Beatriz Rojo-Bezares

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.

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.

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 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.

Tn1546 structures and multilocus sequence typing of vanA-containing enterococci of animal, human and food origin

OBJECTIVES To characterize the Tn1546 structure and to perform the genetic typing of 51 PFGE-unrelated vanA-containing enterococci of different origin (clinical, food and faecal samples of healthy humans and healthy poultry). METHODS Tn1546 structure was characterized by a PCR primer walking strategy and sequencing. Multilocus sequence typing (MLST) was performed for Enterococcus faecium and Enterococcus faecalis strains, and esp and hyl genes were detected by PCR. RESULTS Nine different Tn1546 structures were identified in the studied strains. Type I was the most prevalent structure (75%) (identical to GenBank M97297). Two new Tn1546 structures were identified (in three clinical and animal strains), containing two new insertion sequences (ISs; ISEfa11 disrupting vanS and ISEfa10 disrupting orf1). An additional new Tn1546 structure was found in one animal strain, containing ISEf1 interrupting vanY and IS1542 in the orf2-vanR region. A high diversity of sequence types (STs) was detected among clinical (6 ST/18 strains) and non-clinical E. faecium strains (18 ST/24 strains). STs associated with clonal complexes CC17 and CC9 were mainly detected among clinical and non-clinical E. faecium strains, respectively. Seven new STs were identified in non-clinical strains. The esp and hyl genes were only found among clinical E. faecium strains. CONCLUSIONS A moderate variability in Tn1546 structure has been detected among unrelated vanA-containing enterococci of different origins, showing three new structures including two new ISs. A high diversity of STs was detected among E. faecium strains, especially among non-clinical strains, and new STs have been identified.

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 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.

First detection of blaIMI-2 gene in a clinical Escherichia coli strain

The dissemination of carbapenem-resistant Enterobacteriaceae is increasing worldwide in the last decade, mainly due to the acquisition of beta-lactamase genes encoding carbapenemases. Among class A carbapenemases, KPC is widespread (14); however, IMI enzymes have been described so far only in Enterobacter genus (1, 10, 15). The detected blaIMI genes are linked to a gene encoding a LysR-type transcriptional regulator, and as previously reported, the blaIMI-1 gene is located in the chromosome of Enterobacter cloacae (10), whereas blaIMI-2 is related to plasmids (1, 15). We report here the first description of a blaIMI-2-positive Escherichia coli strain. (Part of this study was presented at the 21st European Congress of Clinical Microbiology and Infectious Diseases Congress, Milan, Italy, 7 to 10 May 2011.) A carbapenem-resistant E. coli strain, W635, was recovered from a blood sample of an elderly oncologic patient (without any history of travel) who was admitted with sepsis and treated with piperacillin-tazobactam at a Spanish hospital in 2010. E. coli W635 was ascribed to a new sequence type registered as ST1998 (http: //mlst.ucc.ie/mlst/dbs/Ecoli) and showed resistance to imipenem (IPM), meropenem (MEM), ertapenem (ETP), doripenem, ampicillin (AMP), ticarcillin, amoxicillin-clavulanic acid (AMC), cephalothin, streptomycin, nalidixic acid, ciprofloxacin, norfloxacin, sulfonamides, and trimethoprim and intermediate resistance to aztreonam and chloramphenicol by the CLSI disk diffusion method (3). A class A carbapenemase phenotype was demonstrated in this strain by double-disk synergy test (5, 9). After multiplex PCR (8) and subsequent sequencing, a partial sequence of the blaIMI-2 gene was detected in E. coli W635. To gain insight into the blaIMI-2 gene and its surrounding structure, the flanking regions were amplified by PCR using specific primers designed in this work (according to GenBank accession number AY780889). The LysR-type regulator gene (blaIMI-2R) was found upstream of the blaIMI-2 gene, and their genetic environment was studied by inverse PCR using PvuII and BglII restriction enzymes. Sequence analysis revealed a total fragment of 6,184 bp that was deposited in GenBank database with the accession number JN412066. A new insertion sequence of 1,321 bp, designed ISEc36 by ISFinder (http://www-is.biotoul.fr/), was detected upstream of the blaIMI-2R gene. This IS belongs to the IS3 family and IS2 group, and it exhibits an identity of 92% with respect to the ISEc27 sequence (GenBank accession number AY857617). Testing for the presence of genes implicated in other antimicrobial resistances (beta-lactams, aminoglycosides, quinolones, trimethoprim, and sulfonamides) and the study of mutations in gyrA and parC genes, as well as the characterization of integrons and sul2 gene environment, were performed by PCR and sequencing (4, 11, 13). Table 1 shows the MICs and genotypic results. Two different transconjugants from W635 were obtained by mating experiments, using E. coli CSH26 as recipient and plates supplemented with either MEM (8 g/ml) or AMP (50 g/ml) and rifampin (100 g/ml). Detection and typing of plasmids of E. coli W635 and transconjugant strains were carried out by PCRbased replicon typing (2, 6). Plasmids of strain W635 and its transconjugants belonging to incompatibility group I1 (IncI1) and F (IncF) were subtyped by plasmid multilocus sequence typing (7, 12). E. coli W635 contained the following typeable plasmids: IncI1 (ST26, CC-26), IncF (F43:A :B ; Y2 variant:A :B ), and ColETP. The location of blaIMI-2 in strain W635 and its transconjugants was studied by pulsed-field gel electrophoresis (PFGE)-S1 nuclease and PFGE-XbaI Southern blotting and hybridization (4) with blaIMI-2, IncI1, IncF, and ColETP probes. The blaIMI-2 gene in E. coli W635 was detected in an IncF plasmid of approximately 48.5 kb. The size of this plasmid is smaller than found in previously reported results that described the blaIMI-2 gene located on transferable plasmids with sizes of 66 kb or 80 kb (1, 15). This is the first report of a carbapenem-resistant E. coli strain carrying the class A carbapenemase IMI-2. The blaIMI-2 gene located in a conjugative plasmid and linked to mobile elements might significantly spread between different members of the Enterobacteriaceae, and therefore this emerging resistance mechanism should be tracked in the future. Nucleotide sequence accession number. The nucleotide sequence of the novel genetic environment of blaIMI-2 gene determined in this study was included in the GenBank database with the accession number JN412066.

Comparison of Local Features from Two Spanish Hospitals Reveals Common and Specific Traits at Multiple Levels of the Molecular Epidemiology of Metallo-β-Lactamase-Producing Pseudomonas spp.

ABSTRACT 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.

Characterisation of plasmids implicated in the mobilisation of extended-spectrum and AmpC β-lactamase genes in clinical Salmonella enterica isolates and temporal stability of the resistance genotype.

Plasmids implicated in the mobilisation of β-lactamase genes in extended-spectrum β-lactamase (ESBL)- and AmpC-producing Salmonella enterica isolates recovered from three Spanish hospitals were characterised. The temporal stability of these plasmids and of the resistance phenotype without antimicrobial pressure was also assessed in the laboratory setting. The resistance determinants and their genetic environments were characterised by PCR sequencing, and their genomic location was analysed by S1 nuclease pulsed-field gel electrophoresis (PFGE) and I-CeuI PFGE, followed by Southern blot hybridisation. The 11 S. enterica studied strains carried blaCTX-M-9 (serovar Virchow, 2 isolates), blaCTX-M-10 (Virchow, 2), blaCTX-M-14 (Enteritidis, 1), blaCTX-M-15 (Gnesta and S. enterica group C, 2), blaSHV-2 (Livingstone, 1), blaSHV-12 (Enteritidis, 1) and blaCMY-2 (Bredeney, 2). The ISEcp1-blaCTX-M-14-IS903 and ISEcp1-blaCTX-M-15-orf477 genetic structures were detected. IncI1 and IncA/C plasmids carried blaCTX-M-14, blaCTX-M-15, blaSHV-2, blaSHV-12 and blaCMY-2 genes. blaCTX-M-9 included in an In60 complex integron and blaCTX-M-10 linked to a phage-related element were found in non-typeable plasmids. Conjugation and temporal stability experiments were performed in vitro through daily passages (100 days) in the absence of antimicrobial pressure. In the stability experiments, 5 of the 11 tested isolates lost the ESBL or AmpC plasmidic genes and this was associated with concomitant loss of the whole or partial plasmid. In conclusion, successful plasmids belonging to different Inc groups mobilise ESBL- and AmpC-encoding genes in S. enterica. Loss of ESBL/AmpC genes in the absence of antimicrobial pressure might explain the low prevalence of these β-lactamases among Salmonella isolates.

Phenotypic and Genotypic Characterization of Salmonella enterica Recovered from Poultry Meat in Tunisia and Identification of New Genetic Traits

Thirty-seven Salmonella enterica isolates obtained from poultry meat in Tunisia were included in this study for characterization of antibiotic resistance mechanisms. High percentages of resistance were detected to ampicillin, sulfonamides, tetracycline, nalidixic acid, and streptomycin (32.4%-89.2%), and lower percentages to amoxicillin-clavulanic acid, kanamycin, amikacin, trimethoprim-sulfamethoxazol, and chloramphenicol (2.7%-18.9%). All strains showed susceptibility to ceftazidime, cefotaxime, gentamicin, and ciprofloxacin. Class 1 integrons were detected in 30% of Salmonella isolates, and four different gene cassette arrangements were detected, including genes implicated in resistance to aminoglycosides (aadA1 and aadA2) and trimethoprim (dfrA1). Four different Pc variants (PcW, PcH1, PcH1(TTN-10), PcW(TGN-10)) with inactive P2 have been found among these isolates. Integron-positive isolates were ascribed to eight different serotypes. A Salmonella Schwarzengrund isolate harbored a new class 1 integron containing the qacH-dfrA1b-aadA1b-catB2 gene cassette arrangement, with the very unusual PcH1(TTN-10) promoter, which has been registered in GenBank (accession no. HQ874651). Different plasmid replicon types were demonstrated among integron-positive isolates: IncI1 (8 isolates), IncN (8), IncP (2), IncFIB (2), and IncFII (2). Ten different pulsed-field gel electrophoresis profiles were detected among the 11 integron-positive isolates and 8 different sequence types were identified by multilocus sequence typing, one of them (registered as ST867) was new, detected in 3 Salmonella Zanzibar isolates. A high diversity of clones is observed among poultry Salmonella isolates and a high proportion of them show a multiresistant phenotype with very diverse mobile genetic structures that could be implicated in bacterial dissemination in different environments.