Analysis of blaSHV-12-carrying Escherichia coli clones and plasmids from human, animal and food sources.

Objectives: This study aimed at characterizing 23 Escherichia coli isolates from various sources and their respective bla SHV-12 -carrying plasmids and sequencing one of these plasmids completely. Methods: Isolates were typed by XbaI-PFGE, MLST and PCR-based phylotyping. Transformed bla SHV-12 -carrying plasmids were examined by replicon typing, S1-nuclease, conjugation, EcoRI-HindIII-BamHI digests and plasmid MLST. Co-located resistance genes and integrons as well as the bla SHV-12 genetic environment were analysed by PCR and sequencing. One IncI1 plasmid was sequenced completely using HiSeq 2500 and gap closure by PCRs and Sanger sequencing. Results: Among the 23 SHV-12-positive E. coli , some isolates from different sources showed the same characteristics: ST23/phylogroup A (human, dog, livestock), ST57/D (wild bird, chicken meat) and ST117/D (chicken meat, chicken). All bla SHV-12 genes were horizontally transferable via 30-120 kb plasmids of incompatibility groups IncI1 ( n = 17), IncK ( n = 3), IncF ( n = 1), IncX3 ( n = 1) and a non-typeable plasmid. IncK plasmids, indistinguishable in size and restriction patterns, were found in isolates from different sources (ST57/D, meat; ST131/B2, meat; ST57/B1, dog). The IncI1- bla SHV-12 -carrying plasmids were mostly assigned to plasmid ST (pST) 26 and pST3. Three plasmids showed novel pSTs (pST214, pST215). The majority of the IncI1 transformants exhibited resistance to ß-lactams, chloramphenicol and streptomycin (in relation with a class 1 integron containing an estX - psp - aadA2 - cmlA1 - aadA1 - qacI gene cassette array), and to tetracycline. A novel bla SHV-12 environment was detected and whole plasmid sequencing revealed a Tn 21 -derived- bla SHV12 -ΔTn 1721 resistance complex. Conclusions: Results from this study suggest that the dissemination of bla SHV-12 genes occurs by vertical (clonal) and horizontal transfer, the latter mainly mediated through IncI1 multidrug-resistance plasmids.

Molecular characterization of antibiotic resistance in Escherichia coli strains from a dairy cattle farm and its surroundings.

BACKGROUND: This study describes the phenotypic and genotypic characteristics of 78 genetically different Escherichia coli recovered from air and exudate samples of a dairy cattle farm and its surroundings in Spain, in order to gain insight into the flow of antimicrobial resistance through the environment and food supply. RESULTS: Antimicrobial resistance was detected in 21.8% of the 78 E. coli isolates analyzed (resistance for at least one of the 14 agents tested). The highest resistance rates were recorded for ampicillin, nalidixic acid, trimethoprim/sulfamethoxazole and tetracycline. The resistance genes detected were as follows (antibiotic (number of resistant strains), gene (number of strains)): ampicillin (9), blaTEM-1 (6); tetracycline (15), tet(A) (7), tet(B) (4), tet(A) + tet(B) (1); chloramphenicol (5), cmlA (2), floR (2); trimethoprim/sulfamethoxazole (10), sul2 (4), sul1 (3), sul3 (2), sul1 + sul2 (1); gentamicin-tobramycin (1), ant(2″) (1). About 14% of strains showed a multidrug-resistant phenotype and, of them, seven strains carried class 1 integrons containing predominantly the dfrA1-aadA1 array. One multidrug-resistant strain was found in both inside and outside air, suggesting that the airborne spread of multidrug-resistant bacteria from the animal housing facilities to the surroundings is feasible. CONCLUSIONS: This study gives a genetic background of the antimicrobial resistance problem in a dairy cattle farm and shows that air can act as a source for dissemination of antimicrobial-resistant bacteria. © 2016 Society of Chemical Industry.

Airborne dissemination of Escherichia coli in a dairy cattle farm and its environment.

There are multiple ways bacteria can be transported from its origin to another area or substrate. Water, food handlers, insects and other animals are known to serve as a vehicle for bacterial dispersion. However, the importance of the air in open areas as a possible way of bacterial dissemination has not been so well analyzed. In this study, we investigated the airborne dissemination of Escherichia coli from the inside of a dairy cattle farm to the immediate environment. The air samples were taken inside the farm (area 0) and from the immediate outside farm surroundings at distance of 50, 100 and 150m in four directions (north, south, east, and west). At each point, the air was collected at different heights: 40cm, 70cm and 1m. The sampling was carried out in two weather seasons (November and July). E. coli was isolated in both inside and outside air, even in samples taken 150m from the farm. A seasonal effect was observed with more bacterial isolates when temperature was higher. Regarding the distribution of the isolates, wind direction appeared as a determining factor. In order to verify that E. coli strains isolated from animal housing facilities were identical to those isolated from the air of the immediate farm environment, their genomic DNA profiles were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion with the endonuclease XbaI. The comparison of genetic profiles suggested that the strains isolated from inside and outside the farm were related, leading to the conclusion that the air is an important vehicle for E. coli dissemination.

Characterization of Beta-lactamases in Faecal Enterobacteriaceae Recovered from Healthy Humans in Spain: Focusing on AmpC Polymorphisms.

The intestinal tract is a huge reservoir of Enterobacteriaceae, some of which are opportunist pathogens. Several genera of these bacteria harbour intrinsic antibiotic resistance genes, such as ampC genes in species of Citrobacter, Enterobacter or Escherichia genera. In this work, beta-lactamases and other resistance mechanisms have been characterized in Enterobacteriaceae isolates recovered from healthy human faecal samples, focusing on the ampC beta-lactamase genes. Fifty human faecal samples were obtained, and 70 Enterobacteriaceae bacteria were isolated: 44 Escherichia coli, 4 Citrobacter braakii, 9 Citrobacter freundii, 8 Enterobacter cloacae, 1 Proteus mirabilis, 1 Proteus vulgaris, 1 Klebsiella oxytoca, 1 Serratia sp. and 1 Cronobacter sp. A high percentage of resistance to ampicillin was detected (57%), observing the AmpC phenotype in 22 isolates (31%) and the ESBL phenotype in 3 isolates. AmpC molecular characterization showed high diversity into bla CMY and bla ACT genes from Citrobacter and Enterobacter species, respectively, and the pulsed-field gel electrophoresis (PFGE) analysis demonstrated low clonality among them. The prevalence of people colonized by strains carrying plasmid-mediated ampC genes obtained in this study was 2%. The unique plasmid-mediated bla AmpC identified in this study was the bla CMY-2 gene, detected in an E. coli isolate ascribed to the sequence type ST405 which belonged to phylogenetic group D. The hybridization and conjugation experiments demonstrated that the ISEcp1-bla CMY-2-blc structure was carried by a ~78-kb self-transferable IncK plasmid. This study shows a high polymorphism among beta-lactamase genes in Enterobacteriaceae from healthy people microbiota. Extensive AmpC-carrier studies would provide important information and could allow the anticipation of future global health problems.

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.

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.

Gilthead seabream (Sparus aurata) as carriers of SHV-12 and TEM-52 extended-spectrum beta-lactamases-containing Escherichia coli isolates.

In recent years, bacterial resistance to beta-lactam antibiotics has risen dramatically in Escherichia coli isolated from animals that could pass through the food chain to humans. One hundred eighteen fecal samples of Sparus aurata were tested for extended-spectrum beta-lactamase (ESBL)-containing E. coli recovery. Susceptibility to 16 antimicrobial agents was performed by disk diffusion. ESBL-phenotypic detection was carried out by double-disk test, and the presence of genes encoding TEM, OXA, SHV, and CTX-M type beta-lactamases was studied by polymerase chain reaction and sequencing. The detection of other antimicrobial resistance mechanisms and phylogenetic groups was also performed in recovered isolates as well as their clonal diversity by pulsed-field gel electrophoresis. Five of the 118 fecal samples harbored ESBL-positive E. coli isolates (4.2%), and one isolate per sample was completely characterized. These five ESBL-positive E. coli isolates contained the bla(TEM-52) or bla(SHV-12) genes, as well as a variety of other resistance genes (cmlA, tetA, aadA, sul1, sul2, and sul3). Four isolates harbored class 1 integrons with the following gene cassettes in their variable region: dfrA1 + aadA1 (one isolate) and sat + psp + aadA2 (three isolates). Four unrelated pulsed-field gel electrophoresis patterns were identified among the five ESBL-positive isolates, and they were ascribed to phylogroups A and B1. The intestinal tract of S. aurata might constitute a reservoir of ESBL-producing E. coli isolates.

Diversity of genetic lineages among CTX-M-15 and CTX-M-14 producing Escherichia coli strains in a Tunisian hospital.

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.

Antimicrobial resistance, integrons and plasmid replicon typing in multiresistant clinical Escherichia coli strains from Enugu State, Nigeria.

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.

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.

Detection of CTX-M-14 and TEM-52 extended-spectrum beta-lactamases in fecal Escherichia coli isolates of captive ostrich in Portugal.

The main aim of this study was to determine the frequency of antibiotic resistance among Escherichia coli isolates recovered in Levine agar plates from 54 fecal samples of captive ostriches from a farm in the South of Portugal. Fifty-four nonselected E. coli isolates were obtained (one/sample) and the phenotypes and genotypes of antibiotic resistance were characterized. The following numbers of isolates showed antibiotic resistance: ampicillin (nine), tetracycline (seven), streptomycin (three), amoxicillin-clavulanic acid, cefoxitin, or gentamicin (one), and cefotaxime, ceftazidime, azthreonam, imipenem, nalidixic acid, ciprofloxacin, and trimethoprim/sulfamethoxazole (zero). The bla(TEM) gene was identified in six out of nine ampicillin-resistant isolates, and the tet(A) or tet(B) genes in five out of seven tetracycline-resistant isolates. Mutations at positions -42, -18, -1, and +58 of ampC promoter region were identified in one cefoxitin-resistant isolate. Further, the occurrence of extended-spectrum beta-lactamase (ESBL)-producing E. coli isolates was estimated in the 54 fecal samples of ostriches using cefotaxime-supplemented Levine agar plates for ESBL-positive E. coli recovery. Three samples contained ESBL-positive E. coli isolates of which one isolate/sample was characterized, leading to the detection of the following beta-lactamases: bla(CTX-M-14a) + bla(TEM-1b) (two isolates) and bla(TEM-52c) (one isolate). The three ESBL-positive isolates were classified into the phylogroup B1, and contained class 1 integrons with the gene cassettes dfrA17 + aadA5 (one isolate) and aadA1 (two isolates). This study adds to our knowledge about the wide dissemination of ESBL-producing E. coli isolates in different ecosystems, including captive ostriches, that could be transferred to humans through the food chain.

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.

Prevalence of broad-spectrum cephalosporin-resistant Escherichia coli isolates in food samples in Tunisia, and characterization of integrons and antimicrobial resistance mechanisms implicated.

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.

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 diversity of the pln locus among oenological Lactobacillus plantarum strains.

A total of 33 Lactobacillus plantarum strains obtained from grape musts and wines during alcoholic and malolactic fermentations were submitted to PCR analysis with specific primers directed to 27 genes of the plantaricin (pln) locus previously described for L. plantarum strains. The number of genes that were detected varied depending on the strain, and cluster analysis of results rendered seven groups, named plantaritypes (similarity within each group >89%) that included all the 33 oenological strains, four L. plantarum type strains (C11, NC8, J23 and J51) that had been previously described, and strain WCFS1 whose genome had been fully sequenced. The common features for most strains (74%) were the presence of the plnABCD regulatory system (which includes genes of the inducing peptide, its coupled membrane-located histidine protein kinase and two response regulators), the two-peptide bacteriocin plnEF genes and the genes of a membrane-bound ABC transport system. The pln locus is shown to be widespread among oenological strains (94% of appearance), as well as to possess a remarkable plasticity and variable regions related to its regulation and bacteriocin production.

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.

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 integrons and associated resistance genes in faecal Escherichia coli isolates of healthy humans in Spain.

OBJECTIVES: To analyse the prevalence and diversity of integrons in faecal Escherichia coli isolates from healthy humans in Spain. METHODS: One hundred E. coli isolates were obtained in Levine agar plates from faecal samples of 100 healthy humans during March to October 2007. Susceptibility to 16 antimicrobial agents was determined by the disc diffusion method. The presence and characterization of class 1, 2 and 3 integrons, as well as the presence of other antimicrobial resistance genes, were performed by PCR and DNA sequencing. RESULTS: Integrases associated with class 1 and/or class 2 integrons were identified in 29 E. coli isolates (intI1 gene in 26 isolates, intI2 in 1 isolate and intI1 + intI2 in 2 isolates), the remaining 71 isolates being free of these integrons. Seven different gene cassette arrangements were demonstrated in 27 of the 28 intI1-positive isolates and were as follows (number of isolates): dfrA1 + aadA1 (12), aadA (8), dfrA17 + aadA5 (3), dfrA7 (1), dfrA5 (1), dfrA1 (1) and dfrA12 + orfF + aadA2 (1). Four isolates presented defective class 1 integrons lacking the 3'-conserved region. The three isolates containing class 2 integrons harboured the dfrA1 + sat + aadA1 gene cassette array in their variable region. Integron-positive isolates showed higher percentages of resistance to streptomycin, ampicillin, tetracycline, trimethoprim, sulfamethoxazole, chloramphenicol and nalidixic acid than integron-negative isolates. Sixty-five percent of the integron-positive isolates belonged to phylogenetic groups A or D. CONCLUSIONS: A high prevalence of integrons was detected in faecal E. coli of healthy humans. Individuals in the community could be a reservoir of integron-containing E. coli isolates.