Céline Coelho

Genetic Detection of Extended-Spectrum β-Lactamase-Containing Escherichia coli Isolates from Birds of Prey from Serra da Estrela Natural Reserve in Portugal

ABSTRACT Extended-spectrum β-lactamase-containing Escherichia coli isolates were detected in 32 of 119 fecal samples (26.9%) from birds of prey at Serra da Estrela, and these isolates contained the following β-lactamases: CTX-M-1 (n = 13), CTX-M-1 plus TEM-1 (n = 14), CTX-M-1 plus TEM-20 (n = 1), SHV-5 (n = 1), SHV-5 plus TEM-1 (n = 2), and TEM-20 (n = 1).

Proteomic characterization of vanA-containing Enterococcus recovered from Seagulls at the Berlengas Natural Reserve, W Portugal

BackgroundEnterococci have emerged as the third most common cause of nosocomial infections, requiring bactericidal antimicrobial therapy. Although vancomycin resistance is a major problem in clinics and has emerged in an important extend in farm animals, few studies have examined it in wild animals. To determine the prevalence of van A-containing Enterococcus strains among faecal samples of Seagulls (Larus cachinnans) of Berlengas Natural Reserve of Portugal, we developed a proteomic approach integrated with genomic data. The purpose was to detect the maximum number of proteins that vary in different enterococci species which are thought to be connected in some, as yet unknown, way to antibiotic resistance.ResultsFrom the 57 seagull samples, 54 faecal samples showed the presence of Enterococcus isolates (94.7%). For the enterococci, E. faecium was the most prevalent species in seagulls (50%), followed by E. faecalis and E. durans (10.4%), and E. hirae (6.3%). VanA-containing enterococcal strains were detected in 10.5% of the 57 seagull faecal samples studied. Four of the vanA-containing enterococci were identified as E. faecium and two as E. durans. The tet(M) gene was found in all five tetracycline-resistant vanA strains. The erm(B) gene was demonstrated in all six erythromycin-resistant vanA strains. The hyl virulence gene was detected in all four van A-containing E. faecium isolates in this study, and two of them harboured the pur K1 allele. In addition these strains also showed ampicillin and ciprofoxacin resistance. The whole-cell proteomic profile of van A-containing Enterococcus strains was applied to evaluate the discriminatory power of this technique for their identification. The major differences among species-specific profiles were found in the positions corresponding to 97-45 kDa. Sixty individualized protein spots for each vanA isolate was identified and suitable for peptide mass fingerprinting measures by spectrometry measuring (MALDI/TOF MS) and their identification through bioinformatic databases query. The proteins were classified in different groups according to their biological function: protein biosynthesis, ATP synthesis, glycolysis, conjugation and antibiotic resistance. Taking into account the origin of these strains and its relation to infectious processes in humans and animals, it is important to explore the proteome of new strains which might serve as protein biomarkers for biological activity.ConclusionsThe comprehensive description of proteins isolated from vancomycin-resistant Enterococcus faecium and E. durans may provide new targets for development of antimicrobial agents. This knowledge may help to identify new biomarkers of antibiotic resistance and virulence factors.

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.

MLST and a genetic study of antibiotic resistance and virulence factors in vanA-containing Enterococcus from buzzards (Buteo buteo)

Aims:  To analyse the occurrence of faecal carriage of vancomycin‐resistant enterococci (VRE) in Buteo buteo and to study the associated resistance and virulence genes.

Prevalence and mechanisms of erythromycin resistance in Streptococcus agalactiae from healthy pregnant women.

We sought to determine the resistance phenotypes for erythromycin and clindamycin and the mechanisms implicated in 93 Streptococcus agalactiae isolates recovered from healthy pregnant women. Susceptibility testing for erythromycin, clindamycin, penicillin, cefotaxime, vancomycin, quinupristin-dalfopristin, choramphenicol, ofloxacin, and meropenen was carried out by disc-diffusion test, and the E-test was also applied for erythromycin and clindamycin. The constitutive MLS(B) resistance (cMLS(B)) and inducible MLS(B) resistance (iMLS(B)) phenotypes, respectively, as well as the M resistance phenotype were determined by the erythromycin-clindamycin double-disc test. The presence of ermA, ermB, ermC, msrA, and mef(A/E) macrolide resistance genes was studied by PCR. Resistance to erythromycin and clindamycin was found in 15% and 9.6% of the isolates, respectively. The resistance phenotypes detected among the 14 erythromycin-resistant isolates were as follows (number of isolates): cMLS(B) (9), iMLS(B) (3), and M (2). The MICs for erythromycin and clindamycin were as follows: cMLS(B) isolates (128-256 and >or=32 mg/L, respectively), iMLS(B) isolates (16-256 and 1 mg/L), and M isolates (2-8 and 1 mg/L). The following combination of genes were detected among isolates with cMLS(B) or iMLS(B) phenotypes: erm(B) (6 isolates), ermA + ermTR (3), ermA + ermB + ermTR (1), and none of these genes (2). The two isolates with M phenotype harbored the mef(A/E), and msrA gene was also found in one of them.