Discovering Tuberosin and Villosol as Potent and Selective Inhibitors of AKT1 for Therapeutic Targeting of Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is a major cause of death in developing countries because of high tobacco consumption. RAC-alpha serine-threonine kinase (AKT1) is considered as an attractive drug target because its prolonged activation and overexpression are associated with cancer progression and metastasis. In addition, several AKT1 inhibitors are being developed to control OSCC and other associated forms of cancers. We performed a screening of the IMPPAT (Indian Medicinal Plants, Phytochemistry and Therapeutics) database to discover promising AKT1 inhibitors which pass through various important filters such as ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties, physicochemical properties, PAINS (pan-assay interference compounds) filters, PASS (prediction of activity spectra for substances) analysis, and specific interactions with AKT1. Molecules bearing admirable binding affinity and specificity towards AKT1 were selected for further analysis. Initially, we identified 30 natural compounds bearing appreciable affinity and specific interaction with AKT1. Finally, tuberosin and villosol were selected as potent and selective AKT1 inhibitors. To obtain deeper insights into binding mechanism and selectivity, we performed an all-atom molecular dynamics (MD) simulation and principal component analysis (PCA). We observed that both tuberosin and villosol strongly bind to AKT1, and their complexes were stable throughout the simulation trajectories. Our in-depth structure analysis suggested that tuberosin and villosol could be further exploited in the therapeutic targeting of OSCC and other cancers after further clinical validations.

Selection and propagation of IncP conjugative plasmids following long-term anthropogenic metal pollution in river sediments.

For a century, the MetalEurop foundry released metals into the river "La Deûle". Previous work revealed higher microbial diversity in metal impacted sediments, and horizontal gene transfer mediated by conjugative plasmids was suggested to drive the community adaptation to metals. We used an integrative state-of-the-art molecular approach coupling quantitative PCR, conjugation assays, flow cytometry, fluorescence activated cell sorting and 16S rRNA gene amplicon sequencing to investigate the presence of conjugative plasmids and their propagation patterns in sediment microbiomes. We highlighted the existence of a native broad-host range IncP conjugative plasmid population in polluted sediments, confirming their ecological importance for microbial adaptation. However, despite incompatibilities and decreased transfer frequencies with our own alien IncP plasmid, we evidenced that a wide diversity of bacterial members was still prone to uptake the plasmid, indicating that sediment microbial communities are still inclined to receive conjugative plasmids from the same group. We observed that metal pollution favoured exogenous plasmid transfer to specific metal-selected bacteria, which are likely coming from upstream sources (e.g. wastewater treatment plant, farms…). Altogether, our results suggest that MetalEurop sediments are hotspots for gene transfer via plasmids, acting as an "environmental reservoir" for microbes and mobile elements released by human activities.

Changes in rumen bacterial and archaeal communities over the transition period in primiparous Holstein dairy cows.

In the present study, we hypothesized that the rumen bacterial and archaeal communities would change significantly over the transition period of dairy cows, mainly as an adaptation to the classical use of low-grain prepartum and high-grain postpartum diets. Bacterial 16S rRNA gene amplicon sequencing of rumen samples from 10 primiparous Holstein dairy cows revealed no changes over the transition period in relative abundance of genera such as Ruminococcus, Butyrivibrio, Clostridium, Coprococcus, and Pseudobutyrivibrio. However, other dominant genus-level taxa, such as Prevotella, unclassified Ruminococcaceae, and unclassified Succinivibrionaceae, showed distinct changes in relative abundance from the prepartum to the postpartum period. Overall, we observed individual fluctuation patterns over the transition period for a range of bacterial taxa that, in some cases, were correlated with observed changes in the rumen short-chain fatty acids profile. Combined results from clone library and terminal-restriction fragment length polymorphism (T-RFLP) analyses, targeting the methyl-coenzyme M reductase α-subunit (mcrA) gene, revealed a methanogenic archaeal community dominated by the Methanobacteriales and Methanomassiliicoccales orders, particularly the genera Methanobrevibacter, Methanosphaera, and Methanomassiliicoccus. As observed for the bacterial community, the T-RFLP patterns showed significant shifts in methanogenic community composition over the transition period. Together, the composition of the rumen bacterial and archaeal communities exhibited changes in response to particularly the dietary changes of dairy cows over the transition period.

The bacterial microbiota in first-void urine from men with and without idiopathic urethritis.

BACKGROUND: Non-gonococcal urethritis (NGU) is a common syndrome in men. NGU may have several causes, but many cases are caused by sexually transmitted infections that may also cause complications in their female partners. Chlamydia trachomatis and Mycoplasma genitalium are the most common causes of NGU, but in up to 35% of the cases, none of the known viral or bacterial causes are found. Traditionally, pathogens have been detected using various culture techniques that may not identify all species present in the urethra. To address this, we used culture-independent methods for analysis of the male urethral microbiota. METHODS: This case-control study analysed first void urine samples, collected at STD clinics in Stockholm, Sweden from men with idiopathic urethritis (IU), i.e. negative for Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Ureaplasma urealyticum, Trichomonas vaginalis, adenovirus, and herpes simplex virus type 1 and -2 together with samples from men without urethritis. Forty-six controls and 39 idiopathic urethritis patients were analysed. RESULTS: The microbiota was highly diverse: None of the 302 operational taxonomic units (OTUs) found in negative controls and IU patients were found in all of the samples or even in all of the samples in one group. More than 50% of the OTUs were only found in one or two of the total of 85 samples. Still the most dominant 1/6 of the genera constituted 79% of the sequences. Hierarchical clustering in a heatmap showed no specific clustering of patients or controls. A number of IU patient samples were dominated by a single genus previously related to urethritis (Gardnerella, Haemophilus, Ureaplasma). CONCLUSION: The male urethra contain a very diverse composition of bacteria, even in healthy controls. NGU may be caused by a number of different bacteria but more studies including a higher number of samples are needed for elucidation of the role of each species.

Deciphering conjugative plasmid permissiveness in wastewater microbiomes.

Wastewater treatment plants (WWTPs) are designed to robustly treat polluted water. They are characterized by ceaseless flows of organic, chemical and microbial matter, followed by treatment steps before environmental release. WWTPs are hotspots of horizontal gene transfer between bacteria via conjugative plasmids, leading to dissemination of potentially hazardous genetic material such as antimicrobial resistance genes (AMRGs). While current focus is on the threat of AMRGs spreading and their environmental maintenance, conjugative plasmid transfer dynamics within and between bacterial communities still remains largely uncharted. Furthermore, current in vitro methods used to assess conjugation in complex microbiomes do not include in situ behaviours of recipient cells, resulting in partial understanding of transfers. We investigated the in vitro conjugation capacities of WWTP microbiomes from inlet sewage and outlet treated water using the broad-host range IncP-1 conjugative plasmid, pKJK5. A thorough molecular approach coupling metagenomes to 16S rRNA DNA/cDNA amplicon sequencing was established to characterize microbiomes using the ecological concept of functional response groups. A broad diversity of recipient bacterial phyla for the plasmid was observed, especially in WWTP outlets. We also identified permissive bacteria potentially able to cross WWTPs and engage in conjugation before and after water treatment. Bacterial activity and lifestyle seem to influence conjugation extent, as treated water copiotrophs were the most represented strategist amongst transconjugants. Correlation analysis highlighted possible plasmid transmission routes into communities between the sewage to the environment, with identification of keystone members (e.g., Arcobacter) potentially involved in cross-border exchanges between distant Gram-positive and Gram-negative phyla.

It is elemental: soil nutrient stoichiometry drives bacterial diversity.

It is well established that resource quantity and elemental stoichiometry play major roles in shaping below and aboveground plant biodiversity, but their importance for shaping microbial diversity in soil remains unclear. Here, we used statistical modeling on a regional database covering 179 locations and six ecosystem types across Scotland to evaluate the roles of total carbon (C), nitrogen (N) and phosphorus (P) availabilities and ratios, together with land use, climate and biotic and abiotic factors, in determining regional scale patterns of soil bacterial diversity. We found that bacterial diversity and composition were primarily driven by variation in soil resource stoichiometry (total C:N:P ratios), itself linked to different land uses, and secondarily driven by other important biodiversity drivers such as climate, soil spatial heterogeneity, soil pH, root influence (plant-soil microbe interactions) and microbial biomass (soil microbe-microbe interactions). In aggregate, these findings provide evidence that nutrient stoichiometry is a strong predictor of bacterial diversity and composition at a regional scale.

The developing hypopharyngeal microbiota in early life.

BACKGROUND: The airways of healthy humans harbor a distinct microbial community. Perturbations in the microbial community have been associated with disease, yet little is known about the formation and development of a healthy airway microbiota in early life. Our goal was to understand the establishment of the airway microbiota within the first 3 months of life. We investigated the hypopharyngeal microbiota in the unselected COPSAC2010 cohort of 700 infants, using 16S rRNA gene sequencing of hypopharyngeal aspirates from 1 week, 1 month, and 3 months of age. RESULTS: Our analysis shows that majority of the hypopharyngeal microbiota of healthy infants belong to each individual's core microbiota and we demonstrate five distinct community pneumotypes. Four of these pneumotypes are dominated by the genera Staphylococcus, Streptococcus, Moraxella, and Corynebacterium, respectively. Furthermore, we show temporal pneumotype changes suggesting a rapid development towards maturation of the hypopharyngeal microbiota and a significant effect from older siblings. Despite an overall common trajectory towards maturation, individual infants' microbiota are more similar to their own, than to others, over time. CONCLUSIONS: Our findings demonstrate a consolidation of the population of indigenous bacteria in healthy airways and indicate distinct trajectories in the early development of the hypopharyngeal microbiota.

Synbiotic Lactobacillus acidophilus NCFM and cellobiose does not affect human gut bacterial diversity but increases abundance of lactobacilli, bifidobacteria and branched-chain fatty acids: a randomized, double-blinded cross-over trial.

Probiotics, prebiotics, and combinations thereof, that is synbiotics, have been reported to modulate gut microbiota of humans. In this study, effects of a novel synbiotic on the composition and metabolic activity of human gut microbiota were investigated. Healthy volunteers (n = 18) were enrolled in a double-blinded, randomized, and placebo-controlled cross-over study and received synbiotic [Lactobacillus acidophilus NCFM (10(9) CFU) and cellobiose (5 g)] or placebo daily for 3 weeks. Fecal samples were collected and lactobacilli numbers were quantified by qPCR. Furthermore, 454 tag-encoded amplicon pyrosequencing was used to monitor the effect of synbiotic on the composition of the microbiota. The synbiotic increased levels of Lactobacillus spp. and relative abundances of the genera Bifidobacterium, Collinsella, and Eubacterium while the genus Dialister was decreased (P < 0.05). No other effects were found on microbiota composition. Remarkably, however, the synbiotic increased concentrations of branched-chain fatty acids, measured by gas chromatography, while short-chain fatty acids were not affected.

Investigating the diversity of pseudomonas spp. in soil using culture dependent and independent techniques.

Less than 1 % of bacterial populations present in environmental samples are culturable, meaning that cultivation will lead to an underestimation of total cell counts and total diversity. However, it is less clear whether this is also true for specific well-defined groups of bacteria for which selective culture media is available. In this study, we use culture dependent and independent techniques to describe whether isolation of Pseudomonas spp. on selective nutrient-poor NAA 1:100 agar-medium can reflect the full diversity, found by pyrosequencing, of the total soil Pseudomonas community in an urban waste field trial experiment. Approximately 3,600 bacterial colonies were isolated using nutrient-poor NAA 1:100 medium from soils treated with different fertilizers; (i) high N-level sewage sludge (SA), (ii) high N-level cattle manure (CMA), and (iii) unfertilized control soil (U). Based on Pseudomonas specific quantitative-PCR and Pseudomonas CFU counts, less than 4 % of Pseudomonas spp. were culturable using NAA 1:100 medium. The Pseudomonas selectivity and specificity of the culture medium were evaluated by 454 pyrosequencing of 16S rRNA gene amplicons generated using Bacteria- and Pseudomonas-specific primers. Pyrosequencing results showed that most isolates were Pseudomonas and that the culturable fraction of Pseudomonas spp. reflects most clusters of the total Pseudomonas diversity in soil. This indicates that NAA 1:100 medium is highly selective for Pseudomonas species, and reveals the ability of NAA 1:100 medium to culture mostly the dominant Pseudomonas species in soil.

Impact of long-term diesel contamination on soil microbial community structure.

Microbial community composition and diversity at a diesel-contaminated railway site were investigated by pyrosequencing of bacterial and archaeal 16S rRNA gene fragments to understand the interrelationships among microbial community composition, pollution level, and soil geochemical and physical properties. To this end, 26 soil samples from four matrix types with various geochemical characteristics and contaminant concentrations were investigated. The presence of diesel contamination significantly impacted microbial community composition and diversity, regardless of the soil matrix type. Clean samples showed higher diversity than contaminated samples (P < 0.001). Bacterial phyla with high relative abundances in all samples included Proteobacteria, Firmicutes, Actinobacteria, Acidobacteria, and Chloroflexi. High relative abundances of Archaea, specifically of the phylum Euryarchaeota, were observed in contaminated samples. Redundancy analysis indicated that increased relative abundances of the phyla Chloroflexi, Firmicutes, and Euryarchaeota correlated with the presence of contamination. Shifts in the chemical composition of diesel constituents across the site and the abundance of specific operational taxonomic units (OTUs; defined using a 97% sequence identity threshold) in contaminated samples together suggest that natural attenuation of contamination has occurred. OTUs with sequence similarity to strictly anaerobic Anaerolineae within the Chloroflexi, as well as to Methanosaeta of the phylum Euryarchaeota, were detected. Anaerolineae and Methanosaeta are known to be associated with anaerobic degradation of oil-related compounds; therefore, their presence suggests that natural attenuation has occurred under anoxic conditions. This research underscores the usefulness of next-generation sequencing techniques both to understand the ecological impact of contamination and to identify potential molecular proxies for detection of natural attenuation.

Mining of unexplored habitats for novel chitinases--chiA as a helper gene proxy in metagenomics.

The main objective of this study was to assess the abundance and diversity of chitin-degrading microbial communities in ten terrestrial and aquatic habitats in order to provide guidance to the subsequent exploration of such environments for novel chitinolytic enzymes. A combined protocol which encompassed (1) classical overall enzymatic assays, (2) chiA gene abundance measurement by qPCR, (3) chiA gene pyrosequencing, and (4) chiA gene-based PCR-DGGE was used. The chiA gene pyrosequencing is unprecedented, as it is the first massive parallel sequencing of this gene. The data obtained showed the existence across habitats of core bacterial communities responsible for chitin assimilation irrespective of ecosystem origin. Conversely, there were habitat-specific differences. In addition, a suite of sequences were obtained that are as yet unregistered in the chitinase database. In terms of chiA gene abundance and diversity, typical low-abundance/diversity versus high-abundance/diversity habitats was distinguished. From the combined data, we selected chitin-amended agricultural soil, the rhizosphere of the Arctic plant Oxyria digyna and the freshwater sponge Ephydatia fluviatilis as the most promising habitats for subsequent bioexploration. Thus, the screening strategy used is proposed as a guide for further metagenomics-based exploration of the selected habitats.

The fate of indigenous microbiota, starter cultures, Escherichia coli, Listeria innocua and Staphylococcus aureus in Danish raw milk and cheeses determined by pyrosequencing and quantitative real time (qRT)-PCR.

The purpose of this work was to study the bacterial communities in raw milk and in Danish raw milk cheeses using pyrosequencing of tagged amplicons of the V3 and V4 regions of the 16S rDNA and cDNA. Furthermore, the effects of acidification and ripening starter cultures, cooking temperatures and rate of acidification on survival of added Escherichia coli, Listeria innocua and Staphylococcus aureus in cheeses at different stages of ripening were studied by pyrosequencing and quantitative real time (qRT)-PCR. A high diversity of bacterial species was detected in raw milk. Lactococcus lactis, Streptococcus thermophilus, Lactobacillus casei and Lactobacillus rhamnosus were the main bacteria detected in raw milk and cheeses. Bacteria belonging to the genera Brevibacterium, Staphylococcus, Escherichia, Weissella, Leuconostoc, Pediococcus were also detected in both 16S rDNA and cDNA obtained from raw milk and cheeses. E. coli, which was added to milk used for production of some cheeses, was detected in both DNA and RNA extracted from cheeses at different stages of ripening showing the highest percentage of the total sequence reads at 7 days of ripening and decreased again in the later ripening stages. Growth of E. coli in cheeses appeared to be affected by the cooking temperature and the rate of acidification but not by the ripening starter cultures applied or the indigenous microbiota of raw milk. Growth of L. innocua and S. aureus added to milks was inhibited in all cheeses at different stages of ripening. The use of 16S rRNA gene pyrosequencing and qRT-PCR allows a deeper understanding of the behavior of indigenous microbiota, starter cultures and pathogenic bacteria in raw milk and cheeses.

Predominant genera of fecal microbiota in children with atopic dermatitis are not altered by intake of probiotic bacteria Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bi-07.

The effect of probiotic bacteria Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 on the composition of the Lactobacillus group, Bifidobacterium and the total bacterial population in feces from young children with atopic dermatitis was investigated. The study included 50 children randomized to intake of one of the probiotic strain or placebo. Microbial composition was characterized by denaturing gradient gel electrophoresis, quantitative PCR and, in a subset of subjects, by pyrosequencing of the 16S rRNA gene. The core population of the Lactobacillus group was identified as Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus oris, Leuconostoc mesenteroides, while the bifidobacterial community included Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum and Bifidobacterium catenulatum. The fecal numbers of L. acidophilus and B. lactis increased significantly after intervention, indicating survival of the ingested bacteria. The levels of Bifidobacterium correlated positively (P=0.03), while the levels of the Lactobacillus group negatively (P=0.01) with improvement of atopic eczema evaluated by the Severity Scoring of Atopic Dermatitis index. This correlation was observed across the whole study cohort and not attributed to the probiotic intake. The main conclusion of the study is that administration of L. acidophilus NCFM and B. lactis Bi-07 does not affect the composition and diversity of the main bacterial populations in feces.

Diversity and characterization of mercury-resistant bacteria in snow, freshwater and sea-ice brine from the High Arctic.

It is well-established that atmospheric deposition transports mercury from lower latitudes to the Arctic. The role of bacteria in the dynamics of the deposited mercury, however, is unknown. We characterized mercury-resistant bacteria from High Arctic snow, freshwater and sea-ice brine. Bacterial densities were 9.4 × 10(5), 5 × 10(5) and 0.9-3.1 × 10(3) cells mL(-1) in freshwater, brine and snow, respectively. Highest cultivability was observed in snow (11.9%), followed by freshwater (0.3%) and brine (0.03%). In snow, the mercury-resistant bacteria accounted for up to 31% of the culturable bacteria, but <2% in freshwater and brine. The resistant bacteria belonged to the Alpha-, Beta- and Gammaproteobacteria, Firmicutes, Actinobacteria, and Bacteriodetes. Resistance levels of most isolates were not temperature dependent. Of the resistant isolates, 25% reduced Hg(II) to Hg(0). No relation between resistance level, ability to reduce Hg(II) and phylogenetic group was observed. An estimation of the potential bacterial reduction of Hg(II) in snow suggested that it was important in the deeper snow layers where light attenuation inhibited photoreduction. Thus, by reducing Hg(II) to Hg(0), mercury-resistant bacteria may limit the supply of substrate for methylation processes and, hence, contribute to lowering the risk that methylmercury is being incorporated into the Arctic food chains.

The presence of embedded bacterial pure cultures in agar plates stimulate the culturability of soil bacteria.

Traditional methods for bacterial cultivation recover only a small fraction of bacteria from all sorts of natural environments, and attempts have been made to improve the bacterial culturability. Here we describe the development of a cultivation method, based on the embedment of pure bacterial cultures in between two layers of agar. Plates containing either embedded Pseudomonas putida or Arthrobacter globiformis resulted in higher numbers of CFUs of soil bacteria (21% and 38%, respectively) after 833 h of incubation, compared to plates with no embedded strain. This indicates a stimulatory effect of the bacterial pure cultures on the cultivation of soil bacteria. Analysis of partial 16S rRNA gene sequences revealed a phylogenetical distribution of the soil isolates into 7 classes in 4 phyla. No difference was observed at the phylum or class level when comparing isolates grouped according to embedded strain. The number of isolates belonging to the same class as the embedded strain was reduced in comparison to that of plates with no embedded strain, indicating that intercellular signalling was unlikely to cause the observed stimulatory effect. Significantly higher fractions of isolates with less than 97% sequence homology to known sequenced isolates in GenBank were recovered from plates with embedded strains than from those without, which indicate a higher number of potential novel soil isolates. This approach for cultivation is therefore a feasible alternative or supplement to traditional cultivation on agar plates in order to enhance bacterial culturability.

Prevalence of Helicobacter pylori vacA and cagA genotypes in Ethiopian dyspeptic patients.

A total of 300 gastric biopsy samples and 50 Helicobacter pylori isolates were collected from Ethiopian adult dyspeptic patients. The vacA and cagA genes were detected in 90 and 79% of biopsy specimens, respectively, and in 100 and 87% of clinical isolates, respectively. Both genes were detected in 84% of the gastric biopsy samples and in 87% of the clinical isolates. Among vacA genotypes, the s1/m1 genotype was the most common in gastric biopsy samples (48%). The vacA and cagA positive H. pylori strains were detected to a higher degree in patients with chronic active gastritis (71%) than patients with other histopathological findings (29%) (P < 0.05).

Investigation of Burkholderia cepacia nosocomial outbreak with high fatality in patients suffering from diseases other than cystic fibrosis.

Over a 1-y period, 26 inpatients at the Jordan University Hospital in Amman were detected with bacteraemia (23 cases) or respiratory tract colonized with B. cepacia (3 cases). A combination of genetic identification and molecular typing has proved that all cases were caused by a single epidemic strain of B. cepacia genomovar IIIa. Nosocomial infections could be documented in 21/26 (81%) patients, mostly with severe underlying or malignant diseases other than cystic fibrosis, but the source of infection was undetected. The overall mortality related to infection with B. cepacia was 42%. All B. cepacia isolates were resistant to ampicillin, amikacin, carbenicillin and gentamicin; and mostly susceptible to piperacillin, chloramphenicol, cotri-moxazole, tetracycline, ceftazidime, and tazocin (62-88%). This study demonstrates the nosocomial and high fatality of B. cepacia genomovar IIIa in Jordanian patients suffering from diseases other than cystic fibrosis.

PCR-denaturing gradient gel electrophoresis and two feces antigen tests for detection of Helicobacter pylori in mice.

PCR-denaturing Gradient Gel Electrophoresis (PCR-DGGE), a method suitable for the detection of microbial species in complex ecosystems, was evaluated for the detection and identification of Helicobacter spp. in feces and stomach tissue of mice. Two commercially available stool antigen tests for clinical diagnostics in humans were also evaluated in the C57Bl/6 mouse model of H. pylori infection. PCR-DGGE detected only Helicobacter ganmani in feces from H. pylori-infected as well as control animals, whereas in stomach specimens it demonstrated the presence of H. pylori in challenged and H. ganmani in control animals. Hence, the method detected DNA only of the predominant Helicobacter spp., which was also shown in cell dilution experiments. The Amplified IDEIA Hp StAR feces antigen test detected H. pylori in feces from all infected animals and generated no false-positive results, whereas the Premier Platinum HpSA-test also detected H. pylori in all infected animals but generated false-positive or equivocal results in 50% of the control animals. Premier Platinum HpSA, as opposed to Hp StAR, cross-reacted with non-pylori Helicobacter spp. in vitro.

Effect of cold starvation, acid stress, and nutrients on metabolic activity of Helicobacter pylori.

Helicobacter pylori can transform, in vivo as well as in vitro, from dividing spiral-shaped forms into nonculturable coccoids, with intermediate forms called U forms. The importance of nonculturable coccoid forms of H. pylori in disease transmission and antibiotic treatment failures is unclear. Metabolic activities of actively growing as well as nonculturable H. pylori were investigated by comparing the concentrations of cellular ATP and total RNA, gene expression, presence of cytoplasmic polyphosphate granules and iron inclusions, and cellular morphology during extended broth culture and nutritional cold starvation. In addition, the effect of exposing broth-cultured or cold-starved cells to a nutrient-rich or acidic environment on the metabolic activities was investigated. ATP was detectable up to 14 days and for at least 25 days after transformation from the spiral form to the coccoid form or U form in broth-cultured and cold-starved cells, respectively. mRNAs of VacA, a 26-kDa protein, and urease A were detected by using reverse transcription-PCR in cells cultured for 2 months in broth or cold starved for at least 28 months. The ATP concentration was not affected during exposure to fresh or acidified broth, while 4- to 12-h exposures of nonculturable cells to lysed human erythrocytes increased cellular ATP 12- to 150-fold. Incubation of nonculturable cold-starved cells with an erythrocyte lysate increased total RNA expression and ureA mRNA transcription as measured by quantitative real-time reverse transcription-PCR. Furthermore, the number of structurally intact starved coccoids containing polyphosphate granules increased almost fourfold (P = 0.0022) under the same conditions. In conclusion, a specific environmental stimulus can induce ATP, polyphosphate, and RNA metabolism in nonculturable H. pylori, indicating viability of such morphological forms.