Laura Vinué

Prevalence of extended-spectrum beta-lactamase-producing Escherichia coli isolates in faecal samples of broilers

Seventy-six faecal samples were obtained from broilers at slaughterhouse level in Portugal. Samples were inoculated on cefotaxime-supplemented Levine agar plates. Cefotaxime-resistant Escherichia coli isolates were recovered from 32 samples (42.1%), obtaining a total of 34 E. coli isolates (one or two isolates per sample). Susceptibility to 16 antibiotics was studied by disk diffusion method, and 85% of the isolates presented a phenotype of multi-resistance that included antimicrobial agents of at least four different families. Extended-spectrum-beta-lactamases (ESBL) of the TEM and CTX-M groups were detected in 31 ESBL-positive E. coli isolates. Twenty-six isolates harboured the bla(TEM-52) gene and two of them also harboured bla(TEM-1b). The bla(CTX-M-14) gene was identified in three isolates (in association with bla(TEM-1b) in one of them), and bla(CTX-M-32) was demonstrated in two additional isolates. Three of the 34 cefotaxime-resistant isolates (9%) did not produce ESBLs, and two of them presented mutations at positions -42 (C-->T), -18 (G-->A), -1 (C-->T), and +58(C-->T) of the promoter/attenuator region of ampC gene. tet(A) and/or tet(B) genes were detected in all 34 tetracycline-resistant isolates, aadA in all 26 streptomycin-resistant isolates; cmlA in 3 of 6 chloramphenicol-resistant isolates, and aac(3)-II or aac(3)-I + aac(3)-IV genes in all 4 gentamicin-resistant isolates. Different combinations of sul1, sul2 and sul3 genes were demonstrated among the 22 trimethoprim-sulfamethoxazole-resistant isolates. Amino acid changes in GyrA and ParC proteins were identified in all 18 ciprofloxacin-resistant isolates. The results of this study indicate that the intestinal tract of healthy poultry is a reservoir of ESBL-positive E. coli isolates.

Seagulls of the Berlengas Natural Reserve of Portugal as Carriers of Fecal Escherichia coli Harboring CTX-M and TEM Extended-Spectrum Beta-Lactamases

ABSTRACT Escherichia coli isolates containing the following extended-spectrum beta-lactamases have been detected in 11 of 57 fecal samples (19.3%) in Berlengas Island seagulls: TEM-52 (eight isolates), CTX-M-1 (one isolate), CTX-M-14a (one isolate), and CTX-M-32 (one isolate). Most of the extended-spectrum beta-lactamase-positive isolates harbored class 1 or class 2 integrons, which included different antibiotic resistance gene cassettes.

Prevalence and diversity of extended-spectrum ß-lactamases in faecal Escherichia coli isolates from healthy humans in Spain

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates were detected in seven of 105 faecal samples from healthy humans, from two Spanish cities, during 2007. In these isolates, five ESBLs were detected, CTX-M-14 (n = 2), CTX-M-1 (n = 2), CTX-M-32 (n = 1), CTX-M-8 (n = 1) and TEM-52 (n = 1). Both bla(CTX-M-14a) (surrounded by ISEcp1-IS903) and bla(CTX-M-14b) variants (included in an integron structure) were identified in this study. This is the first time that the bla(CTX-M-8) gene and ESBLs of the CTX-M-8 group have been found in Europe and Spain, respectively. Faecal E. coli of healthy humans therefore constitute a reservoir of bla(CTX-M) genes with different surrounding genetic elements.

Mechanisms of Antibiotic Resistance in Escherichia coli Isolates Recovered from Wild Animals

Seventy-two fecal samples obtained from wild animals in Portugal were sampled on Levine agar plates (non-supplemented with antibiotics), and Escherichia coli isolates were recovered from 56 of them (78%), obtaining a total of 112 E. coli isolates (two per sample). Susceptibility to 16 antibiotics was studied in these isolates, and the following percentages of resistance were obtained: tetracycline, streptomycin, ampicillin, and trimethoprim-sulfamethoxazole (SXT) (range 19-35%); nalidixic acid (14%); ciprofloxacin (9%); amoxicillin-clavulanic acid, gentamicin, tobramycin, and chloramphenicol (range 4.5-7%); cefotaxime, and aztreonam (1.8%); ceftazidime (0.9%); and amikacin, cefoxitin, and imipenem (0%). A bla(TEM) gene was found in 22 of the 25 ampicillin-resistant isolates, and the gene encoding CTX-M-14 beta-lactamase was identified in the two cefotaxime-resistant isolates (recovered from a common kestrel and a sparrowhawk), associated with bla(TEM-52) gene in one of them. Other resistance genes detected were as follows: aac(3)-II or aac(3)-IV genes in all gentamicin-resistant isolates; aadA1 or aadA2 in 22 of 25 streptomycin-resistant isolates; tet(A) and/or tet(B) in all 39 tetracycline-resistant isolates; and sul1 and/or sul2 and/or sul3 genes in all 21 SXT-resistant isolates. Two amino acid changes in GyrA protein (Ser83Leu + Asp87Asn) and one change in ParC protein (Ser80Ile) were identified in all 10 ciprofloxacin-resistant isolates of our series. The intestinal tract of wild animals is a reservoir of antibiotic resistance genes, especially for ampicillin, tetracycline, streptomycin, and SXT, and it is also remarkable that multiresistant E. coli isolates are detected in some of the tested animals.

Prevalence and Diversity of Integrons and Associated Resistance Genes in Escherichia coli Isolates from Poultry Meat in Tunisia

Fifty-five Escherichia coli isolates were acquired from chicken and turkey meat obtained from two slaughterhouses in Tunis. Eighty-nine percent, 80%, 78%, 67%, 45%, 27%, 7%, 4%, and 2% of these isolates showed resistance to tetracycline, trimethoprim/sulfamethoxazole, streptomycin, nalidixic acid, ampicillin, chloramphenicol, ciprofloxacin, colistine, and gentamicin, respectively. No resistance was detected to cefotaxime, ceftazidime, or amikacin. bla(TEM) gene was found in 22 of 25 ampicillin-resistant isolates, and 1 isolate harbored bla(OXA-1) gene. Tetracycline resistance was predominately mediated by the tetA gene. The sul1, sul2, and sul3 genes, alone or combined, were detected in 46 of 48 sulfonamide-resistant isolates, and sul1 and sul3 were included in class 1 integrons in some cases. Sixty percent of isolates harbored integrons (class 1, 30 isolates; class 2, 5 isolates). Class 2 integrons contained in all cases the dfrA1-sat1-aadA1-orfX gene cassette arrangement. Nine gene cassette arrangements have been detected among class 1 integrons, containing different alleles of dfrA (five alleles) and aadA (2 alleles) genes, which encode trimethoprim and streptomycin resistance, respectively. An uncommon gene cassette array (sat-psp-aadA2-cmlA1-aadA1-qacH-IS440-sul3) has been identified in three class 1 integron-positive isolates, and one additional isolate had this same structure with the insertion of IS26 inside the aadA1 gene (included in GenBank with accession no. FJ160769). The 55 studied isolates belong to the four phylogenic groups of E. coli, and phylogroups A and D were the most prevalent ones. At least one virulence-associated gene (fimA, papC, or aer) was detected in 44 of the 55 (80%) studied isolates. E. coli isolates of poultry origin could be a reservoir of antimicrobial-resistance genes and of integrons, and its evolution should be tracked in the future.

Genetic characterisation of CTX-M-15-producing Klebsiella pneumoniae and Escherichia coli strains isolated from stem cell transplant patients in Tunisia

Characterisation of extended-spectrum beta-lactamase (ESBL) genes and their genetic environments as well as the presence of integrons were analysed in nine Klebsiella pneumoniae and two Escherichia coli ESBL-positive isolates recovered in the Centre of Bone Marrow Transplantation of Tunisia. All strains harboured the bla(CTX-M-15) gene and presented minimum inhibitory concentrations for cefotaxime and ceftazidime of 256-1024 mg L(-1) and 16-512 mg L(-1), respectively, and eight of them showed different pulsed-field gel electrophoresis patterns. The bla(OXA-1) and bla(TEM-1) genes were detected in eight and ten strains, respectively. In addition, bla(SHV-1), bla(SHV-11) and bla(SHV-27) were found in six, one and one K. pneumoniae strains, respectively. The new variant bla(SHV-103) was characterised in one K. pneumoniae strain. The intI1 gene was detected in eight K. pneumoniae strains and the dfrA5+ereA2 and aadA gene cassettes were found in one and five strains, respectively. All strains harboured a 70 kb plasmid, and its transference in addition to bla(CTX-M-15), bla(TEM-1b) and bla(OXA-1) genes was demonstrated from three K. pneumoniae to E. coli. ISEcp1 and orf477 were located upstream and downstream, respectively, of the bla(CTX-M-15) gene in 10 strains. The occurrence of the bla(CTX-M-15) gene in unrelated strains might have originated from the dissemination of mobile genetic elements in which ISEcp1 may have played an important role.

Genetic environment of sul genes and characterisation of integrons in Escherichia coli isolates of blood origin in a Spanish hospital

The prevalence and characterisation of integrons and the genetic environment of sulphonamide resistance genes were studied in 135 Escherichia coli isolates recovered from blood cultures in a Spanish hospital during 2007. Class 1 and 2 integrons were identified in 54 isolates (intI1, 52 isolates; intI2, 1 isolate; and intI1+intI2, 1 isolate). Of the 53 intI1-positive isolates, 36 (67.9%) contained the classic class 1 integron including the qacEDelta1-sul1 region, and 11 different gene cassette arrangements were demonstrated in 33 of these isolates. Seventeen intI1-positive isolates lacked the qacEDelta1-sul1 region, and 8 gene cassette arrangements were demonstrated in 12 of these isolates. Seventy-one isolates showed a sulphonamide-resistant phenotype, 63 of which contained sul genes. The sul1 gene was associated with intI1 in 36 of 42 sul1-positive isolates, and the sul3 gene was associated with non-classic class 1 integrons in 5 of 7 sul3-positive isolates. Finally, sul2 was found associated with strA-strB genes in 32 of 35 sul2-positive isolates, identifying 11 genetic structures, 1 of them presenting the IS150 element disrupting the strB gene; this structure was included in GenBank with accession no. FJ705354. Almost one-half of the E. coli isolates from blood cultures contained integrons and sul genes. Moreover, sul genes were detected in different structures, one of them new, and could be important determinants in antibiotic resistance dissemination.

Resistance mechanisms and farm-level distribution of fecal Escherichia coli isolates resistant to extended-spectrum cephalosporins in pigs in Spain

INTRODUCTION Fecal Escherichia coli isolates showing a phenotype of reduced susceptibility or resistance to extended-spectrum cephalosporins are common among pigs in Spain. The aim of this study was to describe the main beta-lactam resistance mechanisms carried by these strains and their distribution at farm-level. MATERIALS AND METHODS Twenty-nine E. coli isolates showing reduced susceptibility or resistance to extended-spectrum cephalosporins were collected from a sampling frame of 80 pig farms distributed over 13 Spanish provinces. The survey was carried out at the slaughterhouse level in 2004. RESULTS Of the 29 isolates, 21 (72%) met the criteria for a positive phenotypic confirmatory test for extended-spectrum beta-lactamases (ESBL). The following ESBLs were detected: SHV-12 (12 isolates, 41%), CTX-M-1 (three isolates, 10%), CTX-M-9 (three isolates, 10%), and CTX-M-14 (three isolates, 10%). The remaining eight isolates (28%) were phenotypically non-ESBL, with seven of them (24%) showing mutations on the chromosomal ampC gene promoter at positions -42 (C-->T), -18 (G-->A), -1 (C-->T), and +58 (C-->T). A multiplex PCR for detection of plasmidic class C beta-lactamases was negative for all isolates. CONCLUSION Different ESBLs and other mechanisms linked to extended-spectrum cephalosporin resistance are widely distributed among fecal E. coli from slaughter pigs in Spain.

Antimicrobial resistance and phylogenetic groups in isolates of Escherichia coli from seagulls at the Berlengas nature reserve

Fifty-three faecal samples from yellow-legged gulls (Larus cachinnans) at the Berlengas nature reserve in Portugal were cultured on Levine agar plates not supplemented with antimicrobial agents, and one Escherichia coli colony was isolated and identified from each sample. The percentages of resistant isolates for each of the drugs were ampicillin (43·4 per cent), tetracycline (39·6 per cent), nalidixic acid (34·0 per cent), streptomycin (32·1 per cent), trimethoprimsulfamethoxazole (SXT) (26·4 per cent), ciprofloxacin (18·9 per cent), chloramphenicol (18·9 per cent), gentamicin (7·5 per cent), tobramycin (7·5 per cent) amikacin (5·7 per cent) and amoxicillin-clavulanic acid (1·9 per cent). All the isolates were susceptible to cefoxitin, ceftazidime, cefotaxime, aztreonam and imipenem. The following resistance genes were detected: blaTEM (17 of 23 ampicillin-resistant isolates), tet(A) and/or tet(B) (18 of 21 tetracycline-resistant isolates), aadA (12 of 17 streptomycin-resistant isolates), cmlA (all chloramphenicol-resistant isolates), aac(3)-II with or without aac(3)-IV (all four gentamicinresistant isolates), and sul1 and/or sul2 and/or sul3 (all 14 SXT-resistant isolates). The intI1 gene was detected in 10 of 14 SXT-resistant isolates, and three of them also contained class 2 integrons; four different gene cassette arrangements were identified among class 1 integrons (aadA, dfrA1+aadA1, dfrA12+orfF+aadA2 and sat+psp+aadA2) and one among the class 2 integrons (dfrA1+sat+aadA1). Ninety per cent of the isolates were included in the A or B1 phylogenetic groups.

Genetic Characterization of Extended-Spectrum Beta-Lactamases in Escherichia coli Isolates of Pigs from a Portuguese Intensive Swine Farm

There is a great concern by the emergence and the wide dissemination of extended-spectrum beta-lactamases (ESBLs) among animal Escherichia coli isolates. We intended to determinate the carriage level and type of ESBLs in E. coli obtained from fecal samples from pigs raised on an intensive pig farm in Portugal; further to characterize other associated resistance genes and their plasmid content, the phylogenetic groups, and the clonal relationship of ESBL-positive isolates. Sixty-five fecal samples were seeded in Levine media supplemented with cefotaxime for E. coli recovery. Susceptibility to 16 antimicrobial agents was performed by disk diffusion agar. ESBL-phenotypic detection was carried out by double-disk test; and the presence of the genes encoding TEM, OXA, SHV, and CTX-M type beta-lactamases was studied by polymerase chain reaction and sequencing. Other mechanisms of antimicrobial resistance and phylogenetic groups were also determined. Clonal relationship was performed by pulsed-field gel electrophoresis. ESBL-producing E. coli isolates were detected in 16 fecal samples, and one isolate per sample was studied. The CTX-M-1 type ESBL was detected in the 16 isolates. The gene encoding TEM-1 was identified to be associated with eight CTX-M-1-positive isolates. The tet(A) gene was found in 12 of 14 tetracycline-resistant isolates, and the aadA or strA-strB genes were found in the streptomycin-resistant isolates. Fourteen and two ESBL-containing isolates belonged to A and B1 phylogenetic groups, respectively. Clonal relationship of ESBL-containing isolates identified seven unrelated patterns. Swine represent an important reservoir of ESBL-containing E. coli isolates, especially of the CTX-M-1 type.