WO2001088183A2 - Method for the isolation of helicobacter pylori - Google Patents

Abstract

Methods and kits are disclosed for isolating urease-positive bacteria by exposing a sample for 1 to 60 minutes to a media containing urea along with simultaneous or subsequent exposure to pH below 3.0. In one embodiment, the bacteria is H. pylori and the acidic conditions are provided by addition of HC1. These methods and kits are especially useful for isolating or detecting H. pylori in samples, such as saliva samples, contaminated by other microorganisms.

Description

NOVEL METHOD FOR THE ISOLATION OF HELICOBACTER PYLORI FROM HIGHLY CONTAMINATED SPECIMENS

Field of the Invention The present invention relates to methods and kits for isolating H. pylori.

More specifically, this invention relates to methods and kits for isolation of H. pylori from highly contaminated human and environmental samples.

Background Helicobacter pylori infects approximately 50% of the world population

(Goodwin et al., "Helicobacter pylori infection," Lancet 349:265-269 (1997)). Much is now known about this organism's genome, its mechanisms of pathogenesis, and its epidemiology (Malfertheiner et al., "Helicobacter pylori, the year in 1997," Current Opinion in Gastroenterology 13 (suppl 1) 1-62 (1997)). In addition, a number of techniques have been described enabling isolation of H. pylori from gastric biopsies during diagnostic upper gastrointestinal endoscopy (Malfertheiner et al. (1997)). Very few laboratories, however, have been able to report the successful isolation of H. pylori from extra-gastric sites (Banatvala et al., "Helicobacter pylori in dental plaque," Lancet 341 :380 (1993); Furguson et al., "Isolation of Helicobacter pylori from saliva," J Clin. Microbiol. 31 :2802-2804 (1993); Bernarder et al, "Absence of Helicobacter pylori in dental plaque in Helicobacter pylori positive dyspeptic patients," Eur. J. Clin. Microbiol. Infect. Dis. 12:282-285 (1993); Thomas et al., "Isolation of Helicobacter pylori from human feces," Lancet 340:1194-1195 (1992)). Furthermore, attempts to reisolate H. pylori cultures from contaminated samples have met with very little success (Banatvala et al. (1993); Furguson et al. (1993); Bernarder et al. (1993); Thomas et al. (1992); and Jonkers et al., "Influence of oropharyngeal flora and specimen pretreatment on the recovery of Helicobacter pylori " Eur. J. Clin. Microbiol. Infect. Dis. 15:378-382 (1996)). Improved molecular techniques have demonstrated the presence of H pylori DNA in feces, saliva, dental plaque, and water through PCR assays (Song et al., "Helicobacter pylori in the dental plaque. A comparison of different PCR primer sets," Dig. Dis. Sci. 44:479-484 (1999); Song et al., "Characteristic pattern of Helicobacter pylori in dental plaque and saliva detected with nested PCR," J. Medical. Microbiol. 49:349- 353 (2000); Li et al., "High prevalence of Helicobacter pylori in saliva demonstrated by a novel PCR assay," J Clin. Pathol. 48:662-666 (1995); Li et al, "A newly developed PCR assay of H. pylori in gastric biopsy, saliva and feces. Evidence of high prevalence of H. pylori in saliva supports oral transmission," Dig. Dis. Sci. , 41:2142-2149 (1996); Casswall et al., "Evaluation of serology, ¹³C-urea breath test, and polymerase chain reaction of stool samples to detect Helicobacter pylori in Bangladeshi children," J. Pediatric. Gastroenterol. Nutr. 28:31-36 (1999); Gramley et al., "Detection of Helicobacter pylori DNA in fecal samples from infected individuals," J. Clin. Microbiol. 37:2236-2240 (1999); and Ηulten et al.,

"Helicobacter pylori in the drinking water in Peru," Gastroenterology 110:1031- 1035 (1996)). However, molecular techniques, such as PCR assays, only detect a few specific H. pylori DNA sequences. Therefore, there remains a need for the development of other techniques for the isolation of intact H. pylori cells from these samples in order to study the H pylori isolates for other characteristics, e.g., antibiotic resistance, microbiological properties, biochemical properties, and the like.

Current methods utilized by clinical laboratories for the isolation of H. pylori from contaminated specimens use antibiotic-containing selective media (e.g., Skirrows media (Tee et al, "Comparative evaluation of three selective media and nonselective medium for the culture of Helicobacter pylori from gastric biopsies," J. Clin. Microbiol. 29:2587-2589 (1991)). However, the majority, if not all, of these commercially available selective media are only suitable for the isolation of H. pylori from lightly contaminated samples, like the majority of gastric biopsies. There remains a need for a method for the isolation of H. pylori from specimens which are highly contaminated, like saliva, dental plaque, or feces (Jonkers et al. (1996)). Furthermore, attempts to culture H. pylori from gastric biopsies, particularly from patients with decreased gastric acid production, frequently result in excessive overgrowth of other microorganisms even when employing selective media (Jonkers et al. (1996); Stockbrugger et al., "Intragastric nitrites, nitrosamines and bacterial overgrowth during cimetidine treatment," Gut 23:1048-1054 (1982); Verdu et al., "Effect of omeprazole on intrgastric bacterial counts, nitrates, nitrites and nitroso compounds," Gut 35:455-460 (1994); Dent et al., "Evaluation of a new selective medium for Campylobacter pylori ," Eur. J. Clin. Microbiol. Infect. Dis. 7:555-558 (1988); and Stolte et al., "Elimination of Helicobacter pylori under treatment with omeprazole," Zeitschriβ fur Gastroenterologie 28:271-274 (1990)). For example, Jonkers et al. showed that the isolation of low numbers (10³ CFU/ml) of H. pylori in the presence of high numbers of oropharygeal microflora (>10⁴ CFU/ml) was very difficult despite the use of selective media (Jonkers et al. (1996)).

Summary Methods and kits are disclosed for isolating urease-positive bacteria, which involve exposure of a sample to urea during or before exposure to acidic conditions. In one embodiment, the urease-positive bacteria is H. pylori. These methods and kits are especially useful for isolating or detecting H. pylori in cultures or samples highly contaminated with other microorganisms.

Brief Description of The Drawings FIGURE 1 is digital image showing a comparison of the urea/ΗCl method and a standard selective media method using Skirrow's media for isolation of H. pylori from contaminated samples. The left side of the figure, digital images marked "A," provides results for samples processed using the urea/ΗCl method. The right side of the figure, digital images marked "B," provides results for samples after direct application to Skirrow's media and incubation. Sample "a" is 0.1 ml saliva containing initial 1000 CFUs of added H. pylori; sample "b" is a homogenized gastric biopsy; and sample "c" is a highly contaminated H. pylori culture.

Detailed Description of Specific Embodiments H. pylori is a slow-growing and fastidious organism and may exist in extra- gastric sites in very low numbers (Song et al. (1999) and Song et al., "Quantitation of H. pylori by cPCR," J. Clin. Pathol. 53:218-222 (2000)). It is likely, therefore, that in culturing samples such as dental plaque, saliva, feces or water, containing low numbers of H. pylori, successful growth of H. pylori is inhibited by larger populations of fast-growing competitive microorganisms. Thus, H. pylori, although present in these samples, often is not detected. Therefore, there remains a need for a method to isolate H. pylori from samples containing relatively low numbers of H. pylori in the presence of large numbers of background microorganisms. These methods are disclosed herein. An important virulence property of H. pylori is the enzyme urease, which resides both inside the cell cytoplasm and on the surface of the cell membrane (Dunn et al., "Purification and characterization of urease from Helicobacter pylori," J. Biol. Chem. 265:9464-9469 (1990) and Ηawtin et al., "Investigation of the structure and localization of the urease of Helicobacter pylori using monoclonal antibody," J. Gen. Microbiol. 136:1995-2000 (1990)). H. pylori urease can rapidly hydrolyze urea, resulting in a basic "ammonia cloud" around the bacterium, thereby protecting H. pylori from gastric acid in the human stomach (Lee et al., "Basic bacteriology of H. pylori: H. pylori colonization factors," Hunt, R.H. and Tytgat, G.N.J., eds. In Helicobater pylori: Basic mechanisms to clinical cure, Kluwer Academic Publications, Boston 59-72 (1994)). Although a few other bacterial species (e.g., Proteus sp.) also produce urease, they do so at relatively low levels compared to H pylori. Additionally, the H pylori urease binds urea with greater affinity than the urease enzymes produced by other bacteria, which is demonstrated by the low K_m (substrate concentration at which an enzyme-catalyzed reaction proceeds at one-half maximum velocity) of the H pylori urease enzyme (Mobley et al., "Characterization of urease from Campylobacter pylori," J. Clin. Microbiol. 26:831-836 (1988)). Therefore, H. pylori urease confers a higher rate of urea hydrolysis, and subsequently produces larger quantities of ammonia more rapidly than other organisms, resulting in greater acid-tolerance capabilities for H pylori compared to other microorganisms in the presence of urea (Dunn et al. (1990);

Mobley et al. (1988); Hu et al., "Purification and N-terminal analysis of urease from Helicobacter pylori," Infect. Immun. 58:992-998 (1990); Evans et al., "Characterization of the Helicobacter pylori urease and purification of its subunits," Microb. Pathog. 10:15-26 (1991); and Ferrero et al., "The importance of urease in acid protection for the gastric-colonizing bacteria Helicobacter pylori and

Helicobacter felis sp nov," Microb. Ecol. Health Dis. 4:121-134 (1991)). The ability of H. pylori to produce extraordinary quantities of urease has been used in the clinical setting with specific detection assays for H pylori (e.g., CLO-test, urease breath test and serology). However, prior to the present disclosure, the urease enzyme has not been exploited for use in the isolation of this bacterium from contaminated samples. In the absence of urea, the optimal pH for H. pylori growth has been shown to be 5.5 to 8.5 (Morgan et al., "Growth of Campylobacter pylori in liquid media," J. Clin. Microbiol. 25:2123-2125 (1987) and Kangatharalingam et al., "Helicobacter pylori comb. nov. exhibits facultative acidophilism and obligate microaerophilism," Appl. Environ. Microbiol. 60:2176-2179 (1994)). Under these conditions, H. pylori cells demonstrated a high sensitivity to acid (Morgan et al. (1987) and

Kangatharalingam et al. (1994)). Other investigations have suggested that in the presence of urea, H. pylori survives at pH greater than 2.0 (Kangatharalingam et al. (1994); Jiang et al., "Effect of environment and substrate factors on survival and growth of Helicobacter pylori," J. Food Protection 61:929-933 (1998); Clyne et al., "Helicobacter pylori requires an acidic environment to survive in the presence of urea," Infect. Immun. 63:1669-1673 (1995); and Hunt, R.H., "Hp and pH: implication for the eradication of Helicobacter pylori," Scand. J. Gastroenterol. 28 (suppl 196):12-16 (1993)). The addition of 8 mM urea enhanced survival of H. pylori at pΗ 3.5 (Jiang et al. (1998)). However, other studies have shown that H pylori could not survive at pH values less than, or equal to, pH 2.2 even in presence of urea (Kangatharalingam et al. (1994); Jiang et al. (1998); Clyne et al. (1995); Hunt, R.H. (1993)). In the majority of these investigations, H pylori was exposed to low pH conditions for more than 30 minutes (Kangatharalingam et al. (1994); Jiang et al. (1998); Clyne et al. (1995); Hunt, R.H. (1993)). Recent studies demonstrated that when the exposure time to acid was 30 minutes or less, H pylori could survive under conditions as low as pH 2.0 (Krishnamurthy et al., "Helicobacter pylori containing only cytoplasmic urease is susceptible to acid," Infect. Immun. 66:5060- 5066 (1998)). Clyne et al. ("Helicobacter pylori requires an acidic environment to survive in the presence of urea," Infection and Immunity 63:1669 (1995)) analyzed the growth of pure cultures of H. pylori on Columbia blood agar plates after exposure to 10 mM urea at pH values ranging from 2.2 to 10.0. In addition, a mathematical model has been used to investigate a temporary protection of H. pylori by urea if the pH is less than 2.5 (Chen et al., "Helicobacter pylori survival in gastric mucosa by generation of a pH gradient," Biophysical J. 73:1081-1088 (1997)). Krishnamurthy et al. ^ Helicobacter pylori containing only cytoplasmic urease is susceptible to acid," Infection and Immunity, 66:5060, (1998)) provide data indicating that H pylori containing only cytoplasmic, and not surface urease, may be susceptible to acid in the presence of 5 mM urea. These authors analyzed the growth of pure cultures of H. pylori on trypticase soy agar containing sheep blood after exposure to a citric acid-HCl buffer at pH 2.0 for 30 minutes in the presence of 5 mM urea. Disclosed herein are the first methods that utilize urea and an acidic pH to isolate H pylori from a sample. Specifically, the conditions are disclosed herein are of use to isolate H. pylori from samples containing other microorganisms that may interfere with the isolation of H. pylori. Thus, methods and kits are provided for isolating urease-positive bacteria, including H. pylori, from highly contaminated cultures or samples.

A method is disclosed herein for isolating urease-positive bacteria from a sample suspected of containing urease-positive bacteria and which may contain other microorganisms. The method includes: (a) adding to the sample an amount of urea sufficient to protect urease- positive bacteria that may be present in the sample from an acidic pΗ;

(b) adding an acid to the sample to obtain the acidic pΗ;

(c) incubating the sample at the acidic pΗ under a first set of conditions sufficient to render the other microorganisms non- viable, but wherein the urease- positive bacteria remain viable, thereby producing a treated sample;

(d) applying the treated sample to a growth media; and

(e) incubating the treated sample on the growth media under a second set of conditions that support growth of the urease-positive bacteria, thereby isolating the urease-positive bacteria. In one embodiment, the sample is a highly contaminated specimen from a gastric biopsy, saliva, dental plaque, endoscopy equipment, an environmental source, a contaminated Helicobacter pylori culture, or stool. In another embodiment, the method further comprises increasing the pH of the treated sample to a pH that further supports growth of the urease-positive bacteria on the growth media. In a further embodiment, the urease-positive bacteria is selected from the genera consisting of Helicobacter, Proteus, and Yersinia. In one specific, non- limiting example, the urease-positive bacteria is Helicobacter pylori.

In yet another embodiment, referred to hereinafter as the general urea/HCl H pylori method, the urease positive bacteria are H pylori and the acid is HCl. For the general urea/HCl H. pylori method, the source of the sample is saliva. In one embodiment of the general urea/HCl H pylori method, the effective concentration of urea is between about 0.04 mM and about 1 M and HCl is at a concentration of between about 0.002 N and about 0.48 N. In another embodiment of the general urea HCl H pylori method, the effective concentration of urea is between about 0.31 mM and about 500 mM and HCl is at a concentration of between about 0.03 N and about 0.24 N. In yet another embodiment of the general urea/HCl H. pylori method, the effective concentration of urea is between about 5 mM and about 200 mM andHCl is at a concentration of between about 0.06 N and about 0.12 N.

In a further embodiment of the general urea/HCl H. pylori method, the incubation time is between about 1 minute and about 20 minutes. Alternatively, the incubation time is between about 2 minutes and about 10 minutes. In one embodiment of the general urea HCl H pylori method, the growth media is selected from the group consisting of heart infusion agar with rabbit blood and Skirrow's media.

In one specific, non-limiting example, when the source of the sample is saliva, and the general urea/HCl H. pylori method is utilized, the effective concentration of urea is between about 20 mM and about 1 M and the effective concentration of HCl is between about 0.06 N and about 0.24 N. In one embodiment of this embodiment, the growth media is selected form the group consisting of heart infusion agar with rabbit blood and Skirrow's media. In another embodiment, the concentration of urea is between about 20 mM and about 1 M and HCl is at a concentration of between about 0.03 N and about 0.24 N. In another embodiment, the incubation time is between about 1 minute and about 20 minutes, or the incubation time is between about 2 minutes and about 10 minutes. In one embodiment of the general urea/HCl H. pylori method, the effective pH is less than about 2.7. In another embodiment of the general urea/HCl H. pylori method, the acidic pH is less than about 1.5.

In another embodiment of the general urea/HCl H pylori method, the method further includes filtering the sample using a filter before exposure of the sample to an effective concentration of urea. In this embodiment, exposure of the sample to a concentration of urea and exposure of the cells to hydrochloric acid may be carried out on the filter. In yet another embodiment, the method further includes placing the filter on growth media after the sample is exposed to an effective concentration of urea and an effective amount of hydrochloric acid. In one specific, non-limiting example, the filter is hydrophobic. In another specific, non-limiting example, the filter has a grid pattern.

A method is disclosed herein for isolating urease-positive bacteria from a sample suspected of containing urease-positive bacteria and which may contain other microorganisms. The method includes

(a) adding to the sample an amount of urea and an amount of acid, wherein the amount of urea is sufficient to protect urease-positive bacteria that are present in the sample from an acidic pH, and wherein the amount of acid is sufficient to result in an acidic pH; (b) incubating the sample at the acidic pH under a first set of conditions sufficient to render the other microorganisms non-viable, but wherein the urease- positive bacteria remain viable, thereby producing a treated sample;

(c) applying the treated sample to growth media; and

(d) incubating the treated sample on the growth media under a second set of conditions that support growth of the urease-positive bacteria, thereby isolating the urease-positive bacteria. Method for isolating H. pylori from contaminated specimens.

In one embodiment, a method is disclosed for isolating urease-positive bacteria from a sample. The method comprises:

(a) exposing the sample to a concentration of urea in a urea solution, wherein the concentration of urea is sufficient to protect the urease-positive bacteria from an acidic pH;

(b) acidifying the urea solution to the acidic pH using an acid;

(c) incubating the sample at the acidic pH for an incubation time sufficient to inhibit growth of a urease-negative, but not the urease-positive bacteria, thereby producing a treated sample;

(d) applying the treated sample to growth media; and

(e) incubating the treated sample under conditions that support growth of the urease-positive bacteria, thereby isolating the urease-positive bacteria.

After incubating the sample at the acidic pH for an incubation time, the pH of the sample may be increased to facilitate growth of the urease-positive bacteria before application of the bacteria to a growth media. In one embodiment, the method further comprises diluting the treated sample with a diluent before incubating the treated sample on the growth media. This diluent is typically a buffered solution such as phosphate buffered saline (PBS), buffered to a pH sufficient to increase the pH of the sample in urea and acid to about 5.5-7.4, or from about 6.6 to about 7.0. The step of diluting the treated sample with a diluent may result in greater than 100-fold dilution of the acid. In another embodiment, bacteria are pelleted by centrifugation after incubation at the acidic pH for the effective incubation time, and resuspended in a buffered solution, for example PBS, of a pH of between about 6.3 to about 7.2 before incubating the treated sample after application to a growth media.

The step of incubating the sample after application to the growth media is carried out for a time sufficient to detect growth of the urease-positive bacteria. One of ordinary skill in the art using methods known in the art combined with the current disclosure can determine specific protocols including growth conditions for growing urease-positive bacteria such as H pylori in the final step of the method. This protocol may consist of, but is not limited to, applying a sample (e.g., about 100 μl) of the treated sample to a growth media such as heart infustion agar (HIA) plates with 5% rabbit blood (BBL, Cockeysville, MD), and incubating the plated sample for about 5 days at 37° C under microaerobic conditions (N₂ 85%, CO₂ 10%, O₂ 5%). After such incubation, colonies can be counted and numbers of H. pylori can be assessed quantitatively by determining the number of colony forming units (CFU). For studies aimed at the determination of the optimal conditions for isolating particular urease-positive bacteria from a particular source, an ordinary artisan can determine survival of the urease-positive bacteria under a set of conditions, expressed as percent of an initial inoculum. Additionally, or to determine the initial inoculum, parallel controls can be employed under the same conditions to those used for acid challenge, except that bacterial controls are incubated with PBS instead of the acid and urea solutions.

A "growth media" is any media that promotes the growth of the urease- positive bacteria. Many effective growth media for urease-positive bacteria are known in the art and available from many media supply companies (e.g., BBL, Cockeysville, MD). For H. pylori, many effective growth media, including both selective and non-selective growth media, are known. Selective growth media are supplemented with antibiotics and support growth of H. pylori over other organisms. Selective growth media include, but are not limited to, supplemented blood agar media. Examples of blood agar media that are effective growth media for H. pylori include, but are not limited to, heart infusion agar (ΗIA) with rabbit blood at, for example, a concentration of 5% rabbit blood, trypticase soy agar containing sheep blood, Columbia blood agar containing defibrinated horse serum at, for example, a concentration of 7%, sheep blood agar at, for example, a concentration of 7%, and fetal calf serum in agar.

For H. pylori selective media, typically blood agar media are supplemented with antibiotics. Examples of selective H. pylori media include, but are not limited to, Skirrow's medium, Dent's CP medium, and Glupszynski's Brussels campylobacter charcoal (BCC) medium (Tee et al., 1991). When a selective media is used, the effective acidic pΗ may be increased due to the additional inhibitory effect on growth of contaminating bacteria by the selective media. It is more desirable to use selective media where contamination levels in a sample are very high. One of ordinary skill, using this disclosure in combination with known methods, can readily determine an optimal growth media. Although the current method is effective for isolating urease-positive bacteria from any sample, including pure culture samples, the method is particularly useful where the sample is suspected of containing microorganisms other than urease-positive bacteria. In one embodiment, the method is used to isolate urease-positive bacteria where the sample contains relatively large quantities of microorganisms other than urease-positive bacteria (i.e., highly contaminated samples).

"Urease positive" refers to bacteria that express the enzyme urease. Urease is an enzyme which catalyzes the conversion of urea into ammonium and carbon dioxide. Many urease-positive bacteria are known in the art. The present methods and kits may be applicable to any urease-positive bacteria. Candidate genera (urease-positive) are Agrobacterium, Alcaligenes, Anabaena, Anacystis, Arthrobacter, Aspergillus, Bacillus, Bacteroides, Bilophila, Chromatism, Clostridium, Corynebacterium, Cryptococcus, Haemophilus, Helicobacter,

Klebsiella, Microccus, Morganella, Mycobacterium, Neurospora, Paracoccus, Peptostreptococcus, Proteus, Providencia, Pseudomomas, Rhizobium, Rhodotorula, Selenomanas, Sporosarcina, Staphylococcus, Thiocapsa, Thiocystis, Trichosporon, Yersinia, etc. (Mobley, et al. Microbial Ureases. Microbiol Rev. 53:85-108 (1989); Balows A, et al, eds. Manual of Clinical Microbiology, American Society for Microbiology, Washington, DC (1991)). It is likely that the isolation method described herein can be successfully employed for other urease-positive bacterium if milder conditions (i.e., a higher pH or a different acid) are used and the urease enzyme is excreted by the microbe or associated with the cell membrane or periplasmic space to counter-act the acidic environment. However, urease activity can vary widely between genera and between species or even subspecies within a genus. One of ordinary skill can use well-known techniques to optimize assays for specific purposes. In one embodiment, the urease-positive bacteria is selected from the genera consisting of Helicobacter, and Proteus. In one specific, non-limiting example, the urease-positive bacteria is a gastric Helicobacter. These gastric

Helicobacter include, but are not limited to, H pylori, H. heilmannii, H.felis, H mustelae, H. nemestrinae, and H acinonyx (Megraud, "H. pylori species heterogeneity", Hunt, R.H. and Tytgat, G.N.J., eds. In Helicobacter pylori: Basic Mechanisms to clinical cure. Kluwer Academic Publishers, Boston, 28-40 (1994)). In another specific non-limiting example, the urease-positive bacteria is Helicobacter pylori. By "Helicobacter pylori" or "H pylori" is meant all strains of the bacterium

Helicobacter pylori. Helicobacter pylori are microaerophilic, Gram-negative organisms which appear curved in tissue sample, and are more often rod-like, U- shaped, or circular in culture. Helicobacter pylori have a tuft of polar, sheathed flagelli and exhibit optimal motility in a highly viscous milieu such as that found in the gastric mucous. Helicobacter pylori exhibit very high urease and catalase activity and do not metabolize sugars.

A wide- variety of acids are useful for the methods disclosed herein to adjust the pΗ of a solution to the desired acidic pΗ. Many acids are well-known by those of ordinary skill in the art. One of ordinary skill in the art can readily determine which acids can be used to adjust the pΗ of a solution to less than about 2.7. These include, but are not limited to, lactic acid, acetic acid, malic acid, propionic acid, sulfuric acid, nitric acid, and hydrochloric acid. In one embodiment, the acid is lactic acid, acetic acid, malic acid, propanic acid, or hydrochloric acid. In one specific, non-limiting example, the acid is hydrochloric acid. It has been observed that urease-positive bacteria, for example, H pylori, can survive exposure at an effective acidic pΗ provided that the bacteria are exposed to a urea solution before or during acid exposure. The urease-positive bacteria can be isolated after this exposure, with the subsequent elimination of virtually all other microorganisms, (i.e., urease-deficient microorganisms). Although not wishing to be limited by theory, the fundamental mechanism believed to be involved in the survival of urease-positive bacteria under the conditions disclosed herein is the production of large amounts of the enzyme urease by the urease-positive bacteria, with subsequent hydrolysis of urea in the media resulting in the production of ammonia and carbon dioxide. A basic pΗ "ammonia cloud" is formed which surrounds the urease-positive organisms, thus protecting the individual cells from damage by the acidic pΗ of the media. By "acidic pH" is meant a pH at which a particular urease-positive bacteria, but not a urease-negative bacteria, will survive after exposure for an effective incubation time, if the treatment follows, or is simultaneous with, exposure of the bacteria to urea. For H. pylori, the acidic pH is generally less than about 2.7. However, the precise optimal acidic pH is influenced by the strain of H. pylori to be isolated and by the amount of contaminating microorganisms in the sample. One of ordinary skill using this disclosure can readily determine the optimal acidic pH by taking into consideration the H pylori survival rate and the extent of H. pylori inhibition by other microorganisms in the sample. In one embodiment, the acidic pH for H. pylori is less than about 2.4. In another embodiment, the acidic pH for H. pylori is less than about 2.3. In yet another embodiment, for H. pylori, the acidic pH for H pylori is less than about 2.1. For some samples, especially those that are highly contaminated, the acidic pH for H pylori is less than about 1.6. In one specific, non-limiting example, the sample is highly contaminated, and the acidic pH for H. pylori is less than about 1.3. In another specific, non-limiting example, the sample is a highly contaminated samples, the acidic pH for H pylori is less than about 1.0.

The methods and kits disclosed herein are effective for samples isolated from any source in which urease-positive bacteria may exist. In one embodiment of the method for isolating H pylori, the sample is isolated from a source selected from the group consisting of a gastric biopsy, saliva, dental plaque, endoscopy equipment, an environmental source, a contaminated Helicobacter pylori culture, and stool. "Sample" refers to any specimen from which isolation or detection of the urease- positive bacteria is desired. For the general method of isolating H pylori, the sample may take many forms, including, but not limited to, tissue from biopsies, stool samples, liquid samples from environmental water sources, contaminated H. pylori cultures, saliva, swabbed samples from swabbed equipment or samples from dental plaque. Where the urease-positive bacteria is H. pylori, a source of the sample is saliva. The steps of exposing the sample to an effective concentration of urea in a urea solution and acidifying the urea solution to an effective acidic pH using an acid can be carried out in any order. However, it is important for the method that the urease-containing bacteria are not exposed to the effective acidic pH before being exposed to urea. In one embodiment, the urease-containing bacteria are exposed to urea while they are exposed to acid. In one embodiment, the sample is exposed to an effective concentration of urea in the urea solution before the urea solution is brought to an acidic pH with the acid. In another embodiment, the urea solution is brought to an effective acidic pH using the acid before the sample is exposed to an effective concentration of urea in the urea solution.

By "concentration of urea" is meant a concentration of urea that is sufficient to allow at least a portion of a population of urease-positive bacteria to protect themselves from subsequent exposure to an effective acidic pH. The concentration of urea used is dependent on the concentration of acid to which the urease-positive bacteria will be exposed. Where the bacteria isolated by a method disclosed herein are H. pylori, the concentration of urea is between about 0.04 mM and about 1 M when the effective concentration of HCl is between about 0.002 N and about 0.48 N. In one embodiment, the effective concentration of urea is between about 0.3 mM and about 500 mM and the effective concentration of HCl is between about 0.03 N and about 0.24 N. In another embodiment, the concentration of urea is between about 5 mM and about 200 mM and the effective concentration of HCl is between about 0.06 N and about 0.12 N. One of ordinary skill in the art can use these ranges of effective urea concentrations, combined with the pH of HCl solutions at the above-identified ranges as well as the results disclosed in the Example section of this disclosure, to determine effective urea concentrations for acids other than HCl.

By "incubation time" is meant a time at which a particular urease-positive bacteria, but not a urease-negative bacteria, will survive exposure at an effective acidic pH, if the treatment follows, or is simultaneous with, exposure of the bacteria to urea. For H pylori, the incubation time is typically between 1 minute and 60 minutes. In one embodiment, the incubation time is between 2 minutes and 10 minutes. However, the precise optimal incubation time is influenced by the precise pH used for acid exposure. In general, optimal incubation time is shorter as pH is lower for the step of exposure to acid. One of ordinary skill using this disclosure can readily determine the optimal incubation time by taking into consideration H pylori survival rate and inhibition of growth of other organisms in the sample. The step of exposing the sample to a concentration of urea, and the step of incubating the sample at the acidic pH can be carried out at a wide temperature range. The useful temperature range for these steps is limited only in that a portion of a population of urease-positive bacteria must be isolated after exposure to urea and acid in a selected temperature range. Typically, the steps are carried out at a temperature between about 4° C and about 42° C. In one embodiment, the steps are carried out at a temperature between about 15° C and about 37° C.

The acidic pH, concentration of urea, exposure temperatures, and incubation time are set at values that are specific for a particular source and a particular urease- positive bacteria. The specificity is due to the fact that certain types of samples may be highly contaminated, or may contain micro-organisms that are more resistant to exposure to acids, and different species and strains of a species of urease-positive bacteria may be more or less resistant to acid exposure after exposure to urea. One of ordinary skill can determine the concentration of urea, exposure temperature, and incubation time using this disclosure to determine conditions that are necessary to eliminate contaminating microorganisms, but allow survival of the urease-positive bacteria.

For example, where the sample is saliva and the urease-positive bacteria is H pylori, the concentration of urea is between about 20 mM and about 1 M and HCl is at a concentration of between about 0.06 N and about 0.24 N. In one embodiment, this method is carried out at temperatures between about 4° C and about 42° C and the step of incubating the sample at the acidic pH is earned out for between 1 minute and 60 minutes. In another embodiment, this method is carried out a temperature of between about 15° C and about 37° C and the effective incubation time is between about 2 minutes and about 10 minutes. Where the sample is a highly contaminated H. pylori culture sample, one set of conditions for the method includes exposure to a mixture of 0.06 N HCl and 80 mM urea for 5 minutes at 37° C. Filtering embodiment of general method of urease/acid treatment for isolating H. pylori from contaminated specimens.

In one embodiment of the method for the isolation of urease-positive bacteria, the method further comprises the step of filtering the sample through a membrane filter before the step of exposing the sample to an effective concentration of urea. Many filter membranes are known in the art having pore sizes which retain urease-positive bacteria, such as H. pylori. Typically, the pore size of the filter is no larger than about 1.0 μm. A large number of membrane filters with varied pore sizes are commercially available from Millipore, Gelman, Nuclepore, and other companies. For certain biological samples such as saliva and stool, a preclarification step may be used for this filtration embodiment. This preclarification step could be accomplished by filtering a homogenate of the sample through a large-pore membrane or by a brief centrifugation step.

After the filtration step, a series of washes with PBS can be employed to help remove inhibitory compounds and proteins that might be found in the sample. The steps of exposing the sample to an effective concentration of urea and incubating the sample at an effective pΗ can be carried out after the sample is applied to the filter. After the steps of exposure to urea and incubation at an effective acidic pΗ, the filter can be placed directly on growth media such as ΗIA with 5% rabbit blood. In one embodiment the filter is hydrophobic and has a grid pattern, thereby facilitating counting of colonies.

Kits for isolating H. pylori from contaminated specimens.

Another aspect is a kit for the isolation of urease positive bacteria, for example, Helicobacter pylori. The kit comprises:

(a) a first component comprising a urea solution of specified strength; and

(b) a second component comprising an acid solution of a specified type and strength. In one embodiment the acid is hydrochloric acid. In another embodiment the kit further comprises a third component comprising a growth media. In another embodiment of the kit, the first component and the second component are provided together in the kit as a first urea-acid component. In one embodiment the urea-acid component is a urea-HCl component. In a further embodiment, the components are provided in separate containers such as tubes, vials, or bottles.

The kit may contain other reagents and equipment useful in performing isolation of a urease-positive bacteria, including buffers, containers (e.g., test tubes, culture plates). Additionally, the kit may include instructions for the isolation of urease-positive bacteria using kit components.

Without being bound by theory, the following theoretical considerations regarding the present disclosure are provided. The present disclosure provides a new method employing different concentrations of urea, in the presence of hydrochloric acid, to allow H pylori isolation from highly contaminated specimens. Based on the results presented in the examples below, it appears that, in presence of urea, a short exposure to low pH will not damage H pylori cells significantly. Under these conditions, other microorganisms found in environmental or contaminated samples are eliminated, thereby facilitating culturing of H. pylori. Urea, a major constituent of the mucous layer overlying the gastric mucosa, appears to help H. pylori tolerate and survive deleterious acid effects. Data included herein shows that in the presence of appropriate concentrations of urea, H pylori could survive a short duration of exposure to pH values as low as 0.36.

Natural infection of H. pylori may be similar to the experimental conditions described herein. During natural infection, H. pylori and numerous other organisms enter the stomach through the mouth (Verdu et al., "Effect of omeprazole on intragastric bacterial counts, nitrates, nitrites and nitroso compounds," Gut 35:455- 460 (1994) and Drasar et al., "Studies on the intestinal flora," Gastroenterology 56:71-79 (1969)). The median luminal pΗ of the human stomach is about 1.4 (1.0- 6.0) (Rektorschek et al., "Influence of pΗ on metabolism and urease activity of Helicobacter pylori," Gastoenterology 115:628-641 (1998)). Prior to reaching its physiological niche, adherent to the gastric mucosa and under the overlying mucus layer (Ηazell et al., "Campylobacter pyloridis and gastritis. With intercellular spaces and adaptation to an environment of mucus as important factors in colonization of the gastric epithelium," J. Infect. Dis. 153:658-663 (1986); Sarosiek et al., "Breakdown of the mucus layer by H. pylori," Hunt, R.H. and Tytgat, G.NJ. eds. In Helicobater pylori: Basic mechanisms to clinical cure. Kluwer Academic Publications, Boston 59-72 (1994); Engel et al., "Analysis of hydrogen ion concentration in the gastric gel mucus layer," Am. J. Phsiol. 247:G321-338 (1984)), H. pylori must penetrate the acidic gastric mucus layer, probably by utilizing its flagella, urease production, and other mechanisms of acid resistance (Perm et al., "The flagella of Helicobacter pylori : molecular analysis and potential in pathogenesis," G. Gasbarrini and S. Pretolani eds. In Basic and clinical aspects of Helicobacter pylori infection. Springer-Verlag, Berlin 60-64 (1994)). It is postulated that acid exposure in the human stomach is relatively short. During this period it is believed that H. pylori urease hydrolyzes urea found in the gastric mucus and thereby protects the bacterium from ΗC1 damage. If, however, H. pylori is exposed to an extremely low pΗ for an extended period of time, it is felt that urease may become "exhausted" and that new urease cannot be synthesized due to little or no metabolic activity under these conditions (Rektorschek et al. (1998)). Therefore, prolonged exposure to acid in the host gastric lumen minimizes the survival of viable H. pylori.

The results described herein demonstrated that H. pylori cannot survive pΗ values less than about 2 for durations of greater than about an hour even in the presence of urea. Other microorganisms are quickly killed by ΗC1, probably due to a lack of urease in our in vitro experiments, and probably due to a lack of urease and/or a lack of motility (flagella) in the human stomach. In fact, other investigations have demonstrated that urease-negative mutants of Helicobacter sp are unable to colonize animal models (Tsuda et al., "A urease-negative mutant of Helicobacter pylori constructed by allelic exchange mutagenesis lacks the ability to colonize the nude stomach," Infect Immun 62:3586-3589 (1994) and Andrutis et al., "Inability of an isogenic urease-negative mutant strain of Helicobacter mustelae to colonize the fenet stomach," Infect Immun 63:3722-3725 (1995)).

The results described herein (Table 1) and other research (Rektorschek et al. (1998)) suggest that in the absence of urea, even a very short exposure to acid damages H. pylori. Conversely, H. pylori survived exposure to ΗC1 after addition of urea or when both urea and ΗC1 were added concurrently. However, no survival was demonstrated when H. pylori was exposed to acid prior to the addition of urea. Therefore, when extrapolating from our in vitro model to the host, H. pylori is quite sensitive to acid, and the presence of urea appears to be essential for H. pylori transit through the gastric lumen. The studies described herein may represent a model of the natural process of H. pylori colonization of the human stomach by demonstrating transient survival of H. pylori under extremely acidic conditions in the presence of urea.

In this study, changes of H. pylori concentration (range: 10² to 10⁷ CFU/ml) incubated in a mixture of saliva, ΗC1, and urea did not significantly alter its survival rate. This is consistent with previous research, suggesting the buffering action of ammonia produced by H. pylori urease activity takes place in the periplasmic space or on the cell wall surface instead of in the macro environment of the incubation mixture (Rektorschek et al. (1998) and Scott et al., "The role of internal urease in acid resistance of Helicobacter pylori," Gastroenterology 114:58-70 (1998)). H. pylori survival at acidic pΗ may likely be due to elevation of periplasmic or cell surface pΗ and not to the elevation of macro environment pΗ. The appropriate concentration of urea-ΗCl solution can successfully eliminate virtually all background microorganisms in saliva, thereby allowing successful recovery of viable H. pylori. Using this method, H. pylori could consistently be isolated even at very low levels of inoculum (10-100 CFU of H. pylori per 1 ml of saliva, >10 microorganisms/ml). This method provides excellent potential for the isolation of H. pylori from highly contaminated specimens (e.g., saliva, dental plaque, feces, or water) and therefore may be of critical importance in studies concerning the source and transmission route of H. pylori infection. H. pylori could not be isolated from saliva using Skirrow's media alone when the H. pylori inoculum in the sample was lower than 10⁴ CFU/ml due to the overwhelming growth of other microorganisms present in saliva.

This method also provides a simple way to re-isolate contaminated H. pylori cultures in the laboratory. During collection, storage, transportation, inoculation, and incubation of H. pylori specimens, contamination is difficult to avoid completely (Dent et al., "Evaluation of a new selective medium for Campylobacter pylori," Eur. J. Clin. Microbiol. Infect. Dis. 7:555-568 (1988)). Using the short exposure to urea and ΗC1 before inoculation, contamination can easily be eliminated. This new method could replace regular selective commercial media for primary isolation of H. pylori from gastric biopsies and could be very useful for monitoring antibiotic resistance and offering targeted therapy to H. v/oπ-infected patients in the clinical setting.

Acid tolerance and urease activity of the H. pylori type strain (ATCC 43504) is different than clinical strains. Clinical strains of H. pylori appear to have more urease activity and higher acid survival rates than the ATCC type strain. It is very likely that the acidic environment in the stomach induces the stronger urease activity of clinical H. pylori strains, which appears to be critical for H. pylori survival in the acidic gastric environment. The H. pylori type strain apparently lost urease activity due to many passages on non-acid-containing media. H. pylori isolates obtained from sites other than the stomach may have lower urease activities, similar to the type strain of H. pylori, due to a higher pΗ at these sites. Therefore, the method developed in this study, which was optimized using the ATCC type strain, is expected to be suitable for the isolation of H. pylori from sites other than the stomach.

In conclusion, H. pylori can survive short exposures to extremely low pΗ in the presence of high concentrations of urea; based on these results, a novel and simple method has been developed for the isolation of H. pylori microorganisms from highly contaminated samples such as saliva. Using this method, H. pylori added into saliva can be reisolated even when the number of H. pylori is as low as 10 to 100 cells/ml in saliva. Other microorganisms are almost totally inhibited. This method may, therefore, be valuable in determining the source and routes of H. pylori transmission. Morever, this method may also be valuable in the clinical setting, by allowing H. pylori to be isolated from gastric biopsies without the use of selective media containing antibiotics.

The following examples describe and illustrate the methods and compositions disclosed herein. These examples are intended to be merely illustrative only, and not limiting thereof in either scope or spirit. Those of skill in the art will readily understand that variations exist for the materials, conditions, and processes of the procedures described in these examples. EXAMPLE 1 Characterization of the H. pylori Urea-HCl Isolation Method

A characterization was performed of various factors involved in the H pylori Urea-HCl isolation method. H pylori type strain ATCC 43504 (American Type Culture Collection, Manassas, VA) was used in this study. Following a two-day growth, H pylori strain ATCC 43504 was harvested from a heart infusion agar (HIA) media with 5% rabbit blood (BBL, Cockeysville, MD) and suspended in normal saline (150 mM/L of NaCl, Remel, Lenexa, KS) to a turbidity equivalent to a McFarland No. 2 standard (Dade, West Sacramento, CA). Subsequently, 100 μl of the bacterial suspension was diluted with 0.9 ml of normal saline. In order to determine growth kinetics, 10 μl of the diluted suspension (about 10⁵ CFU of H. pylori) was incubated with 5 μl urea (Sigma Chemical Co., St. Louis, MO) solution and 10 μl ΗC1 (Sigma Chemical Co., St. Louis, MO) solution (final concentration, urea 0.04 mM to 1M, ΗC1 0.002 N to 0.48 N) for 5 minutes at room temperature.

The incubation mixture was then serially diluted using PBS buffer (0.017 M, pΗ 7.4, GIBCOBRL, Grand island, NY), resulting in greater than 100-fold dilution of ΗC1, and an increase in pΗ up to about 6.8. ΗIA plates with rabbit blood (BBL, Cockeysville, MD), having 100 1 of each serial dilution plated onto them, were incubated for 5 days at 37° C under microaerobic conditions (N₂ 85%, CO₂ 10%, O₂ 5%). Resulting colonies were counted. In each experiment, parallel controls were employed under identical conditions, except that the control cultures were incubated with PBS instead of ΗC1 and urea solutions. H. pylori survival was assessed quantitatively by determining CFU number, expressed as percentage of the initial inoculum (i.e., CFU after ΗC1 challenge versus CFU in controls). Each experiment was performed in triplicate. The pΗ of the incubation mixtures of normal saline or saliva, urea, and ΗC1 were determined using a Corning model -440 pΗ meter (Corning Co., Corning, NY). Finally, experiments were performed to determine the survival rate of H. pylori strains by varying the days of growth, incubation period, and incubation temperature (4° to 37° C) in urea-ΗCl solutions.

Overall, varying the concentrations of urea and ΗC1 resulted in different rates of H. pylori survival (Table 1). Decreases in ΗC1 concentration, with subsequent pH increases, resulted in increased H pylori survival rates. Brief exposure of H. pylori suspensions to 0.48 N HCl, with a solution pH as low as 0.36, demonstrated that 0.01% of the organisms could survive in the presence of 1M urea. However, the majority of the initial bacterial inoculum was almost completely eliminated in the absence of urea (Table 1). H. pylori did not survive if there was an exposure to greater than 0.008 N HCl (pH less than about 2.1) immediately prior to exposure to urea. However, addition of urea prior to HCl, or addition of both urea and HCl to H. pylori suspensions (i.e., a mixture of HCl and urea) simultaneously, gave similar survival rates (0.01%-70% of initial inoculum under pH 0.36-2.7). Minimal H pylori recovery occuned in solution mixtures with less than or equal to 0.04 mM urea and a pH less than 2. However, high concentrations of urea also resulted in damage to H pylori and less recovery of the initial inoculum (Table 1). HCl concentrations of less than 0.002 N had a minimal effect on H pylori survival in the presence of urea concentrations of 0.3 to 80 mM. Varying incubation times between 1 and 20 minutes in the 0.03 to 0.06 N HCl concentration range with 2.5 to 80 mM urea concentrations (resultant pH 1.2 to 1.5) gave similar H pylori recovery rates (6%-10%). However, if the incubation time increased to more than 1 hour, the recovery rate decreased significantly.

Suspensions of the ATCC 43504 type strain demonstrated decreased cell viability over time. Growth from HIA plus 5% sheep blood plates was suspended in PBS to a turbidity level corresponding to a 2.0 McFarland standard. One-, two-, three-, four-, and five-day growth corresponded to 4 x 10⁸, 2 x 10⁸, 8 10⁷, 8 x 10⁶, and 2 10⁵ CFU/ml, respectively. However, the days of growth on solid media had no effect on the percent of viable cell recovery after urea-HCl challenge (80 mM urea and 0.06 N HCl). Similar survival percentages were observed for H pylori ATCC 43504 from the following days of growth: 1-, 2-, 3-, 4-, or 5-day growth suspensions conesponded to 7.4%, 10.8%, 7%, 10.9% or 10% recovery of the initial inoculum, respectively.

The temperature of incubation during exposure of H. pylori ATCC 43504 suspensions to urea-HCl mixtures influenced the H. pylori survival rate significantly. While holding urea and HCl concentrations constant (80 mM and 0.06 N, respectively), incubation temperatures of 37°C, 25°C and 4°C gave 15.8%, 10.4%, and 0.8% H. pylori recovery rates, respectively (P < 0.001).

The clinical isolates and the H. pylori ATCC 43504 type strain exhibited significantly different tolerances to acid. After exposure to 80 mM urea and 0.06 N ΗC1 at 25° C, 13 clinical strains had survival rates ranging from 14% to 90% of the initial inoculum (median 43%) as compared to the ATCC type strain which resulted in a recovery rate of 8% (Table 2, P O.001).

Table 1. Survival rate of H. pylori in saline after short exposure to HCl and urea"

a. Each test consisted of 10 1 of the type strain H. pylori suspension in saline (about 2 x 10⁵ CFU), 5 1 urea solution, and 10 1 of ΗC1 dilution. Mixtures were incubated at room temperature for 5 min., serially diluted with PBS (pΗ 7.4), and inoculated onto ΗIA plus 5% sheep blood plates. H. pylori colonies were counted after 5- day microaerobic incubation at 37°C. The survival rate of H. pylori is expressed as a percentage of CFU after urea-ΗCl treatment/input as determined by parallel controls with water replacing ΗC1 and urea. All values are means of experiments performed in triplicate. b. Final concentration during incubation of H. pylori suspension, urea and ΗC1 solution before dilution by PBS. c. pΗ value of mixtures of saline, ΗC1, and urea prepared in the same ratio as mentioned above, without H. pylori. Urea concentration had no significant influence on pΗ of the mixture before adding H. pylori.

Table 2. H. pylori urease activity and acid tolerance.

a. Strains 01/001 through 01/013 are clinical sfrains; ATCC 43504 is the H. pylori type strain.

b. Urease activities of intact, log phase, H. pylori cells. Mean value of at least two experiments.

c. Exposed to a mixture of 20 mM urea and 0.06 N HCl at 37 °C for 5 minutes.

Each of the clinical strains (01/001 through 01/013) had a significantly higher acid survival rate than the ATCC 43504 type strain (P < 0.001). EXAMPLE 2 Comparison of H. pylori Urease Activity: Type Strain and Clinical Strains.

Urease activity was compared for the type strain and various clinical strains of H. pylori. Urease activity for all H. pylori strains tested was measured by using a coupled enzyme assay as described by Kaltwasser et al. ("NADΗ-dependent coupled enzyme assay for urease and other ammonia-producing systems," Analytical Biochemistry 16:132-138 (1966)) and Dunn et al.(Dunn et al. (1990)) previously. Each reaction contained 31 mM Tris-ΗCl (pΗ 8.0), 810 μM 2-oxoglutarate (Roche Diagnostics Corp., Indianapolis, IN), 240 μM NADΗ (Roche Diagnostics Corp.), 15 units/ml glutamate dehydrogenase (Roche Diagnostics Corp.), 10 mM urea, 1 mM sodium sulfide (Sigma Chemical Co., St. Louis, MO) and about 10⁶ H. pylori cells. The final reaction volume for each strain tested was 0.6 ml. Absorbance at 340 nM was read every 20 seconds using a DU 640 spectrophotometer (Beckman Instruments Inc., Fullerton, CA). Reactions were performed at room temperature (25° C). A unit of urease was defined as the amount of enzyme catalyzing the formation of 1 μmol of ammonia per minute (Kaltwasser et al. (1966)). Intact (i.e., not broken) H. pylori cells (viable cell suspended in isotonic normal saline) were used. One-day old growth of H. pylori culture on ΗIA + 5% rabbit blood media was used in order to ensure maximum viability of all bacterial cells employed (Benaissa et al., "Changes in Helicobacter pylori ultrastructure and antigens during conversion from the bacillary to the coccoid form," Infect Immun 64:2331-2335 (1996)), which was confirmed in our lab by performing growth curves.

H. pylori suspensions for urease analysis were prepared as follows: Cells were harvested from ΗIA plus 5% rabbit blood plates and suspended in normal saline. Suspensions were then vortexed thoroughly until no evidence of solid matter was observed and adjusted to a turbidity equivalent to a 2.0 McFarland standard. For all urease assays, the immersion time of the bacterial cells in suspension before initiation of the urease assay was held constant at 10 minutes. This technique was employed to minimize experimental error caused by urease enzyme diffusing out of cells into the suspension following different immersion times. All clinical strains used were passed about 5 times on ΗIA plus 5% rabbit blood plates prior to urease analysis. We measured the urease activity of intact H. pylori cells (i.e., representative of urease found in both the periplasmic space and on the cell wall surface), not of non- viable or broken cells. Previous investigations have shown that urease of the periplasmic space and cell wall rather than that inside the cell were the most critical elements for H. pylori survival in an acidic environment (Krishnamurthy et al. (1998).

The urease activity of type strain and clinical strains of H. pylori and their survival rate after exposure to ΗC1 are shown in Table 2. All clinical strains tested demonstrated stronger urease activity and higher survival rates after exposure to ΗC1 than the ATCC type strain. However, enzyme activity did not always correlate with survival rates for every clinical strain tested.

EXAMPLE 3 Isolation of H. pylori Added in Saliva.

Experiments were carried out as described above in Example 1, with the exception of diluting 100 μl of a 2.0 McFarland H. pylori suspension with 0.9 ml of saliva. Through these experiments, the optimal HCl and urea concentrations in saliva incubations were then determined based on H pylori survival rates and inhibition of other microorganisms as described. Subsequently, following the determination of the optimal urea and HCl concentrations, experiments to discover the minimum number of H. pylori that could be spiked into saliva and then recovered were performed. To accomplish this objective, 1 ml saliva and 10 μl H pylori suspension containing either 10, 10², 10³' or 10⁴ CFU of H. pylori were evaluated. In a 1.5-ml Eppendoff tube, 0.5 ml of the saliva-H. pylori mixture was incubated with 0.25 ml urea and 0.5 ml ΗC1 of optimal concentrations (i.e., 0.06 N ΗC1 and 80 mM urea) at 37° C for 2 minutes, then centrifuged in a conventional table-top centrifuge (Eppendorf, Hamburg, Germany) at 37° C for 5 minutes 14,000 rpm. Supernatants were discarded and the pellet was suspended in 1 ml PBS, then spread evenly onto HIA plus 5% rabbit blood plates (0.2 ml/plate, equivalent to 0.1 ml saliva), and incubated under microaerobic conditions for 5 days. H. pylori-like colonies were counted, then subcultured, and species confirmation was performed by biochemical analysis (urease, catalase and oxidase production). The H pylori survival rate was expressed as a percentage of the original inoculum exposed to urea/acid challenge. Controls were performed in parallel using similar H. pylori suspensions without saliva or the urea/ΗCl challenge. In addition, for comparison, saliva spiked with serial dilutions of H. pylori was inoculated onto Skirrow's media (BBL, Cockeysville, MD) to assess the ability to recover added H. pylori cells. Tables 3 and 4 depict the optimal ΗC1 and urea mixtures for isolation of H pylori from saliva. A higher concentration of urea was necessary for maximum recovery of H. pylori in saliva under higher concentrations of ΗC1. As shown in table 3, when employing ΗIA media plus 5% rabbit blood, greater than 0.03 N ΗC1 was generally required to inhibit growth of non-H. pylori oral flora microorganisms found in saliva. Employing Skirrow's media, greater than or equal to 0.015 N ΗC1 was required for inhibition of background organisms. When the ΗC1 concentration was greater than or equal to 0.03N, ΗIA plus 5% rabbit blood and Skirrow's media gave similar results when assessing H. pylori survival rate (Table 5) and the degree of inhibition of background organisms (Table 3). When saliva inoculated with H. pylori was exposed to low ΗC1 concentrations (range: 0.015 to 0.03 N ΗC1) and then plated onto Skirrow's media, H. pylori survival rate did not increase, but, in fact, decreased compared to survival rates at higher ΗC1 concentrations (Table 5).

When inoculums (10 tolO⁴ CFU) of H. pylori were added to 1 ml of saliva and exposed to the optimal urea-ΗCl concentrations (i.e., 0.06 N ΗC1 and 80 mM urea) at 37 °C using the standard protocol, survival rates of H. pylori ranged from 2% to 15% of the initial inoculum with negligible concurrent growth of other microorganisms on ΗIA plus 5% rabbit blood media (Fig. 1 A. a). H. pylori was consistently and readily isolated when the inoculum added was 100 CFU per one milliliter of saliva. Even when the amount of H. pylori was as low as 10 CFU/ml, colonies of H. pylori could still be isolated using the optimal urea-ΗCl protocol.

Without the urea-ΗCl pretreatment before plating, over a 1000-fold dilution of the saliva was necessary and the inoculum size employed could not be over 0.01 μl of saliva plated onto Skirrows agar, because otherwise the numerous background microorganisms would prevent the isolation of H. pylori colonies (Fig. IB. a). When employing Skirrows media without urea-ΗCl challenge, a minimum inoculum of 10⁴ CFU/ml of H. pylori in saliva was necessary for successful isolation. However, achieving successful isolation of H. pylori on selective media alone was very difficult due to excessive overgrowth of other microorganisms similar in colony morphology to H. pylori.

Table 3. Effect of varying the ΗC1 concentration on the survival of background microorganisms found in saliva".

a. Each treatment consisted of 0.5 ml saliva, 0.25 ml urea solution, and 0.5 ml HCl solution. Mixtures were incubated at 37° C for 2 min., then centrifuged at 14,000 rpm in a conventional table-top microcentrifuge for 5 min. The pellet was resuspended in 0.2 ml of PBS buffer, inoculated onto HIA plus 5% rabbit blood or Skirrows media, and incubated microaerobically for 5 days at 37°C. b. Final concentration during incubation of saliva, urea, and HCl solution before dilution by PBS buffer. c. pH values of the mixtures of saliva, HCl, and urea prepared by the same ratio mentioned above. Urea concentration had no significant influence on pH value. Table 4. Survival rate of H. pylori added to saliva after a short exposure to HCl and urea"

a. Each test consisted of 10 1 of saliva (containing 2 x 10⁵ CFU of added H. pylori), 5 1 urea solution, and 10 1 of HCl solution. Mixtures were incubated at room temperature for 5 min., serially diluted with PBS buffer (pH 7.4), and inoculated onto HIA plus 5% rabbit blood plates. H. pylori colonies were counted after 5 days of microaerobic incubation at 37°C. The H. pylori survival rates are expressed as the percentage of colonies after urea-HCl treatment/input colonies as determined by parallel controls with water replacing urea and HCl. All values are means of triplicate trials. b. Final concentration during incubation of H. pylori suspension, urea, and HCl solution before dilution with PBS buffer.

c. pH value of mixtures of saliva, HCl, and urea prepared in the same ratio as mentioned above, without H. pylori. Urea had no significant influence on the pH of mixtures before adding H. pylori.

Table 5. A comparison of HIA plus 5% rabbit blood and Skirrows media on survival rate of H. pylori added to saliva after exposure to urea-HCl mixtures³

a. Each test consisted of 10 1 saliva (containing 2 x 10⁵ CFUs of added H. pylori), 5 1 urea solution and 10 1 HCl dilution, incubated at room temperature for 5 min., then serially diluted by PBS buffer (pH 7.4), inoculated on HIA media. H. pylori CFU was counted after 5-day microaerobic incubation. Survival rate of H. pylori is expressed as percentage of CFUs in parallel control with water replacing ΗC1 and urea. All values were means of triplicate tests. b. Final concentration during incubation of H. pylori suspension, urea and ΗC1 solution before dilution with PBS buffer. c. pΗ value of mixture of saliva, ΗC1 and urea prepared by the same ratio above, without H. pylori. d. Not applicable due to overgrowth of other organisms.

EXAMPLE 4 Isolation of H. pylori from Gastric Biopsy.

An analysis was performed concerning the recovery of H. pylori from gastric biopsies. Gastric biopsies were collected from 24 consecutive adult patients undergoing diagnostic endoscopy at the Department of Veterans Affairs Medical Center in Atlanta, Georgia. Informed consent was obtained at the time of endoscopy and the study was approved by the Emory University and VA Medical Center institutional review boards. Patients were refened for diagnostic upper endoscopy by their treating physician and were enrolled in the H. pylori Antibiotic Resistance Project (Η.A.R.P) monitoring program established and funded by the Foodborne and Diarrheal Diseases Branch of the Centers for Disease Control and Prevention, Atlanta, Georgia. Primary cultures of H. pylori strains were isolated from these biopsies, which were also tested for the presence of H. pylori urease using the CLO- test (Trimed, Kansas city, MO). Biopsies were also stained after being placed in 10% buffered formalin, embedded in paraffin and sectioned for histologic examination for the presence of H. pylori.

Gastric biopsies stored in trypticase soy broth with 20% glycerol (fresh or frozen at -70°C for 1-5 days) were homogenized using a sterile, plastic pestle and microtube (Kontes, Nineland, New Jersey), and suspended in 0.5 ml of the broth. Subsequently, 50 μl of the undiluted homogenate and 10-fold serial dilutions of the homogenate were plated onto Skirrows media. Concurrently, a second 50 μl aliquot of the undiluted homogenate and the serial dilutions were mixed at 37° C with 50 μl of HCl and 25 μl of urea solution using the optimal urea-HCl conditions (i.e., 0.06 N HCl and 80 mM urea) and centrifuged as previously described. Following centrifugation, pellets were resuspended in PBS buffer and 10-fold serial dilutions in PBS were inoculated onto HIA plus 5% rabbit blood plates. Plates were incubated under microaerobic conditions at 37° C for 5 days followed by enumeration of H. pylori colonies.

Direct inoculation onto Skirrows media, compared to urea-HCl challenge followed by inoculation onto HIA plus 5% rabbit blood media, gave similar isolation results: 13 (54%) positive and 11 (46%) negative. Culture results were identical with those obtained by standard histology and rapid urease testing (CLO- test). The number of H. pylori colonies recovered using the optimal urea-ΗCl protocol was 20%-95% (mean = 50%) of the number of H. pylori colonies recovered from Skirrows media without acid challenge. However, 3 biopsies (12.5%) had other contaminating organisms when grown on Skirrows media alone. Conversely, we found virtually no growth of non-H. pylori microorganisms on ΗIA plus 5% rabbit blood media after the urea-ΗCl challenge of the biopsy samples. Additionally, H. pylori was isolated from one additional gastric biopsy shipped to us from Jamaica after employing the optimal urea-ΗCl challenge protocol (Fig. 1 A.b). On the other hand, inoculation of this sample onto Skirrows media alone gave a negative result due to the overgrowth of other microorganisms (Fig. 1.B.b).

EXAMPLE 5 Reisolation of Contaminated H. pylori Culture.

The effectiveness of the urea-ΗCl H pylori isolation procedure for the purification of H. pylori from contaminated cultures was compared to conventional techniques using selective media. Samples of 10 frozen H. pylori cultures known to be highly contaminated (Fig. l.B.c) were positive upon biochemical analysis for urease, catalase, and oxidase, suggesting the presence of H. pylori. These mixed cultures were initially harvested and then exposed to the optimal urea/ΗCl procedure (i.e., exposure to a mixture of 0.06 N ΗC1 and 80 mM urea for 2 minutes at 37°C, then centrifuged in a conventional table-top microcentrifuge at 14, 000 rpm for 5 minutes. The pellet was resuspended in 1 ml PBS and then spread evenly onto ΗIA plus 5% rabbit blood plates followed by incubation at 37°C under microaerobic conditions). Subsequent 5-day growth revealed only H. pylori on the media without further contamination (Fig. 1. A. c). EXAMPLE 6 Isolation of H. mustelae Added in saliva.

The effectiveness of the urea-ΗCl isolation procedure for the purification of H. mustelae was analyzed. Methods were the same as described in Example 3. The optimal ΗC1 and urea concentrations determined in the Example 3 were used. It was shown that the survival rate of H. mustelae after exposure to ΗC1 and urea was about 10% of the initial inoculum with negligible concurrent growth of other organisms on ΗIA plus 5% rabbit blood media.

Throughout this application, various patents, publications, and books, have been cited. The entireties of each of these patents, publications, and books are hereby incorporated by reference into this application.

Claims

ClaimsWHAT IS CLAIMED IS:

1. A method for isolating urease-positive bacteria from a sample suspected of containing urease-positive bacteria and which may contain other microorganisms, said method comprising:

(a) adding to the sample an amount of urea sufficient to protect urease- positive bacteria that may be present in the sample from an acidic pH;

(b) adding an acid to the sample to obtain the acidic pH; (c) incubating the sample at the acidic pH under a first set of conditions sufficient to render the other microorganisms non- viable, but wherein the urease- positive bacteria remain viable, thereby producing a treated sample;

(d) applying the treated sample to a growth media; and

(e) incubating the treated sample on the growth media under a second set of conditions that support growth of the urease-positive bacteria, thereby isolating the urease-positive bacteria.

2. The method of claim 1, wherein the sample is a highly contaminated specimen from a gastric biopsy, saliva, dental plaque, endoscopy equipment, an environmental source, a contaminated urease positive bacteria culture, or a stool sample.

3. The method of claim 2, further comprising increasing the pH of the treated sample to a pH that enhances growth of the urease-positive bacteria on the growth media.

4. The method of claim 3, wherein the urease-positive bacteria are selected from the group consisting of Helicobacter pylori and a Proteus species.

5. The method of claim 4, wherein the urease-positive bacteria are

Helicobacter pylori.

6. The method of claim 5, wherein the acid is HCl.

7. A method for isolating urease-positive bacteria from a sample suspected of containing urease-positive bacteria and which may contain other microorganisms, said method comprising:

(a) adding to the sample an amount of urea and an amount of acid, wherein the amount of urea is sufficient to protect urease-positive bacteria that may be present in the sample from an acidic pH, and wherein the amount of acid is sufficient to obtain the acidic pH; (b) incubating the sample at the acidic pH under a first set of conditions sufficient to render the other microorganisms non- viable, but wherein the urease- positive bacteria remain viable, thereby producing a treated sample;

(c) applying the treated sample to growth media; and

(d) incubating the treated sample on the growth media under a second set of conditions that support growth of the urease-positive bacteria, thereby isolating the urease-positive bacteria.

8. The method of claim 5, wherein the concentration of urea is between about 0.04 mM and about 1 M and the concentration of HCl is between about 0.002 N and about 0.48 N.

9. The method of claim 8, wherein the concentration of urea is between about 0.31 mM and about 500 mM and HCl is present at a concentration of between about 0.03 N and about 0.24 N.

10. The method of claim 9, wherein the concentration of urea is between about 5 mM and about 200 mM and the concentration of HCl is between about 0.06 N and about 0.12 N.

11. The method of claim 5, wherein the effective incubation time is between about 1 minute and about 20 minutes.

12. The method of claim 11, wherein the effective incubation time is between about 2 minutes and about 10 minutes.

13. The method of claim 6, wherein the source is saliva.

14. The method of claim 13, wherein the concentration of urea is between about 20 mM and about 1 M and HCl is at a concentration of between about 0.06 N and about 0.24 N.

15. The method of claim 14, wherein the growth media is selected form the group consisting of heart infusion agar with rabbit blood and Skirrow media.

16. The method of claim 13, wherein the concentration of urea is between about 20 mM and about 1 M and HCl is at a concentration of between about 0.03 N and about 0.24 N.

17. The method of claim 16, wherein the incubation time is between about 1 minute and about 20 minutes.

18. The method of claim 17, wherein the incubation time is between about 2 minutes and about 10 minutes.

19. The method of claim 5, wherein the acidic pH is less than about 2.7.

20. The method of claim 22, wherein the acidic pH is less than about 1.5.

21. The method of claim 5, further comprising the step of filtering the sample using a filter before the step of exposing the sample to a concentration of urea.

22. The method of claim 21, wherein the steps of exposing the sample to a concentration of urea and exposing the cells to hydrochloric acid are carried out on the filter.

23. The method of claim 22, wherein the step of applying the treated sample to growth media comprises placing the filter on growth media.

24. The method of claim 23, wherein the filter is hydrophobic and has a grid pattern.