Technical note: use of transfer RNA-intergenic spacer PCR combined with capillary electrophoresis to identify coagulase-negative Staphylococcus species originating from bovine milk and teat apices.

Coagulase-negative staphylococci (CNS) are the most frequently isolated bacteria in milk samples from cows with and without mastitis. Elucidating their relevance in bovine udder health is hampered because identification at the species level, if done at all, used to be performed based on phenotypic features. To provide a rapid, cheap, and easy-to-use genotypic technique that can be used to identify CNS species from milk and teat apices from cows, the performance of transfer RNA-intergenic spacer PCR (tDNA-PCR) in combination with capillary electrophoresis was evaluated. After updating the tDNA library with CNS reference strains, 288 field isolates were identified with tDNA-PCR and gene sequencing, and the latter was used as the reference method. The field isolates were divided in 2 groups of 144. Isolates of the first group were identified with tDNA-PCR with a typeability of 81.9% and an accuracy of 94.1%. Peak patterns of these isolates were then added to the tDNA library with species identity as determined by DNA sequencing. The second group was identified with the updated tDNA library, resulting in 91.0% typeability and 99.2% accuracy. This study showed that the updated tDNA-PCR in combination with capillary electrophoresis was almost as accurate as gene sequencing but faster and cheaper (only $3 per isolate), and is a useful tool in observational studies concerning the epidemiology of bovine CNS species.

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.

Isolation and characterization of Helicobacter suis sp. nov. from pig stomachs.

A new cultivation method was successfully applied for the in vitro isolation of a hitherto uncultured spiral Helicobacter species associated with ulceration of the non-glandular stomach and gastritis in pigs and formerly described as 'Candidatus Helicobacter suis'. Three isolates, HS1(T), HS2 and HS3, were subcultured from the stomach mucosa of three pigs after slaughter and were analysed using a polyphasic taxonomic approach. The novel isolates grew on biphasic culture plates or very moist agar bases in microaerobic conditions and exhibited urease, oxidase and catalase activities. Sequencing of the 16S rRNA gene, the 23S rRNA gene, the partial hsp60 gene and partial ureAB genes confirmed that the strains present in the gastric mucosa of pigs constituted a separate taxon, corresponding to 'Helicobacter heilmannii' type 1 strains as detected in the gastric mucosa of humans and other primates. For all genes sequenced, the highest sequence similarities were obtained with Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis, Helicobacter species isolated from the gastric mucosa of dogs and cats, which have also been detected in the human gastric mucosa and which are commonly referred to as 'Helicobacter heilmannii' type 2. SDS-PAGE of whole-cell proteins of strains HS1(T), HS2 and HS3 differentiated them from other Helicobacter species of gastric origin. The results of the polyphasic taxonomic analysis confirmed that the novel isolates constitute a novel taxon corresponding to 'Helicobacter heilmannii' type 1 strains from humans and to 'Candidatus H. suis' from pigs. The name Helicobacter suis sp. nov. is proposed for the novel isolates with the type strain HS1(T) (=LMG 23995(T)=DSM 19735(T)).

Evaluation of 16S rRNA gene-based PCR assays for genus-level identification of Helicobacter species.

The inclusivity, exclusivity, and detection limit of six 16S rRNA gene-based Helicobacter genus-specific PCR assays were examined. Five out of six assays were 100% inclusive, but the tests varied considerably in their exclusivity (9.1 to 95.5%). The clinical detection limit varied between 10(3) and 1 viable bacterial cell per reaction mixture.

Helicobacter baculiformis sp. nov., isolated from feline stomach mucosa.

A Gram-negative, microaerophilic slender rod, measuring approximately 10 mum long and approximately 1 microm wide, isolated from the gastric mucosa of a cat and designated strain M50(T), was subjected to a polyphasic taxonomic study. Despite its apparent lack of helical coils, the organism showed a corkscrew-like motion by means of multiple sheathed flagella located at both ends of the cell and by a periplasmic fibril coiled around the body. Strain M50(T) grew preferably on biphasic culture plates or on very moist agar. Coccoid forms predominated in cultures older than 4 days as well as in growth obtained on dry agar plates. The strain grew at 37 degrees C, but not at 25 or 42 degrees C and exhibited urease, oxidase and catalase activities. On the basis of 16S rRNA gene sequence analysis, the novel isolate was identified as a member of the genus Helicobacter and showed about 98 to 99 % sequence similarity to Helicobacter felis, Helicobacter bizzozeronii, Helicobacter salomonis, Helicobacter cynogastricus and 'Candidatus Helicobacter heilmannii', five highly related species previously detected in the feline or canine gastric mucosa. Protein profiling of strain M50(T) using SDS-PAGE revealed a pattern different from those of other Helicobacter species of mammalian gastric origin. Additionally, the urease and HSP60 gene sequences of strain M50(T) were different from those of H. felis, H. bizzozeronii, H. salomonis, H. cynogastricus and 'Ca. H. heilmannii'. It is thus proposed that strain M50(T) (=LMG 23839(T)=CCUG 53816(T)) represents a novel species within this genus, for which the name Helicobacter baculiformis sp. nov. is proposed.

Acute in vivo interactions of Helicobacter equorum with its equine host.

REASONS FOR PERFORMING STUDY: A novel urease-negative Helicobacter species has been isolated from faecal samples of clinically healthy horses, but no information is available about the main sites of colonisation in the equine gastrointestinal tract nor is the pathogenic potential of this microorganism known. An experimental infection in horses was therefore carried out. METHODS: Four horses were infected with H. equorum strain CCUG 52199T and subjected to euthanasia at 10 (n = 2) and 30 days (n = 2) post inoculation. A fifth animal was inoculated with phosphate buffered saline and used as control. Gastrointestinal samples were examined histologically and bacteriologically. These samples, as well as faecal material collected at regular intervals, were also subjected to PCR analysis. RESULTS: All horses remained clinically healthy and no specific macroscopic lesions were identified, nor were there any microscopic changes. H. equorum-DNA was detected in the faeces during the whole experiment in all infected animals but not in the negative control. Sites of colonisation were caecum, colon and rectum. CONCLUSIONS: H. equorum is able to colonise the equine lower bowel and is excreted in faeces without apparent pathology. No association between the presence of the organism and gastrointestinal disease was demonstrated.

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.

An unusual Actinobacillus equuli strain isolated from a rabbit with Tyzzer's disease.

Actinobacillus equuli was isolated in pure culture from the liver and lungs of an adult rabbit with Tyzzer's disease (Clostridium piliforme). Based on the haemolytic features on blood agar plates, a positive reaction in the CAMP-test, hydrolysis of esculin, the inability to ferment l-arabinose, tDNA-PCR and sequencing of the 16S rRNA gene, the isolate was classified as A. equuli subsp. haemolyticus biovar 1. However, the aqxA gene, characteristic for haemolytic A. equuli strains, was not detected by PCR.

Helicobacter equorum sp. nov., a urease-negative Helicobacter species isolated from horse faeces.

Gram-negative, curved, motile bacteria (strains EqF1T and EqF2) were isolated from faecal samples from two clinically healthy horses. Both strains possessed a single, monopolar, sheathed flagellum and were urease-negative. The novel strains grew at 37 degrees C under microaerobic conditions and were positive for oxidase, catalase and alkaline phosphatase activities. The isolates reduced nitrate to nitrite, but gamma-glutamyl transpeptidase activity was not detected. The novel isolates did not grow at 42 degrees C or on media containing 1 % glycine. They were resistant to cephalotin and nalidixic acid and susceptible to metronidazole. Analysis of the 16S and 23S rRNA gene sequences of the two novel strains identified them as representing a single species within the genus Helicobacter. In terms of 16S rRNA gene sequence similarity, Helicobacter pullorum and Helicobacter canadensis were the most closely related species (98 % similarity). 23S rRNA gene sequence analysis also classified strains EqF1T and EqF2 within the enterohepatic division of the genus Helicobacter, but only 94 % similarity was detected with H. pullorum and H. canadensis, which are helicobacters with unsheathed flagella. The most closely related species in terms of 23S rRNA gene sequence similarity was Helicobacter canis (95 %). Numerical analysis of whole-cell protein extracts by SDS-PAGE was performed and the novel isolates were clearly differentiated from H. pullorum, H. canadensis, H. canis and other species of the genus Helicobacter. This finding was also confirmed by sequence analysis of the hsp60 gene. On the basis of these genetic, biochemical and protein data, the isolates are classified as representing a novel species, for which the name Helicobacter equorum sp. nov. is proposed (type strain EqF1T=LMG 23362T=CCUG 52199T).

Prevalence of Helicobacter equorum in faecal samples from horses and humans.

Recently, a new enterohepatic Helicobacter species, H. equorum, was isolated from faecal samples of two clinically healthy horses. At the onset of this study, nothing was known about the prevalence of this organism in horses, nor was there any information available on the possible zoonotic character of this agent. This study aimed to determine the prevalence of H. equorum in faecal samples from equine and human origin. Therefore, faecal samples of 120 healthy privately owned horses, 227 healthy riding-school horses and 239 hospitalised horses were screened for H. equorum-DNA by means of a PCR amplifying a 1074-bp fragment of the 23S rRNA gene with primers specific for H. equorum. The vast majority of the hospitalised horses were under treatment with an antimicrobial agent at the moment of sampling, while the other horses had not been treated with an antimicrobial agent in the 14 days preceding the sampling. Stool samples of 531 humans suffering from gastro-intestinal disease and 100 clinically healthy humans were likewise examined. H. equorum-DNA was demonstrated in faeces from 0.8% of the privately owned horses, 3.1% of the riding-school horses and 7.9% of the hospitalised horses. The prevalence of H. equorum was significantly higher in hospitalised than in healthy, privately owned horses (P=0.02). H. equorum-DNA was not detected in human samples. These results indicate that the prevalence of H. equorum in horses may be influenced by the health status of the investigated horse population and/or by antimicrobial treatment. We may additionally assume that this micro-organism does not commonly infect humans.

Protein variability among Mycoplasma hyopneumoniae isolates.

Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to study the protein variability of Mycoplasma hyopneumoniae isolates. Fifty-six M. hyopneumoniae isolates from 6 different countries and 37 different herds were used. From eight herds, more than one isolate was available. All SDS-PAGE patterns of isolates originating from different herds were clearly divergent. Intra-species protein variability was quantified using the reference strain J and seven field strains all obtained from different herds and classified according to virulence. Between the field strains, a variability of 25% was found, while the culture-adapted strain J was clearly divergent and showed 30% variability with the field strains. No clustering according to virulence was obtained, but a protein band of about 181kDa was present in the two highly virulent isolates whereas this protein band was absent in the moderately and low virulent isolates. Protein patterns of isolates derived from different animals from the same herd, were identical or differed in only a few protein bands. This study clearly indicates that, in agreement with previous studies on genomic diversity of M. hyopneumoniae isolates, proteomic variability within the species is high. Our study did not find clear evidence that more than one M. hyopneumoniae isolate circulates within a herd at a specific time point. The minor differences found between M. hyopneumoniae isolates from the same herd might reflect the organism's ability to alter its proteomic expression profile under field conditions.

Helicobacter cynogastricus sp. nov., isolated from the canine gastric mucosa.

A Gram-negative, microaerophilic helical rod, isolated from the gastric mucosa of a dog and designated strain JKM4(T), was subjected to a polyphasic taxonomic study. The tightly coiled organism, measuring 10-18 mum long and up to 1 mum wide, was motile by means of multiple sheathed flagella located at both ends of the cell and by a periplasmic fibril running along the external side of the helix. Strain JKM4(T) grew preferably on biphasic culture plates or on very moist agar. Coccoid forms predominated in cultures older than 4 days as well as in growth obtained on dry agar plates. The strain grew at 30 and 37 degrees C, but not at 25 or 42 degrees C and exhibited urease, oxidase and catalase activities. On the basis of 16S rRNA gene sequence analysis, the novel isolate was identified as a member of the genus Helicobacter and showed > 97 % similarity to Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis, three species previously isolated from the canine gastric mucosa. Protein profiling of strain JKM4(T) using SDS-PAGE revealed a pattern different from those of other Helicobacter species of mammalian gastric origin and from Helicobacter canis. Additionally, the urease gene sequence of strain JKM4(T) was different from those of urease genes of H. felis, H. bizzozeronii, H. salomonis and "Candidatus Helicobacter heilmannii". It is thus proposed that strain JKM4(T) (=LMG 23188(T)) represents a novel species within this genus, Helicobacter cynogastricus sp. nov.

Low frequency of Helicobacter species in the stomachs of experimental rabbits.

The natural occurrence of established Helicobacter species was investigated in the stomachs of 65 laboratory rabbits, by means of polymerase chain reaction (PCR) (65/65) and histological analysis (51/65). The degree of inflammation in the different regions of the rabbits' stomach was evaluated on haematoxylin and eosin (HE)-stained histological slides. Four rabbits were found positive for Helicobacter species by PCR. Based on 16S ribosomal ribonucleic acid (rRNA) sequences, H. canadensis/H. pullorum organisms were identified in three animals. Bacteria were seen on merely one histological slide from one of these animals. H. felis was identified in one rabbit. Histological examination revealed no inflammation in the stomachs of 40 rabbits, while moderate gastric inflammation was seen in 11 animals, mainly in the antrum. In conclusion, the stomach of the laboratory rabbits included in the study was occasionally found positive for Helicobacter species, which were mostly identified as enterohepatic helicobacters, probably reflecting a mere passage of these bacteria through the stomach.

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.

In vitro antimicrobial susceptibility testing of Helicobacter felis, H. bizzozeronii, and H. salomonis.

The susceptibilities of Helicobacter felis (15 strains), H. bizzozeronii (7 strains), and H. salomonis (3 strains) to 10 antimicrobial agents were investigated by determination of the MIC using the agar dilution method. No consistent differences were noticed between the different Helicobacter species, which were all highly susceptible to ampicillin, clarithromycin, tetracycline, tylosin, enrofloxacin, gentamicin, and neomycin, as demonstrated by low MICs. Higher MICs were obtained for lincomycin (up to 8 microg/ml) and spectinomycin (up to 4 microg/ml). Two H. felis strains showed a MIC of 16 microg/ml for metronidazole, suggesting acquired resistance to this antimicrobial agent.

The inflammatory response in the mouse stomach to Helicobacter bizzozeronii, Helicobacter salomonis and two Helicobacter felis Strains.

The inflammatory response in the mouse stomach was evaluated as a means of distinguishing different non-pylori Helicobacter (H.) strains in terms of virulence. Mice of four strains (BALB/c, SJL, C57BL/6 and CFW) were infected intragastrically with four bacterial strains (H. felis ATCC 49179 and CCUG 37471, H. bizzozeronii and H. salomonis). The animals were killed for gastric examination at 3, 9 or 16 weeks post-inoculation. H. salomonis could not be detected by the polymerase chain reaction, but the other three organisms were detected in all stomach samples at all timepoints. SJL mice consistently showed particularly severe gastric inflammation regardless of bacterial strain. Lymphocytes and occasionally neutrophils were seen in submucosa and lamina propria mucosae. BALB/c mice showed the least severe inflammatory changes. H. bizzozeronii differed from the two H. felis strains in producing less striking pathological changes in mice. Of the two H. felis strains, ATCC 49179 produced the more severe inflammatory changes in SJL mice.

First report on the occurrence of 'Helicobacter heilmannii' in the stomach of rabbits.

Gastric Helicobacter spp. have been described in a wide range of animal species, including dogs, cats, primates, swine, cattle and rodents. However, in lagomorphs--more specifically rabbits--gastric Helicobacter infections have never been reported. Biopsy specimens were collected from different stomach regions of 23 rabbits, including 10 pet rabbits, 10 industrial animals and 3 research animals. These were subjected to a PCR assay for the detection of Helicobacter DNA. Identification up to the species level was based on 16S rRNA sequence analysis and a recently developed multiplex PCR. Seven rabbits (four pet, one research animal and two industrial animals) tested positive in the Helicobacter genus-specific PCR in the stomach, with the corpus being predominantly positive. H. felis and H. salomonis, hitherto presumed to be naturally hosted by cats and dogs, were detected in three animals and one animal, respectively. One of these animals had been completely devoid of any form of contact with cats or dogs. A H. pullorum/H. rappini-like organism (96% 16S rDNA sequence similarity) was found in an industrially held rabbit. The helicobacters of the two remaining rabbits could not be identified up to the species level. To conclude, this is the first report on the occurrence of Helicobacter spp. in the stomach of rabbits. In view of the fact that H. felis and H. salomonis are put forward as having zoonotic potential, further research is necessary to investigate the implications of these findings not only for the rabbit but also for human health.

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.

Multiplex PCR assay for differentiation of Helicobacter felis, H. bizzozeronii, and H. salomonis.

Helicobacter felis, Helicobacter bizzozeronii, and Helicobacter salomonis are frequently found in the gastric mucous membrane of dogs and cats. These large spiral organisms are phylogenetically highly related to each other. Their fastidious nature makes it difficult to cultivate them in vitro, hampering traditional identification methods. We describe here a multiplex PCR test based on the tRNA intergenic spacers and on the urease gene, combined with capillary electrophoresis, that allows discrimination of these three species. In combination with previously described 16S ribosomal DNA-based primers specific for the nonculturable "Candidatus Helicobacter suis," our procedure was shown to be very useful in determining the species identity of "Helicobacter heilmannii"-like organisms observed in human stomachs and will facilitate research concerning their possible zoonotic importance.