Hajer Radhouani - Academia.edu (original) (raw)

Papers by Hajer Radhouani

Research paper thumbnail of In vitro activity of dalbavancin against enterococci isolates from wild animals, pets, poultry and humans in Portugal

Journal of Basic Microbiology, 2008

Dalbavancin is a new new semisynthetic teicoplanin-related lipoglycopeptide with activity against... more Dalbavancin is a new new semisynthetic teicoplanin-related lipoglycopeptide with activity against gram-positive organisms. We investigated the activity of dalbavancin against faecal enterococci isolates from wild animals, pets, poultry and healthy humans in Portugal.The in vitro activity of dalbavancin was determined by the microbroth dilution method according to the Clinical Laboratory Standards Institute (CLSI) guidelines in 589 enterococci of different species and origins. All vancomycin-susceptible Enterococcus spp. were inhibited by ≤0.25 mg/l dalbavancin. Although vancomycin-resistant-enterococci (VRE) showed higher dalbavancin MIC values (16 mg/l), the isolates that exhibited the VanC resistance phenotype were inhibited at dalbavancin concentrations ≤0.125 mg/l. Only van A isolates were not inhibited by low concentrations of dalbavancin since van A strains showed higher dalbavancin MIC values (16 mg/l). (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Research paper thumbnail of Genetic characterization of vancomycin-resistant enterococci isolates from wild rabbits

Journal of Basic Microbiology, 2009

The presence of van A-containing E. faecium isolates was demonstrated in three of 77 faecal sampl... more The presence of van A-containing E. faecium isolates was demonstrated in three of 77 faecal samples (3.9%) of wild rabbits recovered in Portugal. Enterococcal strains with intrinsic vancomycin resistance (van C-1 or van C-2/3 gene) were found in five (6.5%) and three (3.9%) faecal samples, respectively. The mechanisms of resistance for other antibiotics were studied in these vancomycin-resistant isolates. All van A strains showed resistance for tetracycline [with the presence of tet (L) gene, associated or not with tet (M) gene] and for erythromycin [with the presence of the erm (B) gene]. Two isolates were resistant to ciprofloxacin and one to ampicillin. Two van C-1 strains and one van C-2/3 strain were tetracycline resistant [containing the tet (M) gene associated with tet (L) gene] and erythromycin resistant [with erm (B) gene]. Two van C-1 and two van C-2/3 strains were also ciprofloxacin resistant and one van C-1 strain was, additionally, resistant to quinupristin-dalfopristin. The two remaining isolates (van C-1, van C-2/3) did not show resistance for any additional antibiotic. The intestinal tract of wild rabbits could be a reservoir of van A-containing enterococci. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Research paper thumbnail of Gilthead seabream ( Sparus aurata) carrying antibiotic resistant enterococci. A potential bioindicator of marine contamination

Marine Pollution Bulletin, 2011

Antibiotic resistance in bacteria is a growing problem that is not only restricted to the clinica... more Antibiotic resistance in bacteria is a growing problem that is not only restricted to the clinical setting but also to other environments such as marine species that harbor antibiotic resistant bacteria and therefore may serve as reservoirs for antibiotic-resistance genetic determinants. The aim of this study was to evaluate antibiotic resistance phenotypes in enterococci isolated from fecal samples of gilthead seabream and the associated mechanisms of resistance. A collection of 118 samples were analyzed and 73 enterococci were recovered. The strains showed high percentages of resistance to erythromycin and tetracycline (58.9% and 17.8%, respectively). Lower level of resistance (<13%) was detected for quinupristin-dalfopristin, ampicillin, high-level-gentamicin, high-level-streptomycin, high-level-kanamycin, ciprofloxacin and chloramphenicol. The erm(B), tet(L) or tet(M), aac(6')-aph(2″) and…

Research paper thumbnail of MLST and a genetic study of antibiotic resistance and virulence factors in vanA -containing Enterococcu s from buzzards ( Buteo buteo

Letters in Applied Microbiology, 2010

Aims: To analyse the occurrence of faecal carriage of vancomycin-resistant enterococci (VRE) in ... more 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.Methods and Results: The presence of VRE was investigated in 33 faecal samples of B. buteo. Samples were seeded in Slanetz–Bartley agar plates supplemented with vancomycin for VRE recovery. Genes encoding antimicrobial resistance and virulence were studied by polymerase chain reaction. Vancomycin-resistant Enterococcus faecium isolates were characterized by multilocus sequence typing. VRE with an acquired mechanism of resistance (vanA genotype) were detected in 9% of samples analysed (Ent. faecium and Enterococcus durans). In addition, 27% of samples contained VRE with an intrinsic mechanism of resistance (Enterococcus gallinarum, vanC1). All vanA-containing isolates showed resistance to tetracycline and erythromycin and harboured the tet(M) and/or tet(L) genes, in addition to the ermB gene. The vat(E) and/or vat(D), cat(A) and aph(3′)-IIIa genes were identified in quinupristin–dalfopristin-, chloramphenicol-, and kanamycin-resistant vanA-containing strains, respectively. The sequence types ST273 and ST5 were identified in two vanA-positive Ent. faecium isolates, and the presence of hyl, gelE, cylA, cylL and cylM virulence genes and gelatinase activity were identified in Ent. faecium ST5 strain.Conclusions: The intestinal tract of B. buteo could be a reservoir of vanA-positive enterococci.Significance and Impact of the Study: First study focused to define the occurrence of vanA-containing Enterococcus strains in B. buteo.

Research paper thumbnail of Detection of antibiotic resistant E. coli and Enterococcus spp. in stool of healthy growing children in Portugal

Journal of Basic Microbiology, 2009

From stool specimens of 118 healthy children's (1–14 years) in Portugal 92 E. coli and 101 Entero... more From stool specimens of 118 healthy children's (1–14 years) in Portugal 92 E. coli and 101 Enterococcu s spp. strains have been isolated. Almost half (40.2%) of the E. coli isolates were resistant to ampicillin, 25.0% were resistant to tetracycline and 26.1% were resistant to streptomycin. Resistance genes detected by specific PCR included blaTEM and/or blaSHV and/or blaCTX-M (33 of 37 ampicillin and/or cefotaxime resistant isolates), tet (A) and/or tet (B) (16 of 23 tetracycline-resistant isolates), aad A (19 of 24 streptomycin-resistant isolates), cml A (in the two chloramphenicol-resistant isolates), aac (3)-II with/without aac (3)-IV (in the four gentamicin-resistant isolates), sul 1 and/or sul 2 and/or sul 3 (in all trimethoprim/sulfamethoxazole resistant isolates). The majority of the resistant E. coli isolates (69.1%) belonged to phylogenetic group B2. Of the enterococci isolates E. faecium (n = 53), E. faecalis (n = 41), E. hirae (n = 4) and E. durans (n = 3) more than one-fourth (28.7%) of the isolates were resistant to tetracycline; 21.8% were resistant to erythromycin and 8.9% were resistant to kanamycin. Resistance genes detected by PCR in enterococci included aph (3)′-IIIa (in all kanamycin-resistant isolates), aac (6′) (in all gentamicin-resistant isolates), tet (M) and/or tet (L) (26 of 29 tetracycline-resistant isolates), erm (B) (17 of 22 erythromycin-resistant isolates). This survey showed that faecal bacteria such as E. coli and enterococci of healthy growing children's could be a reservoir of antimicrobial resistance genes. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Research paper thumbnail of Detection of Escherichia coli harbouring extended-spectrum -lactamases of the CTX-M classes in faecal samples of common buzzards (Buteo buteo

Journal of Antimicrobial Chemotherapy, 2010

Research paper thumbnail of Detection and genetic characterisation of vanA-containing Enterococcus strains in healthy Lusitano horses: vanA-containing enterococcus in Lusitano horses

Equine Veterinary Journal, 2010

Lusitano horses were investigated in order to detect the presence of vancomycin-resistant enteroc... more Lusitano horses were investigated in order to detect the presence of vancomycin-resistant enterococci. vanA isolates showed high level vancomycin (Minimum inhibitory concentration; MIC ≥128 mg/l) and teicoplanin resistance (MIC 64 mg/l), as well as resistance to ciprofloxacin, erythromycin and tetracycline. The tet(L) and erm(B) genes, associated with tetracycline and erythromycin resistance, respectively, were found in all vanA isolates. The intestinal tract of Lusitano horses can be a potential reservoir for vanA-containing enterococci.

Research paper thumbnail of Genetic Characterization of Antibiotic Resistance in Enteropathogenic Escherichia coli Carrying Extended-Spectrum β-Lactamases Recovered from Diarrhoeic Rabbits

Zoonoses and Public Health, 2010

A total of 52 Escherichia coli strains isolated from diarrhoeic rabbits were investigated for the... more A total of 52 Escherichia coli strains isolated from diarrhoeic rabbits were investigated for their enteropathogenic E. coli (EPEC) pathotype by PCR amplification of eae and bfp virulence genes. A total of 22 EPEC isolates were identified, serotyped and studied for antibiotic resistance and screened for the detection of extended-spectrum β-lactamases (ESBLs). The EPEC isolates belonged to three serogroups (O26, O92 and O103). The most common serogroup (O103:K-:H2) was observed among 17 EPEC strains, the O92:K-serogroup in three isolates (the antibiotic sensitive ones) and the remaining O26:K-serogroup in two isolates (the ESBLs isolates). Resistances to ampicillin and tetracycline were the most frequent and detected followed by resistance to nalidixic acid, streptomycin, trimethoprim–sulphamethoxazole, cefoxitin, gentamicin and ciprofloxacin. All the isolates were sensitive for amikacin, ceftazidime, aztreonam, imipenem, chloramphenicol, tobramycin and amoxicillin + clavulanic acid. Two isolates recovered from two adult animals showed an intermediate susceptibility to cefotaxime, and a positive screening test for ESBL was demonstrated in both. The blaTEM gene was demonstrated in the majority of ampicillin-resistant isolates. The aac(3)-II or aac(3)-IV genes were detected in the four gentamicin-resistant isolates. In addition, the aadA gene was detected in 60% of streptomycin-resistant isolates. The tet(A) or tet(B) genes were identified in all tetracycline-resistant isolates. A total of nine EPEC isolates showed the phenotype SXT-resistant, and the sul1 and/or sul2 and/or sul3 genes were detected in all of them. Our findings showed that the molecular detection by the eae and bfp genes by PCR followed by serotyping is useful for monitoring trends in EPEC infections of rabbits allowing the identification of their possible reservoirs. The detection of genes involved in the resistance to antibiotics of different families in a relatively high proportion of faecal E. coli isolates of rabbits is of great interest and could be considered a serious public health problem.

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

Applied and Environmental Microbiology, 2010

Research paper thumbnail of Prevalence and Mechanisms of Erythromycin Resistance in Streptococcus agalactiae from Healthy Pregnant Women

Microbial Drug Resistance, 2009

We sought to determine the resistance phenotypes for erythromycin and clindamycin and the mechani... more 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.

Research paper thumbnail of Antimicrobial activity of doripenem against bacterial isolates from humans and animals

Journal of Antibiotics, 2010

Research paper thumbnail of Molecular characterization of antimicrobial resistance in enterococci and Escherichia coli isolates from European wild rabbit ( Oryctolagus cuniculus

Science of The Total Environment, 2010

A total of 44 Escherichia coli and 64 enterococci recovered from 77 intestinal samples of wild Eu... more A total of 44 Escherichia coli and 64 enterococci recovered from 77 intestinal samples of wild European rabbits in Portugal were analyzed for resistance to antimicrobial agents. Resistance in E. coli isolates was observed for ampicillin, tetracycline, sulfamethoxazole/trimethoprim, streptomycin, gentamicin, tobramycin, nalidixic acid, ciprofloxacin and chloramphenicol. None of the E. coli isolates produced extended-spectrum beta-lactamases (ESBLs). The bla(TEM), aadA, aac(3)-II, tet(A) and/or tet(B), and the catA genes were demonstrated in all ampicillin, streptomycin, gentamicin, tetracycline, and chloramphenicol-resistant isolates respectively, and the sul1 and/or sul2 and/or sul3 genes in 4 of 5 sulfamethoxazole/trimethoprim resistant isolates. Of the enterococcal isolates, Enterococcus faecalis was the most prevalent detected species (39 isolates), followed by E. faecium (21 isolates) and E. hirae (4 isolates). More than one-fourth (29.7%) of the isolates were resistant to tetracycline; 20.3% were resistant to erythromycin, 14.1% were resistant to ciprofloxacin and 10.9% were resistant to high-level-kanamycin. Lower level of resistance (<10%) was detected for ampicillin, quinupristin/dalfopristin and high-level-gentamicin, -streptomycin. No vancomycin-resistance was detected in the enterococci isolates. Resistance genes detected included aac(6')-aph(2''), ant(6)-Ia, tet(M) and/or tet(L) in all gentamicin, streptomycin and tetracycline-resistant isolates respectively. The aph(3')-IIIa gene was detected in 6 of 7 kanamycin-resistant isolates, the erm(B) gene in 11 of 13 erythromycin-resistant isolates and the vat(D) gene in the quinupristin/dalfopristin-resistant E. faecium isolate. This survey showed that faecal bacteria such as E. coli and enterococci of wild rabbits could be a reservoir of antimicrobial resistance genes.

Research paper thumbnail of HIGH PREVALENCE OF EXTENDED-SPECTRUM β-LACTAMASES ESCHERICHIA COLI AND VANCOMYCIN-RESISTANT ENTEROCOCCI ISOLATES FROM CHICKEN PRODUCTS. A PROBLEM OF PUBLIC HEALTH

Journal of Food Safety, 2010

ABSTRACTTwenty-nine chicken products were acquired from different supermarkets in Portugal during... more ABSTRACTTwenty-nine chicken products were acquired from different supermarkets in Portugal during September to December 2007 and were analyzed for extended-spectrum β-lactamases (ESBL) Escherichia coli and vancomycin-resistant enterococci (VRE). Cefotaxime-resistant E. coli isolates were recovered in 27 of 29 chicken samples representing 93% of the analyzed samples. The highest percentages of resistance (more than 50% of the isolates) were detected for ampicillin, nalidixic acid and tetracycline. VRE isolates were detected in 17 of 29 samples of chicken origin (59%) and were identified as Enterococcus durans (n = 15) and E. faecium (n = 2) with the highest percentages of resistance being detected for erythromycin, tetracycline, ampicillin and ciprofloxacin. Seven E. durans and the two E. faecium isolates recovered from chicken wings, gizzard and skin show gelatinase activity.The high rate of colonization of chicken products by these bacteria supports other studies suggesting that the food chain could be a source of ESBL and VRE colonization in humans representing a public health problem.Twenty-nine chicken products were acquired from different supermarkets in Portugal during September to December 2007 and were analyzed for extended-spectrum β-lactamases (ESBL) Escherichia coli and vancomycin-resistant enterococci (VRE). Cefotaxime-resistant E. coli isolates were recovered in 27 of 29 chicken samples representing 93% of the analyzed samples. The highest percentages of resistance (more than 50% of the isolates) were detected for ampicillin, nalidixic acid and tetracycline. VRE isolates were detected in 17 of 29 samples of chicken origin (59%) and were identified as Enterococcus durans (n = 15) and E. faecium (n = 2) with the highest percentages of resistance being detected for erythromycin, tetracycline, ampicillin and ciprofloxacin. Seven E. durans and the two E. faecium isolates recovered from chicken wings, gizzard and skin show gelatinase activity.The high rate of colonization of chicken products by these bacteria supports other studies suggesting that the food chain could be a source of ESBL and VRE colonization in humans representing a public health problem.PRACTICAL APPLICATIONSThe data indicate that chicken products may be contaminated with a high prevalence of extended-spectrum β-lactamases Escherichia coli and vancomycin-resistant enterococci (VRE). It is important to mention that the isolates present a diversity of phenotypes of antimicrobial resistance, and half of the VRE isolates show also gelatinase activity, indicating that these animals may be a reservoir of bacteria showing virulence and increased resistance to antimicrobial agents, raising special concerns about their transmission to humans through the food chain.The data indicate that chicken products may be contaminated with a high prevalence of extended-spectrum β-lactamases Escherichia coli and vancomycin-resistant enterococci (VRE). It is important to mention that the isolates present a diversity of phenotypes of antimicrobial resistance, and half of the VRE isolates show also gelatinase activity, indicating that these animals may be a reservoir of bacteria showing virulence and increased resistance to antimicrobial agents, raising special concerns about their transmission to humans through the food chain.

Research paper thumbnail of Proteomic characterization of van A-containing Enterococcu s recovered from Seagulls at the Berlengas Natural Reserve, W Portugal

Proteome Science, 2010

Background Enterococci have emerged as the third most common cause of nosocomial infections, requ... more Background Enterococci 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. Results From 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. Conclusions The 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.

Research paper thumbnail of Detection of vancomycin-resistant enterococci from faecal samples of Iberian wolf and Iberian lynx, including Enterococcus faecium strains of CC17 and the new singleton ST573

Science of The Total Environment, 2011

The aim of this study was to perform the molecular characterization of vancomycin resistant enter... more The aim of this study was to perform the molecular characterization of vancomycin resistant enterococci (VRE) within the faecal flora of Iberian wolf and Iberian lynx. The association with other resistance genes and the detection of virulence genes were also analysed. From 2008 to 2010, 365 faecal samples from Iberian wolf and Iberian lynx were collected and tested for VRE recovery. Mechanisms of resistance to vancomycin and other antibiotics, as well as genes encoding virulence factors were detected through PCR. Multilocus Sequence Typing (MLST) was performed for Enterococcus faecium strains. VRE were recovered in 8 of the 365 analysed samples. The vanA gene was identified in two E. faecium isolates recovered from Iberian wolf faecal samples and the remaining six showed intrinsic resistance (3 vanC1-E. gallinarum and 3 vanC2-E. casseliflavus, from Iberian wolf and Iberian lynx faecal samples, respectively). One vanA-containing isolate showed tetracycline and erythromycin resistance [with erm(B) and tet(L) genes] and the other one also exhibited ampicillin and kanamycin resistance [with erm(B), tet(M) and aph(3')-III genes]. One of the vanA-isolates revealed a new sequence type named ST573 and the other one belonged to the CC17 clonal complex (ST18). The hyl gene was detected in one E. casseliflavus and three E. gallinarum but not among vanA-positive isolates, and the occurrence of cylA and cylL genes was confirmed in two E. casseliflavus isolates. A low prevalence of VRE has been detected in faecal samples of Iberian wolf and Iberian lynx and strains with an acquired mechanism of resistance to vancomycin have not been detected among Iberian lynx.

Research paper thumbnail of Wild boars as reservoirs of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli of different phylogenetic groups

Journal of Basic Microbiology, 2009

ESBL-producing E. coli isolates have been isolated from eight of seventy seven faecal samples (10... more ESBL-producing E. coli isolates have been isolated from eight of seventy seven faecal samples (10.4%) of wild boars in Portugal. The ESBL types identified by PCR and sequencing were blaCTX-M-1 (6 isolates) and blaCTX-M-1 + blaTEM1-b (2 isolates). Further resistance genes detected included tet (A) or tet (B) (in three tetracycline-resistant isolates), aad A (in three streptomycin-resistant isolates), cml A (in one chloramphenicol-resistant isolate), sul 1 and/or sul 2 and/or sul 3 (in all sulfonamide-resistant isolates). The intI 1 gene encoding class 1 integrase was detected in all ESBL-producing E. coli isolates. One isolate also carried the intI 2 gene, encoding class 2 integrase. The ESBL-producing E. coli isolates could be assigned to phylogenetic groups B1 (3 isolates), B2 (3 isolates) or A (2 isolates). Amino acid change in GyrA protein (Ser83Leu or Asp87Tyr) was detected in three nalidixic acid-resistant and ciprofloxacin-susceptible isolates. Two amino acid changes in GyrA (Ser83Leu + Asp87Asn) and one in ParC (Ser80Ile) were identified in two nalidixic acid- and ciprofloxacin-resistant isolates. As evidenced by this study wild boars could be a reservoir of antimicrobial resistance genes. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Research paper thumbnail of Genomic and proteomic evaluation of antibiotic resistance in Salmonella strains

Journal of Proteomics, 2010

Using Salmonella strains identical to those present in the gastrointestinal tract of different an... more Using Salmonella strains identical to those present in the gastrointestinal tract of different animals we aim to determine and compare the proteome of two serotypes, Salmonella Typhimurium and Enteritidis recovered from faecal samples of wild boars and wild rabbits, respectively. The presence of genes responsible for antibiotic resistance was detected by PCR. Proteomes of the two distinct serotypes were determined using 2-DE in order to identify proteins associated with antibiotic resistance or virulence. Through 2-DE we obtained a total of 229 spots from both strains. All were suitable for MALDI-TOF/TOF and, in correlation with bioinformatic databases, allowed accurate identification and characterization of proteins. S.

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

Proteome Science, 2010

Background: Enterococci have emerged as the third most common cause of nosocomial infections, req... more Background: Enterococci 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 vanA-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.

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

Foodborne Pathogens and Disease, 2010

There is a great concern by the emergence and the wide dissemination of extended-spectrum beta-la... more 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 streptomycinresistant 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. .

Research paper thumbnail of Vancomycin-resistant enterococci from Portuguese wastewater treatment plants

Journal of Basic Microbiology, 2010

The objective of this study was to evaluate the incidence of vancomycin resistant enterococci in ... more The objective of this study was to evaluate the incidence of vancomycin resistant enterococci in sludge and sewage of urban and poultry-slaughterhouse wastewater treatment plants. A total of 17 vancomycin resistant enterococci (eight vanA -containing Enterococcus faecium and nine vanC1/vanC2 -containing Enterococcus gallinarum/casseliflavus) were found among 499 isolates of sewage and sludge samples of 14 urban and nine poultry-slaughterhouse wastewater treatment plants. These seventeen VRE isolates showed resistance to kanamycin (n = 8), tetracycline (n = 7), erythromycin (n = 7), ciprofloxacin (n = 7), ampicillin (n = 7), streptomycin (n = 6), and gentamicin (n = 2). The tetM gene, related with tetracycline resistance, was found in six of eight van A-containing isolates, in all seven vanC-1 isolates and in one of two vanC-2 isolates. The ermB gene in seven erythromycin-resistant isolates; and the aac6 ′-aph2 ″ gene in the two high-level-gentamicin-resistant isolates. Moreover, two vanA -containing E. faecium isolates harbored the hyl virulence gene, and three isolates the entA bacteriocin gene. The purK-1 allele was detected in our urban vanA -containing E. faecium isolate, and we found as well the purK-6 allele in one poultry-slaughterhouse vanA -containing E. faecium isolate. This study suggests that the wastewater treatment plants might be an important source of dissemination of antibiotic-resistant enterococci in Portugal. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Research paper thumbnail of In vitro activity of dalbavancin against enterococci isolates from wild animals, pets, poultry and humans in Portugal

Journal of Basic Microbiology, 2008

Dalbavancin is a new new semisynthetic teicoplanin-related lipoglycopeptide with activity against... more Dalbavancin is a new new semisynthetic teicoplanin-related lipoglycopeptide with activity against gram-positive organisms. We investigated the activity of dalbavancin against faecal enterococci isolates from wild animals, pets, poultry and healthy humans in Portugal.The in vitro activity of dalbavancin was determined by the microbroth dilution method according to the Clinical Laboratory Standards Institute (CLSI) guidelines in 589 enterococci of different species and origins. All vancomycin-susceptible Enterococcus spp. were inhibited by ≤0.25 mg/l dalbavancin. Although vancomycin-resistant-enterococci (VRE) showed higher dalbavancin MIC values (16 mg/l), the isolates that exhibited the VanC resistance phenotype were inhibited at dalbavancin concentrations ≤0.125 mg/l. Only van A isolates were not inhibited by low concentrations of dalbavancin since van A strains showed higher dalbavancin MIC values (16 mg/l). (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Research paper thumbnail of Genetic characterization of vancomycin-resistant enterococci isolates from wild rabbits

Journal of Basic Microbiology, 2009

The presence of van A-containing E. faecium isolates was demonstrated in three of 77 faecal sampl... more The presence of van A-containing E. faecium isolates was demonstrated in three of 77 faecal samples (3.9%) of wild rabbits recovered in Portugal. Enterococcal strains with intrinsic vancomycin resistance (van C-1 or van C-2/3 gene) were found in five (6.5%) and three (3.9%) faecal samples, respectively. The mechanisms of resistance for other antibiotics were studied in these vancomycin-resistant isolates. All van A strains showed resistance for tetracycline [with the presence of tet (L) gene, associated or not with tet (M) gene] and for erythromycin [with the presence of the erm (B) gene]. Two isolates were resistant to ciprofloxacin and one to ampicillin. Two van C-1 strains and one van C-2/3 strain were tetracycline resistant [containing the tet (M) gene associated with tet (L) gene] and erythromycin resistant [with erm (B) gene]. Two van C-1 and two van C-2/3 strains were also ciprofloxacin resistant and one van C-1 strain was, additionally, resistant to quinupristin-dalfopristin. The two remaining isolates (van C-1, van C-2/3) did not show resistance for any additional antibiotic. The intestinal tract of wild rabbits could be a reservoir of van A-containing enterococci. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Research paper thumbnail of Gilthead seabream ( Sparus aurata) carrying antibiotic resistant enterococci. A potential bioindicator of marine contamination

Marine Pollution Bulletin, 2011

Antibiotic resistance in bacteria is a growing problem that is not only restricted to the clinica... more Antibiotic resistance in bacteria is a growing problem that is not only restricted to the clinical setting but also to other environments such as marine species that harbor antibiotic resistant bacteria and therefore may serve as reservoirs for antibiotic-resistance genetic determinants. The aim of this study was to evaluate antibiotic resistance phenotypes in enterococci isolated from fecal samples of gilthead seabream and the associated mechanisms of resistance. A collection of 118 samples were analyzed and 73 enterococci were recovered. The strains showed high percentages of resistance to erythromycin and tetracycline (58.9% and 17.8%, respectively). Lower level of resistance (<13%) was detected for quinupristin-dalfopristin, ampicillin, high-level-gentamicin, high-level-streptomycin, high-level-kanamycin, ciprofloxacin and chloramphenicol. The erm(B), tet(L) or tet(M), aac(6')-aph(2″) and…

Research paper thumbnail of MLST and a genetic study of antibiotic resistance and virulence factors in vanA -containing Enterococcu s from buzzards ( Buteo buteo

Letters in Applied Microbiology, 2010

Aims: To analyse the occurrence of faecal carriage of vancomycin-resistant enterococci (VRE) in ... more 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.Methods and Results: The presence of VRE was investigated in 33 faecal samples of B. buteo. Samples were seeded in Slanetz–Bartley agar plates supplemented with vancomycin for VRE recovery. Genes encoding antimicrobial resistance and virulence were studied by polymerase chain reaction. Vancomycin-resistant Enterococcus faecium isolates were characterized by multilocus sequence typing. VRE with an acquired mechanism of resistance (vanA genotype) were detected in 9% of samples analysed (Ent. faecium and Enterococcus durans). In addition, 27% of samples contained VRE with an intrinsic mechanism of resistance (Enterococcus gallinarum, vanC1). All vanA-containing isolates showed resistance to tetracycline and erythromycin and harboured the tet(M) and/or tet(L) genes, in addition to the ermB gene. The vat(E) and/or vat(D), cat(A) and aph(3′)-IIIa genes were identified in quinupristin–dalfopristin-, chloramphenicol-, and kanamycin-resistant vanA-containing strains, respectively. The sequence types ST273 and ST5 were identified in two vanA-positive Ent. faecium isolates, and the presence of hyl, gelE, cylA, cylL and cylM virulence genes and gelatinase activity were identified in Ent. faecium ST5 strain.Conclusions: The intestinal tract of B. buteo could be a reservoir of vanA-positive enterococci.Significance and Impact of the Study: First study focused to define the occurrence of vanA-containing Enterococcus strains in B. buteo.

Research paper thumbnail of Detection of antibiotic resistant E. coli and Enterococcus spp. in stool of healthy growing children in Portugal

Journal of Basic Microbiology, 2009

From stool specimens of 118 healthy children's (1–14 years) in Portugal 92 E. coli and 101 Entero... more From stool specimens of 118 healthy children's (1–14 years) in Portugal 92 E. coli and 101 Enterococcu s spp. strains have been isolated. Almost half (40.2%) of the E. coli isolates were resistant to ampicillin, 25.0% were resistant to tetracycline and 26.1% were resistant to streptomycin. Resistance genes detected by specific PCR included blaTEM and/or blaSHV and/or blaCTX-M (33 of 37 ampicillin and/or cefotaxime resistant isolates), tet (A) and/or tet (B) (16 of 23 tetracycline-resistant isolates), aad A (19 of 24 streptomycin-resistant isolates), cml A (in the two chloramphenicol-resistant isolates), aac (3)-II with/without aac (3)-IV (in the four gentamicin-resistant isolates), sul 1 and/or sul 2 and/or sul 3 (in all trimethoprim/sulfamethoxazole resistant isolates). The majority of the resistant E. coli isolates (69.1%) belonged to phylogenetic group B2. Of the enterococci isolates E. faecium (n = 53), E. faecalis (n = 41), E. hirae (n = 4) and E. durans (n = 3) more than one-fourth (28.7%) of the isolates were resistant to tetracycline; 21.8% were resistant to erythromycin and 8.9% were resistant to kanamycin. Resistance genes detected by PCR in enterococci included aph (3)′-IIIa (in all kanamycin-resistant isolates), aac (6′) (in all gentamicin-resistant isolates), tet (M) and/or tet (L) (26 of 29 tetracycline-resistant isolates), erm (B) (17 of 22 erythromycin-resistant isolates). This survey showed that faecal bacteria such as E. coli and enterococci of healthy growing children's could be a reservoir of antimicrobial resistance genes. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Research paper thumbnail of Detection of Escherichia coli harbouring extended-spectrum -lactamases of the CTX-M classes in faecal samples of common buzzards (Buteo buteo

Journal of Antimicrobial Chemotherapy, 2010

Research paper thumbnail of Detection and genetic characterisation of vanA-containing Enterococcus strains in healthy Lusitano horses: vanA-containing enterococcus in Lusitano horses

Equine Veterinary Journal, 2010

Lusitano horses were investigated in order to detect the presence of vancomycin-resistant enteroc... more Lusitano horses were investigated in order to detect the presence of vancomycin-resistant enterococci. vanA isolates showed high level vancomycin (Minimum inhibitory concentration; MIC ≥128 mg/l) and teicoplanin resistance (MIC 64 mg/l), as well as resistance to ciprofloxacin, erythromycin and tetracycline. The tet(L) and erm(B) genes, associated with tetracycline and erythromycin resistance, respectively, were found in all vanA isolates. The intestinal tract of Lusitano horses can be a potential reservoir for vanA-containing enterococci.

Research paper thumbnail of Genetic Characterization of Antibiotic Resistance in Enteropathogenic Escherichia coli Carrying Extended-Spectrum β-Lactamases Recovered from Diarrhoeic Rabbits

Zoonoses and Public Health, 2010

A total of 52 Escherichia coli strains isolated from diarrhoeic rabbits were investigated for the... more A total of 52 Escherichia coli strains isolated from diarrhoeic rabbits were investigated for their enteropathogenic E. coli (EPEC) pathotype by PCR amplification of eae and bfp virulence genes. A total of 22 EPEC isolates were identified, serotyped and studied for antibiotic resistance and screened for the detection of extended-spectrum β-lactamases (ESBLs). The EPEC isolates belonged to three serogroups (O26, O92 and O103). The most common serogroup (O103:K-:H2) was observed among 17 EPEC strains, the O92:K-serogroup in three isolates (the antibiotic sensitive ones) and the remaining O26:K-serogroup in two isolates (the ESBLs isolates). Resistances to ampicillin and tetracycline were the most frequent and detected followed by resistance to nalidixic acid, streptomycin, trimethoprim–sulphamethoxazole, cefoxitin, gentamicin and ciprofloxacin. All the isolates were sensitive for amikacin, ceftazidime, aztreonam, imipenem, chloramphenicol, tobramycin and amoxicillin + clavulanic acid. Two isolates recovered from two adult animals showed an intermediate susceptibility to cefotaxime, and a positive screening test for ESBL was demonstrated in both. The blaTEM gene was demonstrated in the majority of ampicillin-resistant isolates. The aac(3)-II or aac(3)-IV genes were detected in the four gentamicin-resistant isolates. In addition, the aadA gene was detected in 60% of streptomycin-resistant isolates. The tet(A) or tet(B) genes were identified in all tetracycline-resistant isolates. A total of nine EPEC isolates showed the phenotype SXT-resistant, and the sul1 and/or sul2 and/or sul3 genes were detected in all of them. Our findings showed that the molecular detection by the eae and bfp genes by PCR followed by serotyping is useful for monitoring trends in EPEC infections of rabbits allowing the identification of their possible reservoirs. The detection of genes involved in the resistance to antibiotics of different families in a relatively high proportion of faecal E. coli isolates of rabbits is of great interest and could be considered a serious public health problem.

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

Applied and Environmental Microbiology, 2010

Research paper thumbnail of Prevalence and Mechanisms of Erythromycin Resistance in Streptococcus agalactiae from Healthy Pregnant Women

Microbial Drug Resistance, 2009

We sought to determine the resistance phenotypes for erythromycin and clindamycin and the mechani... more 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.

Research paper thumbnail of Antimicrobial activity of doripenem against bacterial isolates from humans and animals

Journal of Antibiotics, 2010

Research paper thumbnail of Molecular characterization of antimicrobial resistance in enterococci and Escherichia coli isolates from European wild rabbit ( Oryctolagus cuniculus

Science of The Total Environment, 2010

A total of 44 Escherichia coli and 64 enterococci recovered from 77 intestinal samples of wild Eu... more A total of 44 Escherichia coli and 64 enterococci recovered from 77 intestinal samples of wild European rabbits in Portugal were analyzed for resistance to antimicrobial agents. Resistance in E. coli isolates was observed for ampicillin, tetracycline, sulfamethoxazole/trimethoprim, streptomycin, gentamicin, tobramycin, nalidixic acid, ciprofloxacin and chloramphenicol. None of the E. coli isolates produced extended-spectrum beta-lactamases (ESBLs). The bla(TEM), aadA, aac(3)-II, tet(A) and/or tet(B), and the catA genes were demonstrated in all ampicillin, streptomycin, gentamicin, tetracycline, and chloramphenicol-resistant isolates respectively, and the sul1 and/or sul2 and/or sul3 genes in 4 of 5 sulfamethoxazole/trimethoprim resistant isolates. Of the enterococcal isolates, Enterococcus faecalis was the most prevalent detected species (39 isolates), followed by E. faecium (21 isolates) and E. hirae (4 isolates). More than one-fourth (29.7%) of the isolates were resistant to tetracycline; 20.3% were resistant to erythromycin, 14.1% were resistant to ciprofloxacin and 10.9% were resistant to high-level-kanamycin. Lower level of resistance (<10%) was detected for ampicillin, quinupristin/dalfopristin and high-level-gentamicin, -streptomycin. No vancomycin-resistance was detected in the enterococci isolates. Resistance genes detected included aac(6')-aph(2''), ant(6)-Ia, tet(M) and/or tet(L) in all gentamicin, streptomycin and tetracycline-resistant isolates respectively. The aph(3')-IIIa gene was detected in 6 of 7 kanamycin-resistant isolates, the erm(B) gene in 11 of 13 erythromycin-resistant isolates and the vat(D) gene in the quinupristin/dalfopristin-resistant E. faecium isolate. This survey showed that faecal bacteria such as E. coli and enterococci of wild rabbits could be a reservoir of antimicrobial resistance genes.

Research paper thumbnail of HIGH PREVALENCE OF EXTENDED-SPECTRUM β-LACTAMASES ESCHERICHIA COLI AND VANCOMYCIN-RESISTANT ENTEROCOCCI ISOLATES FROM CHICKEN PRODUCTS. A PROBLEM OF PUBLIC HEALTH

Journal of Food Safety, 2010

ABSTRACTTwenty-nine chicken products were acquired from different supermarkets in Portugal during... more ABSTRACTTwenty-nine chicken products were acquired from different supermarkets in Portugal during September to December 2007 and were analyzed for extended-spectrum β-lactamases (ESBL) Escherichia coli and vancomycin-resistant enterococci (VRE). Cefotaxime-resistant E. coli isolates were recovered in 27 of 29 chicken samples representing 93% of the analyzed samples. The highest percentages of resistance (more than 50% of the isolates) were detected for ampicillin, nalidixic acid and tetracycline. VRE isolates were detected in 17 of 29 samples of chicken origin (59%) and were identified as Enterococcus durans (n = 15) and E. faecium (n = 2) with the highest percentages of resistance being detected for erythromycin, tetracycline, ampicillin and ciprofloxacin. Seven E. durans and the two E. faecium isolates recovered from chicken wings, gizzard and skin show gelatinase activity.The high rate of colonization of chicken products by these bacteria supports other studies suggesting that the food chain could be a source of ESBL and VRE colonization in humans representing a public health problem.Twenty-nine chicken products were acquired from different supermarkets in Portugal during September to December 2007 and were analyzed for extended-spectrum β-lactamases (ESBL) Escherichia coli and vancomycin-resistant enterococci (VRE). Cefotaxime-resistant E. coli isolates were recovered in 27 of 29 chicken samples representing 93% of the analyzed samples. The highest percentages of resistance (more than 50% of the isolates) were detected for ampicillin, nalidixic acid and tetracycline. VRE isolates were detected in 17 of 29 samples of chicken origin (59%) and were identified as Enterococcus durans (n = 15) and E. faecium (n = 2) with the highest percentages of resistance being detected for erythromycin, tetracycline, ampicillin and ciprofloxacin. Seven E. durans and the two E. faecium isolates recovered from chicken wings, gizzard and skin show gelatinase activity.The high rate of colonization of chicken products by these bacteria supports other studies suggesting that the food chain could be a source of ESBL and VRE colonization in humans representing a public health problem.PRACTICAL APPLICATIONSThe data indicate that chicken products may be contaminated with a high prevalence of extended-spectrum β-lactamases Escherichia coli and vancomycin-resistant enterococci (VRE). It is important to mention that the isolates present a diversity of phenotypes of antimicrobial resistance, and half of the VRE isolates show also gelatinase activity, indicating that these animals may be a reservoir of bacteria showing virulence and increased resistance to antimicrobial agents, raising special concerns about their transmission to humans through the food chain.The data indicate that chicken products may be contaminated with a high prevalence of extended-spectrum β-lactamases Escherichia coli and vancomycin-resistant enterococci (VRE). It is important to mention that the isolates present a diversity of phenotypes of antimicrobial resistance, and half of the VRE isolates show also gelatinase activity, indicating that these animals may be a reservoir of bacteria showing virulence and increased resistance to antimicrobial agents, raising special concerns about their transmission to humans through the food chain.

Research paper thumbnail of Proteomic characterization of van A-containing Enterococcu s recovered from Seagulls at the Berlengas Natural Reserve, W Portugal

Proteome Science, 2010

Background Enterococci have emerged as the third most common cause of nosocomial infections, requ... more Background Enterococci 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. Results From 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. Conclusions The 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.

Research paper thumbnail of Detection of vancomycin-resistant enterococci from faecal samples of Iberian wolf and Iberian lynx, including Enterococcus faecium strains of CC17 and the new singleton ST573

Science of The Total Environment, 2011

The aim of this study was to perform the molecular characterization of vancomycin resistant enter... more The aim of this study was to perform the molecular characterization of vancomycin resistant enterococci (VRE) within the faecal flora of Iberian wolf and Iberian lynx. The association with other resistance genes and the detection of virulence genes were also analysed. From 2008 to 2010, 365 faecal samples from Iberian wolf and Iberian lynx were collected and tested for VRE recovery. Mechanisms of resistance to vancomycin and other antibiotics, as well as genes encoding virulence factors were detected through PCR. Multilocus Sequence Typing (MLST) was performed for Enterococcus faecium strains. VRE were recovered in 8 of the 365 analysed samples. The vanA gene was identified in two E. faecium isolates recovered from Iberian wolf faecal samples and the remaining six showed intrinsic resistance (3 vanC1-E. gallinarum and 3 vanC2-E. casseliflavus, from Iberian wolf and Iberian lynx faecal samples, respectively). One vanA-containing isolate showed tetracycline and erythromycin resistance [with erm(B) and tet(L) genes] and the other one also exhibited ampicillin and kanamycin resistance [with erm(B), tet(M) and aph(3')-III genes]. One of the vanA-isolates revealed a new sequence type named ST573 and the other one belonged to the CC17 clonal complex (ST18). The hyl gene was detected in one E. casseliflavus and three E. gallinarum but not among vanA-positive isolates, and the occurrence of cylA and cylL genes was confirmed in two E. casseliflavus isolates. A low prevalence of VRE has been detected in faecal samples of Iberian wolf and Iberian lynx and strains with an acquired mechanism of resistance to vancomycin have not been detected among Iberian lynx.

Research paper thumbnail of Wild boars as reservoirs of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli of different phylogenetic groups

Journal of Basic Microbiology, 2009

ESBL-producing E. coli isolates have been isolated from eight of seventy seven faecal samples (10... more ESBL-producing E. coli isolates have been isolated from eight of seventy seven faecal samples (10.4%) of wild boars in Portugal. The ESBL types identified by PCR and sequencing were blaCTX-M-1 (6 isolates) and blaCTX-M-1 + blaTEM1-b (2 isolates). Further resistance genes detected included tet (A) or tet (B) (in three tetracycline-resistant isolates), aad A (in three streptomycin-resistant isolates), cml A (in one chloramphenicol-resistant isolate), sul 1 and/or sul 2 and/or sul 3 (in all sulfonamide-resistant isolates). The intI 1 gene encoding class 1 integrase was detected in all ESBL-producing E. coli isolates. One isolate also carried the intI 2 gene, encoding class 2 integrase. The ESBL-producing E. coli isolates could be assigned to phylogenetic groups B1 (3 isolates), B2 (3 isolates) or A (2 isolates). Amino acid change in GyrA protein (Ser83Leu or Asp87Tyr) was detected in three nalidixic acid-resistant and ciprofloxacin-susceptible isolates. Two amino acid changes in GyrA (Ser83Leu + Asp87Asn) and one in ParC (Ser80Ile) were identified in two nalidixic acid- and ciprofloxacin-resistant isolates. As evidenced by this study wild boars could be a reservoir of antimicrobial resistance genes. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Research paper thumbnail of Genomic and proteomic evaluation of antibiotic resistance in Salmonella strains

Journal of Proteomics, 2010

Using Salmonella strains identical to those present in the gastrointestinal tract of different an... more Using Salmonella strains identical to those present in the gastrointestinal tract of different animals we aim to determine and compare the proteome of two serotypes, Salmonella Typhimurium and Enteritidis recovered from faecal samples of wild boars and wild rabbits, respectively. The presence of genes responsible for antibiotic resistance was detected by PCR. Proteomes of the two distinct serotypes were determined using 2-DE in order to identify proteins associated with antibiotic resistance or virulence. Through 2-DE we obtained a total of 229 spots from both strains. All were suitable for MALDI-TOF/TOF and, in correlation with bioinformatic databases, allowed accurate identification and characterization of proteins. S.

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

Proteome Science, 2010

Background: Enterococci have emerged as the third most common cause of nosocomial infections, req... more Background: Enterococci 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 vanA-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.

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

Foodborne Pathogens and Disease, 2010

There is a great concern by the emergence and the wide dissemination of extended-spectrum beta-la... more 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 streptomycinresistant 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. .

Research paper thumbnail of Vancomycin-resistant enterococci from Portuguese wastewater treatment plants

Journal of Basic Microbiology, 2010

The objective of this study was to evaluate the incidence of vancomycin resistant enterococci in ... more The objective of this study was to evaluate the incidence of vancomycin resistant enterococci in sludge and sewage of urban and poultry-slaughterhouse wastewater treatment plants. A total of 17 vancomycin resistant enterococci (eight vanA -containing Enterococcus faecium and nine vanC1/vanC2 -containing Enterococcus gallinarum/casseliflavus) were found among 499 isolates of sewage and sludge samples of 14 urban and nine poultry-slaughterhouse wastewater treatment plants. These seventeen VRE isolates showed resistance to kanamycin (n = 8), tetracycline (n = 7), erythromycin (n = 7), ciprofloxacin (n = 7), ampicillin (n = 7), streptomycin (n = 6), and gentamicin (n = 2). The tetM gene, related with tetracycline resistance, was found in six of eight van A-containing isolates, in all seven vanC-1 isolates and in one of two vanC-2 isolates. The ermB gene in seven erythromycin-resistant isolates; and the aac6 ′-aph2 ″ gene in the two high-level-gentamicin-resistant isolates. Moreover, two vanA -containing E. faecium isolates harbored the hyl virulence gene, and three isolates the entA bacteriocin gene. The purK-1 allele was detected in our urban vanA -containing E. faecium isolate, and we found as well the purK-6 allele in one poultry-slaughterhouse vanA -containing E. faecium isolate. This study suggests that the wastewater treatment plants might be an important source of dissemination of antibiotic-resistant enterococci in Portugal. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)