Genetic background of Escherichia coli and extended-spectrum beta-lactamase type.
ABSTRACT: To assess the implication of the genetic background of Escherichia coli strains in the emergence of extended-spectrum-Beta -lactamases (ESBL), 55 TEM-, 52 CTX-M-, and 22 SHV-type ESBL-producing clinical isolates involved in various extraintestinal infections or colonization were studied in terms of phylogenetic group, virulence factor (VF) content (pap, sfa/foc, hly, and aer genes), and fluoroquinolone resistance. A factorial analysis of correspondence showed that SHV type, and to a lesser extent TEM type, were preferentially observed in B2 phylogenetic group strains that exhibited numerous VFs but were fluoroquinolone-susceptible, whereas the newly emerged CTX-M type was associated with the D phylogenetic group strains that lacked VF but were fluoroquinolone-resistant. Thus, the emergence of ESBL-producing E. coli seems to be the result of complex interactions between the type of ESBL, genetic background of the strain, and selective pressures in ecologic niches.
Project description: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.
Project description:The accurate identification of Extended-Spectrum ?-Lactamase (ESBL) genes in Gram-negative bacteria is necessary for surveillance and epidemiological studies of transmission through foods. We report a novel rapid, cheap, and accurate closed tube molecular diagnostic tool based on two multiplex HRM protocols for analysis of the predominant ESBL families encountered in foods. The first multiplex PCR assay targeted blaCTX-M including phylogenetic groups 1 (CTX-M-1-15, including CTX-M-1, CTX-M-3 and CTX-M-15), 2 (CTX-M-2), and 9 (CTX-M-9-14, including CTX-M-9 and CTX-M-14). The second assay involved blaTEM /bla CTX-M /blaSHV, including TEM variants (TEM-1 and TEM-2), SHV-1-56 (SHV-1, SHV-2 and SHV-56), and CTX-M-8-41 (CTX-M-8, CTX-M-25, CTX-M-26 and CTX-M-39 to CTX-M-41). The individual melting curves were differentiated by a temperature shift according to the type of ESBL gene. The specificity and sensitivity of the first assay were 100% and 98%, respectively. For the second assay, the specificity and sensitivity were 87% and 89%, respectively. The detection of ESBL variants or mutations in existing genes was also demonstrated by the subtyping of a variant of the CTXM-1-15. The HRM is a potential tool for the rapid detection of present ?-lactamase genes and their characterization in a highly sensitive, closed-tube, inexpensive method that is applicable in high throughput studies.
Project description: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.
Project description:BACKGROUND: The impact of food animals as a possible reservoir for extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae, and the dissemination of such strains into the food production chain need to be assessed. In this study 334 fecal samples from pigs, cattle, chicken and sheep were investigated at slaughter. Additionally, 100 raw milk samples, representing bulk tank milk of 100 different dairy farms, 104 minced meat (pork and beef) samples and 67 E. coli isolates from cattle E. coli mastitis were analyzed. RESULTS: As many as 15.3% of the porcine, 13.7% of the bovine, 8.6% of the sheep and 63.4% of the chicken fecal samples yielded ESBL producers after an enrichment step. In contrast, none of the minced meat, none of the bulk tank milk samples and only one of the mastitis milk samples contained ESBL producing strains. Of the total of 91 isolates, 89 were E. coli, one was Citrobacter youngae and one was Enterobacter cloacae. PCR analysis revealed that 78 isolates (85.7%) produced CTX-M group 1 ESBLs while six isolates (6.6%) produced CTX-M group 9 enzymes. Five detected ESBLs (5.5%) belonged to the SHV group and 2 isolates (2.2%) contained a TEM-type enzyme. A total of 27 CTX-M producers were additionally PCR-positive for TEM-beta-lactamase. The ESBL-encoding genes of 53 isolates were sequenced of which 34 produced CTX-M-1, 6 produced CTX-M-14, 5 produced CTX-M-15 and also 5 produced SHV-12. Two isolates produced TEM-52 and one isolate expressed a novel CTX-M group 1 ESBL, CTX-M-117. One isolate--aside from a CTX-M ESBL-- contained an additional novel TEM-type broad-spectrum beta-lactamase, TEM-186. CONCLUSIONS: The relatively high rates of ESBL producers in food animals and the high genetic diversity among these isolates are worrisome and indicate an established reservoir in farm animals.
Project description:Three clinical Klebsiella pneumoniae strains, KpARG74, KpARG220 and KpARG185, isolated from a hospital in Algeria, carried the novel ?-lactamases SHV-98, SHV-99 and SHV-100, respectively, and co-expressed TEM-1 and either CTX-M-3 or CTX-M-15. In contrast, transformed cells possessing the genes for these novel ?-lactamases, i.e. EcDH5?-SHV-98, EcDH5?-SHV-99 and EcDH5?-SHV-100, respectively, carried unique sequence features of bla(SHV) gene variants, enabling oxyimino-cephalosporin susceptibility and confirming that none of the transformants exhibited extended-spectrum ?-lactamase (ESBL) properties. SHV-100 is apparently functional, despite differing from the SHV-1 sequence by duplication of 13 amino acids. The SHV-99 enzyme differed from the parental SHV-1 by the amino acid substitution Asp104?Gly, which is an important position in the development of the ESBL phenotype in TEM ?-lactamases. This is the first time, to our knowledge, that this mutation has been reported in clinically occurring isolates. Thus, kinetic characterization of the SHV-99 enzyme was performed. The SHV-99 enzyme showed higher affinity (K(m) of 196 µM), catalytic activity (k(cat) of 0.5 s?¹) and catalytic efficiency (k(cat)/K(m) of 0.003 µM?¹ s?¹) than SHV-1 ?-lactamase against aztreonam. These results showed that the neutral glycine at residue 104 increased the affinity of the enzyme to aztreonam, but was unable to develop the ESBL phenotype in SHV enzymes. As the emergence of new threatening combinations of resistance determinants among nosocomial pathogens is further possible, this study has highlighted the need to reverse the spread of initial mutations.
Project description:In 2010 the Clinical and Laboratory Standards Institute (CLSI) lowered the susceptibility breakpoints of some cephalosporins and aztreonam for Enterobacteriaceae and eliminated the need to perform screening for extended-spectrum ?-lactamases (ESBLs) and confirmatory tests. The aim of this study was to determine how many ESBL-producing strains of three common species of Enterobacteriaceae test susceptible using the new breakpoints. As determined with the CLSI screening and confirmatory tests, 382 consecutive ESBL-producing strains were collected at Huashan Hospital between 2007 and 2008, including 158 strains of Escherichia coli, 164 of Klebsiella pneumoniae, and 60 of Proteus mirabilis. Susceptibility was determined by the CLSI agar dilution method. CTX-M-, TEM-, and SHV-specific genes were determined by PCR amplification and sequencing. bla(CTX-M) genes alone or in combination with bla(SHV) were present in 92.7% (354/382) of these ESBL-producing strains. Forty-two (25.6%) strains of K. pneumoniae harbored SHV-type ESBLs alone or in combination. No TEM ESBLs were found. Utilizing the new breakpoints, all 382 strains were resistant to cefazolin, cefotaxime, and ceftriaxone, while 85.0 to 96.7% of P. mirabilis strains tested susceptible to ceftazidime, cefepime, and aztreonam, 41.8 to 45.6% of E. coli strains appeared to be susceptible to ceftazidime and cefepime, and 20.1% of K. pneumoniae were susceptible to cefepime. In conclusion, all ESBL-producing strains of Enterobacteriaceae would be reported to be resistant to cefazolin, cefotaxime, and ceftriaxone by using the new CLSI breakpoints, but a substantial number of ESBL-containing P. mirabilis and E. coli strains would be reported to be susceptible to ceftazidime, cefepime, and aztreonam, which is likely due to the high prevalence of CTX-M type ESBLs.
Project description:The contribution of integrons to the dissemination of extended-spectrum beta-lactamases (ESBL) was analyzed on all ESBL-producing Escherichia coli isolates from 1988 to 2000 at Ramon y Cajal Hospital. We studied 133 E. coli pulsed-field gel electrophoresis types: (i) 52 ESBL-producing clinical strains (C-ESBL) (16 TEM, 9 SHV, 21 CTX-M-9, 1 CTX-M-14, and 5 CTX-M-10); (ii) 43 non-ESBL blood clinical strains (C-nESBL); and (iii) 38 non-ESBL fecal isolates from healthy volunteers (V-nESBL). Class 1 integrons were more common among C-ESBL (67%) than among C-nESBL (40%) or V-nESBL (26%) (P < 0.001) due to the high number of strains with bla(CTX-M-9), which is linked to an In6-like class 1 integron. Without this bias, class 1 integron occurrence would be similar in C-ESBL and C-nESBL groups (47% versus 40%). Occurrence of class 2 integrons was similar among clinical and community isolates (13 to 18%). No isolates contained class 3 integrons. The relatively low rate of class 1 integrons within transferable elements carrying bla(TEM) (23%) or bla(SHV) (33%) and the absence of class 2 integrons in all ESBL transconjugants mirror the assembly of translocative pieces containing bla(TEM) or bla(SHV) on local available transferable elements lacking integrons. The low diversity of class 1 integrons (seven types recovered in all groups) might indicate a wide dissemination of specific genetic elements in which they are located. In our environment, the spread of genetic elements encoding ESBL has no major impact on the dispersion of integrons, nor do integrons have a major impact on the spread of ESBL, except when bla(ESBL) genes are within an integron platform such as bla(CTX-M-9).
Project description:A survey carried out in 2005 among members of a healthy population of children living in Bolivia and Peru revealed that fecal carriage of Escherichia coli strains resistant to expanded-spectrum cephalosporins was remarkably increased compared to that observed in the same settings in 2002 (1.7% in 2005 versus 0.1% in 2002). In this work, we demonstrated that this phenomenon was mainly related to the dissemination of CTX-M-type extended-spectrum beta-lactamase (ESBL) determinants among commensal E. coli strains. Of 50 ESBL-producing isolates collected in the 2005 survey, 44 harbored a CTX-M-type and 6 an SHV-type (SHV-2 or SHV-12) ESBL. Compared to 2002 results, an increased diversity of CTX-M-type ESBLs was also observed: members of the CTX-M-1 group (CTX-M-15) emerged in Bolivia (where only CTX-M-2 was observed in 2002), while members of the CTX-M-9 group (CTX-M-14 and CTX-M-24) emerged in Peru (where only CTX-M-15 and CTX-M-2 were observed in 2002). A new CTX-M-2 variant named CTX-M-56 was also detected. Molecular characterization of the CTX-M-producing isolates and gene transfer experiments suggested that different mechanisms could be involved in the spreading of different CTX-M group determinants and revealed that additional resistance determinants for non-beta-lactam antibiotics were preferentially carried by plasmids encoding certain CTX-M variants (CTX-M-15 and variants of the CTX-M-2 group). Three CTX-M-15-encoding conjugative plasmids from Peruvian isolates carried the new fluoroquinolone resistance gene aac(6')-Ib-cr. To our best knowledge, this is the first report of the detection of aac(6')-Ib-cr in Latin America.
Project description:Klebsiella pneumoniae is a major pathogen implicated in nosocomial infections. Extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae isolates are a public health concern. We aim to characterize the type of β-lactamases and the associated resistance mechanisms in ESBL-producing K. pneumoniae isolates obtained from blood cultures in a Portuguese hospital, as well as to determine the circulating clones. Twenty-two cefotaxime/ceftazidime-resistant (CTX/CAZR) K. pneumoniae isolates were included in the study. Identification was performed by MALDI-TOF MS and the antimicrobial susceptibility testing by disk-diffusion. The screening test for ESBL-production was performed and ESBL-producer isolates were further characterized. The presence of different beta-lactamase genes (blaCTX-M, blaSHV, blaTEM, blaKPC, blaNDM, blaVIM, blaOXA-48, blaCMY-2, blaDHA-1, blaFOX, blaMOX, and blaACC) was analyzed by PCR/sequencing in ESBL-producer isolates, as well as the presence of other resistance genes (aac(6')-Ib-cr, tetA/B, dfrA, qnrA/B/S, sul1/2/3) or integron-related genes (int1/2/3). Multilocus-sequence-typing (MLST) was performed for selected isolates. ESBL activity was detected in 12 of the 22 CTX/CAZR K. pneumoniae isolates and 11 of them carried the blaCTX-M-15 gene (together with blaTEM), and the remaining isolate carried the blaSHV-106 gene. All the blaCTX-M-15 harboring isolates also contained a blaSHV gene (blaSHV-1, blaSHV-11 or blaSHV-27 variants). Both blaSHV-27 and blaSHV-106 genes correspond to ESBL-variants. Two of the CTX-M-15 producing isolates carried a carbapenemase gene (blaKPC2/3 and blaOXA-48) and showed imipenem resistance. The majority of the ESBL-producing isolates carried the int1 gene, as well as sulphonamide-resistance genes (sul2 and/or sul3); the tetA gene was detected in all eight tetracycline-resistant isolates. Three different genetic lineages were found in selected isolates: ST348 (one CTX-M-15/TEM/SHV-27/KPC-2/3-producer isolate), ST11 (two CTX-M-15/TEM/SHV-1- and CTX-M-15-TEM-SHV-11-OXA-48-producer isolates) and ST15 (one SHV-106/TEM-producer isolate). ESBL enzymes of CTX-M-15 or SHV-type are detected among blood K. pneumoniae isolates, in some cases in association with carbapenemases of KPC or OXA-48 type.
Project description:Extended-spectrum beta-lactamases (ESBL) of the TEM, SHV, or CTX-M type confer resistance to beta-lactam antibiotics in gram-negative bacteria. The activity of these enzymes against beta-lactam antibiotics and their resistance against inhibitors can be influenced by genetic variation at the single-nucleotide level. Here, we describe the development and validation of an oligonucleotide microarray for the rapid identification of ESBLs in gram-negative bacteria by simultaneously genotyping bla(TEM), bla(SHV), and bla(CTX-M). The array consists of 618 probes that cover mutations responsible for 156 amino acid substitutions. As this comprises unprecedented genotyping coverage, the ESBL array has a high potential for epidemiological studies and infection control. With an assay time of 5 h, the ESBL microarray also could be an attractive option for the development of rapid antimicrobial resistance tests in the future. The validity of the DNA microarray was demonstrated with 60 blinded clinical isolates, which were collected during clinical routines. Fifty-eight of them were characterized phenotypically as ESBL producers. The chip was characterized with regard to its resolution, phenotype-genotype correlation, and ability to resolve mixed genotypes. ESBL phenotypes could be correctly ascribed to ESBL variants of bla(CTX-M) (76%), bla(SHV) (22%), or both (2%), whereas no ESBL variant of bla(TEM) was found. The most prevalent ESBLs identified were CTX-M-15 (57%) and SHV-12 (18%).