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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)

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 bla(CTX-M-1) (6 isolates) and bla(CTX-M-1) + bla(TEM1-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.

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 bla(TEM) and/or bla(SHV) and/or bla(CTX-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.

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., (Copyright 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 < or =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 < or =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).