Amino Acid Substitutions and Differential Gene Expression of Outer Membrane Proteins in Adherent-Invasive Escherichia coli.

Variations in the sequence and/or the expression of outer membrane proteins (OMPs) may modulate bacterial virulence. OmpA and OmpC have been involved in the interaction of adherent-invasive Escherichia coli (AIEC) strain LF82 with intestinal epithelial cells (IECs). Scarce data exist about OMPs sequence variants in a collection of AIEC strains, and no study of OMPs expression during infection exists. We aimed to determine whether particular mutations or differential expression of OMPs are associated with AIEC virulence. The ompA, ompC, and ompF genes in 14 AIEC and 30 non-AIEC strains were sequenced by Sanger method, and the protein expression profile was analyzed by urea-SDS-PAGE. Gene expression was determined during in vitro bacterial infection of intestine-407 cells by RT-qPCR. The distribution of amino acid substitutions in OmpA-A200V, OmpC-S89N, V220I, and W231D associated with pathotype and specific changes (OmpA-A200V, OmpC-V220I, D232A, OmpF-E51V, and M60K) correlated with adhesion and/or invasion indices but no particular variants were found specific of AIEC. OMPs protein levels did not differ according to pathotype when growing in Mueller-Hinton broth. Interestingly, higher OMPs gene expression levels were reported in non-AIEC growing in association with cells compared with those non-AIEC strains growing in the supernatants of infected cultures (p < 0.028), whereas in AIEC strains ompA expression was the only increased when growing in association with cells (p = 0.032), but they did not significantly alter ompC and ompF expression under this condition (p > 0.146). Despite no particular OMPs sequence variants have been found as a common and distinctive trait in AIEC, some mutations could facilitate a better interaction with the host. Moreover, the different behavior between pathotypes regarding OMPs gene expression at different stages of infection could be related with the virulence of the strains.

Prevalence of Aminoglycoside-Modifying Enzymes in Escherichia coli and Klebsiella pneumoniae Producing Extended Spectrum ß-Lactamases Collected in Two Multicenter Studies in Spain.

The in vitro activity of amikacin, gentamicin, kanamycin, tobramycin, neomycin, and netilmicin against 420 Escherichia coli producing extended spectrum ß-lactamases (Ec-ESBLs) and 139 Klebsiella pneumoniae producing extended spectrum ß-lactamase (Kp-ESBL) collected in two multicenter studies performed in Spain in 2000 and 2006 was determined. The presence of genes encoding aminoglycoside-modifying enzymes (AMEs) and 16S ribosomal RNA (rRNA) methylases [aac(3)-Ia, aac(3)-IIa, aac(3)-IVa, aac(6')-Ib, ant(2")-Ia, ant(4')-IIa, aph(3')-Ia, aph(3')-IIa, armA, rmtB, and rmtC] was also investigated. The resistance to (one or more) aminoglycosides was significantly higher in Kp-ESBL (104/139, 74.8%) than in Ec-ESBL (171/420, 40.7%; p < 0.0001). The lowest resistance rates for both species in the two studies were observed for amikacin. The prevalence of AME genes was significantly different in Ec-ESBL (161/420, 38.3%) than in Kp-ESBL (115/139, 82.7%; p < 0.0001). The most prevalent AME genes in Ec-ESBL and Kp-ESBL were aac(6')-Ib (16.2% and 44.6%) and aac(3)-IIa (14.7% and 43.1%), respectively. The expected phenotypic profile correlated with the found AMEs encoding genes in 59.6% Ec-ESBL and 26.1% Kp-ESBL. In Ec-ESBL, aac(6')-Ib was usually associated in 2000 with blaSHV (26.6%), but with blaCTX-M-1 group (34.8%) in 2006, while aac(3)-IIa was coincident in 2000 with blaTEM (14.6%) and with blaCTX-M-1 group (16.3%) in 2006. Among Kp-ESBL, the aac(6')-Ib and aac(3)-IIa genes were more frequent in strains with blaTEM (22.0% and 44.0%) in 2000 and with blaCTX-M-1 group (46.4% and 34.0%) in 2006. Resistance to aminoglycosides in Ec-ESBL and Kp-ESBL is frequent and related to production of AMEs; this limits the clinical use of aminoglycosides against these organisms.

Degenerate primer MOB typing of multiresistant clinical isolates of E. coli uncovers new plasmid backbones.

Degenerate Primer MOB Typing is a PCR-based protocol for the classification of γ-proteobacterial transmissible plasmids in five phylogenetic relaxase MOB families. It was applied to a multiresistant E. coli collection, previously characterized by PCR-based replicon-typing, in order to compare both methods. Plasmids from 32 clinical isolates of multiresistant E. coli (19 extended spectrum beta-lactamase producers and 13 non producers) and their transconjugants were analyzed. A total of 95 relaxases were detected, at least one per isolate, underscoring the high potential of these strains for antibiotic-resistance transmission. MOBP12 and MOBF12 plasmids were the most abundant. Most MOB subfamilies detected were present in both subsets of the collection, indicating a shared mobilome among multiresistant E. coli. The plasmid profile obtained by both methods was compared, which provided useful data upon which decisions related to the implementation of detection methods in the clinic could be based. The phylogenetic depth at which replicon and MOB-typing classify plasmids is different. While replicon-typing aims at plasmid replication regions with non-degenerate primers, MOB-typing classifies plasmids into relaxase subfamilies using degenerate primers. As a result, MOB-typing provides a deeper phylogenetic depth than replicon-typing and new plasmid groups are uncovered. Significantly, MOB typing identified 17 plasmids and an integrative and conjugative element, which were not detected by replicon-typing. Four of these backbones were different from previously reported elements.

OXA-207, a novel OXA-24 variant with reduced catalytic efficiency against carbapenems in Acinetobacter pittii from Spain.

A carbapenem-resistant Acinetobacter pittii strain carrying an OXA-24-like enzyme was isolated in northern Spain in 2008. Sequence analysis confirmed the presence of the novel bla(OXA-207) gene flanked by the site-specific XerC/XerD-like recombination binding sites and showing a unique Gly222Val substitution compared to OXA-24. Cloning and kinetic analysis showed that OXA-207 presents a reduction in the catalytic efficiency against carbapenems and a noticeable increase for oxacillin.

Production of HlyA and ClyA haemolysins among quinolone-resistant Escherichia coli isolated from clinical samples.

Most Escherichia coli resistant to quinolones are not haemolytic. The objective of this study was to determine the phylogroup, clonal relationship, mechanism of quinolone resistance and virulence factors in 70 haemolytic E. coli resistant to nalidixic acid. Sixty-six isolates contained the hlyA gene, belonged to phylogroup B2, and 61 of them presented low-level resistance to fluoroquinolones. Four isolates presented high-level resistance to fluoroquinolones, contained the clyA gene and were included in phylogroup D. One single isolate (phylogroup D, with low level resistance to fluoroquinolones) contained both cytotoxins.

Molecular characterization of multiresistant Escherichia coli producing or not extended-spectrum ß-lactamases.

BACKGROUND: The prevalence and type of plasmids, resistance genes and integrons carried by two collections of multiresistant E. coli producing or not extended-spectrum ß-lactamases have been compared. Rep-PCR was used to determine the clonal relationship of the organisms. Plasmids were classified according to their incompatibility. Class 1 and Class 2 integrons and antibiotic resistance genes were analysed by PCR and sequencing. RESULTS: Both collections of organisms contained a large diversity of unrelated strains with some clones distributed in both groups of isolates. Large plasmids were identified in the two groups of organisms. Plasmids with replicons repK and repColE were more frequent among ESBL-producing isolates, while repFIA, repFII and repA/C replicons were more frequent in isolates lacking ESBL. Conjugative plasmids with repK and repA/C replicons coded for CTX-M-14 and CMY-2 ß-lactamases, respectively. No significant differences were observed in the distribution of class 1 and class 2 integrons among multiresistant E. coli producing or not ESBL, and dfrA17-ant(3")-Ie was the cassette arrangement most commonly found. CONCLUSIONS: In the concrete temporal and geographical context of this study, multiresistant E. coli producing ESBL or other mechanisms of resistance were largely clonally diverse and present some differences in the types of harboured plasmids. Still, some clones were found in both ESBL-producing and -lacking isolates.

Caracterización molecular de aislados clínicos de Pseudomonas aeruginosa productores de la metalobetalactamasa VIM-2 en Cantabria, España / Molecular characterization of Pseudomonas aeruginosa isolates in Cantabria, Spain, producing VIM-2 metallo-beta-lactamase

Introducción En España el aislamiento de cepas de Pseudomonas aeruginosa productoras de metalobetalactamasas (MBL) es poco frecuente. En este artículo se describe la caracterización de 9 aislados clínicos de P. aeruginosa multirresistentes clonalmente relacionados, aislados en Cantabria (España) que poseen el gen (..) (AU)

Introduction Pseudomonas aeruginosa strains producing (..) (AU)

[Molecular characterization of Pseudomonas aeruginosa isolates in Cantabria, Spain, producing VIM-2 metallo-beta-lactamase]. / Caracterización molecular de aislados clínicos de Pseudomonas aeruginosa productores de la metalobetalactamasa VIM-2 en Cantabria, España.

INTRODUCTION: Pseudomonas aeruginosa strains producing metallo-beta-lactamases (MbetaL) are uncommon in Spain. This study describes the characterization of 9 new clonally related multiresistant P. aeruginosa isolates possessing the bla(VIM-2) gene in Cantabria (Northern Spain). METHODS: P. aeruginosa clinical strains (1 per patient) were isolated in the Microbiology Service of Marqués de Valdecilla University Hospital between January 2004 and December 2006. Identification and preliminary susceptibility studies were performed with the MicroScan WalkAway system (Dade Behring, Sacramento, CA) and results were verified by a microdilution reference method. RESULTS: MICs of imipenem and meropenem for the 9 isolates ranged from 32 to 128 and 16 to 64 microg/mL, respectively. Nine isolates had a single Rep-PCR pattern and were intermediate or resistant to ceftazidime, cefepime, gentamicin, tobramycin, amikacin and ciprofloxacin. Eight of the 9 isolates were susceptible to aztreonam. Hydrolysis activity of imipenem in MbetaL-positive isolates ranged from 162+/-18 to 235+/-28 pmol/min/microg protein and was abolished in the presence of 5 mM EDTA. All isolates possessed an integron with genes aac(6')32, bla(VIM-2) and a putative transposase-encoding gene, flanked by the conserved 5'CS and 3'CS regions. CONCLUSION: In the clinical isolates studied, the presence of MbetaL VIM-2 sufficed to explain their resistance to carbapenems.