Carlos Araújo

Vancomycin‐resistant enterococci from Portuguese wastewater treatment plants

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)

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:u2002 To analyse the occurrence of faecal carriage of vancomycin‐resistant enterococci (VRE) in Buteo buteo and to study the associated resistance and virulence genes.

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

From stool specimens of 118 healthy childrens (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 childrens could be a reservoir of antimicrobial resistance genes. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Characterization of vancomycin-resistant enterococci isolated from fecal samples of ostriches by molecular methods.

The aim of this study was to estimate the occurrence of vancomycin-resistant enterococci (VRE) in fecal samples of ostriches from a farm of Southern Portugal, the mechanisms implicated, and the associated virulence factors, 13 years after the banning of the glycopeptide avoparcin as animal growth promoter in the European Union. Fifty-four fecal samples of ostriches were inoculated in Slanetz-Bartley supplemented with vancomycin (4 microg/mL) for VRE recovery. Susceptibility to 11 antibiotics was performed by disk-diffusion agar method in recovered VRE isolates. The mechanism of resistance to vancomycin and to other antibiotics and the presence of the esp and hyl virulence genes were determined by polymerase chain reaction and sequencing. VRE were detected in 7 of the 54 ostrich fecal samples (13%); Enterococcus durans isolates with the vanA genotype were found in 4 of the 54 fecal samples (7.4%), and Enterococcus gallinarum with the intrinsic vanC1 genotype in the remaining three VRE-positive samples. All vanA-containing E. durans isolates showed resistance to tetracycline and erythromycin, and one of them also to ciprofloxacin; they harbored the erm(B) and tet(M) genes, as well as the specific sequences of Tn916 and Tn5397 transposons, but not the esp or hyl virulence genes. Two of the three vanC1 isolates showed resistance to tetracycline [with the tet(M) gene] and one to erythromycin [with the erm(B) gene], and all three contained the hyl gene. Fecal samples of ostriches represent a reservoir of vanA-containing enterococci that could be transmitted to humans through the food chain.

Influence of oral hygiene in patients with fixed appliances in the oral carriage of antimicrobial-resistant Escherichia coli and Enterococcus isolates.

OBJECTIVESnThe aim was to study the oral carriage of Enterococcus and Escherichia coli isolates and their content in antimicrobial-resistance and virulence genes in patients with fixed appliances and in healthy volunteers.nnnSTUDY DESIGNnSamples from supragingival plaques/tooth surfaces/fixed orthodontic appliances were taken in patients with fixed appliances (n = 46) and in healthy volunteers (n = 55). Samples were seeded on specific media for enterococcal and E. coli recovery, and 1 isolate of each type per sample was selected. Antimicrobial susceptibility and the presence of genes encoding antimicrobial resistance, bacteriocins, and virulence factors were checked by polymerase chain reaction.nnnRESULTSnEnterococci or E. coli were not recovered from healthy volunteers. Nevertheless, 10 isolates (5 E. faecium, 3 E. faecalis, and 2 E. coli) were obtained from 19.5% of patients with fixed appliances, and poor oral hygiene was evidenced in all of the these patients. Percentages of antimicrobial resistance and the resistance genes detected among the enterococci were: erythromycin: 100%, erm(B); kanamycin: 75%, aph(3)-IIIa; tetracycline: 50%, tet(L) with/without tet(M); streptomycin: 37%, ant(6)-Ia; chloramphenicol: 12%, catA. One E. coli isolate showed a phenotype of multiresistance containing 5 resistance genes and class 1 and 2 integrons. All enterococci produced gelatinase, and 4 isolates contained genes encoding enterocins L50A/B and P. The esp virulence gene was found in 1 multiresistant E. faecalis isolate.nnnCONCLUSIONSnPoor or improper oral hygiene in individuals with fixed appliances favors the oral carriage of antimicrobial-resistant E. coli and enterococci. Additional investigations are needed to assess its implication in human health.

Genetic Detection and Multilocus Sequence Typing of vanA-Containing Enterococcus Strains from Mullets Fish (Liza ramada)

Enterococci have emerged as important nosocomial and community-acquired pathogens in humans. The presence of vanA-enterococci was investigated in 103 fecal samples recovered from mullets fish (Liza ramada). All fecal samples were inoculated in Slanetz-Bartley agar plates supplemented with 4 mg/L of vancomycin for vancomycin-resistant enterococci (VRE) recovery and two isolates/sample were characterized. Antibiotic susceptibility was tested for 11 antibiotics by disk diffusion and agar dilution methods. VRE identification was performed by biochemical and molecular methods. Additionally, the mechanisms of resistance to glycopeptides (vanA, vanB, vanC1, vanC2, and vanD) and other antibiotics [erm(A), erm(B), tet(L), tet(M), aph(2)-aac(6), aph(3)-IIIa, ant(6), vat(D), vat(E)] as well as the presence of enterococcal surface protein (esp) and hyl virulence factors were investigated. vanA-Enterococcus faecium isolates were recovered from 4 of 103 tested samples, and they showed glycopeptide and erythromycin resistances. Three of them were also ampicillin resistant, two showed resistance to tetracycline, ciprofloxacin, and kanamycin, and one showed resistance to gentamicin. The tet(M) and erm(B) genes were found in all tetracycline- and erythromycin-resistant strains, respectively. The aph(3)-III and aph(2)-aac(6) genes were identified in the kanamycin- and gentamicin-resistant isolates, respectively. The IS1216 element was identified within vanX-vanY region of Tn1546 in two vanA isolates. The hyl and esp virulence genes were found in four and two isolates, respectively. vanA-strains were ascribed to sequence types ST280 (two isolates) and ST273 (two isolates), including both lineages into the clonal complex CC17. Mullets fish can excrete VRE in their feces and may be a reservoir for such resistant bacteria that can be transmitted to other animals including humans.

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.

Virulence factors in enterococci from partridges (Alectoris rufa) representing a food safety problem.

Forty-three Enterococcus isolates recovered from fecal samples of partridges, during the partridge hunting season, were studied for gelatinase and β-hemolysis activities. The presence of fsr-gelE genes and the cyl operon was studied by polymerase chain reaction and correlated with gelatinase and β-hemolysis production, respectively. In addition, genes encoding additional virulence factors (cpd, hyl, agg, esp, and ace) was also analyzed in all enterococci. Most of our Enterococcus faecalis isolates showed gelatinase activity (10 of 15 isolates), and this activity was not present in the other enterococcal species. All enterococci that showed gelatinase activity harbored the gelE and fsr genes. A large proportion of our isolates harbored genes of the cyl operon (19 of 43 isolates), although only 1 isolate contained the five cyl tested genes (E. faecalis), being the only one that expressed β-hemolysis. From the additional virulence factors (cpd, hyl, agg, esp, and ace), at least one virulence gene was detected in 13 of the 15 E. faecalis isolates, with cpd being the most frequently detected gene (9 isolates), followed by agg (5 isolates) and ace (4 isolates) genes. These virulence genes were not detected in the other enterococcal species with the exception of one E. faecium and E. casseliflavus isolate that harbored the hyl and cpd genes, respectively. Moreover, the esp gene was not detected in any of our isolates. In conclusion, this study showed the presence of virulence factors in enterococci of partridges and the possible transmission to humans through the food chain.

In vitro activity of ceftobiprole against Gram-positive and Gram-negative bacteria isolated from humans and animals

Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal; Veterinary and Animal Science Research Centre (CECAV), Vila Real, Portugal; University of Trás-os-Montes and Alto Douro, Department of Genetics and Biotechnology, Vila Real, Portugal; Institute for Biotechnology and Bioengineering, Centre of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal