Caracterización de mecanismos de resistencia a carbapenémicos en aislados clínicos de Pseudomonas aeruginosa en un hospital español / Characterisation of carbapenem-resistance mechanisms in clinical Pseudomonas aeruginosa isolates recovered in a Spanish hospital

INTRODUCCIÓN: Los carbapenémicos son los antibióticos betalactámicos con mayor espectro de actividad en el tratamiento de infecciones por Pseudomonas aeruginosa. El objetivo de este trabajo fue caracterizar molecularmente una colección de aislados de P. aeruginosa resistentes a carbapenémicos (PARC). MÉTODOS: Se recogieron 85 aislados PARC de 60 pacientes en el Hospital San Pedro, Logroño (período 2008-2011). La relación clonal se determinó por electroforesis en gel de campo pulsado (PFGE), la sensibilidad a 15 antipseudomónicos por método de difusión en disco y las alteraciones en oprD, la caracterización de integrones y la tipificación molecular (MLST) por PCR y secuenciación. RESULTADOS: Las 85 PARC se clasificaron en 35 perfiles diferentes de PFGE. Se seleccionaron 61 cepas de los 60 pacientes y se observó que eran multirresistentes, aunque ninguna mostró fenotipo carbapenemasa. Se detectó un gran polimorfismo de OprD, destacando que el 59% de las cepas presentaban un codón de finalización prematuro. ISPa1328 e ISPsp4 truncaban el gen oprD en 2 cepas (GenBank KF517097 y KF517098). El 67% de las cepas presentó integrones de clase 1 con genes codificantes de enzimas modificantes de aminoglucósidos, 2 de las cuales portaban un nuevo integrón: aac(3)-Ia+aadA1h (nombrado In272, GenBank GQ144317). Se detectaron 4 secuencias tipo (ST) (número de cepas): ST175 (35), ST308 (3), ST235 (2) y ST639 (1). CONCLUSIÓN: La multirresistencia, el alto polimorfismo de oprD, el alto porcentaje de integrones, la moderada relación clonal de las cepas y la elevada diseminación epidémica de clones de alto riesgo son aspectos de gran preocupación clínica para erradicar la diseminación de PARC

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 60 patients in the Hospital San Pedro, Logroño (period 2008-2011). Clonal relationship was determined using pulsed-field gel electrophoresis (PFGE), susceptibility testing to 15 anti-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 61 selected strains from 60 patients 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

Characterisation of carbapenem-resistance mechanisms in clinical Pseudomonas aeruginosa isolates recovered in a Spanish hospital. / Caracterización de mecanismos de resistencia a carbapenémicos en aislados clínicos de Pseudomonas aeruginosa en un hospital español.

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.

Diversity of species and antimicrobial resistance determinants of staphylococci in superficial waters in Spain.

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), catpc194 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.

Characteristics of ciprofloxacin-resistant Enterobacteriaceae isolates recovered from wastewater of an Algerian hospital.

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.

Characterization of staphylococci in urban wastewater treatment plants in Spain, with detection of methicillin resistant Staphylococcus aureus ST398.

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.

Genetic Lineages and Antimicrobial Resistance in Pseudomonas spp. Isolates Recovered from Food Samples.

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.

Faecal carriage of Pseudomonas aeruginosa in healthy humans: antimicrobial susceptibility and global genetic lineages.

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.

Carbapenem-resistant Pseudomonas aeruginosa strains from a Spanish hospital: characterization of metallo-beta-lactamases, porin OprD and integrons.

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.

Genetic lineages, antimicrobial resistance, and virulence in Staphylococcus aureus of meat samples in Spain: analysis of immune evasion cluster (IEC) genes.

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 ß-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.

Comparison of local features from two Spanish hospitals reveals common and specific traits at multiple levels of the molecular epidemiology of metallo-ß-lactamase-producing Pseudomonas spp.

Twenty-seven well-characterized metallo-ß-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.

Characterization of ESBLs and associated quinolone resistance in Escherichia coli and Klebsiella pneumoniae isolates from an urban wastewater treatment plant in Algeria.

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.

ESBL, plasmidic AmpC, and associated quinolone resistance determinants in coliforms isolated from hospital effluent: first report of qnrB2, qnrB9, qnrB19, and blaCMY-4 in Algeria.

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.

Lineages and virulence gene content among extended-spectrum ß-lactamase-producing Escherichia coli strains of food origin in Tunisia.

Nineteen extended-spectrum ß-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.

First detection of CTX-M-1, CMY-2, and QnrB19 resistance mechanisms in fecal Escherichia coli isolates from healthy pets in Tunisia.

Our objective was to analyze the carriage rate of extended-spectrum ß-lactamase (ESBL)- and plasmidic AmpC ß-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 ß-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 ß-lactamase and quinolone resistance genes of special interest in human health that potentially could be transmitted to humans in close contact with them.

Molecular characterization of extended-spectrum-beta-lactamase-producing Escherichia coli isolates from red foxes in Portugal.

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 ß-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.

Prevalence and characterization of extended-spectrum beta-lactamase (ESBL)- and CMY-2-producing Escherichia coli isolates from healthy food-producing animals in Tunisia.

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.

Antibiotic resistance and extended-spectrum ß-lactamases in isolated bacteria from seawater of Algiers beaches (Algeria).

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 ß-lactamase showed the presence of CTX-M-15 ß-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.