enterobacter isolates obtained in Tel Aviv, Israel

The extended-spectrum beta-lactamase (ESBL)-producing phenotype is frequent among Enterobacter isolates at the Tel Aviv Sourasky Medical Center, Tel Aviv, Israel. We examined the clonal relatedness and characterized the ESBLs of a collection of these strains. Clonal relatedness was determined by pulsed-field gel electrophoresis. Isoelectric focusing (IEF) and transconjugation experiments were performed. ESBL gene families were screened by colony hybridization and PCR for bla(TEM), bla(SHV), bla(CTX-M), bla(IBC), bla(PER), bla(OXA), bla(VEB), and bla(SFO); and the PCR products were sequenced. The 17 Enterobacter isolates studied comprised 15 distinct genotypes. All isolates showed at least one IEF band (range, one to five bands) whose appearance was suppressed by addition of clavulanate; pIs ranged from 5.4 to > or = 8.2. Colony hybridization identified at least one family of beta-lactamase genes in 11 isolates: 10 harbored bla(TEM) and 9 harbored bla(SHV). PCR screening and sequence analysis of the PCR products for bla(TEM), bla(SHV), and bla(CTX-M) identified TEM-1 in 11 isolates, SHV-12 in 7 isolates, SHV-1 in 1 isolate, a CTX-M-2-like gene in 2 isolates, and CTX-M-26 in 1 isolate. In transconjugation experiments with four isolates harboring bla(TEM-1) and bla(SHV-12), both genes were simultaneously transferred to the recipient strain Escherichia coli HB101. Plasmid mapping, PCR, and Southern analysis with TEM- and SHV-specific probes demonstrated that a single transferred plasmid carried both the TEM-1 and the SHV-12 genes. The widespread presence of ESBLs among Enterobacter isolates in Tel Aviv is likely due not to clonal spread but, rather, to plasmid-mediated transfer, at times simultaneously, of genes encoding several types of enzymes. The dominant ESBL identified was SHV-12.     

Monitoring and characterization of extended-spectrum beta-lactamases in Escherichia coli strains from healthy and sick animals in Spain in 2003

Genes encoding CTX-M-14, CTX-M-9, CTX-M-1, CTX-M-32, SHV-12, TEM-52, or CMY-2 beta-lactamases were detected in 21 Escherichia coli strains recovered during 2003 from sick animals (11 of 459 [2.4%] strains) and healthy animals (10 of 158 [6.3%] strains) in Spain. Twelve of these strains harbored bla(CTX-M) genes and showed unrelated pulsed-field gel electrophoresis patterns.     

Beta-lactamases in ampicillin-resistant Escherichia coli isolates from foods,humans, and healthy animals

TEM-, SHV-, and OXA-type beta-lactamases were studied by PCR with 124 ampicillin-resistant (AMP(r)) Escherichia coli isolates recovered from foods of animal origin (n = 20) and feces of humans (n = 49) and healthy animals (n = 55). PCR showed that 103 isolates were positive for TEM and negative for SHV and OXA. Three E. coli isolates showed a positive reaction for OXA, and one showed a positive reaction for SHV. The remaining 17 E. coli isolates were negative for the three enzymes by PCR. Fifty-seven of the 103 bla(TEM) amplicons were sequenced. Different molecular variants of bla(TEM-1) were found in 52 isolates: bla(TEM-1a) (n = 9), bla(TEM-1b) (n = 36), bla(TEM-1c) (n = 6), and bla(TEM-1f) (n = 1). Four inhibitor-resistant TEM (IRT) beta-lactamase-encoding genes were also detected: bla(TEM-30c) (IRT-2), bla(TEM-34b) (IRT-6), bla(TEM-40b) (IRT-11), and bla(TEM-51a) (IRT-15). A new bla(TEM) gene, named bla(TEM-95b), which showed a mutation in amino acid 145 (P-->A) was detected. It was found in a food isolate of chicken origin (AMP(r), amoxicillin-clavulanic acid susceptible). The promoter region in 24 bla(TEM) amplicons was analyzed, and the weak P3 promoter was found in 23 of them (bla(TEM-1) in 20 amplicons and bla(TEM-51a), bla(TEM-30c), and bla(TEM-95b) in 1 amplicon each). The strong Pa/Pb promoter was found only in the bla(TEM-34b) gene. No extended-spectrum beta-lactamases were detected. Mutations at position -42 or -32 in the ampC gene promoter were demonstrated in 4 of 10 E. coli isolates for which the cefoxitin MIC was >/=16 micro g/ml. Different variants of bla(TEM-1) and IRT bla(TEM) genes were found among the AMP(r) E. coli isolates from foods and the feces of humans and healthy animals, and a new gene, bla(TEM-95b) (P3), was detected.     

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.     

Incidence of class A extended-spectrum beta-lactamases in Champagne-Ardenne (France): a 1 year prospective study

OBJECTIVES: To assess the frequency and diversity of extended spectrum beta-lactamases (ESBLs) in the Champagne-Ardenne region France, and to identify genetic elements associated with the bla(CTX-M) genes. METHODS: During 2004, all the non-duplicate isolates of Pseudomonas aeruginosa and Acinetobacter baumannii resistant to ceftazidime and of Enterobacteriaceae intermediate or resistant to ceftazidime and/or cefotaxime, screening samples excluded, were collected in 10 public hospitals and 3 private clinics. bla genes were sequenced and bla(CTX-M) environment characterized by PCR mapping. RESULTS: In Enterobacteriaceae (138/21 861; 0.6%), ESBLs were predominantly TEM-24 (n = 52; 37.7%) and CTX-M-15 (n = 37; 26.8%). Three new enzymes were identified, CTX-M-61 (CTX-M-1 group), TEM- and SHV-type. A. baumannii (n = 5) produced VEB-1 and P. aeruginosa (n = 2) SHV-2a. ISEcp1 was detected in 22/27 strains, disrupted in 7 of them. The IS903-like element was downstream of bla(CTX-M-14) and bla(CTX-M-16). ISCR1 was found upstream of bla(CTX-M-2) and bla(CTX-M-9), and ISCR1 and bla(CTX-M-2) were located on a sul1-type class 1 integron. In comparison with 2001-02, ESBL distribution among Enterobacteriaceae showed an increase in CTX-M-type (44.9% vs 3.7% P < 10(-7)) due to Escherichia coli CTX-M-15 and to the almost total disappearance of TEM-3 (0.9% vs 51.2%). E. coli was the most frequent species (50.0% vs 5.1% in 1998) despite a similar prevalence to that in 1998 (0.5% vs 0.2%). CONCLUSIONS: A careful detection of bla(CTX-M)-type spread to other species would help to anticipate clonal endemics such as those observed in Enterobacter aerogenes TEM-24.     

Predominance and genetic diversity of community- and hospital-acquired CTX-M extended-spectrum beta-lactamases in York, UK

OBJECTIVES: This study was conducted to detect the presence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae within the faecal flora of both community- and hospital-based patients in York and to characterize the bla(TEM), bla(SHV) and bla(CTX-M) genes present in these isolates. METHODS: One thousand faeces samples were collected and screened at York Hospital during October-December 2003. Ninety-five non-duplicate Enterobacteriaceae isolates resistant to third-generation cephalosporins were recovered; 22 isolates were selected for further study on the basis of a positive double disc diffusion test for ESBL production. Antibiotic susceptibility testing was performed to a range of antibiotics. The TEM, SHV and CTX-M genes were detected by PCR and the DNA sequenced. RESULTS: The distribution of ESBL-positive isolates from the hospital and community was 1.4:1. These included nine Escherichia coli, seven Enterobacter cloacae, four Citrobacter freundii and a single isolate each of Klebsiella spp. and Salmonella spp. A total of 17 isolates contained bla(CTX-M) (five bla(CTX-M-15), three bla(CTX-M-14) and nine bla(CTX-M-9)). ISEcp1 was present in isolates expressing CTX-M-14 and -15, but was absent upstream of In60-associated bla(CTX-M-9). E. coli isolates also contained either a bla(TEM-1) or bla(TEM-2), whereas six of the E. cloacae carried bla(SHV-12) and the Klebsiella spp. bla(SHV-36) in addition to bla(CTX-M-9). The single Salmonella spp. carried bla(SHV-12). CONCLUSIONS: The overall prevalence of ESBL in isolates of Enterobacteriaceae from York was 1.9%. ESBL-producing isolates were found in both the community and hospital, with the CTX-M type most common. This is also the first report of an ESBL-producing Salmonella in the UK.