ummary Eighteen methicillin (cloxacillin)-resistant Staphylococcus aureus isolates were obtained from 17 cows (2.2%) out of 776 which yielded S. aureus positive milk samples. Resistant strains were found on 12 (5.2%) of 232 farms. The physiological characteristics (production of haemolysins and fibrinolysin) of 17 of these isolates were similar to those of the common bovine strains. Their antibiotic resistance characteristics were similar to those of the methicillin-resistant strains which have been described from human sources. The epidemiology of methicillin-resistant S. aureus strains and the methods which can be used in their routine detection are briefly discussed. Resume Souches de staphylocoques dores resistantes a la methicilline (cloxacilline) isolees chez les bovins Au cours de l'examen de 776 echantillons de lait contenant des staphylocoques dores, on a trouve 18 souches resistantes a la methicilline (cloxacilline) chez 17 bovins (2,2%). Les souches resistantes provenaient de 12 fermes (sur les 232 examinees au total) (soit 5,2%). Les proprietes physiologiques de ces souches sont les memes que celles des souches trouvees habituellement chez les bovins. Le developpement de la resistance aux antibiotiques ne differe pas de celui des souches resistantes a la methicilline isolees chez l'homme. On discute brievement l'epidemiologie des souches de S. aureus resistantes a la methicilline, ainsi que les methodes pour leur mise en evidence de routine. Resumen Cepas de Staphylococcus aureus resistentes a la meticilina (cloxacilina) isoladas en casos de mastitis bovina Al analizar 776 muestras de leche, positivas de Staphylococcus aureus, se hallaron 18 estirpes resistentes a la meticilina (cloxacilina) en 17 vacas (2,3%). Las estirpes resistentes procedian de 12 (5,2%) explotaciones entre un total de 232 examinadas. Las propiedades fisiologicas de estas cepas eran las mismas que las normales ubicadas en vacas. La conformacion de su resistencia frente a los antibioticos no se diferencio de las estirpes resistentes a la meticilina aisladas en personas. Se discuten de forma sucinta la epidemiologia de las estirpes S. aureus resistentes a la meticilina y las tecnicas para su hallada rutinaria. Zusammenfassung Bei der Untersuchung von 776 Staphylococcus aureus-positiven Milchproben wurden 18 Methicillin (cloxacillin)-resistente Stamme bei 17 Rindern (2,2%) gefunden. Die resistenten Stamme stammten aus 12 (5,2%) von insgesamt 232 untersuchten Farmen. Die physiologischen Eigenschaften dieser Stamme waren die gleichen wie die normaler, beim Rind vorkommender Stamme. Die Ausbildung ihrer Anti-biotikaresistenz unterschied sich nicht von der beim Menschen isolierter Methicillin-resistenter Stamme. Die Epidemiologie von Methicillin-resistenten S. aureus-Stammen sowie Methoden zu ihrer routinemafiigen Auffindung werden kurz diskutiert.

Eighty-seven Lactobacillus strains isolated from cloacal swabs of broiler chickens derived from 20 different farms in Belgium were identified to species level and tested for susceptibility to macrolide and lincosamide antibiotics. Five different Lactobacillus species were identified as being predominantly present in the cloacae of broilers: Lactobacillus crispatus, Lactobacillus salivarius subsp. salivarius, Lactobacillus amylovorus, Lactobacillus gallinarum and Lactobacillu sreuteri. Acquired resistance prevalence to macrolides and lincosamides was very high in the investigated lactobacilli: 89% of the strains were resistant to either or both lincosamide and macrolide class antibiotics. The vast majority of these resistant strains (96%) displayed constitutive resistance. More than one-half of the macrolide and/or lincosamide resistant strains carried an erm(B), erm(C), mef(A), lnu(A) gene or a combination of these genes.

The in vitro activity of 13 antimicrobial agents against 23 Helicobacter pullorum strains from poultry (21) and human (two) origin, and one human H. canadensis strain was tested by the agar dilution method. With the H. pullorum strains, monomodal distributions of Minimum Inhibitory Concentrations (MICs) were seen with lincomycin, doxycycline, gentamicin, tobramycin, erythromycin, tylosin, metronidazole, and enrofloxacin in concentration ranges considered as indicating susceptibility in other bacteria. The normal susceptibility level for nalidixic acid was situated at or slightly above the MIC breakpoints proposed for Campylobacteriaceae. Ampicillin, ceftriaxone, and sulphamethoxazole-trimethoprim showed poor activity against H. pullorum. For the H. canadensis strain, a similar susceptibility pattern was seen, except for nalidixic acid and enrofloxacin, whose MIC of >512 and 8 microg/ml, respectively, indicated resistance of this agent. With spectinomycin, a bimodal distribution of the MICs was noted for the tested strains; eight H. pullorum isolates originating from one flock showed acquired resistance (MIC>512 microg/ml).

Members of the Staphylococcus sciuri group (S. sciuri, S. lentus, and S. vitulinus) are coagulase-negative, novobiocin-resistant staphylococci that could be distinguished from other staphylococci on the basis of positive oxidase activity. In the present study, a scheme based on conventional methods and utilization of various carbohydrates was evaluated for the identification of oxidase-positive staphylococci, and validated using two molecular techniques. Of the 173 oxidase-positive staphylococcal tested strains, 161 were identified as S. sciuri, 9 as S. lentus, 2 as S. vitulinus, and one as S. fleurettii by our scheme. The level of agreement with tRNA intergenic length polymorphism analysis (tDNA-PCR) was high (97.5–100% correlation). The accuracy and ease of use of this protocol suggest that it may be useful and valuable in microbiological laboratories for the identification of members of this group.

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.

Two enterococcal strains LMG 16607(T) and LMG 16612 originating from sea water were analysed in a polyphasic taxonomic study. Both strains, assigned as Enterococcus sp. in the BCCM/LMG culture collection, possessed analogous protein profiles, but these were different from all other enterococcal species. 16S rRNA gene sequence analysis of one strain showed the highest similarity, 96(.)9-96(.)1 %, with its closest phylogenetic neighbours Enterococcus saccharolyticus, Enterococcus sulfureus, Enterococcus saccharominimus and Enterococcus italicus. Further genomic analysis by (GTG)(5)-PCR fingerprinting and sequence analysis of the housekeeping gene phenylalanyl-tRNA synthase (pheS) and distinct biochemical features confirmed that the two strains represent a novel enterococcal species for which the name Enterococcus aquimarinus sp. nov. is proposed. The type strain is LMG 16607(T) (=CCM 7283(T)).

The taxonomic position of strain LMG 13590(T), originally isolated from dog faeces and classified as Enterococcus dispar in the BCCM/LMG Bacteria Catalogue, was reinvestigated. This strain and 12 recent isolates from faecal samples of healthy dogs occupied a clearly separate position when investigated with multilocus sequence analysis (MLSA) of the genes encoding the alpha subunit of ATP synthase (atpA), RNA polymerase alpha subunit (rpoA) and phenylalanyl-tRNA synthase alpha subunit (pheS). The 16S rRNA gene sequence of one representative strain showed highest similarities of 98-99% with E. dispar LMG 13521(T), Enterococcus canis LMG 12316(T) and Enterococcus asini LMG 18727(T). A further polyphasic taxonomic study based on whole-cell protein fingerprinting, DNA-DNA hybridization and biochemical features demonstrated that the 13 enterococcal dog faecal strains represent a single, novel Enterococcus species for which the name Enterococcus canintestini sp. nov. is proposed. The type strain is LMG 13590(T) (=CCM 7285(T)).

This study was conducted to investigate the effect of avilamycin used as a growth promoter on the number of E. faecium and on avilamycin-resistant E. faecium in the intestines of broilers over time. Avilamycin was added at 13.6 ppm to the feed of chickens during 28 days or during a typical growth period of 42 days; a nonmedicated group was included. Three hundred twenty-four Ross broiler chickens were equally distributed over the different groups in a treatment trial and kept in three isolation rooms. In each room, two replicates of the three experimental groups were kept in separate pens. At various time points, samples from different intestinal compartments or the feces were serially diluted and plated on avilamycin-supplemented and on unsupplemented Slanetz and Bartley (SL) media, and E. faecium counts were recorded. Only in the feces and only on the last sampling day was a significant decrease noted in the E. faecium counts in chickens treated with avilamycin for 42 days. Intermediate resistant (MIC 4-8 microg/ml) and resistant strains (MIC>or=16 microg/ml) were isolated from all groups, and there was a rise in prevalence over time. Pulsed-field gel electrophoresis profiles of these strains indicated clonal spread from one pen to another within the same room. The ratio between the counts of E. faecium isolated on antibiotic-supplemented to unsupplemented plates was significantly higher at the end of the trial in the feces samples from the group fed avilamycin for 42 days compared to the other groups, indicating a selective effect of avilamycin.

Four staphylococcal isolates from clinical and necropsy specimens from a cat, a dog, a horse and a parrot (Psittacus erithacus timneh) were found to constitute a distinct taxon. 16S rRNA gene sequence analysis revealed that its closest phylogenetic relatives are Staphylococcus intermedius and Staphylococcus delphini. Growth characteristics, biochemical features and DNA-DNA hybridizations demonstrated that the strains differ from these and other known species and that they represent a single, novel Staphylococcus species for which the name Staphylococcus pseudintermedius sp. nov. is proposed. The novel species is commonly confused with S. intermedius in routine diagnostic veterinary bacteriology. Although the strains described were isolated from lesions and show several characteristics typical of pathogenic staphylococci, such as coagulase, DNase and beta-haemolysin production, the pathogenic significance of the novel species remains unclear. The type strain, LMG 22219(T) (=ON 86(T)=CCUG 49543(T)), was isolated from lung tissue of a cat.

ABSTRACT A total of 28 staphylococcal isolates from human clinical specimens belonging to the Staphylococcus sciuri group were identified and characterized. The API Staph and ID32 STAPH correctly identified S. sciuri and S. lentus but not S. vitulinus strains. Identification to the subspecies level was possible only by a PCR-based method.

To obtain better insights into the possible exchange of resistance genes between human and animal streptococci, the sequences of the erm (B) genes of streptococcal isolates from humans, pigs, pork carcasses, chickens, and calves were compared. Identical erm (B) gene sequences were present in strains from humans, pigs, pork carcasses, and calves. During in vitro mating experiments, the erm (B) gene was exchanged between porcine Streptococcus suis and human S. pneumoniae, S. pyogenes, and S. oralis strains. The presence of different tetracycline resistance genes and the int Tn 1545 gene was determined in animal streptococci carrying the erm (B) gene. Although tet(M) and int Tn 1545 genes were detected in 24% of the porcine and pork carcass streptococcal strains, the tet(O) gene was the predominant tetracycline resistance gene in these strains (81%). The latter gene was co-transferred with the erm (B) gene from porcine S. suis strains to human streptococci in the mating experiments. These results show that, identical erm (B) gene sequences were present in animal and human streptococci and that transfer of the erm (B) gene from porcine S. suis to human streptococci and vice versa is possible, but probably occurs at a low frequency.

Faecal samples from 50 pigeons all originating from different lofts were screened for the presence of macrolide and lincosamide (ML)-resistant isolates of Streptococcus gallolyticus and Enterococcus columbae by plating the samples onto selective media. Sixty-eight ML-resistant E. columbae strains were recovered from the faecal samples of 29 animals. Two of these samples also harboured ML-resistant S. gallolyticus strains. The erm(B) gene was detected in 58 E. columbae and in five S. gallolyticus isolates. Four of these E. columbae isolates also carried the mef(A) gene. Five E. columbae strains possessed the mef(A) gene in the absence of erm(B). On the basis of the sequence of the complete erm(B) gene, 10 E. columbae isolates clustered together in six groups. In two of these isolates, the erm(B) gene sequence was identical to that of S. gallolyticus strains, indicating that exchange of resistance genes might occur between pathogenic and non-pathogenic bacterial species belonging to the pigeon's intestinal flora.

Four isolates, which were obtained from Belgian, Moroccan and Romanian dairy products, constituted a homogeneous but unidentified taxon after screening with whole-cell protein fingerprinting. Complete 16S rRNA gene sequence analysis classified representative strains in the genus Enterococcus. Highest sequence similarities of 98.6 and 98.0 % were obtained with the species Enterococcus sulfureus and Enterococcus saccharolyticus, respectively. Growth characteristics, biochemical features, tRNA intergenic length polymorphism analysis, DNA-DNA hybridization and DNA G+C contents of selected strains demonstrated that they represent a single, novel Enterococcus species. It differs phenotypically from other enterococci in characteristics commonly considered as typical of this genus: no growth in 6.5 % NaCl or 0.4 % sodium azide, and no acid production from a wide range of carbohydrates. The name Enterococcus saccharominimus sp. nov. is proposed for this novel species; the type strain (LMG 21727(T)=CCM 7220(T)) was isolated from contaminated pasteurized cow's milk.

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.

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.

Clostridium perfringens strains isolated in 2002 from the intestines of broiler chickens from 31 different farms located in Belgium were tested for susceptibility to 12 antibiotics used for therapy, growth promotion or prevention of coccidiosis. All strains were uniformly sensitive to the ionophore antibiotics monensin, lasalocid, salinomycin, maduramycin and narasin. All were sensitive to avilamycin, tylosin and amoxicillin, while flavomycin (bambermycin) showed low or no activity. Chlortetracycline and oxytetracycline were active at very low concentrations, but low-level acquired resistance was detected in 66% of the strains investigated. Fifty percent of these strains carried the tetP(B) resistance gene, while the tet(Q) gene was detected in only one strain. One strain with high-level resistance against tetracyclines carried the tet(M) gene. Sixty-three percent of the strains showed low-level resistance to lincomycin. The lnu(A) and lnu(B) genes were each only found in one strain. Compared with a similar investigation carried out in 1980, an increase was seen in resistance percentages with lincomycin (63% against 49%) and a slight decrease with tetracycline (66% against 74%).

Earlier field observations suggest that teat apex colonization by Staphylococcus chromogenes pre-partum in dairy heifers protects udder quarters against elevated somatic cell counts early post-partum. To explain these findings, the in vitro inhibitory capability of S. chromogenes from teat apices of heifers towards some major mastitis pathogens was tested using a modified cross-streaking method. Two out of 10 S. chromogenes isolates, both originating from two different teats from the same heifer, consistently inhibited growth of all Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis strains, but none of the Escherichia coli strains. The present study, therefore, supports the protective effect of teat apex colonization by S. chromogenes by in vitro production of inhibitory substances.

Fifty-six Staphylococcus aureus isolates recovered between 1998 and 2003 from 31 rabbit farms with and without problems of chronic staphylococcosis, were screened for resistance to enrofloxacin, erythromycin, gentamicin, lincomycin, neomycin, penicillin and tetracyclines using the agar dilution test. For penicillin, a disk diffusion test was also performed. The detection of tetP(B), tet(K), tet(L), tet(M), tet(O), tet(T), tet(W), erm(A), erm(B), erm(C) and mec(A) genes was done via a PCR assay. Four isolates showed resistance to erythromycin and lincomycin. These isolates were positive for the erm(C) gene in the PCR. Eleven strains were resistant to tetracyclines and all harboured the tet(K) gene. In the agar dilution test, five isolates showed resistance to penicillin, whereas in the disk diffusion test 12 isolates showed resistance. None of these 12 resistant isolates carried the mec(A) gene. Only one strain showed resistance to gentamicin, and all strains were susceptible to enrofloxacin and neomycin. This study demonstrates that resistance to antimicrobial agents in S. aureus isolates originating from rabbits is relatively rare compared to resistance in S. aureus isolates originating from other animals and humans.

The MICs of five antimicrobial agents were determined by the agar dilution method for 98 Arcobacter butzleri and 28 Arcobacter cryaerophilus strains from humans, and poultry. With gentamicin, a MIC of 16 microg/ml was recorded for one A. butzleri strain isolated from poultry, whereas for the other strains MICs ranged from 0.25 to 4 microg/ml. With ciprofloxacin, a bimodal distribution of susceptibility levels was seen for human A. butzleri isolates (0.015-0.03 versus 0.12-0.25), whereas MICs for 65 of the 68 A. butzleri poultry strains ranged from 0.12 to 0.5 microg/ml and three strains from three different broilers were resistant with a MIC of 16 microg/ml. One A. cryaerophilus strain from poultry was resistant to erythromycin at a MIC of 128 microg/ml, whereas MICs for the other Arcobacter strains ranged from 2 to 32 microg/ml. No difference in susceptibility or resistance among the human and poultry strains tested was observed with doxycycline and nalidixic acid. The presence of acquired resistance to erythromycin and ciprofloxacin among poultry isolates is a matter of concern, because the two antimicrobials are generally prescribed as first-line drugs for the treatment of infections with Campylobacteraceae in humans.