Optimizing the growth of stressed Helicobacter pylori.

Helicobacter pylori is a gram-negative bacterium that colonizes the human stomach and is responsible for causing gastric ulcers. H. pylori is known to become stressed and nonculturable after exposure to unfavorable conditions. In this study, we enhanced previously published resuscitation procedures, characterized conditions under which stressed H. pylori can be recovered, and formulated a selective and differential resuscitation medium. Results showed that a specialized broth supplemented with trace minerals and lysed human erythrocytes and serum is required for the recovery of nonculturable H. pylori. The type of stress was an important factor in the efficacy of resuscitation, with cells exposed to atmospheric oxygen more readily resuscitated than nutrient deprived cells. After resuscitation, culturable cells were recovered from previously nonculturable oxygen stressed cells (24 and 72 h of exposure) and nonculturable nutrient deprived cells (24 h of exposure). The length of time the cells were exposed to the stress was also an important factor in the recovery of stressed H. pylori. RNA levels were quantified and transcription of the cell division related gene, cdrA (HP0066), was assessed by qRT-PCR. The low levels of RNA detected in stressed cells, after resuscitation, support the idea that a small population of viable cells may be responsible for the colonies recovered on solid agar. The modification of the resuscitation broth into a selective and differential slant culture medium also allowed the recovery of stressed H. pylori. The methods presented here highlight the benefits and limitations of using human blood products for recovering nonculturable H. pylori.

Related papers:

title: Helicobacter and Gastric Malignancies
abstract: Over the past year Helicobacter pylori has been confirmed as the most important risk factor for non‐cardia gastric adenocarcinomas and gastric mucosa‐associated lymphoid tissue (MALT) lymphomas. Eradication therapy has been proven to be beneficial when given prior to the development of intestinal metaplasia, but is less efficacious when administered later. However, the best data from clinical trials indicate that H. pylori eradication alone will have only a moderate effect on gastric cancer incidence worldwide. The mechanisms responsible for H. pylori‐associated gastric carcinogenesis continue to be dissected. Accumulating evidence suggests that some H. pylori may be able to invade through the gastric epithelial barrier, though pro‐carcinogenic effects may also be related to the complex and evolving pathways of altering signal transduction pathways within gastric epithelial cells that are stimulated by adherence and translocation of H. pylori products through its type IV secretory system. Determinants of the host response to H. pylori infection continue to focus on polymorphisms in genes related to the innate and acquired immune responses, including NOD2, COX‐2, and TLR‐4. H. pylori eradication is indicated for low‐grade gastric B‐cell MALT lymphoma and may even provide “cure” in some apparently H. pylori‐negative cases. How and why does H. pylori promote lymphomagenesis? Some evidence from human and murine models points to specific chromosomal translocations and host genetic polymorphisms as relating to the outcome of infection. Finally, Helicobacter hepaticus infection has been linked to both intestinal and breast tumorigenesis in susceptible strains of female mice – a provocative and novel finding warranting further investigation.

title: Helicobacter pylori occurrence and transmission: A family affair?
abstract: About half of the world's population is estimated to be infected with Helicobacter pylori, a gastric bacterium that contributes to the development of peptic ulcer disease and gastric cancer. H. pylori is more prevalent in low-income areas of the world and social and economic development decreases the prevalence as reflected in comparisons both within and between countries. The infection is typically acquired in early childhood and once established commonly persists throughout life unless treated. Person-to-person transmission within the family appears to be the predominant mode of transmission, particularly from mothers to children and among siblings, indicating that intimate contact is important. The route of transmission is uncertain, but the gastro-oral, oral-oral and faecal-oral routes are likely possibilities. Hence, gastroenteritis may facilitate dissemination of the infection. The community and environment may play additional roles for H. pylori transmission in some (low-income) settings. Furthermore, host and bacterial factors may modify the probabilities of acquisition and persistence of the infection. The understanding of H. pylori occurrence and transmission is of practical importance if future study deems prevention of the infection desirable in some high-prevalence populations. The present paper reviews aspects of H. pylori occurrence and transmission with an emphasis on household factors.

title: A conceptual model of water's role as a reservoir in Helicobacter pylori transmission: a review of the evidence
abstract: Helicobacter pylori infection plays a role in the development of chronic gastritis, peptic ulcer and gastric cancer, yet the route of transmission into susceptible hosts remains unknown. Studies employing microbiological techniques have demonstrated that H. pylori has the ability to survive when introduced into water and that H. pylori is present in water and other environmental samples all over the world. Epidemiological studies have shown that water source and exposures related to water supply, including factors related to sewage disposal and exposure to animals, are risk factors for infection. This review describes the microbiological and epidemiological evidence for, and proposes a model of, waterborne H. pylori transmission outlining important features in the transmission cycle. In the model, humans and animals shed the bacteria in their faeces and the mechanisms for entry into water, and for survival, ingestion and infection are dependent upon a range of environmental influences. Verification of the proposed model pathways has important implications for public-health prevention strategies.

title: Helicobacter pylori in the natural environment.
abstract: The presence of Helicobacter pylori (H. pylori) in the natural environment has been demonstrated in a number of studies. However, its route of infection into humans is unknown. To study this further, we attempted to detect H. pylori in the natural environment in a region of Japan with a high infection rate. Tap and well water and field soil samples were collected from around the residences of subjects who had participated in an epidemiological survey in 1996. Samples of water from rivers and ponds, and specimens of flies and cow faeces were collected in the region. DNA was extracted from the water, field soil and faecal samples after selective collection of H. pylori by the immunomagnetic-bead separation technique. H. pylori-specific DNA was detected in water, field soil, flies and cow faeces by nested polymerase chain reaction (PCR), and the ureA partial sequences of the PCR products were aligned. The nucleotide sequences of the samples amplified by PCR were highly homologous (96-100%) with the H. pylori sequence in the GenBank database and the H. pylori-specific DNA sequences were highly homologous with each other. These findings suggest the existence of H. pylori in the natural environment and a possible transmission route.

title: Plural transformation-processes from spiral to coccoid Helicobacter pylori and its viability.
abstract: Helicobacter pylori (H. pylori), present in a half of the world's population, is a very successful pathogen. The infection by this bacterium causes several gastric diseases in human. H. pylori is morphologically divided into two types; a spiral and a coccoid form. Both types are observed in human stomach. Although the former is converted into the latter in vitro, the process of coccoid formation remains obscure. Furthermore, whether coccoid forms possess viability arouses much controversy among scientists. We investigated both the process of coccoid formation by electron microscopy and the viability of coccoid H. pylori.



A laboratory strain, ATCC43504, was cultured in liquid medium (Brucella Broth medium with 10% heat inactivated horse serum) for seven days. In each culture day, the organisms were observed by scanning-, conventional transmission- and immuno-electron microscopy and were simultaneously examined the culturability of H. pylori to identify the viability of coccoid form.



As the days went by, spiral forms were replaced by coccoid forms in each medium which possessed culturability to some degree until the 4th day. By the detailed observation of ultrastructural features, coccoid forms were classified into two types on the ground that represents different transformation-processes and distinctive ultrastructures. One was the coccoid form that we named Type A with an irregularly surface- and intracytoplasmic-structure which adhered to one another. The other was Type B having a smooth surface with flagella coiled around its own body and the strictly membranous structure. This type was not adhered to another organism. In later days, each type came to be similar external structure and lost the culturability.



The coccoid forms of H. pylori can be classified into two types by electron microscopy which represent different transformation-processes and consist of the dying bacteria, the living ones with culturability and the viable but non-culturable ones.

title: The coccoid forms of Helicobacter pylori. Criteria for their viability.
abstract: The fact that Helicobacter pylori can revert to a coccoid form has stimulated speculation about its role in transmission and as a possible cause of reinfection in duodenal ulcer disease. Bismuth subcitrate (32 micrograms/ml), bismuth subsalicylate (64 micrograms/ml), amoxicillin (0.05 micrograms/ml) and erythromycin (4 micrograms/ml) inhibited the growth of H. pylori and stimulated the formation of basically respiring but non-culturable coccoid structures. The presence of polyphosphates as energy and phosphorus source permits a certain level of endogenous metabolism to preserve RNA and DNA, as well as structural components like cell wall, cell membrane and cytoplasma for at least 3 months. However, the applied standard laboratory methods were insufficient for regrowth of H. pylori out of the coccoid form.

title: Detection of Helicobacter pylori DNA in drinking water biofilms: implications for transmission in early life
abstract: To provide evidence of water quality as a risk factor for acquisition of Helicobacter pylori in early life, and to identify evidence for its presence within pots used to store drinking water.

title: Failure To Detect Helicobacter pylori DNA in Drinking and Environmental Water in Dhaka, Bangladesh, Using Highly Sensitive Real-Time PCR Assays
abstract: The main transmission pathway of Helicobacter pylori has not been determined, but several reports have described detection of H. pylori DNA in drinking and environmental water, suggesting that H. pylori may be waterborne. To address this possibility, we developed, tested, and optimized two complementary H. pylori-specific real-time PCR assays for quantification of H. pylori DNA in water. The minimum detection level of the assays including collection procedures and DNA extraction was shown to be approximately 250 H. pylori genomes per water sample. Using our assays, we then analyzed samples of drinking and environmental water (n = 75) and natural water biofilms (n = 21) from a high-endemicity area in Bangladesh. We could not identify H. pylori DNA in any of the samples, even though other pathogenic bacteria have been found previously in the same water samples by using the same methodology. A series of control experiments were performed to ensure that the negative results were not falsely caused by PCR inhibition, nonspecific assays, degradation of template DNA, or low detection sensitivity. Our results suggest that it is unlikely that the predominant transmission route of H. pylori in this area is waterborne.

title: Coccoid Form of Helicobacter pylori as a Morphological Manifestation of Cell Adaptation to the Environment
abstract: After characterization of preferred conditions for Helicobacter pylori survival in the sessile state, it was observed that the bacterium transforms from spiral to coccoid under mild circumstances, whereas under extreme ones it is unable to undergo shape modification. This strongly supports the view that transformation into the coccoid form is an active, biologically led process, switched on by the bacterium as a protection mechanism.

title: Recent findings on the viable but nonculturable state in pathogenic bacteria.
abstract: Many bacteria, including a variety of important human pathogens, are known to respond to various environmental stresses by entry into a novel physiological state, where the cells remain viable, but are no longer culturable on standard laboratory media. On resuscitation from this 'viable but nonculturable' (VBNC) state, the cells regain culturability and the renewed ability to cause infection. It is likely that the VBNC state is a survival strategy, although several interesting alternative explanations have been suggested. This review describes the VBNC state, the various chemical and physical factors known to induce cells into this state, the cellular traits and gene expression exhibited by VBNC cells, their antibiotic resistance, retention of virulence and ability to attach and persist in the environment, and factors that have been found to allow resuscitation of VBNC cells. Along with simple reversal of the inducing stresses, a variety of interesting chemical and biological factors have been shown to allow resuscitation, including extracellular resuscitation-promoting proteins, a novel quorum-sensing system (AI-3) and interactions with amoeba. Finally, the central role of catalase in the VBNC response of some bacteria, including its genetic regulation, is described.

title: Survival of Helicobacter pylori in a Natural Freshwater Environment
abstract: The mode by which Helicobacter pylori, the causative agent of most gastric ulcers, is transmitted remains undetermined. Epidemiological evidence suggests these organisms are waterborne; however, H. pylori has rarely been grown from potential water sources. This may be due to the ability of this organism to rapidly enter the viable but nonculturable (VBNC) state. Our investigation examines the entrance of H. pylori into this state in laboratory cultures and a natural freshwater environment as well as the relationship between morphology and culturability. To this end, membrane diffusion chambers were utilized to expose the cells to the natural fluctuations of a freshwater stream. In both the laboratory and environment, samples were assayed for culturability using plate counts and stained using a LIVE/DEAD BacLight assay for viability and morphological determinations. Additionally, water samples were collected, six environmental parameters were measured, and resuscitation conditions were examined. H. pylori was observed to lose culturability in the laboratory and stream, although viability was maintained. While the results of our study agree with those of previous studies which suggested that there is a transition in morphology from rods to cocci as culturability is lost, the morphological distribution of cells did not change as culturability was lost in the environment. The majority of cells in the VBNC state in the laboratory are cocci; however, all morphological forms were present in the environment. The results of these studies suggest that H. pylori persists in laboratory cultures and the environment in the VBNC state and that cells in this state represent a public health hazard.

title: Morphological Changes Leading to the Nonculturable State
abstract: Specific morphological changes occur when bacterial cells are introduced into nutrient-depleted environments. In addition, bacteria that are indigenous to these conditions tend to be smaller than bacteria found in nutrient-rich environments. Changes in bacterial morphology during starvation-survival have been previously reviewed (45, 61). The conditions under which bacteria alter their morphology are discussed, with reasons for the changes suggested.

title: Molecular Genetic Methods for Detection and Identification of Viable but Nonculturable Microorganisms
abstract: The problems associated with culture-based methods for detection and identification of microorganisms in clinical and environmental samples have motivated the development of alternative methods which do not require cultivation of the target organisms. Molecular genetic methods, which specifically target microbial nucleic acids, have become important tools for the identification of both cultured and uncultured microorganisms. The sensitivity of these methods approaches that of culture-based methods and it is arguable that the specificity of these methods generally exceeds that of culture-based methods. Indeed, molecular genetic methods have become a powerful set of tools used by many investigators to detect and identify culturable microorganisms in their viable but nonculturable state, as well as organisms which have yet to be brought into culture.

title: Coccoid forms of Helicobacter pylori are the morphologic manifestation of cell death
abstract: Helicobacter pylori can transform from its normal helical bacillary morphology to a coccoid morphology. Since this coccoid form cannot be cultured in vitro, it has been speculated that it is a dormant form potentially involved in the transmission of H. pylori and in a patient's relapse after antibiotic therapy. In this study we determined the effects of aging, temperature, aerobiosis, starvation, and antibiotics on the morphologic conversion rate and culturability of H. pylori. Aerobiosis and the addition of a bactericidal antibiotic to the culture medium resulted in the highest conversion rate. During the conversion to coccoid forms, the cultures always lost culturability at the stage where 50% of the organisms were still in bacillary form; this result indicated that culturability and coccoid morphology are two separate but related entities. Independent of the conditions used to induce the conversion into coccoids, the morphological conversion was accompanied by several marked antigenic and ultrastructural changes. Also, both the total amounts and the integrity of RNA and DNA were significantly reduced in coccoid forms. With the potential-sensitive probe diOC(5)-3, a clear loss of membrane potential in coccoid forms was observed. Inhibition of protein or RNA synthesis by the addition of bacteriostatic antibiotics did not prevent the conversion to coccoid forms but resulted in an increased conversion rate. Hence, we conclude that conversion of H. pylori from the bacillary to the coccoid form is a passive process that does not require protein synthesis. Our data suggest that the coccoid form of H. pylori is the morphologic manifestation of bacterial cell death.

title: Growth and morphological transformations of Helicobacter pylori in broth media
abstract: Helicobacter pylori, a cause of peptic ulcer disease and certain types of gastric cancers, has usually been cultured on diverse agar-based media, resulting in a requirement for 2 to 4 days of growth at 37 degrees C. We have developed a novel broth medium consisting of a base medium supplemented with 2% newborn calf serum, Mg2+, Cu2+, Fe2+, Zn2+, Mn2+, and 1 mg of lysed human erythrocytes per ml. This medium supports rapid growth of H. pylori, with a doubling time of about 50 min. Optimal growth was obtained in a pH range higher than that supporting most other gram-negative bacteria (at pH 8.5). H. pylori cultured in this supplemented broth retains the spiral morphology seen in both histological sections and cultures from agar-based media and also retains a high urease activity. After 18 h in this broth, H. pylori transforms to a coccal form with a complete loss of urease activity. Previously these cocci have been reported to be senescent, since they could not be subcultured on agar medium. Our experiments suggest that some of the cocci can revert back to the spiral morphology with full recovery of urease activity when subcultured in fresh microaerobic broth medium.

title: Recovery of Helicobacter pylori ATCC43504 from a viable but not culturable state: regrowth or resuscitation?
abstract: 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.

title: [Resuscitation from the viable but nonculturable state of Helicobacter pylori].
abstract: It is well known that the change of state from the culturable to not culturable on pathogenic organisms could easily occurred. These states of bacteria were so called viable but nonculturable (VNC). The coccoid form of H. pylori is seems to be in this state. The possibility of resuscitation of H. pylori was examined in vitro. The coccoid form bacteria was treated with Ammonium Sulfate and heat shock before culture, and cultured in Brucella broth containing, sodium pyruvate, laked human erythrocyte and serum. The growth of the rod spiral form was found in the coccoid form bacteria, and further the bacteria could grow on blood agar and Skirrow agar as well as the original strain. It was strongly suggested that the resuscitation from VNC state of cells to culturable form consists of two processes, stimulation and supplemented of appropriate nutrition.

title: Oxidative metabolism in nonculturable Helicobacter pylori and Vibrio vulnificus cells studied by substrate-enhanced tetrazolium reduction and digital image processing
abstract: Growing and nonculturable cells of Helicobacter pylori and Vibrio vulnificus were studied for the capacity to reduce tetrazolium salts in order to elucidate the possible physiological basis for the proposed "viable but nonculturable" (VNC) state. Initial difficulties in obtaining consistent reduction of rho-iodonitrotetrazolium violet (INT) by H. pylori led us to develop a method for studying the effect of adding exogenous substrates on these reactions. The established procedure provided a profile of substrate enhancement of oxidative activity revealed by INT reduction which was related to both the identity and physiological state of the organism studied. Representation and interpretation of these enhancement profiles were facilitated by digital image processing. Nonculturable cells of H. pylori produced by carbon and nitrogen starvation in air lost all INT-reducing capacity in 24 h when stored at 37 degrees C, while 99% of those produced at 4 degrees C retained oxidative activity for at least 250 days when tested in the presence but not in the absence of succinate, alpha-ketoglutarate, or aspartate. Activity was detected at similar levels in cells with coccoid and spiral shapes. In contrast, only 1% of nonculturable cells of V. vulnificus, produced under conditions previously reported to induce the VNC state in this organism, retained intrinsic INT-reducing capacity; no substrate-enhanced activity occurred in the remainder of the population. Thus, there was no common pattern of oxidative activity indicative of a VNC state in both test organisms. Nonculturable cells of H. pylori can retain several different oxidative enzyme activities; whether these indicate viability or the persistence of cells as "bags of enzymes" remains to be established.

title: Detection of mRNA by Reverse Transcription-PCR as an Indicator of Viability in Escherichia coliCells
abstract: The relationship between the detection of mRNA and cellular viability in Escherichia coli was investigated in cells killed by heat or ethanol. Reverse transcription-PCR (RT-PCR) methods were developed for detecting mRNA from rpoH,groEL, and tufA genes. mRNA from all three genes was detected immediately after the cells had been killed by heat or ethanol but gradually disappeared with time when dead cells were held at room temperature. In heat-killed cells, some mRNA targets became undetectable after 2 to 16 h, whereas after ethanol treatment, mRNA was still detected after 16 h. In contrast, 16S rRNA was detected by RT-PCR in all samples containing dead cells and did not disappear during a subsequent incubation of 16 h at room temperature. Of the different types of nucleic acid, mRNA is the most promising candidate for an indicator of viability in bacteria, but its persistence in dead cells depends on the inactivating treatment and subsequent holding conditions.

title: In Vitro Aging of Helicobacter pylori: Changes in Morphology, Intracellular Composition and Surface Properties
abstract: During the conversion from the bacillary into the coccoid form, Helicobacter pylori organisms are known to change extensively. The aim of this study was to determine some of the changes that occur regarding morphology, intracellular composition and surface properties during the aging of bacteria in vitro.

title: Comparison of the nucleic acids of helical and coccoid forms of Helicobacter pylori
abstract: The nucleic acids of the helical and coccoid forms of Helicobacter pylori were studied to determine if the coccoid forms are "viable (capable of growing) but nonculturable." Using a reference strain (NCTC 11638) and five clinical strains, the nucleic acid contents, DNA integrity, and results of PCR and reverse transcription-PCR (RT-PCR) were compared for helical H. pylori and coccoid forms induced using glycochenodeoxycholic acid or bismuth citrate. The DNA and RNA contents of the coccoid forms were respectively 6.8- and 8.1-fold lower than those of helical H. pylori after 3 days of induction and 11.5- and 14.7-fold lower after 7 days. Agarose gel electrophoresis of DNA extracted from the coccoid forms after 3 days of induction showed a smear pattern indicating DNA cleavage, whereas DNA from helical H. pylori showed a single band with a high molecular mass. After 12 days of induction, all RNA samples from 100% coccoid cultures were negative for the mRNA of urease A or the 26-kDa species-specific protein by RT-PCR. However, most RNA samples obtained after 3 or 7 days of induction were positive at low levels despite the lack of recovery from these cultures. These results suggest that the coccoid form of H. pylori has impaired genomic DNA and is in the process of cellular degeneration, thus being still alive but nonincreasable.

title: Nucleotide Sequence and Characterization of cdrA, a Cell Division-Related Gene of Helicobacter pylori
abstract: We identified cell division-related gene cdrA in Helicobacter pylori HPK5. The putative gene product, CdrA, is a 367-amino-acid polypeptide that exhibited a high level of homology to conserved hypothetical ATP-binding protein HP0066 of H. pylori 26695, except in the N-terminal region, and showed some similarity to the FtsK/SpoIIIE family proteins. We isolated a cdrA-disrupted mutant by allelic exchange mutagenesis. Because of the low transformation frequency, the possibility that a suppressing mutation would be found in the obtained cdrA mutant was discussed. A repressive role for CdrA on cell division was suggested by the observations that the wild-type strain formed filamentous cells in a high-salt level medium at early stationary phase, while a cdrA-disrupted mutant did not show such an abnormality. In addition, the wild-type strain adopted coccoid forms in the stationary phase, whereas the cdrA-disrupted mutant remained mostly as short rods. Furthermore, the cdrA-disrupted mutant regained the filamentation phenotype when the intact cdrA gene was introduced by allelic exchange. Taken together, these observations show that the cdrA gene plays an important role in the cell growth of H. pylori.

title: Development of a Plating Medium for Selection of Helicobacter pylori from Water Samples
abstract: The goal of this study was to develop a simple plating medium to allow large-scale screening of water samples for the presence of Helicobacter pylori. Five conventional plating media (brain heart infusion, brucella agar, Columbia blood agar base, campylobacter agar kit Skirrow, and HPSPA medium), each containing a commercial antibiotic supplement, were initially evaluated. Eight strains selected as common waterborne organisms (Acinetobacter, Aeromonas, Bacillus, Escherichia coli, Enterobacter, Enterococcus, Helicobacter pylori, and Pseudomonas strains) were individually plated onto each of these media. Three organisms (Acinetobacter, E. coli, and H. pylori) were able to grow on all five media. This growth was unacceptable since Helicobacter grows very slowly and competing organisms must be inhibited for up to 7 days. Therefore, a more selective medium (HP agar) containing a novel mixture of growth supplements plus amphotericin B and polymyxin B was developed. This medium also included a phenol red color indicator for urease production. Aliquots of nonsterile well water that contained native flora (Flavobacterium, Serratia, Citrobacter, Pasteurella, Ochrobactrum, Rahnella, and unidentified molds) and were further adulterated with the eight strains listed above (106 CFU of each strain per 100 ml) were spiked with H. pylori and were plated. In spite of the heavy mixed microbial load, only H. pylori colonies grew during 7 days of incubation at 37°C. The color indicator system allowed presumptive identification of H. pylori colonies sooner (12 to 20 h) than the conventional media tested allowed. The HP formulation developed in this study provides a medium with superior selectivity for H. pylori from mixed microbial populations in water and reduces the time required to complete the assay.

title: Development of a Selective Medium for Isolation ofHelicobacter pylori from Cattle and Beef Samples
abstract: Helicobacter pylori has been isolated from the human stomach with media containing only minimal selective agents. However, current research on the transmission and sources of infection requires more selective media due to the higher numbers of contaminants in environmental, oral, and fecal samples. The objective of this study was to develop and evaluate detection techniques that are sufficiently selective to isolate H. pylori from potential animal and food sources. Since H. pylori survives in the acidic environment of the stomach, low pH with added urea was studied as a potential selective combination. H. pylori grew fairly well on H. pylori Special Peptone plating medium supplemented with 10 mM urea at pH 4.5, but this pH did not sufficiently inhibit the growth of contaminants. Various antibiotic combinations were then compared, and a combination consisting of 10 mg of vancomycin per liter, 5 mg of amphotericin B per liter, 10 mg of cefsulodin per liter, 62,000 IU of polymyxin B sulfate per liter, 40 mg of trimethoprim per liter, and 20 mg of sulfamethoxazole per liter proved to be highly selective but still allowed robust colonies of H. pylori to grow. This medium was highly selective for recovering H. pylori from cattle and beef samples, and it is possible that it could be used to enhance the recovery of this bacterium from human and environmental samples, which may be contaminated with large numbers of competing microorganisms.

title: Resuscitation of Vibrio cholerae O1 strain TSI-4 from a viable but nonculturable state by heat shock.
abstract: Vibrio cholerae strain TSI-4 was incubated in an M9 salt solution at 15 degrees C for more than 100 days. The plate counts showed no viable cells on day 30, but a broth culture from that day showed the growth of bacteria. However, after 35 days the bacteria entered the nonculturable state, based on the assessment of both the plate counts and broth culture. A portion of the culture was heated at 45 degrees C for 1 min in a water bath and subsequently plated onto a nutrient agar plate. More than 1000 colonies were recovered after this heat-shock treatment. The recovered cells showed the same chromosomal DNA pattern in the restriction map and the same outer membrane protein pattern in SDS-PAGE. Recovery of viable cells by heat-shock was achieved in cultures grown on M9 salt but not from cultures grown in phosphate-buffered saline. This suggests that the presence of NH4Cl in the M9 salt solution may support the growth of the bacteria in a low nutrient medium, while also playing an important role in resuscitation.

title: Helicobacter pylori Growth and Urease Detection in the Chemically Defined Medium Ham's F-12 Nutrient Mixture
abstract: Obstacles continue to hinder in vitro studies of the gastric human pathogen Helicobacter pylori, including difficulty culturing the organism in the absence of serum or blood, rapid loss of viability following exponential growth due to autolysis, and the necessity for using high starting inocula. We demonstrate thatH. pylori grows in the chemically defined broth medium Ham's F-12 nutrient mixture (F-12) in the absence of fetal bovine serum (FBS); this represents a breakthrough for studies in which serum components or proteins interfere with interpretation of results. Cultures can be continually passaged in fresh, FBS-free F-12 medium at an initial inoculum of only ∼103 CFU/ml. AllH. pylori strains (n = 21), including fresh clinical isolates, grew in serum-free F-12. H. pylori grew poorly in the related medium, F-10, unless additional zinc was supplied. Enhanced growth of H. pylori in F-12 broth was obtained by addition of bovine serum albumin (BSA) (1 mg/ml), β-cyclodextrin (200 μg/ml), or cholesterol (50 μg/ml). H. pylori also grew in several simplified versions of F-12 broth lacking glucose and most vitamins but containing hypoxanthine, pyruvate, and all 20 amino acids. On F-12 medium solidified with agar, H. pylori only grew when BSA (98% pure; 1 mg/ml), cholesterol (50 μg/ml), β-cyclodextrin (200 μg/ml), or FBS (2 to 4%) was added; addition of urea and phenol allowed colorimetric detection of urease activity. Thus, F-12 agar plus cholesterol or β-cyclodextrin represents the first transparent chemically defined agar and the first urease indicator agar forH. pylori. Several lines of evidence suggested that BSA itself is not responsible for H. pylori growth enhancement in F-12 containing BSA or FBS. Taken together, these innovations represent significant advances in the cultivation and recovery of H. pylori using chemically defined media. Use of F-12 or its derivatives may lead to improved understanding ofH. pylori metabolism, virulence factors, and transmission, and result in improved recovery and identification ofH. pylori from clinical specimens.

title: Nutritional Requirements and Antibiotic Resistance Patterns of Helicobacter Species in Chemically Defined Media
abstract: The growth of the gastric pathogen Helicobacter pylori in the absence of serum remains challenging, and nutritional requirements have only partially been defined, while almost nothing is known about nutritional requirements of other Helicobacter spp. Although previous data showed that H. pylori grows in the chemically defined medium F-12, but not in other tissue culture media examined, the specific components responsible for growth were not entirely understood. Here we describe the optimization of amino acids, metals, and sodium chloride for H. pylori. Iron, zinc, and magnesium were critical for growth; copper was not required. Optimization of sodium chloride was further beneficial. Nutritional requirements and antibiotic resistance patterns of several other Helicobacter spp. revealed that all except H. felis grew in serum-free, unsupplemented F-12. All Helicobacter spp. were resistant to at least six antimicrobial agents when cultured in the presence of serum. However, in the absence of serum, H. pylori, H. mustelae, and H. muridarum became sensitive to polymyxin B and/or trimethoprim. Much of the data were obtained using a convenient ATP assay to quantify growth. H. pylori has surprisingly few absolute requirements for growth: 9 amino acids, sodium and potassium chloride, thiamine, iron, zinc, magnesium, hypoxanthine, and pyruvate. These data suggest that H. pylori and other Helicobacter spp. are not as fastidious as previously thought. The data also suggest that chemically defined media described herein could yield the growth of a wide range of Helicobacter spp., allowing a more detailed characterization of Helicobacter physiology and interactions with host cells.

title: Factors affecting growth and susceptibility testing of Helicobacter pylori in liquid media
abstract: In order to increase the database for in vitro growth and/or susceptibility testing in liquid media, we evaluated the growth of Helicobacter pylori in broth media containing 5% sheep blood. We also compared the effect of bismuth on the growth of H. pylori in broth media containing 10% fetal calf serum with the effect on growth in media containing 0.5% starch. In contrast to the result seen with agar, we found that sheep blood, whether whole or laked, inhibited the growth of H. pylori in broth media. In addition, we found that bismuth inhibited growth in media with starch but that this inhibition was negated in media with serum.

title: The effect of oxygen on the growth and cell morphology of Helicobacter pylori.
abstract: The in vitro effect of progressive oxygen decrease on the growth and morphology of Helicobacter pylori was studied. H. pylori ATCC 43,504 was used for the experiments. The strain inoculated in Brucella broth plus fetal calf serum was incubated under a controlled atmosphere with oxygen concentration from 5 to 0%. CFU ml-1 and bacterial morphology were detected at the time of spreading and at 24 h, 72 h, 7 days and 14 days. A detailed ultrastructural investigation of the bacterial cells, grown in different experimental conditions, was performed by scanning electron microscopy. Oxygen deprivation produced a rapid reduction of CFU ml-1. In particular, a significant reduction of viable bacteria was recorded at 72 h of incubation in the presence of 1% oxygen and anaerobiosis, and 0 CFU ml-1 was found after 7 days of incubation at the above mentioned oxygen concentrations. The coccoid phenotype was already prevalent after 24 h of incubation with a progressive tendency to aggregate in clusters. These clusters were progressively larger, depending on the reduction of oxygen concentration, since the aggregation phenomenon can be the expression of a hypothesized mechanism of protection among bacterial cells.

title: Adhesion of water stressed Helicobacter pylori to abiotic surfaces
abstract: The main aim of this work was to study and compare the adhesion of water exposed Helicobacter pylori to six different substrata and correlate any changes in morphology, physiology, ability to form aggregates and cultivability when in the planktonic or in the sessile phase.

title: Nutrient Shock and Incubation Atmosphere Influence Recovery of Culturable Helicobacter pylori from Water
abstract: Three different media—Columbia agar, Wilkins-Chalgren agar, and Helicobacter pylori special peptone agar—were prepared in a diluted version and compared to the standard medium formulation in order to study a possible nutrient shock effect observed when recovering H. pylori from water by counting the number of CFU. This same parameter was subsequently used to evaluate the influence of the incubation atmosphere by using a modular atmosphere-controlled system to provide different atmospheres and by employing an established gas generation kit as a control. Both a low nutrient content of the media and a rapidly achieved microaerophilic incubation atmosphere proved to increase the numbers of environment-stressed H. pylori organisms recovered. An atmosphere of 5% CO2, 5% O2, and 3% H2 is recommended, although other atmospheres with a low oxygen concentration are also acceptable. Besides highlighting and assessing the importance of several factors in the culturability of H. pylori, this paper demonstrates the potential ability to develop an optimized technique for recovery of this pathogen from water.