Colonization Dynamics of Altered Schaedler Flora Is Influenced by Gender, Aging, and Helicobacter hepaticus Infection in the Intestines of Swiss Webster Mice

The distribution and colonization levels of the altered Schaedler flora (ASF) in their natural hosts are poorly understood. Intestinal colonization levels of the eight ASF strains in outbred Swiss Webster mice with or without Helicobacter hepaticus infection were characterized by real-time quantitative PCR. All ASF strains were detected in the cecum and colon, but some strains displayed significant variation in colonization levels with host age, gender, and H. hepaticus infection status.     

Long-term colonization levels of Helicobacter hepaticus in the cecum of hepatitis-prone A/JCr mice are significantly lower than those in hepatitis-resistant C57BL/6 mice

Helicobacter hepaticus infection causes hepatitis in A/JCr mice but mild or no disease in C57BL/6 mice. Colonization of H. hepaticus in the cecum of experimentally infected A/JCr and C57BL/6 mice was quantified by use of real-time polymerase chain reaction (PCR) analysis with primers for the H. hepaticus cdtB gene and mouse 18srRNA. Eight-week-old mice were experimentally (n = 48) or sham (n = 24) infected with H. hepaticus, then were necropsied 6 months after infection. Liver specimens from experimentally infected mice had negative results of PCR analysis for H. hepaticus; thus, real-time quantification was not attempted. Quantitative PCR analysis of H. hepaticus in cecal specimens indicated that C57BL/6 mice were colonized to a greater extent than were A/JCr mice (P < 0.006). Appreciable typhlitis was not observed, but was consistent with that of previous reports; A/JCr mice developed more severe parenchymal necrosis, portal inflammation, and phlebitis in the liver (P < 0.0001), with mild disease observed in infected C57BL/6 mice. Thus, hepatitis in A/JCr mice caused by H. hepaticus infection is associated with significantly lower colonization levels of H. hepaticus in the cecum, compared with those of hepatitis-resistant C57BL/6 mice. Host responses of A/JCr mice that limit cecal colonization with H. hepaticus may have important roles in the pathogenesis of hepatic lesions.     

Evaluation of Helicobacter hepaticus bacterial shedding in fostered and sex-segregated C57BL/6 mice

Neonatal fostering has been evaluated as a means of eliminating Helicobacter hepaticus infection in laboratory mouse colonies. The purpose of the present study was to evaluate cross-fostering of neonatal C57BL/6 pups from experimentally infected dams after male-absent parturition and to determine the effects of sex and housing strategy on H. hepaticus populations. Approximately 20 C57BL/6 mice (age, 1 to 4 days) were fostered daily. In all fostered mice, fecal samples collected at 21 and 42 days of age and cecal samples collected at 42 days of age tested negative for H. hepaticus by polymerase chain reaction analysis. Our results demonstrate that removal of the male prior to parturition extends the fostering period to yield Helicobacter-free mice. In a second experiment, the effects of time of infection, housing strategy, and sex on fecal H. hepaticus shedding and cecal colonization were evaluated. Neither time nor housing strategy affected bacterial shedding. In contrast, fecal and cecal bacterial loads were higher in male mice versus female mice. A novel predictive algorithm was developed to predict cecal bacterial colonization levels in light of fecal bacterial loads. Our findings likely will prove useful in Helicobacter eradication efforts and in studies designed to further elucidate the role of H. hepaticus in disease.     

Spatial and temporal colonization dynamics of segmented filamentous bacteria is influenced by gender,age and experimental infection with Helicobacter hepaticus in Swiss Webster mice

In this study, we examined colonization dynamics of segmented filamentous bacteria (SFB) in intestine of Swiss Webster (SW) mice infected with Helicobacter hepaticus (Hh). At 8 weeks post-inoculation with Hh (WPI), cecal and colonic SFB levels in the control males were significantly lower compared to those at 16 WPI. Hh infection in both genders did not alter SFB levels in the jejunum and ileum, but increased SFB levels in the cecum and colon of males compared to the controls (P < 0.05) at 8 WPI. At 16 WPI, the Hh-infected females contained lower levels of SFB in the jejunum, cecum and colon compared to the female controls. Irrespective of gender, aging and Hh infection, the Il-17A mRNA levels decreased from the small intestine to the cecum and then to the colon, whereas the Foxp3 mRNA levels were comparable in these intestinal regions. There were significant differences in Il-17A mRNA levels in the ileum (P < 0.05, R² = 0.31), with females having greater Il-17A mRNA levels than males, and higher SFB colonization levels related to more Il-17A mRNA. These results indicate that aging and gender play an important role in colonization dynamics of intestinal SFB and ileal SFB-associated Th17 response.     

Spatial distribution and stability of the eight microbial species of the altered schaedler flora in the mouse gastrointestinal tract

The overall complexity of the microbial communities in the gastrointestinal (GI) tracts of mammals has hindered observations of dynamics and interactions of individual bacterial populations. However, such information is crucial for understanding the diverse disease-causing and protective roles that gut microbiota play in their hosts. Here, we determine the spatial distribution, interanimal variation, and persistence of bacteria in the most complex defined-flora (gnotobiotic) model system to date, viz., mice colonized with the eight strains of the altered Schaedler flora (ASF). Quantitative PCR protocols based on the 16S rRNA sequence of each ASF strain were developed and optimized to specifically detect as few as 10 copies of each target. Total numbers of the ASF strains were determined in the different regions of the GI tracts of three C.B-17 SCID mice. Individual strain abundance was dependent on oxygen sensitivity, with microaerotolerant Lactobacillus murinus ASF361 present at 10(5) to 10(7) cells/g of tissue in the upper GI tract and obligate anaerobic ASF strains being predominant in the cecal and colonic flora at 10(8) to 10(10) cells/g of tissue. The variation between the three mice was small for most ASF strains, except for Clostridium sp. strain ASF502 and Bacteroides sp. strain ASF519 in the cecum. A comparison of the relative distribution of the ASF strains in feces and the colon indicated large differences, suggesting that fecal bacterial levels may provide a poor approximation of colonic bacterial levels. All ASF strains were detected by PCR in the feces of C57BL/6 restricted flora mice, which had been maintained in an isolator without sterile food, water, or bedding for several generations, providing evidence for the stability of these strains in the face of potential competition by bacteria introduced into the gut.     

Spatial Distribution and Stability of the Eight Microbial Species of the Altered Schaedler Flora in the Mouse Gastrointestinal Tract

The overall complexity of the microbial communities in the gastrointestinal (GI) tracts of mammals has hindered observations of dynamics and interactions of individual bacterial populations. However, such information is crucial for understanding the diverse disease-causing and protective roles that gut microbiota play in their hosts. Here, we determine the spatial distribution, interanimal variation, and persistence of bacteria in the most complex defined-flora (gnotobiotic) model system to date, viz., mice colonized with the eight strains of the altered Schaedler flora (ASF). Quantitative PCR protocols based on the 16S rRNA sequence of each ASF strain were developed and optimized to specifically detect as few as 10 copies of each target. Total numbers of the ASF strains were determined in the different regions of the GI tracts of three C.B-17 SCID mice. Individual strain abundance was dependent on oxygen sensitivity, with microaerotolerant Lactobacillus murinus ASF361 present at 10⁵ to 10⁷ cells/g of tissue in the upper GI tract and obligate anaerobic ASF strains being predominant in the cecal and colonic flora at 10⁸ to 10¹⁰ cells/g of tissue. The variation between the three mice was small for most ASF strains, except for Clostridium sp. strain ASF502 and Bacteroides sp. strain ASF519 in the cecum. A comparison of the relative distribution of the ASF strains in feces and the colon indicated large differences, suggesting that fecal bacterial levels may provide a poor approximation of colonic bacterial levels. All ASF strains were detected by PCR in the feces of C57BL/6 restricted flora mice, which had been maintained in an isolator without sterile food, water, or bedding for several generations, providing evidence for the stability of these strains in the face of potential competition by bacteria introduced into the gut.     

Helicobacter hepaticus HHGI1 is a pathogenicity island associated with typhlocolitis in B6.129-IL10 tm1Cgn mice

Helicobacter hepaticus strain 3B1 (H. hepaticus) contains a genomic island of approximately 71 kb, HHGI1, with some of the common features shared among known bacterial pathogenicity islands. In this study, we characterized the pathogenic potential of HHGI1 by infecting B6.129-IL10 tm1Cgn (IL10-/-) mice with an isogenic mutant (namely HhPAId1) lacking 19 predicted genes within HHGI1. In contrast to H. hepaticus (P<0.001), HhPAId1 did not cause typhlocolitis and hyperplasia in IL10-/- mice. Colonization levels of HhPAId1 were significantly higher in the cecum (P<0.007) and similar in the colon (P=0.27) when compared to H. hepaticus by 13 or 16 weeks post inoculation (WPI). The magnitude of the Th1-associated IgG2c response against HhPAId1 was less than that against H. hepaticus (P<0.004). There was no significant difference in Th2-associated IgG1 responses against these two strains. Cecal and colonic mRNA levels of proinflammatory cytokines IFN-gamma, TNF-alpha and IL-17a in the HhPAId1-infected mice were significantly lower than those in the H. hepaticus-infected mice (P<0.05) at 13 WPI. These results demonstrate that genes in the HHGI1 contribute to the pathogenicity of H. hepaticus, at least in part via up-regulation of proinflammatory mediators IFN-gamma, TNF-alpha and IL-17a.     

Pathogenesis of mouse hepatitis virus infection in gamma interferon-deficient mice is modulated by co-infection with Helicobacter hepaticus

Gamma interferon-deficient (IFN-gamma KO) mice developed a wasting syndrome and were found to be co-infected with Helicobacter sp., and a new isolate of mouse hepatitis virus (MHV) designated MHV-G. The disease was characterized by pleuritis, peritonitis, hepatitis, pneumonia, and meningitis. Initial experiments used a cecal homogenate inoculum from the clinical cases that contained H. hepaticus and MHV-G to reproduce the development of peritonitis and pleuritis in IFN-gamma KO mice. In contrast, immunocompetent mice given the same inoculum developed an acute, self-limiting infection and remained clinically normal. This result confirmed the importance of IFN-gamma in preventing chronic infection and limiting viral dissemination. To understand the role of both agents in the development of peritonitis and pleuritis, IFN-gamma KO mice were infected with either agent or were co-infected with H. hepaticus and MHV-G. Infection with MHV-G induced a multisystemic infection similar to that described in the original cases, with multifocal hepatic necrosis, acute necrotizing and inflammatory lesions of the gastrointestinal tract, and acute peritonitis and pleuritis with adhesions on the serosal surfaces of the viscera. However, mice given H. hepaticus alone had minimal pathologic changes even though the organism was consistently detected in the cecum or feces. Although co-infection with H. hepaticus and MHV-G induced lesions similar to those associated with MHV-G alone, the pathogenesis of the MHV infection was modified. Helicobacter hepaticus appeared to reduce the severity of MHV-induced lesions during the acute phase of infection, and exacerbated hepatitis and meningitis at the later time point. We conclude that infection of IFN-gamma KO mice with MHV-G results in multisystemic infection with peritonitis, pleuritis, and adhesions due to the aberrant immune response in these mice. In addition, co-infection of these mice with H. hepaticus results in alterations in the pathogenesis of MHV-G infection.     

In vitro and in vivo characterization of alkyl hydroperoxide reductase mutant strains of Helicobacter hepaticus

Mutant strains in the tsaA gene encoding alkyl hydroperoxide reductase were more sensitive to O(2) and to oxidizing agents (paraquat, cumene hydroperoxide and t-butylhydroperoxide) than the wild type, but were markedly more resistant to hydrogen peroxide. The mutant strains resistance phenotype could be attributed to a 4-fold and 3-fold increase in the catalase protein amount and activity, respectively compared to the parent strain. The wild type did not show an increase in catalase expression in response to sequential increases in O(2) exposure or to oxidative stress reagents, so an adaptive compensatory mutation has probably occurred in the mutants. In support of this, chromosomal complementation of tsaA mutants restored alkyl hydroperoxide reductase, but catalase was still up-expressed in all complemented strains. The katA promoter sequence was the same in all mutant strains and the wild type. Like its Helicobacter pylori counterpart strain, a H. hepaticus tsaA mutant contained more lipid hydroperoxides than the wild type strain. Hepatic tissue from mice inoculated with a tsaA mutant had lesions similar to those inoculated with the wild type, and included coagulative necrosis of hepatocytes. The liver and cecum colonizing abilities of the wild type and tsaA mutant were comparable. Up-expression of catalase in the tsaA mutants likely permits the bacterium to compensate (in colonization and virulence attributes) for the loss of an otherwise important oxidative stress-combating enzyme, alkyl hydroperoxide reductase. The use of erythromycin resistance insertion as a facile way to screen for gene-targeted mutants, and the chromosomal complementation of those mutants are new genetic procedures for studying H. hepaticus.     

Review of successful treatment for Helicobacter species in laboratory mice

Three variations of the amoxycillin-based triple therapy (amoxycillin, metronidazole and bismuth) were administered in the diet, by oral gavage or in the diet in conjunction with cross-fostering on to Helicobacter-free foster mothers to mice naturally infected with H. hepaticus and/or H. bilis. The presence of Helicobacter species was determined by polymerase chain reaction (PCR) analysis of faecal pellets. Helicobacter infection was eliminated in 50% of strains of mice treated by oral gavage; 57% of strains of mice treated by medicated diet alone and 100% of strains of mice treated with the medicated diet in conjunction with cross-fostering on to Helicobacter-free foster mothers. Eight strains of mice were successfully treated for Helicobacter infection over a two-year period. The mouse colony has been maintained Helicobacter free, as determined by PCR analysis and has remained off treatment from December 2002 to March 2005.     

Optimal age at fostering for derivation of Helicobacter hepaticus-free mice

Helicobacter hepaticus is well established as an unwanted variable in laboratory rodent colonies. Historically, cesarean section and embryo transfer have been used to derive Helicobacter-free mouse colonies. Neonatal transfer of newborn mice onto Helicobacter-free foster dams was recently reported as an alternative method of deriving Helicobacter-free mice, but until now, the age by which pups must be fostered to remain Helicobacter-free was unknown. The purpose of the study reported here was to determine the age by which mouse pups must be fostered to remain free of H. hepaticus. Beginning on the day of birth, 20 C57BL/6 mice were fostered from H. hepaticus-positive parents onto Helicobacter-free BALB/c dams each day for 14 days for a total of 280 pups fostered. Fecal specimens collected at weaning, and fecal, liver, and cecal specimens collected at euthanasia were analyzed by use of polymerase chain reaction (PCR) analysis. No pup fostered within 24 h of birth became infected with H. hepaticus; however, many of those fostered after 24 h became infected. These results were supported by those of a large field trial, in which 201 litters representing 71 strains of mice were fostered within 24 h of birth. Follow-up fecal PCR analysis was performed on 52 mice or their progeny that were randomly sampled from the 201 fostered litters. All mice tested remained free of H. hepaticus approximately 100 days after fostering. The results indicate that mouse pups must be fostered within 24 h of birth to remain free of H. hepaticus. In addition, cecal and fecal PCR analyses detected more infections, than did liver PCR analysis, thus indicating that those specimens are preferred for detection of H. hepaticus infection.     

Phylogeny of the defined murine microbiota: altered Schaedler flora.

The "altered Schaedler flora" (ASF) was developed for colonizing germfree rodents with a standardized microbiota. The purpose of this study was to identify each of the eight ASF strains by 16S rRNA sequence analysis. Three strains were previously identified as Lactobacillus acidophilus (strain ASF 360), Lactobacillus salivarius (strain ASF 361), and Bacteroides distasonis (strain ASF 519) based on phenotypic criteria. 16S rRNA analysis indicated that each of the strains differed from its presumptive identity. The 16S rRNA sequence of strain ASF 361 is essentially identical to the 16S rRNA sequences of the type strains of Lactobacillus murinis and Lactobacillus animalis (both isolated from mice), and all of these strains probably belong to a single species. Strain ASF 360 is a novel lactobacillus that clusters with L. acidophilus and Lactobacillus lactis. Strain ASF 519 falls into an unnamed genus containing [Bacteroides] distasonis, [Bacteroides] merdae, [Bacteroides] forsythus, and CDC group DF-3. This unnamed genus is in the Cytophaga-Flavobacterium-Bacteroides phylum and is most closely related to the genus Porphyromonas. The spiral-shaped strain, strain ASF 457, is in the Flexistipes phylum and exhibits sequence identity with rodent isolates of Robertson. The remaining four ASF strains, which are extremely oxygen-sensitive fusiform bacteria, group phylogenetically with the low-G+C-content gram-positive bacteria (Firmicutes, Bacillus-Clostridium group). ASF 356, ASF 492, and ASF 502 fall into Clostridium cluster XIV of Collins et al. Morphologically, ASF 492 resembles members of this cluster, Roseburia cecicola, and Eubacterium plexicaudatum. The 16S rRNA sequence of ASF 492 is identical to that of E. plexicaudatum. Since the type strain and other viable original isolates of E. plexicaudatum have been lost, strain ASF 492 is a candidate for a neotype strain. Strain ASF 500 branches deeply in the low-G+C-content gram-positive phylogenetic tree but is not closely related to any organisms whose 16S rRNA sequences are currently in the GenBank database. The 16S rRNA sequence information determined in the present study should allow rapid identification of ASF strains and should permit detailed analysis of the interactions of ASF organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms.     

Defective activation of ERK in macrophages lacking the p50/p105 subunit of NF-kappaB is responsible for elevated expression of IL-12 p40 observed after challenge with Helicobacter hepaticus

Helicobacter hepaticus is an enterohepatic Helicobacter species that induces lower bowel inflammation in susceptible mouse strains, including those lacking the p50/p105 subunit of NF-kappaB. H. hepaticus-induced colitis is associated with elevated levels of IL-12 p40 expression, and p50/p105-deficient macrophages express higher levels of IL-12 p40 than wild-type macrophages after challenge with H. hepaticus. However, the molecular mechanisms by which the p50/p105 subunit of NF-kappaB suppresses IL-12 p40 expression have not yet been elucidated. In this study we have demonstrated that H. hepaticus challenge of macrophages induces ERK activation, and this event plays a critical role in inhibiting the ability of H. hepaticus to induce IL-12 p40. Activation of ERK requires both p50/p105 and the MAPK kinase kinase, Tpl-2. Inhibition of the induction of IL-12 p40 by ERK was independent of c-Rel, a known positive regulator of IL-12 p40. Instead, it was linked to the induction of c-Fos, a known inhibitor of IL-12 p40 expression. These results suggest that H. hepaticus induces ERK activation by a pathway dependent upon Tpl-2 and p105, and that activation of ERK inhibits the expression of IL-12 p40 by inducing c-Fos. Thus, a defect in ERK activation could play a pivotal role in the superinduction of IL-12 p40 observed after challenge of macrophages lacking the p50/p105 subunit of NF-kappaB with H. hepaticus.     

Natural colonization with Helicobacter species and the development of inflammatory bowel disease in interleukin-10-deficient mice

BACKGROUND: The interleukin-10-deficient (IL-10-/-) mice maintained in specific-pathogen-free (SPF) conditions develop typhlocolitis when experimentally infected with Helicobacter species. However, there is limited information regarding the role of Helicobacter species that naturally colonize IL-10-/- mice in typhlocolitis development. The aim of this study was to examine in SPF IL-10-/- mice the association between natural colonization specific Helicobacter species and typhlocolitis development. MATERIAL AND METHODS: Cecum and proximal colon from 72 C57BL/6 x 129/Ola IL-10-/- mice (8-20 weeks old) were removed for DNA extraction and histologic evaluation. Genus-specific polymerase chain reaction- denaturing gradient gel electrophoresis (PCR-DGGE) and species-specific PCR were used to detect Helicobacter species. Mice were grouped by age, sex, and Helicobacter colonization status, and their histologic scores were compared. The development of clinical typhlocolitis was observed in a further 12 mice. RESULTS: Species-specific PCR showed that mice were colonized with Helicobacter ganmani and/or Helicobacter hepaticus. The PCR-DGGE detected H. ganmani, H. hepaticus and an H. ganmani-like organism. The histologic scores in mice colonized with H. hepaticus were significantly higher than that in mice colonized with H. ganmani. Male mice showed significantly higher histologic scores than female mice. Four of the 12 mice developed clinical typhlocolitis in 38 weeks. CONCLUSION: Natural colonization with different Helicobacter species was found in IL-10-/- mice within the same breeding colony. The severity of typhlocolitis differed according to the colonizing Helicobacter species. Furthermore, the rate of typhlocolitis development in IL-10-/- mice naturally colonized with Helicobacter species was significantly slower than that reported in experimentally infected mice.     

Detection of Helicobacter hepaticus in Human Bile Samples of Patients with Biliary Disease

Background:  Since the discovery of Helicobacter pylori , various enterohepatic Helicobacter spices have been detected in the guts of humans and animals. Some enterohepatic Helicobacters have been associated with inflammatory bowel disease or liver disease in mice. However the association of these bacteria with human diseases remains unknown.
Materials and Methods:  We collected 126 bile samples from patients with cholelithiasis, cholecystitis, gallbladder polyp, and other nonbiliary diseases. Samples were screened for the presence of enterohepatic Helicobacter spp. using cultures, nested PCR, or in situ hybridization. We tested for antibodies to H. pylori and H. hepaticus by Western blot analysis.
Results:  Attempts at cultivation were unsuccessful. However, H. hepaticus was detected in bile samples with nested PCR whereas H. bilis was not. Helicobacter hepaticus in the bile was confirmed by in situ hybridization, but H. hepaticus from bile samples was coccoid in appearance. We detected immunoglobulin G antibodies to H. hepaticus in bile samples by Western blotting. Helicobacter hepaticus was detected in 40 (32%) of total 126 samples as H. hepaticus positive if at least one of the three methods with nested PCR, in situ, or Western blotting. Patients with cholelithiasis (41%) and cholecystitis with gastric cancer (36%) had significantly higher ( p  = .029) prevalence of H. hepaticus infection than samples from patients with other diseases.
Conclusion:  Helicobacter hepaticus may closely associate with diseases of the liver and biliary tract in humans.     

Experimental Infection of Ornithodoros erraticus sensu stricto with Two Portuguese African Swine Fever Virus Strains. Study of Factors Involved in the Dynamics of Infection in Ticks

African swine fever (ASF) is a frequently devastating hemorrhagic disease of domestic pigs and wild boar and Ornithodoros erraticus sensu stricto argasid ticks are the only biological vectors of African swine fever virus (ASFV) known to occur in Europe. Recently this disease emerged in Eastern Europe and Russian Federation, showing a huge potential for a rapid spread between countries. There is some risk of re-emergence of ASF in the countries where these ticks exist, that can contribute for the persistence of infection and compromise control measures. In this study we aimed to identify factors that determine the probability of infection and its dynamics in the tick vector Ornithodoros erraticus sensu stricto, with two Portuguese strains of ASFV. Our results suggest that these ticks have a high likelihood of excreting the two haemadsorbing ASF viruses of different host origins and that, in field surveys, the analysis of adults and 5^th nymphal stage can provide the best chance of detecting virus infection. The results also indicate that infection of pigs with highly virulent ASF viruses will promote higher rates of infection and a higher likelihood for virus excretion by ticks. Nevertheless, there is also a risk, although lower, that ticks can become infected on pigs that have overcome the acute phase of infection, which was simulated in our study by membrane feeding ticks with low titres of virus. We believe these results can be valuable in designing and interpreting the results of ASF control programmes, and future work can also be undertaken as our dataset is released under open access, to perform studies in risk assessment for ASFV persistence in a region where O. erraticus sensu stricto ticks are present.     

Phylogeny of the Defined Murine Microbiota: Altered Schaedler Flora

The “altered Schaedler flora” (ASF) was developed for colonizing germfree rodents with a standardized microbiota. The purpose of this study was to identify each of the eight ASF strains by 16S rRNA sequence analysis. Three strains were previously identified as Lactobacillus acidophilus (strain ASF 360), Lactobacillus salivarius (strain ASF 361), and Bacteroides distasonis (strain ASF 519) based on phenotypic criteria. 16S rRNA analysis indicated that each of the strains differed from its presumptive identity. The 16S rRNA sequence of strain ASF 361 is essentially identical to the 16S rRNA sequences of the type strains of Lactobacillus murinis and Lactobacillus animalis (both isolated from mice), and all of these strains probably belong to a single species. Strain ASF 360 is a novel lactobacillus that clusters with L. acidophilus and Lactobacillus lactis. Strain ASF 519 falls into an unnamed genus containing [Bacteroides] distasonis, [Bacteroides] merdae, [Bacteroides] forsythus, and CDC group DF-3. This unnamed genus is in the Cytophaga-Flavobacterium-Bacteroides phylum and is most closely related to the genus Porphyromonas. The spiral-shaped strain, strain ASF 457, is in the Flexistipes phylum and exhibits sequence identity with rodent isolates of Robertson. The remaining four ASF strains, which are extremely oxygen-sensitive fusiform bacteria, group phylogenetically with the low-G+C-content gram-positive bacteria (Firmicutes, Bacillus-Clostridium group). ASF 356, ASF 492, and ASF 502 fall into Clostridium cluster XIV of Collins et al. Morphologically, ASF 492 resembles members of this cluster, Roseburia cecicola, and Eubacterium plexicaudatum. The 16S rRNA sequence of ASF 492 is identical to that of E. plexicaudatum. Since the type strain and other viable original isolates of E. plexicaudatum have been lost, strain ASF 492 is a candidate for a neotype strain. Strain ASF 500 branches deeply in the low-G+C-content gram-positive phylogenetic tree but is not closely related to any organisms whose 16S rRNA sequences are currently in the GenBank database. The 16S rRNA sequence information determined in the present study should allow rapid identification of ASF strains and should permit detailed analysis of the interactions of ASF organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms.     

Genomic characterization of Helicobacter hepaticus: ordered cosmid library and comparative sequence analysis

Helicobacter hepaticus is an important pathogen in laboratory mice and induces the development of liver tumors and gastrointestinal disease in susceptible strains of mice. In this study, a miniset of 36 cosmid clones from a genomic library of H. hepaticus was ordered and grouped into four large contigs representing approximately 1 Mb of the H. hepaticus genome using PCR, DNA sequencing, Southern and dot-blot hybridization and pulsed-field gel electrophoresis. From the 200-300 terminal nucleotide sequences of 38 cosmid clones, 56 coding regions were predicted, of which 51 were found to have orthologs in the public databases and five appeared to be unique to H. hepaticus. Of these 51 genes, 36 have orthologs in Helicobacter pylori and 25 display the highest sequence similarity to H. pylori. However, chromosomal positions of these genes are not conserved between these two helicobacters. In addition, 10 H. hepaticus genes had the highest sequence similarity to orthologs in Campylobacter jejuni. The GC content in a randomly selected 21-kb H. hepaticus genomic sequence was 35.8%, which approximates the average between H. pylori (39%) and C. jejuni (30.6%). These results demonstrate that: (1) H. hepaticus is more closely related to H. pylori than C. jejuni; (2) significant genomic alterations exist between H. hepaticus and H. pylori, including gene organization, protein sequences and GC content, probably in part due to specific adaptation to distinct ecological niches.