Gastric helicobacters in domestic animals and nonhuman primates and their significance for human health.

Helicobacters other than Helicobacter pylori have been associated with gastritis, gastric ulcers, and gastric mucosa-associated lymphoid tissue lymphoma in humans. These very fastidious microorganisms with a typical large spiral-shaped morphology were provisionally designated "H. heilmannii," but in fact they comprise at least five different Helicobacter species, all of which are known to colonize the gastric mucosa of animals. H. suis, which has been isolated from the stomachs of pigs, is the most prevalent gastric non-H. pylori Helicobacter species in humans. Other gastric non-H. pylori helicobacters colonizing the human stomach are H. felis, H. salomonis, H. bizzozeronii, and the still-uncultivable "Candidatus Helicobacter heilmannii." These microorganisms are often detected in the stomachs of dogs and cats. "Candidatus Helicobacter bovis" is highly prevalent in the abomasums of cattle but has only occasionally been detected in the stomachs of humans. There are clear indications that gastric non-H. pylori Helicobacter infections in humans originate from animals, and it is likely that transmission to humans occurs through direct contact. Little is known about the virulence factors of these microorganisms. The recent successes with in vitro isolation of non-H. pylori helicobacters from domestic animals open new perspectives for studying these microorganisms and their interactions with the host.

Non-Helicobacter pylori helicobacters detected in the stomach of humans comprise several naturally occurring Helicobacter species in animals.

Besides the well-known gastric pathogen Helicobacter pylori, other Helicobacter species with a spiral morphology have been detected in a minority of human patients who have undergone gastroscopy. The very fastidious nature of these non-Helicobacter pylori helicobacters (NHPH) makes their in vitro isolation difficult. These organisms have been designated 'Helicobacter heilmannii'. However, sequencing of several genes detected in NHPH-infected tissues has shown that the 'H. heilmannii' group comprises at least five different Helicobacter species, all of them known to colonize the stomach of animals. Recent investigations have indicated that Helicobacter suis is the most prevalent NHPH species in human. This species has only recently been isolated in vitro from porcine stomach mucosa. Other NHPH that colonize the human stomach are Helicobacter felis, Helicobacter bizzozeronii, Helicobacter salomonis and 'Candidatus Helicobacter heilmannii'. In numerous case reports of human gastric NHPH infections, no substantial information is available about the species status of the infecting strain, making it difficult to link the species with certain pathologies. This review aims to clarify the complex nomenclature of NHPH species associated with human gastric disease and their possible animal origin. It is proposed to use the term 'gastric NHPH' to designate gastric spirals that are morphologically different from H. pylori when no identification is available at the species level. Species designations should be reserved for those situations in which the species is defined.

Protective immunization with homologous and heterologous antigens against Helicobacter suis challenge in a mouse model.

Helicobacter (H.) suis colonizes the stomach of more than 60% of slaughter pigs and is also of zoonotic importance. Recently, this bacterium was isolated in vitro, enabling the use of pure cultures for research purposes. In this study, mice were immunized intranasally or subcutaneously with whole bacterial cell lysate of H. suis or the closely related species H. bizzozeronii and H. cynogastricus, and subsequently challenged with H. suis. Control groups consisted of non-immunized and non-challenged mice (negative control group), as well as of sham-immunized mice that were inoculated with H. suis (positive control group). Urease tests on stomach tissue samples at 7 weeks after challenge infection were negative in all negative control mice, all intranasally immunized mice except one, and in all and 3 out of 5 animals of the H. cynogastricus and H. suis subcutaneously immunized groups, respectively. H. suis DNA was detected by PCR in the stomach of all positive control animals and all subcutaneously immunized/challenged animals. All negative control animals and some intranasally immunized/challenged mice were PCR-negative. In conclusion, immunization using antigens derived from the same or closely related bacterial species suppressed gastric colonization with H. suis, but complete protection was only achieved in a minority of animals following intranasal immunization.

tDNA-PCR followed by automated fluorescent capillary electrophoresis for identification of bacterial species.

Transfer RNA intergenic spacer length polymorphism analysis (tDNA-PCR) is a simple and reproducible polymerase chain reaction (PCR) technique for identification of bacteria at the species or even subspecies level. The primers used in the PCR are based on conserved sequences located at the edges of the tRNA genes. Because the selected consensus primers are directed outwardly, the intergenic spacers are amplified rather than the genes themselves. With each PCR, several amplicons of different lengths are obtained, because several intergenic spacers are present in each bacterial genome. The patterns thus obtained are identical within species, but differ between distinct species, and as a result, can be used for identification of bacterial species. The amplicons are separated using high-resolution (1 bp) electrophoresis (e.g., fluorescent capillary electrophoresis) and immediately digitized as tables composed of numerical lengths (expressed in base pairs) and peak intensities. For identification, the resulting peak pattern can be compared with a large database of patterns of well-identified bacterial strains, using an in-house-developed software package that is available online. New patterns (linked to the correct species name, which can be obtained, e.g., after 16S rRNA gene sequence determination) can be added to expand the database further. This protocol describes tDNA-PCR, followed by automated fluorescent capillary electrophoresis to identify bacterial species.

Antimicrobial susceptibility pattern of Edwardsiella ictaluri isolates from natural outbreaks of bacillary necrosis of Pangasianodon hypophthalmus in Vietnam.

The purpose of this study was to assess the in vitro susceptibility of 64 Vietnamese isolates of Edwardsiella ictaluri, the causal agent of the infectious disease Bacillus Necrosis Pangasius in Pangasianodon hypophthalmus, using the agar dilution technique. All isolates originated from different farms and were collected between 2002 and 2005. None of the isolates displayed acquired resistance to amoxicillin, amoxicillin-clavulanic acid, chloramphenicol, florfenicol, gentamicin, kanamycin, neomycin, and nitrofurantoin. Acquired resistance to streptomycin was detected in 83%, to oxytetracycline in 81%, and to trimethoprim in 71% of the isolates, as indicated by a bimodal distribution of the minimal inhibitory concentrations (MICs) of these antimicrobials. The MICs of enrofloxacin displayed a monomodal distribution with tailing toward the higher MIC values, possibly indicating reduced susceptibility of a minority of isolates (3 out of the 64). For the quinolone antimicrobial agents flumequin and oxolinic acid, acquired resistance was encountered in 8% and 6% of the strains, respectively. All strains were intrinsically resistant to the polypeptide antimicrobial agent colistin. Seventy-three percent of the isolates were shown to have acquired resistance to at least three antimicrobial agents. The results of this study emphasize the strict need to control both the prophylactic and curative use of antimicrobial agents in Vietnamese aquaculture.

Recovery of Moraxella ovis from the bovine respiratory tract and differentiation of Moraxella species by tDNA-intergenic spacer PCR.

The purpose of the present study was to identify Moraxella (M.)--like organisms recovered from calves suffering from respiratory disease down to species level by means of tDNA-intergenic spacer length polymorphism analysis (tDNA-PCR), and to perform antimicrobial susceptibility testing of these isolates using an agar dilution technique. A total of 16 isolates originating from 12 unrelated occasions were identified as Moraxella ovis, and tDNA fingerprinting showed clear delineation from other Moraxella species. The minimal inhibitory concentrations (in microg/mL) for 90% of the investigated isolates were < or =0.03 for ampicillin; 0.25 for ceftiofur; 0.5 for oxytetracycline; 8 for gentamicin; 64 for spectinomycin; 0.5/9.5 for the combination trimethoprim-sulfonamides; 4 for erythromycin; 8 for tilmicosin; 1 for florfenicol and 0.125 for enrofloxacin.

Helicobacter pullorum in chickens, Belgium.

A total of 110 broilers from 11 flocks were tested for Helicobacter pullorum by polymerase chain reaction; positive samples were reexamined with a conventional isolation method. H. pullorum isolates were examined by amplified fragment length polymorphism (AFLP) fingerprinting for interstrain genetic diversity and relatedness. Sixteen isolates from cecal samples from 2 different flocks were obtained. AFLP analysis showed that these isolates and 4 additional isolates from a different flock clustered according to their origin, which indicates that H. pullorum colonization may occur with a single strain that disseminates throughout the flock. Strains isolated from different hosts or geographic sources displayed a distinctive pattern. H. pullorum is present in approximately one third of live chickens in Belgium and may represent a risk to human health.

Staphylococcus pseudintermedius sp. nov., a coagulase-positive species from animals.

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.

Identification of non-Helicobacter pylori spiral organisms in gastric samples from humans, dogs, and cats.

Tightly coiled bacteria are a rare cause of gastric pathology in humans and represent a mixture of species for which a zoonotic origin is suspected. Similar organisms are common inhabitants of the gastric mucosae of carnivores and pigs. It was the goal of the present study to determine the actual occurrence of each individual Helicobacter species in human, canine, and feline stomachs in order to better understand the possible zoonotic significance. Gastric biopsy samples from humans with histological evidence of non-Helicobacter pylori spiral bacteria (n = 123) and samples from the gastric antrum, corpus, and cardia from dogs (n = 110) and cats (n = 43) were subjected to a multiplex PCR, enabling the identification of Helicobacter felis, Helicobacter bizzozeronii, Helicobacter salomonis, and "Candidatus Helicobacter suis." A PCR for detecting H. pylori was applied to all human samples. Single infections with "Candidatus Helicobacter suis," H. felis, H. bizzozeronii, H. salomonis, a hitherto unknown genotype of a non-H. pylori spiral organism (Helicobacter-like organism 135 [HLO135]), and H. pylori were identified in 30.9%, 8.9%, 2.4%, 11.4%, 7.3%, and 8.9% of the human biopsy samples, respectively. Mixed infections (16.3%) with two or even three of these were also found. In the canine stomach, H. bizzozeronii (70.0%) was encountered as the main spiral organism, while H. felis (62.7%) and HLO135 (67.4%) were the predominant Helicobacter species found in the feline gastric mucosa. Although the majority of human non-H. pylori organisms are Helicobacter species naturally occurring in the stomachs of pigs, cats, and dogs, the frequent identification of H. salomonis in human gastric biopsy samples is in contrast to its rare identification in pet carnivore samples, urging us to suspect other sources of infection.

Presence and mechanism of macrolide-lincosamide resistance in Enterococcus columbae strains belonging to the intestinal flora of pigeons.

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.

tRNA-intergenic spacer PCR for the identification of Pasteurella and Mannheimia spp.

tRNA-intergenic spacer PCR (tDNA-PCR) was evaluated for its effectiveness in differentiating Pasteurella and Mannheimia (sub)species predominantly of ruminant origin. For this purpose, 38 reference strains and 13 field isolates belonging to both genera were investigated. tDNA-PCR enabled discrimination of all Pasteurella species tested (Pasteurella (P.) aerogenes, P. avium, P. canis, P. lymphangitidis, P. multocida, P. trehalosi). For the differentiation of the subspecies of P. multocida, an additional dulcitol reaction was required. Two of the five so far-defined Mannheimia species, M. granulomatis and M. varigena, had a distinct fingerprinting profile. The remaining three phylogenetically highly related species (M. haemolytica, M. glucosida, and M. ruminalis) clustered together. Nevertheless, M. ruminalis is non-haemolytic, and M. haemolytica and M. glucosida can be differentiated on the basis of two additional phenotypic characteristics (beta-glucosidase and aesculin hydrolysis). In conclusion, tDNA-PCR is a useful tool in differentiating organisms belonging to the genera Pasteurella and Mannheimia.

Lactobacillus ingluviei sp. nov., isolated from the intestinal tract of pigeons.

Lactic acid bacteria were isolated from the crop and intestines of pigeons. One group of strains, showing similar genomic patterns after screening with tRNA intergenic spacer PCR, could not be identified to the species level. Sequencing of the 16S rRNA gene of one representative strain revealed about 96% similarity to sequences from Lactobacillus fermentum and Lactobacillus mucosae. Determination of the DNA base composition, DNA-DNA hybridization experiments, SDS-PAGE of whole-cell proteins and biochemical testing confirmed that the seven strains studied constitute a single novel Lactobacillus species, for which the name Lactobacillus ingluviei sp. nov. is proposed. The type strain is strain KR3T (=LMG 20380T =CCUG 45722T).

Emended description of Streptococcus ferus isolated from pigs and rats.

Streptococcus ferus is a mutans-like streptococcus originally isolated from wild rats fed with sugar cane. Taxonomically, this species has not been studied extensively. Ten Gram-positive coccal strains, isolated from the tonsils and nasal conchae of piglets, were identified as S. ferus by 16S rDNA sequencing, tRNA-intergenic spacer length polymorphism analysis (tDNA-PCR), whole-cell protein profiling using SDS-PAGE, G+C content determination and extensive biochemical testing. In all these tests, the type strain of S. ferus (LMG 16520T), from a rat, was included. The results of the tests are described and an emended species description is presented.

Comparison of susceptibility to antimicrobials of the enterococcal species isolated from pigeons (Columba livia).

Antimicrobial resistance profiles were assessed in Enterococcus faecium, Enterococcus faecalis, and Enterococcus columbae strains isolated from racing pigeons (Columba livia). E. columbae is the most prevalent enterococcal species in the intestines of pigeons. Acquired resistance against the antimicrobials bambermycin, vancomycin, monensin, narasin, virginiamycin (cross-resistant with quinupristin/dalfopristin), avilamycin (cross-resistant with evernimicin), and ampicillin was not seen. Only for the antimicrobials tetracycline and tylosin was a frequent occurrence of resistance found in all three enterococcal species tested. Resistance to the fluoroquinolone antimicrobial enrofloxacin was present in E. columbae. Bacitracin resistance was seen in the species E. faecalis and E. faecium but not in E. columbae. High-level streptomycin resistance was found in E. columbae and in one E. faecium strain, whereas high-level gentamicin resistance was mainly associated with E. faecalis. The differences noted between the different species illustrate the difficulties related to the choice of indicator bacteria for antimicrobial resistance in the intestinal flora.

Study of the intra- and interlaboratory reproducibility of partial single base C-sequencing of the 16S rRNA gene and its applicability for the identification of members of the genus Streptococcus.

The use of Single Base C-Sequencing of the first 500 bases of the 16S rRNA-gene (SBCS) combined with capillary electrophoresis was evaluated for the identification of reference strains of 30 different species within the genus Streptococcus. For SBCS, only dd-CTP's are used in the sequencing reactions instead of the four dideoxy bases and the primer is fluorescently labeled. The reproducibility, interlaboratory exchangeability and discriminative power of this method were studied by comparing the patterns obtained in three laboratories under highly standardized conditions. The interlaboratory reproducibility proved to be high, enabling the construction of a common database for the identification of strains belonging to the streptococcal species studied. Most of the examined species generated distinguishable profiles. SBCS did not differentiate between the closely related species S. constellatus and S. intermedius. Also S. thermophilus and S. vestibularis as well as S. mitis and S. pneumoniae showed highly resembling profiles. The previously reported heterogeneity within the species S. equinus was reflected by SBCS. For all other species, strains belonging to the same species generated indistinguishable patterns. In conclusion, Single Base C-sequencing of the first 500 bases of the 16S rRNA-gene could be a useful and widely applicable method for the identification of bacteria at the species level, with the added advantage of being more rapid and easier to automatize than full sequence determination.

Identification of Lactobacillus species using tDNA-PCR.

tDNA intergenic spacer PCR (tDNA-PCR) using consensus primers complementary to the conserved edges of the tRNA genes can amplify the intergenic spacers. Separation of the PCR products with capillary electrophoresis enables discrimination between fragments differing only one basepair in length. This method was applied to a collection of 82 Lactobacillus strains belonging to 37 species in order to evaluate the discriminatory power of this technique within this genus. Twenty-one species could be distinguished to species level on the basis of a unique tDNA fingerprint pattern. The other species grouped by two (e.g. L. fermentum and L. cellobiosus) or three (L. acidophilus, L. gallinarum and L. helveticus). Inclusion of the resulting fingerprints in a numerical database containing fingerprints of numerous other Gram-positive and Gram-negative species makes the identification of unknown strains possible.