An experimental rodent model was used to demonstrate the viability of the coccoid form of Helicobacter pylori. Concentrated suspensions were prepared for the two different morphologies: at 2 days incubation for the bacillary forms and at 20 days incubation for the "dormant" forms. The strains used for incubation were two fresh isolates from humans with duodenal ulceration, and two collection strains. Five hundred microliters of culture (OD550= 5 Mc Farland) of Helicobacter pylori with bacillary (2-5 x 109 CFU/ml) and coccoid (O CFU/ml) morphology were inoculated intragastrically in BALB/c mice. The gastric mucosa of the mice was colonized by Helicobacter pylori with the administration of fresh bacillary and coccoid cultures and not with the established cultures. Helicobacter pylori was isolated at 1 week after inoculation with the administration of fresh bacillary cultures, while fresh coccoid Helicobacter pylori was recovered in mice stomachs after 2 weeks of inoculation. After colonization, histopathologic changes occurred after 1 month from inoculation; all colonized mice showed a systemic antibody response to Helicobacter pylori. These results support the thesis of the viability of coccoid Helicobacter pylori non-culturable in vitro and confirm that concentrated bacterial suspensions are able to colonize and to produce gastric alterations in this suitable animal model.

The morphologic changes from bacillary to coccoid forms of Helicobacter pylori were studied. These form changes were analyzed by bacterial growth in Brucella broth plus 2% fetal calf serum. The coccoid forms were observed at five days of incubation and a rapid decrease of CFU/ml was recorded. At two weeks of microaerophilic incubation, all coccoid forms observed were not culturable in vitro. The coccoid morphology was observed earlier when the culture of H. pylori was incubated in aerobic conditions and with subinhibitory concentrations of omeprazole and roxithromycin. To evaluate the possibility of resistance of coccal forms, before plating, the cultures were heated to 80 C for 10 min and sonicated. In the absence of these treatments the cultures did not show growth in vitro. The proteic patterns of the same strains of two different morphologies were studied revealing significant differences.

of Helicobacter pylori ATCC43504 from a viable but not culturable state: regrowth or resuscitation? APMIS 106: 571-579, 1998. Studies were conducted following the formation and characterization of the coccoid morphology of Helicobacter pylori. H. pylori ATCC43504 was incubated in brucella broth plus 2% fetal calf serum at three different temperatures: 37"C, room temperature and 4°C in a microaerophilic environment, and readings were taken at 2, 7, 15, 30 and 45 days. At control times, the total and the viable count, viability tests with tetrazolium salts, and ultrastructural studies were carried out. On solid media, H . pylori became nonculturable after 7 days of incubation at room temperature and 4"C, and after 15 days of incubation at 37°C. At these times of incubation, after subculturing in liquid medium under the same conditions, the growth of H. pylori was detected until the 15th day from cultures incubated at 4°C and until the 30th day from cultures stored at 37"C, and at room temperature. Ultrastructural studies showed a gradual reduction of integrity of bacterial cells that remained stable at 30 and 45 days of incubation: 30% of whole cells of bacteria incubated at 37°C and room temperature and 50% in bacteria incubated at 4°C. The viability of the VNC (viable nonculturable) state was assessed by studying the reduction of tetrazolium salts INT (p-iodonitrophenyl tetrazolium violet) and CTC (cyanoditolyl tetrazolium chloride) to their respective formazans and this was linked to the cellular respiration. At 45 days of incubation, when bacterial regrowth was not observed in solid or in liquid medium, different resuscitation methods were applied to evaluate a possible resuscitation of VNC H . pylori. No significant growth on solid medium was observed.

Four yellow-pigmented group D enterococci of uncertain taxonomic position were isolated from several humans with severe infections. The results of DNA composition, DNA-DNA hybridization, fatty acid content, and biochemical property studies demonstrated that these organisms were slightly related to other previously described yellow-pigmented enterococcal species and constitute a new species, for which we propose the name Enterococcusjlavescens. The type strain of E. jlavescens is strain CCM 439.Until recently, the taxonomy of the group D streptococci was uncertain and not well defined. In 1984 Schleifer and Kilpper-Balz proposed that the genus Enterococcus should include the species Enterococcus faecalis and Enterococcus faecium (17). Since then, other species have been transferred to this genus, and new species have also been described. The results of biochemical tests, DNA base composition determinations, DNA-DNA hybridization experiments, penicillin-binding protein determinations, and long-chain fatty acid content determinations have been used to describe as many as 17 different enterococcal species Three yellow pigment-producing species belonging to the enterococcus group have been described previously; these are Enterococcus casseliflavus, Enterococcus mundtii, and Enterococcus sulfireus (2, 3,12). These species have been isolated mainly from environmental materials (e.g., grass silage, plants, and soil, etc.); rarely, they have had a clinical origin (4, 16).During a study on the taxonomy of enterococci, four strains of yellow-pigmented group D enterococci (CA-YPE strains) were isolated from clinical specimens. These organisms differed consistently from the previously described species. Biochemical tests, antibiotic susceptibility tests, and plasmid profiles of these human CA-YPE strains revealed several peculiarities (14). In this study, the results of DNA-DNA hybridization tests and fatty acid content determinations indicated that the four atypical CA-WE strains, which were isolated from several humans with severe infections, occupy a unique taxonomic position and represent a new species, for which we propose the name Enterococcus flavescens.(1-3, 9, 11, 16, 17, 19).J. Coyette, Lihge, Belgium, and some strains (CA strains) were derived from the collection of the Cagliari Institute of Microbiology. Test strains of four recently described species were kindly provided by R. R. Facklam, Centers for Disease Control, Atlanta, Ga.Biochemical tests. Biochemical tests were performed by using previously described methods (8,12,13,16) and by using API 20 Strep and API 50CH systems (Ayerst Italiana) following the manufacturer's instructions. Additional tests were performed as described by Facklam (5). Bacteriolytic activity was determined in double-layer Micrococcus luteus agar plates as described previously by Pompei et al. (15).Numerical taxonomy. The similarity coefficient which we used was the simple matching coefficient, and cluster analysis was performed by using the average linkage method (unweighted pair gr...

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