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Background: We evaluated a strategy to shorten the time from admission to surgery in patients with proximal femur fractures on chronic antiplatelet therapy. We reported a 12-month follow-up on complications and quality of life (QoL)., Methods: Multicentre, open-label, randomized, parallel clinical trial. Patients were randomized to either early platelet function-guided surgery (experimental group) or delayed surgery (control group). Medical and surgical complications and QoL (EQ-5D-5L questionnaire) were assessed during the hospital stay, and after hospital discharge at 30 days, and 6 and 12 months., Results: From 156 randomized patients, 143 patients underwent surgery. The mean age was 85.5 (7.8) years and 68.0% were female. After hospital discharge, 5.7% of patients had surgical wound complications and 55.9% had medical complications, with 42.7% having serious adverse events. QoL improved significantly after surgery, with the best scores at the six-month follow-up. The overall mortality was 32.2%. There were no differences between early and delayed surgery groups in any assessed outcomes., Conclusion: It seems safe to reduce the time of surgery under neuraxial anaesthesia in patients with hip fractures on chronic antiplatelet therapy by platelet function testing. QoL in particular improves in the first six months after surgery.

Introduction: Carbapenems are the beta-lactam antibiotics with the best spectrum of activity in the treatment of Pseudomonas aeruginosa infections. The objective of this study was to molecularly characterise a collection of carbapenem-resistant P. aeruginosa isolates (PARC)., Methods: A total of 85 PARC isolates were recovered from 60patients in the Hospital San Pedro, Logroño (period 2008-2011). Clonal relationship was determined using pulsed-field gel electrophoresis (PFGE), susceptibility testing to 15anti-pseudomonal agents was performed using the disk diffusion method, and alterations in oprD, characterisation of integrons and molecular typing (MLST) using PCR and sequencing., Results: The 85 PARC were classified into 35 different PFGE profiles. Of the 61selected strains from 60patients all of them were multiresistant, although none of them showed a carbapenemase phenotype. High polymorphism was detected in OprD, emphasising that 59% of the strains had a premature stop codon. ISPa1328 and ISPsp4 insertion sequences truncated oprD gene into 2 strains (GenBank KF517097 and KF517098). Two-thirds (67%) of the strains showed class 1 integrons with genes encoding aminoglycoside modifying enzymes, and 2 of them carried a new integron: aac(3)-Ia+aadA1h, named In272, GenBank GQ144317. Four sequence types were detected (Strain Nos.): ST175 (35), ST308 (3), ST235 (2), and ST639 (1)., Conclusion: Multidrug resistance, high polymorphism in oprD, a high percentage of integrons, moderate clonal relationship of strains, and the high epidemic dissemination of high-risk clones are clinical aspects of great concern in order to eradicate the spread of PARC., (Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

The objectives were to determine the presence and diversity of staphylococcal species in surface waters in La Rioja region (Spain), and to characterize recovered isolates. Staphylococci were detected in 42 of 47 evaluable samples, and 72 isolates were obtained, of which 13 were coagulase-positive (CoPS) and 59 were coagulase-negative (CoNS). Twelve CoPS were identified as S. aureus and typed as follows (number of strains): t002/t502/ST5 (four), t10668/ST425 (one), t10712//ST1643 (one), t843/ST130 (one), t10855/ST2461 (one), t3369/ST2657 (one), t1166/ST133 (one), t8083/ST2049 (one) and t045/ST2460 (one); and one as S. pseudintermedius ST147. Virulence genes tst, cna and lukS/F-I were detected, and one strain showed the immune evasion cluster type F. Regarding CoNS, 12 different species were recovered (number of strains): S. epidermidis (11), S. vitulinus (10), S. sciuri (nine), S. fleurettii (seven), S. lentus (six), S. simulans (five), S. xylosus (four), S. chromogenes (two), S. hominis (two), and S. equorum, S. succinus and S. warneri (one each). Fourteen CoNS isolates presented a multidrug resistance phenotype, with the following resistance genes: blaZ, mecA, fusB, fusC, erm(C), mph(C), erm(A), msr(A)/(B), mph(C), ant(4')-Ia, tet(K), tet(L), cat pc194 and str The high diversity of staphylococcal species, as well as multiple resistance and virulence genes, highlights the importance of surface waters as a temporary reservoir and source of transmission., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Hospital effluents are a source of environmental pollution by drugs, antibiotic-resistant bacteria, and resistance genes. Quinolones, particularly ciprofloxacin, are commonly detected in these effluents, contributing to the emergence of antimicrobial resistance. The objective of this study was to characterize ciprofloxacin-resistant Enterobacteriaceae in hospital effluents., Methodology: Isolates were selected on Tergitol-7 agar supplemented with ciprofloxacin and genotyped by ERIC-PCR. Antibiotic susceptibility testing was done using the disk diffusion method, and minimum inhibitory concentrations were determined using the agar dilution method. Resistance genes, integrons, phylogenetic groups, and sequence types were identified by PCR and sequencing., Results: A total of 17 ciprofloxacin-resistant isolates were characterized: Escherichia coli, Escherichia vulneris, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter freundii, and Citrobacter koseri/farmeri. Isolates presented concomitant resistance to nalidixic acid, ciprofloxacin, ofloxacin, and pefloxacin. A diversity in mutation patterns in gyrA and parC genes and new amino-acid substitutions in GyrA subunit were observed. Quinolone plasmidic resistance genes qnrB1, qnrB2, qnrB5/19, qnrS1, and aac(6')-Ib-cr were detected. Resistance to other antibiotic classes was observed. Class 1 integrons and resistance genes blaCTX-M-15, blaOXA-1, sul1, sul2, sul3, tetA, tetB, aadA1/2, aadA5, aph(3')-Ia, aac(3)II, dfrA1, dfrA5, dfrA7, and dfrA12 were detected. Bacterial tolerance to cadmium, zinc, and mercury was observed with the presence of the merA gene. E. coli isolates belonged to phylogenetic groups A, B1, and D and to sequence types ST405, ST443, ST101, ST10, and ST347., Conclusions: This study highlighted bacterial multidrug resistance linked to ciprofloxacin and, consequently, the risk of bacterial exposure to this antibiotic.

The objective of this study was to determine the prevalence of Staphylococcus in urban wastewater treatment plants (UWTP) of La Rioja (Spain), and to characterize de obtained isolates. 16 wastewater samples (8 influent, 8 effluent) of six UWTPs were seeded on mannitol-salt-agar and oxacillin-resistance-screening-agar-base for staphylococci and methicillin-resistant Staphylococcus aureus recovery. Antimicrobial susceptibility profile was determined for 16 antibiotics and the presence of 35 antimicrobial resistance genes and 14 virulence genes by PCR. S. aureus was typed by spa, agr, and multilocus-sequence-typing, and the presence of immune-evasion-genes cluster was analyzed. Staphylococcus spp. were detected in 13 of 16 tested wastewater samples (81%), although the number of CFU/mL decreased after treatment. 40 staphylococci were recovered (1-5/sample), and 8 of them were identified as S. aureus being typed as (number of strains): spa-t011/agr-II/ST398 (1), spa-t002/agr-II/ST5 (2), spa-t3262/agr-II/ST5 (1), spa-t605/agr-II/ST126 (3), and spa-t878/agr-III/ST2849 (1). S. aureus ST398 strain was methicillin-resistant and showed a multidrug resistance phenotype. Virulence genes tst, etd, sea, sec, seg, sei, sem, sen, seo, and seu, were detected among S. aureus and only ST5 strains showed genes of immune evasion cluster. Thirty-two coagulase-negative Staphylococcus of 12 different species were recovered (number of strains): Staphylococcus equorum (7), Staphylococcus vitulinus (4), Staphylococcus lentus (4), Staphylococcus sciuri (4), Staphylococcus fleurettii (2), Staphylococcus haemolyticus (2), Staphylococcus hominis (2), Staphylococcus saprophyticus (2), Staphylococcus succinus (2), Staphylococcus capitis (1), Staphylococcus cohnii (1), and Staphylococcus epidermidis (1). Five presented a multidrug resistance phenotype. The following resistance and virulence genes were found: mecA, lnu(A), vga(A), tet(K), erm(C), msr(A)/(B), mph(C), tst, and sem. We found that Staphylococcus spp. are normal contaminants of urban wastewater, including different lineages of S. aureus and a high diversity of coagulase-negative species. The presence of multiple resistance and virulence genes, including mecA, in staphylococci of wastewater can be a concern for the public health., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Raw food is a reservoir of Pseudomonas isolates that could be disseminated to consumers. The presence of Pseudomonas spp. was studied in food samples, and the phenotypic and genotypic characterizations of the recovered isolates were analyzed. Two samples of meat (3%, turkey and beef) and 13 of vegetables (22%, 7 green peppers and 6 tomatoes) contained Pseudomonas spp. A total of 20 isolates were identified, and were classified as follows (number of isolates): P. aeruginosa (5), P. putida (5), P. nitroreducens (4), P. fulva (2), P. mosselli (1), P. mendocina (1), P. monteilii (1), and Pseudomonas sp. (1). These 20 Pseudomonas isolates were clonally different by pulsed-field-gel-electrophoresis, and were resistant to the following antibiotics: ticarcillin (85%), aztreonam (30%), cefepime (10%), imipenem (10%), and meropenem (5%), but were susceptible to ceftazidime, piperacillin, piperacillin-tazobactam, doripenem, gentamicin, tobramycin, amikacin, ciprofloxacin, norfloxacin, and colistin. Only one strain (Ps158) presented a class 1 integron lacking the 3' conserved segment. The five P. aeruginosa strains were typed by multilocus sequence typing in five different sequence-types (ST17, ST270, ST800, ST1455, and ST1456), and different mutations were detected in protein OprD that were classified in three groups. One strain (Ps159) showed a new insertion sequence (ISPa47) truncating the oprD gene, and conferring resistance to imipenem.

The aim of this study was to analyse the Pseudomonas aeruginosa faecal carriage rate in 98 healthy humans and to perform the phenotypic and genotypic characterization of recovered isolates. The genetic relatedness among the isolates was analysed by pulsed-field gel electrophoresis and multilocus sequence typing that was compared with worldwide epidemic clones. Pseudomonas aeruginosa was isolated from eight healthy individuals (8.2%), and two of them remained colonized after 5 months (in one case by the same clone). All 10 isolates (one/sample) were susceptible to 14 tested antipseudomonal agents and lacked integron structures. Six pulsed-field gel electrophoresis patterns and six sequence types (ST245, ST253, ST254, ST274, ST663 and the new one, ST1059) were identified among them. Four groups of OprD alterations were detected based on mutations and deletions related to PAO1 reference strain in our carbapenem-susceptible strains. This is the first study focused on P. aeruginosa from faecal samples of healthy humans that provides additional insights into the antimicrobial resistance and genetic diversity of P. aeruginosa. Although the isolates were antimicrobial susceptible, most of the sequence types detected were genetically related to Spanish epidemic clones or globally spread sequence types, such as ST274 and ST253., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

The objective of this study was to determine the rate of contamination by Staphylococcus aureus in 100 meat samples obtained during 2011-2012 in La Rioja (Northern Spain), to analyze their content in antimicrobial resistance and virulence genes, as well as in immune evasion cluster (IEC) genes, and to type recovered isolates. Seven of 100 samples (7%) contained S. aureus: 6 samples harbored methicillin-susceptible S. aureus (MSSA) and 1 pork sample harbored methicillin-resistant S. aureus (MRSA). The MRSA isolate corresponded to the ST398 genetic lineage with a multidrug resistance profile and the absence of human IEC genes, which pointed to a typical livestock-associated MRSA profile. MRSA isolate was ascribed to the spa-type t011, agr-type I, and SCCmec-V and showed resistance to erythromycin, clindamycin, tetracycline, and streptomycin, in addition to β-lactams. The remaining six MSSA strains belonged to different sequence types and clonal complexes (three isolates ST45/CC45, one ST617/CC45, one ST5/CC5, and one ST109/CC9), being susceptible to most antibiotics tested but showing a wide virulence gene profile. Five of the six MSSA strains (except ST617/CC45) contained the enterotoxin egc-cluster or egc-like-cluster genes, and strain ST109/CC9 contained eta gene (encoding exfoliatin A). The presence of human IEC genes in MSSA strains (types B and D) points to a possible contamination of meat samples from an undefined human source. The presence of S. aureus with enterotoxin genes and MRSA in food samples might have implications in public health. The IEC system could be a good marker to follow the S. aureus contamination source in meat food products.

Molecular typing and mechanisms of carbapenem resistance such as alterations in porin OprD and presence of metallo-beta-lactamases (MBLs), as well as integrons have been studied in a collection of carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates from a Spanish hospital. One hundred and twenty-three CRPA isolates were recovered from different samples of 80 patients. Clonal relationship among CRPA was analyzed by SpeI-PFGE. Susceptibility testing to 11 antibiotics and MBL phenotype was determined by microdilution, IP/IPI E-test and double disc method. The oprD gene was studied by PCR and sequencing, and mutations were determined comparing with P. aeruginosa PAO1 sequence. Characterization of MBLs, and class 1 and 2 integrons were studied by PCR and sequencing. SDS-PAGE analysis of outer membrane proteins of selected strains was performed. Seventy-four-per-cent of patients with CRPA were hospitalised in the ICU setting and 50% had long hospitalization stays. Sixty-four different PFGE patterns were detected, and 87 CRPA strains were further analyzed. MBL phenotype was detected in 43 of 87 strains (49.4%), which contained blaVIM-2 gene inside class 1 integrons. VIM-2-producing strains belonged to lineages ST175, ST235, and ST973. A great diversity of nucleotide insertions, deletions, and mutations in oprD gene, and the presence of a new insertion sequence (ISPa45) truncating oprD were identified among CRPA strains. Class 1 integrons were detected in 75% of CRPA strains, blaVIM-2 and the new arrangement aac(3)-Ia+ISPa34+aadA1 (named as In661) being the most frequent gene-cassette arrays detected. Other gene cassettes detected in integrons were: aadB, aadA6, aadA7, aac(6')-Ib', and blaOXA-46., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The characterization of extended-spectrum beta-lactamases , plasmidic AmpC (pAmpC), and associated plasmid-mediated quinolone resistance (PMQR) determinants in cefotaxime-resistant coliforms isolated from hospital effluent in Algiers showed blaCTX-M genes in 89%, blaTEM-1 in 79·8%, and pAmpC genes (blaCIT) in 2·7% isolates. Association of ISEcp1B with blaCTX-M was found in all CTX-M+ isolates, and 97·2% harboured class 1 integrons. Sequencing showed blaCTX-M-15, blaCTX-M-3, and blaCMY-4 genes. blaCTX-M-3 and blaCTX-M-15 were located in Inc L/M conjugative plasmids. The PMQR determinants identified were qnrB1, qnrB2, qnrB9, qnrB19, qnrS2, and aac(6')-Ib-cr. qnrB2, qnrB9, qnrB19, and blaCMY-4 are described for the first time in Algeria and qnrB19 for the first time in non-clinical environments. This study highlights the major potential role of hospital effluents as providers of resistance genes to natural environments.

The aim of the study was the characterization of extended spectrum beta-lactamases (ESBLs) and quinolone resistance in cefotaxime-resistant coliform isolates from a wastewater treatment plant (WWTP). ESBLs were detected in 19 out of 24 isolates (79%) from raw water and in 21 out of 24 isolates (87.5%) from treated water, identified as Klebsiella pneumoniae and Escherichia coli. Molecular characterization of ESBLs and quinolone resistance showed allele profiles CTX-M-15 (3), CTX-M-3 (5), CTX-M-15+qnrB1 (1), CTX-M-3+qnrB1 (1), CTX-M-15+aac-(6')-Ib-cr (4), and CTX-M-15+qnrB1+aac-(6')-Ib-cr (7). A double mutation S83L and D87N (GyrA) and a single mutation S80I (ParC) were detected in ciprofloxacin-resistant E. coli isolates. In K. pneumoniae, mutations S83I (GyrA)+S80I (ParC) or single S80I mutation were detected in ciprofloxacin-resistant isolates, and no mutation was observed in ciprofloxacin-susceptible isolates. bla(CTX-M), qnrB1, and aac-(6')-Ib-cr were found, respectively, in these genetic environments: ISEcp1-bla(CTX-M)-orf477, orf1005-orf1-qnrB1, and Tn1721-IS26-aac-(6')-Ib-cr-bla(OXA-1)-catB4. bla(CTX-M-15) was located on IncF plasmid in E. coli and bla(CTX-M-3) on IncL/M plasmid in both species (E. coli and K. pneumoniae). E. coli isolates were affiliated to the phylogroups/MLST: D/ST405 (CC405), A/ST10 (CC10), A/ST617 (CC10), and B1/ST1431. K. pneumoniae isolates belonged to phylogroup KpI and to sequence types ST15, ST17, ST36, ST48, ST54, and ST147. The study showed a multi-drug resistance at the inflow and outflow of the WWTP, with ESBL production, plasmid-mediated quinolones resistance, and mutations in topoisomerases. The findings highlight the similarity of antibiotic resistance mechanisms in the clinical setting and the environment, and the role of the latter as a source of dissemination of resistance genes.

Twenty-seven well-characterized metallo-β-lactamase (MBL)-producing Pseudomonas strains from two distantly located hospitals were analyzed. The results revealed specific features defining the multilevel epidemiology of strains from each hospital in terms of species, clonality, predominance of high-risk clones, composition/diversity of integrons, and linkages of Tn402-related structures. Therefore, despite the global trends driving the epidemiology of MBL-producing Pseudomonas spp., the presence of local features has to be considered in order to understand this threat and implement proper control strategies.

The presence of broad-spectrum-cephalosporin-resistant Escherichia coli isolates and the implicated mechanisms of resistance and virulence factor genes were investigated in red fox (Vulpes vulpes) in Portugal. Cefotaxime-resistant E. coli isolates were isolated from two of 52 fecal samples (4 %), being both ESBL producers. The β-lactamase genes found in the two isolates were bla(SHV-12) + bla(TEM-1b). The tet(A) and sul2 genes were also detected in these isolates, together with the non-classical class 1 integron (intI1-dfrA12-orfF-aadA2-cmlA1-aadA1-qacH-IS440-sul3) with the PcH1 promoter. The two isolates belonged to the phylogroup A. Amino acid changes in GyrA (S83L + D87G) and ParC (S80I) proteins were identified in our study. Concerning MLST typing, both isolates were assigned to ST1086, never found before in wild animals, and they presented closely related PFGE patterns. This study reveals the presence of ESBL-producing E. coli isolates, in a wild ecosystem, which could be disseminated through the environment to other niches.

Nineteen extended-spectrum β-lactamase (ESBL)-positive Escherichia coli strains recovered from food samples in Tunisia were characterized by multilocus sequence typing and phylogenetic typing, and the virulence gene and plasmid content were also determined. These strains presented unrelated pulsed-field gel electrophoresis patterns and contained genes coding for the following ESBLs (the number of strains is in parentheses): CTX-M-1 (15), CTX-M-14 (2), CTX-M-8 (1), and SHV-5 (1). Twelve different sequence types (STs) were identified among the 19 ESBL-positive strains, which included two new STs (ST2022 in 2 bla(CTX-M-14)-containing strains and ST1970 in 2 bla(CTX-M-1)-containing strains). ST155 and ST602 were detected in four and three bla(CTX-M-1)-containing strains, respectively, and ST405 was detected in one bla(CTX-M-8)-producing strain. All ESBL-positive strains were ascribed to the phylogenetic groups A and B1. Most of the bla(CTX-M-1)-containing strains harbored an IncI1 plasmid, except for the four bla(CTX-M-1)-positive strains of beef origin and ST155, which harbored an IncN plasmid. The two bla(CTX-M-14)-containing strains contained an IncI1 plasmid. The virulence gene fimA was detected in all strains. Most strains also carried the aer gene, and six strains carried the eae gene. All strains were negative for the virulence genes sxt, papG-III, papC, hly, cnf1, and bfp. We conclude that ESBL-producing E. coli strains of food origin in Tunisia show high diversity and that plasmids harboring ESBL genes could be implicated in the dissemination of this resistance phenotype.

Our objective was to analyze the carriage rate of extended-spectrum β-lactamase (ESBL)- and plasmidic AmpC β-lactamase (pAmpC)-producing Escherichia coli isolates in fecal samples of healthy pets (dogs and cats) and to characterize the recovered isolates for the presence of other resistance genes and integrons. Eighty fecal samples of healthy pets were inoculated in MacConkey agar plates supplemented with cefotaxime (2 μg/mL) for cefotaxime-resistant (CTX(R)) E. coli recovery. CTX(R) E. coli isolates were detected in 14 of the 80 fecal samples (17.5%) and the following β-lactamase genes (number of isolates) were detected: bla(CTX-M-1) (8), bla(CTX-M-1)+bla(TEM-1b) (3)(,) bla(CTX-M-1)+bla(TEM-1c) (1), bla(CTX-M-1)+bla(TEM-135) (1), and bla(CMY-2)+bla(TEM-1b) (1). The 14 E. coli were distributed into the phylogroups B1 (6 isolates), A (5), and D (3). The qnrB19 gene was detected in one CTX-M-1-producing strain of phylogroup D. Five isolates contained class 1 integrons with the following arrangements: dfrA17-aadA5 (2 isolates), dfrA1-aadA1 (1), and dfrA17-aadA5/ dfrA1-aadA1 (2 isolates). The virulence genes fimA and/or aer were detected in all CTX(R) strains. In this study, the pet population harbored β-lactamase and quinolone resistance genes of special interest in human health that potentially could be transmitted to humans in close contact with them.

The prevalence of extended-spectrum beta-lactamase (ESBL)- and plasmidic AmpC-beta-lactamase (pAmpC-BL)-producing Escherichia coli isolates has been studied in food-producing animals at the farm level in Tunisia, and recovered isolates were characterized for the presence of other resistance genes and integrons. Eighty fecal samples of food-producing animals (23 sheep, 22 chickens, 22 cattle, six horses, five rabbits, and two dromedaries) were obtained from 35 different farms in Tunisia in 2011. Samples were inoculated onto MacConkey agar plates supplemented with cefotaxime (2 mg/L) for cefotaxime-resistant (CTX(R)) E. coli recovery. CTX(R) E. coli isolates were detected in 11 out of 80 samples (13.8%), and one isolate per sample was further characterized (10 from chickens and one from a dromedary). The 11 CTX(R) isolates were distributed into phylogroups: B1 (five isolates), A (two isolates), D (three isolates), and B2 (one isolate). The following beta-lactamase genes were detected: bla(CTX-M-1) (seven isolates), bla(CTX-M-1)+bla(TEM-135) (one isolate), bla(CTX-M-1)+bla(TEM-1b) (one isolate), and bla(CMY-2) (two isolates). All ESBL- and pAmpC-BL-producing E. coli strains showed unrelated pulsed-field gel electrophoresis patterns. Seven isolates contained class 1 integrons with four gene cassette arrangements: dfrA17-aadA5 (three isolates), dfrA1-aadA1 (two isolates), dfrA15-aadA1 (one isolate), and aadA1 (one isolate). All isolates showed tetracycline resistance and contained the tet(A) +/- tet(B) genes. Virulence genes detected were as follows (number of isolates in parentheses): fimA (10); aer (eight); papC (two); and papGIII, hly, cnf, and bfp (none). Chicken farms constitute a reservoir of ESBL- and pAmpC-BL-producing E. coli isolates of the CTX-M-1 and CMY-2 types that potentially could be transmitted to humans via the food chain or by direct contact.

The intensive use of antibiotics in human and veterinary medicine, associated with mechanisms of bacterial genetic transfer, caused a selective pressure that contributed to the dissemination of antimicrobial resistance in different bacteria groups and throughout different ecosystems. Iberian wolf, due to his predatory and wild nature, may serve as an important indicator of environmental contamination with antimicrobial resistant bacteria. The aim of this study was to characterize the diversity of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates within the fecal microbiota of Iberian wolf. Additionally, the identification of other associated resistance genes, phylogenetic groups, and the detection of virulence determinants were also focused on in this study. From 2008 to 2009, 237 fecal samples from Iberian wolf were collected in Portugal. E. coli isolates with TEM-52, SHV-12, CTX-M-1, and CTX-M-14-type ESBLs were detected in 13 of these samples (5.5%). This study reveals the presence of ESBL-producing E. coli isolates, in a wild ecosystem, which could be disseminated through the environment. Moreover, the presence of resistant genes in integrons and the existence of virulence determinants were shown. The association between antibiotic resistance and virulence determinants should be monitored, as it constitutes a serious public health problem.

The aim of the study was to evaluate bacterial antibiotic resistance in seawater from four beaches in Algiers. The most significant resistance rates were observed for amoxicillin and ticarcillin, whereas they were relatively low for ceftazidime, cefotaxime and imipenem. According to sampling sites, the highest resistance rates were recorded for 2 sites subjected to chemical and microbiological inputs (amoxicillin, 43% and 52%; ticarcillin, 19.6% and 47.7%), and for 2 sites relatively preserved from anthropogenic influence, resistance rates were lowest (amoxicillin, 1.5% and 16%; ticarcillin, 0.8% and 2.6%). Thirty-four bacteria resistant to imipenem (n=14) or cefotaxime (n=20) were identified as Pseudomonas aeruginosa (n=15), Pseudomonas fluorescens (7), Stenotrophomonas maltophilia (4), Burkholderia cepacia (2), Bordetella sp. (1), Pantoea sp. (1), Acinetobacter baumannii (1), Chryseomonas luteola (1), Ochrobactrum anthropi (1) and Escherichia coli (1). Screening for extended spectrum β-lactamase showed the presence of CTX-M-15 β-lactamase in the E. coli isolate, and the encoding gene was transferable in association with the IncI1 plasmid of about 50 kbp. Insertion sequence ISEcp1B was located upstream of the CTX-M-15 gene. This work showed a significant level of resistance to antibiotics, mainly among environmental saprophytic bacteria. Transmissible CTX-M-15 was detected in E. coli; this may mean that contamination of the environment by resistant bacteria may cause the spread of resistance genes.

Fourteen broad-spectrum-cephalosporin-resistant Escherichia coli isolates were recovered between June and December 2007 in a Tunisian hospital. Genes encoding extended-spectrum-beta-lactamases (ESBL) and other resistance genes were characterized by PCR and sequencing. The following ESBL genes were identified: bla (CTX-M-15) (12 isolates), bla (CTX-M-14a) (one isolate), and bla (CTX-M-14b) (one isolate). The bla (OXA-1) gene was detected in 13 bla (CTX-M)-producing strains and a bla (TEM-1) gene in 6 of them. The ISEcp1 sequence was found upstream of bla (CTX-M) genes in 8 of 14 strains, and orf477 or IS903 downstream of this gene in 13 strains. Nine of the strains carried class 1 integrons and five different gene cassette arrangements were detected, dfrA17-aadA5 being the most common. One of the strains (bla (CTX-M-14a)-positive) harbored three class 1 integrons, and one of them was non-previously described containing as gene cassettes new variants of aac(6')-Ib and cmlA1 genes and it was linked to the bla (CTX-M-14a) gene flanked by a truncated ISEcp1 sequence (included in GenBank with accession number JF701188). CTX-M-15-producing strains were ascribed to phylogroup B2 (six isolates) and D (six isolates). Multilocus-sequence-typing revealed ten different sequence-types (STs) among ESBL-positive E. coli strains with prevalence of ST405 (four strains of phylogroup D) and ST131 types (two strains of phylogroup B2 and serogroup O25b). A high clonal diversity was also observed among studied strains by pulsed-field-gel-electrophoresis (11 unrelated profiles). CTX-M-15 is an emergent mechanism of resistance in the studied hospital and the world-disseminated 0:25b-ST131-B2 and ST405-D clones have been identified among CTX-M-15-producing isolates.

Eleven multiresistant Escherichia coli strains of animal and human origin were assayed for the presence of antimicrobial resistance genes, integrons and associated gene cassettes, as well as plasmid content. Ciprofloxacin-resistant strains were screened for amino acid changes in GyrA and ParC proteins. The E. coli strains were found to harbor a variety of genes including cmlA, aac (3)-II, aac (3)-IV, aadA, strA-strB, tet (A), tet (B), bla(TEM), sul1, sul2 and sul3. Four of the eight int I1-positive strains were also positive for qacE Δ1 -sul1 region and the following gene cassettes were detected: dfrA7, dfrA12 + orfF + aadA2 and bla(OXA1)+ aadA1. Five strains contained class 1 integrons lacking the qacE Δ1 -sul1 region and they showed a single type of gene cassette arrangement (estX + psp + aadA2 + cmlA + aadA1 + qacH + IS440 + sul3). The two int I2-positive strains carried the same type of gene cassette arrangement (dfrA1 + sat + aadA1). The seven ciprofloxacin-resistant E. coli strains exhibited a Ser-83-Leu substitution in GyrA protein and a Ser-80-Ile substitution in ParC protein; six of these strains presented an additional substitution in GyrA (Asp-87-Gly or Asp-87-Asn) and one strain in ParC (Glu-84-Gly). Eight different plasmid-replicon-types were detected among the 11 E. coli strains, IncF being the most frequent one detected, found in nine strains; other plasmid replicon types detected were IncX, IncI1, IncY, IncW, IncFIC, IncB/O, and IncK. Antimicrobial resistance in the E. coli strains studied was mediated by a variety of genes, some of them included in integrons, as well as by mutations gyr A and par C genes.

The presence of broad-spectrum-cephalosporin-resistant Escherichia coli isolates and the implicated mechanisms of resistance were investigated in 79 food samples of animal origin obtained in different supermarkets and local butcheries in Tunisia. Ten of these samples (12.6%) harbored extended-spectrum beta-lactamase (ESBL) producing E. coli isolates and 13 ESBL-positive isolates were recovered (one or two/sample), which exhibited nine different Pulsed-Field-Gel-Electrophoresis (PFGE) patterns. ESBLs detected were the following: CTX-M-1 (10 strains), CTX-M-1+TEM-1b (2 strains) and CTX-M-1+TEM-20 (1 strain). The orf477 sequence was identified downstream of bla(CTX-M-1) gene in all 13 strains and ISEcp1 upstream in 9 strains. All ESBL-positive strains were included into phylogenetic group A or B1 (4 and 9 strains, respectively). Three of the 79 food samples (3.8%) contained broad-spectrum-cephalosporin-resistant and ESBL-negative E. coli isolates with AmpC phenotype. One isolate per sample was studied, and they showed unrelated PFGE patterns. The CMY-2 type beta-lactamase was identified in one of these 3 strains and specific point mutations in the promoter/attenuator region of ampC gene (at positions -42, -18, -1 and +58) were detected in the remaining two strains. Twelve ESBL-positive and one ESBL-negative E. coli strains contained class 1 integrons with the following gene cassette arrangements: dfrA1+aadA (6 strains) and dfrA17+aadA5 (7 strains). E. coli strains from food samples could represent a reservoir of ESBL-encoding genes and integrons that could be transmitted to humans through the food chain., ((c) 2009 Elsevier B.V. All rights reserved.)

ESBL-producing E. coli isolates have been isolated from eight of seventy seven faecal samples (10.4%) of wild boars in Portugal. The ESBL types identified by PCR and sequencing were bla(CTX-M-1) (6 isolates) and bla(CTX-M-1) + bla(TEM1-b) (2 isolates). Further resistance genes detected included tet (A) or tet (B) (in three tetracycline-resistant isolates), aad A (in three streptomycin-resistant isolates), cml A (in one chloramphenicol-resistant isolate), sul 1 and/or sul 2 and/or sul 3 (in all sulfonamide-resistant isolates). The intI 1 gene encoding class 1 integrase was detected in all ESBL-producing E. coli isolates. One isolate also carried the intI 2 gene, encoding class 2 integrase. The ESBL-producing E. coli isolates could be assigned to phylogenetic groups B1 (3 isolates), B2 (3 isolates) or A (2 isolates). Amino acid change in GyrA protein (Ser83Leu or Asp87Tyr) was detected in three nalidixic acid-resistant and ciprofloxacin-susceptible isolates. Two amino acid changes in GyrA (Ser83Leu + Asp87Asn) and one in ParC (Ser80Ile) were identified in two nalidixic acid- and ciprofloxacin-resistant isolates. As evidenced by this study wild boars could be a reservoir of antimicrobial resistance genes.