Screening of bovine coagulase-negative staphylococci from milk for superantigen-encoding genes.

A collection of 102 coagulase-negative staphylococci (CNS), isolated from cases of subclinical and clinical bovine mastitis and belonging to 10 different species, were screened by PCR for the presence of genes encoding enterotoxins and enterotoxin-like toxins (sea, seb, sec, sed, see, seg, seh, sei, sej, selk, sell, selm, seln, selo, selp, selq and selu), toxic shock syndrome toxin-1 (tst), and exfoliative toxins A and B (eta and etb). No toxin gene sequences were amplified from any of the isolates, indicating that superantigens encoded by genes detectable by the PCR tests used were not involved in the development of subclinical and clinical mastitis in cattle infected with the CNS isolates tested.

High prevalence of tetracycline resistance in Enterococcus isolates from broilers carrying the erm(B) gene.

A total of 73 isolates of Enterococcus spp. carrying the erm(B) gene were obtained from cloacal swabs of broiler chickens derived from 13 different farms in Belgium. The erm(B) gene encodes resistance to macrolides, lincosamides and streptogramin B antibiotics (MLS(B)). The isolates belonged to eight different species: Enterococcus avium (eight isolates), Enterococcus casseliflavus (11 isolates), Enterococcus cecorum (eight isolates), Enterococcus durans (seven isolates), Enterococcus faecalis (10 isolates), Enterococcus faecium (17 isolates), Enterococcus gallinarum (seven isolates) and Enterococcus hirae (five isolates). Acquired resistance to tetracycline was detected in 68 of the isolates, and in 62 of these it was associated with the presence of the resistance genes tet(L), tet(M), tet(O) or tet(S). In three E. faecium isolates that were phenotypically susceptible to tetracycline, tet(L) or tet(M) was present. The transposon integrase gene (int gene) of the Tn916/Tn1545 transposon family was detected in 18 of the 54 isolates that contained the tet(M) gene. It was concluded that acquired resistance to tetracycline antibiotics is often present in enterococci from poultry carrying the erm(B) gene. The use of tetracyclines in poultry may therefore co-select for resistance to MLS(B) antibiotics, which may be important as alternative therapy for enterococcal infections in humans.

Prevalence and mechanism of resistance against macrolides, lincosamides, and streptogramins among Enterococcus faecium isolates from food-producing animals and hospital patients in Belgium.

The prevalence of acquired resistance to streptogramins, macrolides, and lincosamides and the genetic background of this resistance was investigated in Enterococcus faecium strains isolated from food-producing animals and hospital patients 4-5 years after the ban of streptogramins as growth promoters. The minimum inhibitory concentrations (MICs) of quinupristin/dalfopristin (Q/D), virginiamycin M1 (virgM1), erythromycin (ery), tylosin (tyl), and lincomycin (lin) were determined by the agar dilution method for E. faecium isolates derived from pigs (80), broilers (45), and hospitalized patients (103). Resistance or susceptibility was interpreted using a microbiological criterion and breakpoints recommended by the Clinical Laboratory Standards Institute (CLSI), if available. The isolates were also screened by PCR for erm(B), lnu(A), lnu(B), mef(A/E), vat(D), vat(E), vga(A), vga(B), and vgb(A) genes. Acquired resistance to Q/D, virgM1, ery, tyl, and lin was detected in 34%, 96%, 46%, 46%, and 69% of the porcine strains, respectively. For broiler strains this was 15% (Q/D), 98% (virgM1), 69% (ery), 71% (tyl), and 89% (lin) and for human strains 23% (Q/D), 65% (virgM1), 54% (ery), 52% (tyl), and 60% (lin). Strains showing cross-resistance against macrolides and lincosamides almost always carried the erm(B) gene. This gene was present in 64% of the Q/D-resistant isolates. Only in two human and three broiler Q/D- and virgM1-resistant isolates, a combination of the erm(B) and vat(D) or vat(E) genes was found. The genetic background of resistance could not be determined in the other Q/D- or virgM1-resistant strains. This study demonstrates that streptogramin resistance is frequently present in strains from hospitalized patients and food-producing animals, but the genetic basis hitherto mostly remains obscure.

Acquired antimicrobial resistance in the intestinal microbiota of diverse cat populations.

The aim of this study was to investigate the prevalence of acquired antimicrobial resistance in the resident intestinal microbiota of cats and to identify significant differences between various cat populations. Escherichia coli, Enterococcus faecalis, E. faecium and Streptococcus canis were isolated as faecal indicator bacteria from rectal swabs of 47 individually owned cats, 47 cattery cats and 18 hospitalised cats, and submitted through antimicrobial sensitivity tests. The results revealed that bacteria isolated from hospitalised and/or cattery cats were more frequently resistant than those from individually owned cats. E. coli isolates from hospitalised cats were particularly resistant to ampicillin, tetracycline and sulfonamide. Both enterococci and streptococci showed high resistance to tetracycline and in somewhat lesser extent to erythromycin and tylosin. Most E. faecium isolates were resistant to lincomycin and penicillin. One E. faecalis as well as one E. faecium isolate from hospitalised cats showed 'high-level resistance' (MIC > 500 microg/ml) against gentamicin, a commonly used antimicrobial agent in case of human enterococcal infections. The results of this research demonstrate that the extent of acquired antimicrobial resistance in the intestinal microbiota of cats depends on the social environment of the investigated population. It is obvious that the flora of healthy cats may act as a reservoir of resistance genes.

Identification of enterococcal, streptococcal and Weissella species in the faecal flora of individually owned dogs.

AIMS: To improve the limited information on the composition of the faecal Gram-positive coccal flora of healthy dogs by the use of a molecular identification method. METHODS AND RESULTS: Faecal swabs were collected for the selective isolation of Gram-positive coccal strains. Colonies with enterococcal- and streptococcal-like morphology were identified by tRNA intergenic length polymorphism analysis (tDNA-PCR). Fourteen known species belonging to three genera (Enterococcus, Streptococcus and Weissella) and one alleged new enterococcal species were found. CONCLUSIONS: The faecal flora of dogs comprises an unusually broad diversity of culturable Gram-positive coccal species with Enterococcus faecalis being most frequently present followed by not less than six other species of about equal importance. SIGNIFICANCE AND IMPACT OF THE STUDY: Many human- and animal-associated enterococci and streptococci are also present in dog faeces together with the largely uncharacterized Weissella cibaria and a group of strains resembling Enterococcus dispar, but representing a distinct and hitherto unknown species. Phenotypic characteristics of the latter two species were determined and the test results were compared with the species descriptions of W. cibaria and E. dispar respectively.

Molecular analysis of human, porcine, and poultry Enterococcus faecium isolates and their erm(B) genes.

Fifty-nine erm(B)-positive Enterococcus faecium strains isolated from pigs, broilers, and humans were typed using multilocus sequence typing (MLST), and the coding sequence of the erm(B) gene was determined. Identical erm(B) gene sequences were detected in genetically unrelated isolates. Furthermore, genetically indistinguishable strains were found to contain different erm(B) alleles. This may suggest that horizontal exchange of the erm(B) gene between animal and human E. faecium strains or the existence of a common reservoir of erm(B) genes might be more important than direct transmission of resistant strains.

Antibiotic resistance among fecal indicator bacteria from healthy individually owned and kennel dogs.

Escherichia coli and Enterococcus faecalis strains isolated from anal swabs of clinically healthy dogs were examined for the presence of acquired antimicrobial resistance. The strains originated from dogs of 92 different owners and from eight breeding kennels. The purpose of the present study was to evaluate the resistance situation in the intestinal flora of the dog to assess the possible role of the dog flora as a reservoir of antimicrobial resistance. Multiple resistance was rarely found in E. coli strains collected from individually owned dogs, in contrast with strains from kennel dogs. Resistance to ampicillin, trimethoprim, and sulfamethoxazole was significantly less prevalent in E. coli from privately owned dogs than in strains from kennel dogs. Resistance rates against tetracycline and macrolides were unexpectedly high in E. faecalis strains. Two and three E. faecalis strains from individually owned dogs and kennel dogs, respectively, were resistant to gentamicin, an antibiotic often used for treating enterococcal infections in humans. This study demonstrates that resistance percentages may fluctuate with the choice of dog population. The observed antimicrobial resistance percentages indicate that the flora of healthy dogs may act as a reservoir of resistance genes.

Streptococcus minor sp. nov., from faecal samples and tonsils of domestic animals.

Nine isolates, which were obtained from tonsils, anal swabs and faeces of dogs and from tonsils of a cat and a calf, constituted a homogeneous but unidentified taxon after screening with tRNA intergenic length polymorphism analysis and whole-cell protein fingerprinting. 16S rDNA sequence analysis classified representative strains in the genus Streptococcus. Highest sequence similarity (95.9 %) was obtained with Streptococcus ovis. Growth characteristics, biochemical features, DNA-DNA hybridization and DNA G+C contents of selected strains demonstrated that they represent a single, novel streptococcal species. The name Streptococcus minor sp. nov. is proposed for the novel species; the type strain (ON59(T)=LMG 21734(T)=CCUG 47487(T)) was isolated from a dog tonsil.

Description of Enterococcus canis sp. nov. from dogs and reclassification of Enterococcus porcinus Teixeira et al. 2001 as a junior synonym of Enterococcus villorum Vancanneyt et al 2001.

Strains from anal swabs and chronic otitis externa in dogs were shown to be phylogenetically related to the Enterococcus faecium species group. They shared a number of phenotypic characteristics with these species, but they could be easily differentiated by biochemical reactions. In addition, the canine strains were unusual in their nearly complete failure to grow on sodium azide-containing enterococci-selective media and in their Voges-Proskauer reactions (usually negative). By using 16S rRNA sequencing and DNA-DNA hybridization of representative strains, as well as tDNA interspacer gene PCR and SDS-PAGE of whole-cell proteins, the group of canine strains was shown to constitute a novel enterococcal species. The name Enterococcus canis sp. nov. is proposed for this species, with LMG 12316T (= CCUG 46666T) as the type strain. Concurrently, the taxonomic situation and nomenclatural position of Enterococcus porcinus were investigated. As no phenotypic or genotypic differences were found between this species and Enterococcus villorum, the name E. porcinus is considered to be a junior synonym of E. villorum.