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)).

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.

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.

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.

Prevalence and mechanism of resistance against macrolides and lincosamides in Streptococcus suis isolates.

Eighty-seven Streptococcus suis isolates recovered in 1999-2000 from diseased pigs, all from different farms, were screened for resistance against macrolide and lincosamide antibiotics by the disk diffusion and agar dilution test and a PCR assay, amplifying the ermB gene and the mefA/E gene. Seventy-one percent of the isolates showed constitutive resistance to macrolide and lincosamide antibiotics (MLS(B)-phenotype). All these isolates were positive for the ermB gene in the PCR, but negative for the mefA/E gene. For all strains minimum inhibitory concentrations (MIC) against five other antimicrobial agents were determined. All strains were susceptible to penicillin. Ninety-nine percent of the isolates were susceptible to enrofloxacin and tiamulin. Eighty-five percent of the strains were resistant to doxycycline. A 540bp fragment of the ermB genes of eight S. suis strains was sequenced and compared with ermB genes of five S. pneumoniae and five S. pyogenes strains of human origin. A 100% homology was found between these fragments in seven S. suis, one S. pneumoniae and three of the S. pyogenes isolates. This study demonstrates that resistance against macrolides, lincosamides and streptogramin B is widespread in S. suis and mediated by ribosome methylation, encoded by the ermB gene.

Enterococcus haemoperoxidus sp. nov. and Enterococcus moraviensis sp. nov., isolated from water.

A polyphasic taxonomic approach was used to study atypical enterococci isolated from surface waters. All strains were characterized by physiological and biochemical tests as well as by genotyping. The results of biochemical tests and tRNA intergenic length polymorphism analysis (tDNA-PCR) divided all studied strains uniformly into two groups. Because these groups were clearly separated from all enterococcal species described to date, 16S rDNA sequence analysis, DNA base composition analysis and DNA-DNA hybridization of representative strains were done to elucidate the taxonomic position of the analysed groups. On the basis of the results obtained, the names Enterococcus haemoperoxidus (type strain CCM 4851T = LMG 19487T) and Enterococcus moraviensis (type strain CCM 4856T = LMG 19486T) are proposed for the two hitherto undescribed species. The type strains and reference cultures have been deposited in the Czech Collection of Microorganisms (CCM), Masaryk University, Brno, Czech Republic, and in the BCCM/LMG Culture Collection, Ghent University, Belgium.

Enterococcus villorum sp. nov., an enteroadherent bacterium associated with diarrhoea in piglets.

The taxonomic positions of five enteroadherent bacterial pig isolates, showing phenotypic characteristics most similar to those of Enterococcus durans and Enterococcus hirae, were investigated in a polyphasic study that included 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base-ratio determinations, whole-cell protein fingerprinting, D11344-primed PCR typing and an extensive examination of phenotypic properties. The results demonstrated that the organisms represent a new species in the Enterococcus faecium species group, for which the name Enterococcus villorum sp. nov. is proposed. The type strain is LMG 12287T (= CCM 4887T).