Phylogeny of Helicobacter felis sp. nov., Helicobacter mustelae, and related bacteria.

Strain CS1T (T = type strain) is a gram-negative, microaerophilic, urease-positive, spiral-shaped bacterium that was isolated from the gastric mucosa of a cat. Additional strains which possessed biochemical and ultrastructural characteristics similar to those of strain CS1T were isolated from the gastric mucosa of cats and dogs. The guanine-plus-cytosine content of the DNA of strain CS1T was 42.5 mol%. The 16S rRNA sequences of strain CS1T, strain DS3 (a spiral-shaped isolate from a dog), and Helicobacter mustelae were determined by direct RNA sequencing, using a modified Sanger method. These sequences were compared with the 16S rRNA sequences of Helicobacter pylori, "Flexispira rappini," Wolinella succinogenes, and 11 species of campylobacters. A dendrogram was constructed based upon sequence similarities. Strains CS1T and DS3 were very closely related (level of similarity, 99.3%). Two major phylogenetic groups were formed; one group consisted of strains CS1T and DS3, H. mustelae, H. pylori, "F. rappini," and W. succinogenes, and the other group contained the true campylobacters. The average level of similarity between members of these two groups was 84.9%. Within the first group, strains CS1T and DS3, H. pylori, and H. mustelae formed a cluster of organisms with an interspecies similarity level of 94.5%. The phylogenetic positions of W. succinogenes and "F. rappini" were just outside this cluster. On the basis of the results of this study, we believe that strains CS1T (= ATCC 49179T) and DS3 represent a new species of the genus Helicobacter, for which we propose the name Helicobacter felis.     

In Vitro Culture of Previously Uncultured Oral Bacterial Phylotypes

Around a third of oral bacteria cannot be grown using conventional bacteriological culture media. Community profiling targeting 16S rRNA and shotgun metagenomics methods have proved valuable in revealing the complexity of the oral bacterial community. Studies investigating the role of oral bacteria in health and disease require phenotypic characterizations that are possible only with live cultures. The aim of this study was to develop novel culture media and use an in vitro biofilm model to culture previously uncultured oral bacteria. Subgingival plaque samples collected from subjects with periodontitis were cultured on complex mucin-containing agar plates supplemented with proteose peptone (PPA), beef extract (BEA), or Gelysate (GA) as well as on fastidious anaerobe agar plus 5% horse blood (FAA). In vitro biofilms inoculated with the subgingival plaque samples and proteose peptone broth (PPB) as the growth medium were established using the Calgary biofilm device. Specific PCR primers were designed and validated for the previously uncultivated oral taxa Bacteroidetes bacteria HOT 365 and HOT 281, Lachnospiraceae bacteria HOT 100 and HOT 500, and Clostridiales bacterium HOT 093. All agar media were able to support the growth of 10 reference strains of oral bacteria. One previously uncultivated phylotype, Actinomyces sp. HOT 525, was cultivated on FAA. Of 93 previously uncultivated phylotypes found in the inocula, 26 were detected in in vitro-cultivated biofilms. Lachnospiraceae bacterium HOT 500 was successfully cultured from biofilm material harvested from PPA plates in coculture with Parvimonas micra or Veillonella dispar/parvula after colony hybridization-directed enrichment. The establishment of in vitro biofilms from oral inocula enables the cultivation of previously uncultured oral bacteria and provides source material for isolation in coculture.     

Phylogeny of 54 representative strains of species in the family Pasteurellaceae as determined by comparison of 16S rRNA sequences.

Virtually complete 16S rRNA sequences were determined for 54 representative strains of species in the family Pasteurellaceae. Of these strains, 15 were Pasteurella, 16 were Actinobacillus, and 23 were Haemophilus. A phylogenetic tree was constructed based on sequence similarity, using the Neighbor-Joining method. Fifty-three of the strains fell within four large clusters. The first cluster included the type strains of Haemophilus influenzae, H. aegyptius, H. aphrophilus, H. haemolyticus, H. paraphrophilus, H. segnis, and Actinobacillus actinomycetemcomitans. This cluster also contained A. actinomycetemcomitans FDC Y4, ATCC 29522, ATCC 29523, and ATCC 29524 and H. aphrophilus NCTC 7901. The second cluster included the type strains of A. seminis and Pasteurella aerogenes and H. somnus OVCG 43826. The third cluster was composed of the type strains of Pasteurella multocida, P. anatis, P. avium, P. canis, P. dagmatis, P. gallinarum, P. langaa, P. stomatis, P. volantium, H. haemoglobinophilus, H. parasuis, H. paracuniculus, H. paragallinarum, and A. capsulatus. This cluster also contained Pasteurella species A CCUG 18782, Pasteurella species B CCUG 19974, Haemophilus taxon C CAPM 5111, H. parasuis type 5 Nagasaki, P. volantium (H. parainfluenzae) NCTC 4101, and P. trehalosi NCTC 10624. The fourth cluster included the type strains of Actinobacillus lignieresii, A. equuli, A. pleuropneumoniae, A. suis, A. ureae, H. parahaemolyticus, H. parainfluenzae, H. paraphrohaemolyticus, H. ducreyi, and P. haemolytica. This cluster also contained Actinobacillus species strain CCUG 19799 (Bisgaard taxon 11), A. suis ATCC 15557, H. ducreyi ATCC 27722 and HD 35000, Haemophilus minor group strain 202, and H. parainfluenzae ATCC 29242. The type strain of P. pneumotropica branched alone to form a fifth group. The branching of the Pasteurellaceae family tree was quite complex. The four major clusters contained multiple subclusters. The clusters contained both rapidly and slowly evolving strains (indicated by differing numbers of base changes incorporated into the 16S rRNA sequence relative to outgroup organisms). While the results presented a clear picture of the phylogenetic relationships, the complexity of the branching will make division of the family into genera a difficult and somewhat subjective task. We do not suggest any taxonomic changes at this time.     

Cholangiohepatitis and inflammatory bowel disease induced by a novel urease-negative Helicobacter species in A/J and Tac:ICR:HascidfRF mice

Helicobacter bilis and H. hepaticus, both urease-positive intestinal helicobacters of mice, have been shown experimentally to induce proliferative typhlocolitis in scid mice. We recently isolated a urease-negative Helicobacter sp. (H. sp.) that also induced proliferative typhlocolitis in pilot studies in scid mice. To determine the pathogenic potential of H. sp. in immunocompromised and immunocompetent mice, 5-week old male A/J or Tac:Icr:Ha(ICR)-scidfRF mice were inoculated by intraperitoneal (IP) injection with approximately 3 x 10(7) colony-forming units (CFU) of H. sp. Mice were necropsied at various time points postinoculation (PI). Sham-inoculated mice had no clinical, gross, or histopathological lesions. In contrast, scid mice inoculated IP with H. sp. had severe hemorrhagic diarrhea and decreased weight gain at 2, 7, and 18 weeks postinoculation (PI), with severe proliferative typhlocolitis, phlebothrombosis, and hepatitis. A/J mice had no clinical signs, but had mild to moderate proliferative typhlocolitis and moderate to marked cholangiohepatitis at 7 and 24 weeks PI. A/J mice infected with H. sp. developed robust immune responses of a predominant Th1 type. This report demonstrates that infection with a urease-negative helicobacter can cause inflammatory bowel disease (IBD) and hepatitis in scid and immunocompetent A/J mice. These results provide a new model of IBD and cholangio-hepatitis associated with a specific urease-negative, novel H. species.     

Toward a consensus on radiobiology teaching to radiation oncology residents

There are approximately 82 radiation oncology residency programs in the United States, which provide training opportunities for about 400 residents. All accredited radiation oncology residency programs must have at least one basic scientist on the faculty, and it is these individuals who often assume, wholly or in part, the responsibility of teaching radiation and cancer biology to radiation oncology residents in preparation for the American College of Radiology (ACR) In-Training Examination in Radiation Oncology and the American Board of Radiology (ABR) written examinations. In response to a perceived lack of uniformity in radiation and cancer biology curricula currently being taught to residents and a perceived lack of guidance for instructors in formulating course content for this population, a special session was presented at the Forty-eighth Annual Radiation Research Society meeting on April 23, 2001. The session, entitled "Toward a Consensus on Radiobiology Teaching to Radiation Oncology Residents", was focused on issues related to teaching radiobiology to radiation oncology residents and targeted for individuals who actively teach radiation and cancer biology as well as coordinators of residency training programs. The speakers addressed current challenges and future problems facing instructors and programs. Among these were lack of feedback on resident performance on ABR and ACR written examinations and on course content, uncertainty about what topics residents must know to pass the ABR examination, and, in the near future, a reduction (due to retirement) of instructors qualified to teach radiobiology. This article provides a synopsis of the information that was presented during that session, offers a glimpse into how the ABR and ACR examinations are prepared and details of the content of past and future examinations, and summarizes the activities of the Joint Working Group on Radiobiology Teaching which was formed to educate instructors, to establish a consensus for course curricula, and to improve the overall quality of resident teaching.     

Identification of Rothia bacteria as gluten-degrading natural colonizers of the upper gastro-intestinal tract

Background

Gluten proteins, prominent constituents of barley, wheat and rye, cause celiac disease in genetically predisposed subjects. Gluten is notoriously difficult to digest by mammalian proteolytic enzymes and the protease-resistant domains contain multiple immunogenic epitopes. The aim of this study was to identify novel sources of gluten-digesting microbial enzymes from the upper gastro-intestinal tract with the potential to neutralize gluten epitopes.

Methodology/Principal Findings

Oral microorganisms with gluten-degrading capacity were obtained by a selective plating strategy using gluten agar. Microbial speciations were carried out by 16S rDNA gene sequencing. Enzyme activities were assessed using gliadin-derived enzymatic substrates, gliadins in solution, gliadin zymography, and 33-mer α-gliadin and 26-mer γ-gliadin immunogenic peptides. Fragments of the gliadin peptides were separated by RP-HPLC and structurally characterized by mass spectrometry. Strains with high activity towards gluten were typed as Rothia mucilaginosa and Rothia aeria. Gliadins (250 µg/ml) added to Rothia cell suspensions (OD₆₂₀ 1.2) were degraded by 50% after ∼30 min of incubation. Importantly, the 33-mer and 26-mer immunogenic peptides were also cleaved, primarily C-terminal to Xaa-Pro-Gln (XPQ) and Xaa-Pro-Tyr (XPY). The major gliadin-degrading enzymes produced by the Rothia strains were ∼70–75 kDa in size, and the enzyme expressed by Rothia aeria was active over a wide pH range (pH 3–10).

Conclusion/Significance

While the human digestive enzyme system lacks the capacity to cleave immunogenic gluten, such activities are naturally present in the oral microbial enzyme repertoire. The identified bacteria may be exploited for physiologic degradation of harmful gluten peptides.     

The effect of darbepoetin alfa on growth, oxygenation and radioresponsiveness of a breast adenocarcinoma

Tumor hypoxia is associated with poor clinical outcome in a variety of tumors, including cervical, head/neck and breast cancer. Administration of erythropoietic factors has been suggested as a means of improving tumor oxygenation (pO2). This study randomized rats to treatment with low-dose or high-dose darbepoetin alfa or a placebo to determine the effect of darbepoetin alfa on the pO2, growth and response to radiation therapy of R3230 mammary adenocarcinoma. Rats received 3 microg/kg (high dose) or 0.2 microg/kg (low dose) darbepoetin alfa or placebo for eight doses prior to either (1) pO2 measurement and pimonidazole staining or (2) irradiation or sham irradiation on post-transplant day 20. In the animals randomized to radiation treatment, placebo or darbepoetin alfa administration at a reduced dose was continued for 9 weeks or until the tumor diameter exceeded 15 mm (defined as failure for survival analysis). Treatment with high-dose and low-dose darbepoetin alfa produced hematocrits of 68 and 56% compared to 44 and 45% in their respective control groups (both P < 10(-5)). At 18 days post-transplant, tumor volume was not different for either darbepoetin alfa group compared to the placebo group. Tumor oxygenation, as measured by the fraction of pO2 measurement <10 mmHg and the intensity of pimonidazole staining, was significantly improved in the high-dose group (P = 0.046 and 0.03, respectively, compared with controls) but not in the low-dose group. Growth delay curves after irradiation did not differ significantly for high- or low-dose darbepoetin alfa compared to placebo. In this nonanemic animal model of mammary adenocarcinoma, darbepoetin alfa does not significantly alter tumor growth or radioresponsiveness, even though it improves oxygenation when administered at high doses.     

Helicobacter anseris sp. nov. and Helicobacter brantae sp. nov., isolated from feces of resident Canada geese in the greater Boston area

Numbers of nonmigratory Canada geese have increased substantially in the past decade, and they have become a nuisance in some urban areas. Because of their close contact with humans in parks and areas adjacent to surface waterways, contact with their feces poses a zoonotic risk. A total of 97 geese from 10 separate geographic locales in the greater Boston area had their feces sampled for detection of Helicobacter spp. Identification of Helicobacter spp. based on 16S rRNA genus-specific helicobacter primers was noted in 39 of 97 (40.2%) DNA fecal extracts. Twenty-seven (27.8%) of these geese had helicobacters isolated from their feces. A urease-positive novel species, Helicobacter anseris, based on phenotypic, biochemical, and 16S rRNA analyses, was isolated from 20 geese from seven different flocks. A second, novel, urease-negative Helicobacter sp., H. brantae, was identified in seven geese. Four geese had both novel Helicobacter spp. cultured from their feces. Whether these two novel helicobacters pose a zoonotic risk, similar to other enteric helicobacters (e.g., H. canadensis, previously isolated from diarrheic and bacteremic humans and from geese in Europe), will require further studies.