Characterization of extended-spectrum β-lactamases produced by Escherichia coli isolated from hospitalized and nonhospitalized patients : emergence of CTX-M-15-producing strains causing urinary tract infections

Extended-spectrum β-lactamase-producing Escherichia coli isolates were obtained from hospitalised and non-hospitalised patients in Belgium between August 2006 and November 2007. The antimicrobial susceptibility of these isolates was determined and their ESBL genes were characterized. Clonal relationships between the CTX-M-producing E. coli isolates causing urinary tract infections were also studied. A total of 90 hospital- and 45 community-acquired cephalosporin-resistant E. coli isolates were obtained. Tetracycline, enrofloxacine, gentamicin and trimethoprim-sulfamethaxozole resistance rates were significantly different between the community-onset and hospital-acquired isolates. A high diversity of different ESBLs was observed among the hospital-acquired E. coli isolates whereas CTX-M-15 was dominating among the community-acquired E. coli isolates (n=28). Thirtheen different PFGE profiles were observed in the community-acquired CTX-M-15-producing E. coli indicating that multiple clones have acquired the blaCTX-M-15 gene. All community-acquired CTX-M-15-producing E. coli isolates of phylogroups B2 and D were assigned to the sequence type ST131. The hospital-acquired CTX-M-15-producing E. coli isolates of phylogroups B2, B1, A and D corresponded to ST131, ST617, ST48 and ST405, respectively. In conclusion, CTX-M-type ESBLs have emerged as the predominant class of ESBLs produced by E. coli isolates in the hospital and community in Belgium. Of particular concern is the predominant presence of the CTX-M-15 enzyme in ST131 community-acquired E. coli

Characterization of two new CTX-M-25-group extended-spectrum β-lactamase variants identified in Escherichia coli isolates from Israel.

OBJECTIVES: We characterized two new CTX-M-type extended-spectrum β-lactamase (ESBL) variants in Escherichia coli isolates from stool samples of two elderly patients admitted at the Tel Aviv Sourasky Medical Center, Israel. Both patients underwent treatment with cephalosporins prior to isolation of the E. coli strains. METHODS: ESBLs were detected by the double-disk synergy test and PCR-sequencing of β-lactamase genes. The bla(CTX-M) genes were cloned into the pCR-BluntII-TOPO vector in E. coli TOP10. The role of amino-acid substitutions V77A and D240G was analyzed by site-directed mutagenesis of the bla(CTX-M-94) and bla(CTX-M-100) genes and comparative characterization of the resulting E. coli recombinants. MICs of β-lactams were determined by Etest. Plasmid profiling, mating experiments, replicon typing and sequencing of bla(CTX-M) flanking regions were performed to identify the genetic background of the new CTX-M variants. RESULTS: The novel CTX-M β-lactamases, CTX-M-94 and -100, belonged to the CTX-M-25-group. Both variants differed from CTX-M-25 by the substitution V77A, and from CTX-M-39 by D240G. CTX-M-94 differed from all CTX-M-25-group enzymes by the substitution F119L. Glycine-240 was associated with reduced susceptibility to ceftazidime and leucine-119 with increased resistance to ceftriaxone. bla(CTX-M-94) and bla(CTX-M-100) were located within ISEcp1 transposition units inserted into ∼93 kb non-conjugative IncFI and ∼130 kb conjugative IncA/C plasmids, respectively. The plasmids carried also different class 1 integrons. CONCLUSIONS: This is the first report on CTX-M-94 and -100 ESBLs, novel members of the CTX-M-25-group

CTX-M-14 and CTX-M-15 enzymes are the dominant type of extended-spectrum β-lactamase in clinical isolates of Escherichia coli from Korea

This study was performed to assess the prevalence and genotypes of plasmid-borne extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases in Escherichia coli in Korea. A total of 576 isolates of E. coli was collected from 12 Korean hospitals during May and July 2007. A phenotypic confirmatory test detected ESBLs in 82 (14.2 %) of the 576 E. coli isolates. The most common types of ESBLs identified were CTX-M-14 (n=32) and CTX-M-15 (n=27). The prevalence and diversity of the CTX-M mutants, including CTX-M-15, CTX-M-27 and CTX-M-57, with significant hydrolytic activity against ceftazidime were increased. PCR experiments detected genes encoding plasmid-borne AmpC β-lactamases in 15/56 cefoxitin-intermediate or cefoxitin-resistant isolates, and the most common type of AmpC β-lactamase identified was DHA-1 (n=10). These data suggest that the incidence of ESBLs in E. coli has increased as a result of the dissemination of CTX-M enzymes in Korea. In addition, CTX-M-22, CTX-M-27 and CTX-M-57 have appeared in Korea

Characterization of plasmid-mediated beta-lactamases in fecal colonizing patients in the hospital and community setting in Spain

Aim: Active surveillance of plasmid-mediated ß-lactamase-producing Enterobacteriaceae (PMBL-E) in fecal carriers in the hospital and in the community setting in a non-outbreak period of time. Methods: Patients were screened for carriage of Enterobacteriaceae resistant to expanded-spectrum cephalosporins and PMBL-E were characterized (extended-spectrum-ß-lactamase [ESBL], plasmid-mediated AmpC ß-lactamase [pAmpC], and carbapenemases) by PCR and sequencing. Results: The prevalence of ESBL and pAmpC carriers was 5.06% and 0.59%, respectively. Overall, CTX-M-like enzymes were the ESBL dominate enzymes (96.15%). The group CTX-M-9 was the most prevalent (81, 54%) [CTX-M-14 (74, 91.35%), CTX-M-9 (5, 6.17%), CTX-M-24 (1, 1.23%), and CTX-M-27 (1, 1.23%)] followed by the group CTX-M-1 (64, 42.67%) [CTX-M-15 (42, 65.63%), CTX-M-1 (13, 20.31%), CTX-M-32 (8, 12.5%), and CTX-M-3 (1, 1.56%)]. One CTX-M-10, one CTX-M-59, and three CTX-M-8 were also found. A very small representation of SHV or TEM ESBL enzymes was found (3.2% and 0.64%, respectively). pAmpC characterization revealed a predominance of CMY-2 (81.25%), followed by DHA-1 (18.75%). We did not detect the presence of carbapenemase producers. Conclusions: The prevalence of ESBL-producers from fecal carriers is stable in our area, but colonization by pAmpC producers has emerged recently as we have confirmed. Periodic active surveillance is useful to identify these human reservoirs and control the evolution of PMBL carriage in a community over time

New CTX‐M group conferring β‐lactam resistance: a compendium of phylogenetic insights from biochemical, molecular, and structural biology

The production of extended‐spectrum β‐lactamases (ESBLs) is the main defense mechanism found in Gram negative bacteria. Among all the ESBLs, the CTX‐M enzymes appear as the most efficient in terms of dissemination in different epidemiological contexts. CTX‐M enzymes exhibit a striking plas-ticity, with a large number of allelic variants distributed in several sublineages, which can be associated with functional heterogeneity of clinical relevance. This observational analytical study provides an update of this family, currently with more than 200 variants described, from a phylogenetic, molecular, and structural point of view through homology in amino acid sequences. Our data, combined with described literature, provide phylogenetic and structural evidence of a new group. Thus, herein, we propose six groups among CTX‐M enzymes: the already stablished CTX‐M‐1, CTX‐M‐2, CTX‐M‐8, CTX‐M‐9, and CTX‐M‐25 clusters, as well as CTX‐M‐151 as the new cluster.info:eu-repo/semantics/publishedVersio

Molecular Characterization of Extended-Spectrum Beta-Lactamases in Escherichia coli and Klebsiella pneumoniae in Accra, Ghana

Extended-spectrum beta-lactamases (ESBLs) are plasmid-mediated beta lactamases that are capable of hydrolysing beta-lactams except carbapenems and cephamycins. The ESBL types include SHV, TEM and CTX-M, OXA, PER and VEB-1. The most common ones isolated from clinical specimen are the CTX-M, SHV and TEM.  The specific ESBL-producing organisms have different genetic characteristics which mark their identification at the molecular level. This work sought to determine the genetic characterization of ESBL-producing K. pneumoniae and E. coli in Accra. The molecular investigations of the ESBL-coding genes included extraction of 100 DNA templates of phenotypic ESBL-producing isolates by boiling method, preparation of the PCR reaction mixture using appropriate primers, standard PCR reaction in a thermocycler,   agarose gel electrophoresis, bands visualization by ultraviolet trans-illumination and bands photography using a Kodak EDAS 290 gel documentation system. The results significantly (p<0.05) indicated that of the 100 ESBL producers, 90(90%) possess CTX-M genes and 25(25%) had TEM genes. None of the ESBL producers possesses SHV genes. Seventy (70%) of the ESBL producers possess only CTX-M genes and 5(5%) had only TEM genes. Twenty (20%) of the isolates had both CTX-M and TEM genes. Of the 100 ESBL phenotypes, 78(78%) and 2(2%) were positive for CTX-M-1group and CTX-M-9group ESBL genes respectively. Organisms producing CTX-M-type ESBL are more prevalent in Accra than other ESBL types. CTX-M-1group producing isolates dominated the ESBL phenotypes with CTX-M-15 likely to be the dominate CTX-M-type ESBL. There is the need for further studies into the characteristic transmission, pathogenesis, antibiotic resistance expression, and infection control of CTX-M-type ESBL and TEM-type ESBL in Accra. Keywords: Extended spectrum beta-lactamase, CTX-M genes, TEM genes, SHV genes, Molecula

Molecular epidemiology of CTX-M-producing escherichia coli isolates at a tertiary medical center in western pennsylvania

A combination of phenotypic and genotypic methods was used to investigate 70 unique Escherichia coli clinical isolates identified as producing extended-spectrum beta-lactamases (ESBLs) at a medical center in Pittsburgh, PA, between 2007 and 2008. Fifty-seven isolates (81%) produced CTX-M-type ESBLs, among which CTX-M-15 was predominant (n = 46). Isolates producing CTX-M-2, -9, -14, and -65 were also identified. One CTX-M-producing isolate coproduced CMY-2 cephalosporinase. Ten isolates (14%) produced SHV-type ESBLs, either SHV-5 or SHV-7. Two isolates produced only CMY-2 or -32. Pulsed-field gel electrophoresis revealed the presence of two major clusters of CTX-M-15-producing E. coli isolates, one in phylotype B2 (n = 15) and the other in phylotype A (n = 19). Of four phylotype B2 isolates that were able to transfer the bla(CTX-M-15)-carrying plasmids, three showed fingerprints related (>60%) to those of plasmids from phylotype A isolates. In phylotype B2, all CTX-M-15-producing isolates, as well as three isolates producing CTX-M-14, two producing SHV-5, and one producing SHV-7, belonged to the international epidemic clone defined by serotype O25:H4 and sequence type 131. The plasmids from eight of nine CTX-M-15-producing E. coli isolates of phylotype A that were examined were highly related to each other and were also found in two isolates belonging to phylotype D, suggesting horizontal transfer of this bla(CTX-M-15)-carrying plasmid between phylotypes. Our findings underscore the need to further investigate the epidemiology and virulence of CTX-M-producing E. coli in the United States

CTX-M Enzymes: Origin and Diffusion

CTX-M β-lactamases are considered a paradigm in the evolution of a resistance mechanism. Incorporation of different chromosomal blaCTX-M related genes from different species of Kluyvera has derived in different CTX-M clusters. In silico analyses have shown that this event has occurred at least nine times; in CTX-M-1 cluster (3), CTX-M-2 and CTX-M-9 clusters (2 each), and CTX-M-8 and CTX-M-25 clusters (1 each). This has been mainly produced by the participation of genetic mobilization units such as insertion sequences (ISEcp1 or ISCR1) and the later incorporation in hierarchical structures associated with multifaceted genetic structures including complex class 1 integrons and transposons. The capture of these blaCTX-M genes from the environment by highly mobilizable structures could have been a random event. Moreover, after incorporation within these structures, β-lactam selective force such as that exerted by cefotaxime and ceftazidime has fueled mutational events underscoring diversification of different clusters. Nevertheless, more variants of CTX-M enzymes, including those not inhibited by β-lactamase inhibitors such as clavulanic acid (IR-CTX-M variants), only obtained under in in vitro experiments, are still waiting to emerge in the clinical setting. Penetration and the later global spread of CTX-M producing organisms have been produced with the participation of the so-called “epidemic resistance plasmids” often carried in multi-drug resistant and virulent high-risk clones. All these facts but also the incorporation and co-selection of emerging resistance determinants within CTX-M producing bacteria, such as those encoding carbapenemases, depict the currently complex pandemic scenario of multi-drug resistant isolates

Nationwide Survey of Klebsiella Pneumoniae Strains Producing CTX-M Extended-spectrum b-lactamases in Croatia

Extended-spectrum β-lactamases (ESBL) producing bacteria have been increasingly reported in both hospital and community patients. Production of ESBLs is the major mechanism of resistance to oxymino-cephalosporins and aztreonam in Gram-negative bacteria 1,2. Recently a new family of ESBLs with predominant activity against cefotaxime (CTX-M β-lactamases) has been reported. Over 80 CTX-M enzymes have been described so far, which can be grouped into five main subgroups according to amino acid sequence identity (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25) 3 . In some countries, CTX-M β-lactamases are the most prevalent types of ESBLs, for instance in Russia 4, Greece 5 , Spain 6 , Switzerland 7, Japan 8, Taiwan 9, China 10 and Argentina 11 . These enzymes have been identified in countries near Croatia such is Italy 12, Hungary13 and Austria14 The aim of this study was to determine the prevalence and the types of CTX-M β lactamases produced by Klebsiella pneumoniae clinical isolates collected from October 2006 to January 2007 from both community- and hospital –based isolates were included (Figure 1.). 128 ESBL isolates were subjected to further analysis: screening with double disc diffusion test and confirmed by ESBL E test 15

Boronic Acid Transition State Inhibitors as Potent Inactivators of KPC and CTX-M β-Lactamases: Biochemical and Structural Analyses

Design of novel beta-lactamase inhibitors (BLIs) is one of the currently accepted strategies to combat the threat of cephalosporin and carbapenem resistance in Gram-negative bacteria. Boronic acid transition state inhibitors (BATSIs) are competitive, reversible BLIs that offer promise as novel therapeutic agents. In this study, the activities of two alpha-amido-beta-triazolylethaneboronic acid transition state inhibitors (S02030 and MB_076) targeting representative KPC (KPC-2) and CTX-M (CTX-M-96, a CTX-M-15-type extended-spectrum beta-lactamase [ESBL]) beta-lactamases were evaluated. The 50% inhibitory concentrations (IC(50)s) for both inhibitors were measured in the nanomolar range (2 to 135 nM). For S02030, the k(2)/K for CTX-M-96 (24,000 M-1 s(-1)) was twice the reported value for KPC-2 (12,000 M-1 s(-1)); for MB_076, the k(2)/K values ranged from 1,200 M-1 s(-1) (KPC-2) to 3,900 M-1 s(-1) (CTX-M-96). Crystal structures of KPC-2 with MB_076 (1.38-& ANGS; resolution) and S02030 and the in silico models of CTX-M-96 with these two BATSIs show that interaction in the CTX-M-96-S02030 and CTX-M-96-MB_076 complexes were overall equivalent to that observed for the crystallographic structure of KPC-2-S02030 and KPC-2-MB_076. The tetrahedral interaction surrounding the boron atom from S02030 and MB_076 creates a favorable hydrogen bonding network with S70, S130, N132, N170, and S237. However, the changes from W105 in KPC-2 to Y105 in CTX-M-96 and the missing residue R220 in CTX-M-96 alter the arrangement of the inhibitors in the active site of CTX-M-96, partially explaining the difference in kinetic parameters. The novel BATSI scaffolds studied here advance our understanding of structure-activity relationships (SARs) and illustrate the importance of new approaches to beta-lactamase inhibitor design