Development of two PCR-based techniques for detecting helical and coccoid forms of Helicobacter pylori.

The primary mode of transmission of Helicobacter pylori, a human pathogen carried by more than half the population worldwide, is still unresolved. Some epidemiological data suggest water as a possible transmission route. H. pylori in the environment transforms into a nonculturable, coccoid form, which frequently results in the failure to detect this bacterium in environmental samples by conventional culture techniques. To overcome limitations associated with culturing, molecular approaches based on DNA amplification by PCR have been developed and used for the detection of H. pylori in clinical and environmental samples. Our results showed the glmM gene as the most promising target for detection of H. pylori by PCR amplification. Under optimal amplification conditions, glmM-specific primers generated PCR-amplified products that were specific for H. pylori and some other Helicobacter species. Genome sequence analysis revealed the existence of a conserved region linked to a hypervariable region upstream of the 16S rRNA gene of H. pylori. Selective PCR primer sets targeting this sequence were evaluated for the specific detection of H. pylori. One primer set, Cluster2 and B1J99, were shown to be highly specific for H. pylori strains and did not produce any PCR products when other Helicobacter species and other bacterial species were analyzed. In tests with 32 strains of H. pylori, 6 strains of other Helicobacter species, 8 strains of Campylobacter jejuni, and 21 strains belonging to different genera, the primers for glmM were selective for the Helicobacter genus and the primers containing the region flanking the 16S rRNA gene were selective for H. pylori species only. The combination of two sensitive PCR-based methods, one targeting the glmM gene and the other targeting a hypervariable flanking region upstream of the 16S rRNA gene, are complementary to each other. Whereas the glmM-specific primers provide a rapid, sensitive presumptive assay for the presence of H. pylori and closely related Helicobacter spp., the primers for sequences flanking the 16S rRNA gene can confirm the presence of H. pylori and locate the potential source of this bacterium.

Antibacterial activity of black myrobalan (Terminalia chebula Retz) against Helicobacter pylori.

The effect of ether, alcoholic and water extracts of black myrobalan (Teminalia chebula Retz) on Helicobactor pylori were examined using an agar diffusion method on Columbia Agar. Water extracts of black myrobalan showed significant antibacterial activity and had a minimum inhibitory concentration (MIC) and minimum bacteriocidal concentration (MBC) of 125 and 150 mg/l, respectively. The extract was active after autoclaving for 30 min at 121 degrees C. Plant powder (incorporated in agar) gave higher MIC and MBC values (150 and 175 mg/l, respectively). Water extracts of the black myrobalan at a concentration of 1-2.5 mg/ml inhibited urease activity of H. pylori. The results show that black myrobalan extracts contain a heat stable agent(s) with possible therapeutic potential. Other bacterial species were also inhibited by black myrobalan water extracts.

Cellulomonas persica sp. nov. and Cellulomonas iranensis sp. nov., mesophilic cellulose-degrading bacteria isolated from forest soils.

Two newly described species of mesophilic, cellulose-degrading, aerobic bacteria were isolated from forest humus soils along the southern border of the Caspian Sea. Cellulomonas persica and Cellulomonas iranensis are proposed as new specific epithets based on comparative sequence analyses of 16S rDNA, DNA-DNA hybridization and phenotypic characteristics. Formal species descriptions are provided.

Continuous source outbreak of campylobacteriosis traced to chicken.

Poultry is a source of human campylobacteriosis, but a large continuous source outbreak, heretofore, has not been attributed to both a single source of poultry and single serotype of Campylobacter. Here we report an outbreak of C. jejuni affecting 6 catering college trainees and 13 patrons of a restaurant in southern England. An epidemiological investigation successfully tracked the outbreak source to the farm of origin. Frequency of occurrence of campylobacters and outbreak serotype distribution were determined in index cases, the local population, and local chicken suppliers. The source farm was investigated and the effect of interventions assessed. A single outbreak serotype of C. jejuni was isolated from trainee chefs, patrons, and chicken supplied to the college by Wholesaler A. The Campylobacter isolation rate for Wholesaler A was 89% (98% outbreak serotype), compared to 40% for non-Wholesaler A (10% outbreak serotype). The isolation rate for 14 months averaged 85% (99% outbreak serotype) in chickens grown on two farms (X and Y) supplying Wholesaler A, contributing approximately 40% to all local cases. In the research reported here, a specific strain and hygiene practice were found to be important for understanding transmission of Campylobacter from poultry to humans in this outbreak.

[Evaluation of the effect of ecologically hazardous pollutants on the bacteriolytic activity of the predatory bacterium Bdellovibrio]. / Otsenka vliianiia ékologicheski znachimykh polliutantov na funktsional'nuiu aktivnost' bakterial'nykh khishchnikov, Bdellovibrio.

We studied the effect of various concentrations of ecologically hazardous pollutants, urea, phenol, diuron, and cadmium ions, on the physiological activity and survival of the parasitic bacterium Bdellovibrio. Experiments showed that the survival of bdellovibrios in the presence of the pollutants was two times higher when they were cultivated on agar than when they were cultivated in liquid medium. The data obtained are in agreement with the recent concept of the surface-associated state as a survival strategy of bdellovibrios in various ecosystems.

Effect of aerosolization on culturability and viability of gram-negative bacteria.

Estimations of the bacterial content of air can be more easily made now than a decade ago, with colony formation the method of choice for enumeration of airborne bacteria. However, plate counts are subject to error because bacteria exposed to the air may remain viable yet lose the ability to form colonies, i.e., they become viable but nonculturable. If airborne bacteria exhibit this phenomenon, colony formation data will significantly underestimate the bacterial populations in air samples. The objective of the study reported here was to determine the effect of aerosolization on viability and colony-forming ability of Serratia marcescens, Klebsiella planticola, and Cytophaga allerginae. A collision nebulizer was used to spray bacterial suspensions into an aerosol chamber, after which duplicate samples were collected in all-glass impingers over a 4-h period. Humidity was maintained at ca. 20 to 25%, and temperature was maintained at 20 to 22 degrees C for each of two replicate trials per microorganism. Viability was determined by using a modified direct viable count method, employing nalidixic acid or aztreonam and p-iodonitrotetrazolium violet (INT). Cells were stained with acridine orange and observed by epifluorescence microscopy to enumerate total and viable cells. Viable cells were defined as those elongating in the presence of antibiotic and/or reducing INT. CFU were determined by plating on tryptic soy agar and R2A agar. It was found that culture techniques did not provide an adequate description of the bacterial burdens of indoor air (i.e., less than 10% of the aerosolized bacteria were capable of forming visible colonies). It is concluded that total cell count procedures provide a better approximation of the number of bacterial cells in air and that procedures other than plate counting are needed to enumerate bacteria in aerosol samples, especially if the public health quality of indoor air is to be estimated.

Molecular techniques reveal high prevalence of Legionella in dental units.

Legionella bacteria are ubiquitous in freshwater aquatic systems, and humans are infected by them primarily through inhalation of contaminated aerosols. This study analyzed a total of 47 water samples from dental lines in private dental offices and university and hospital dental clinics for Legionella using the polymerase chain reaction, direct fluorescent antibody staining and culture techniques. The typical temperature of dental waterlines (23 C) combined with Legionella's ability to form biofilms, stagnation of the water in the lines and a low chlorine residual all potentially create a unique niche for this microorganism.

Potential virulence of viable but nonculturable Shigella dysenteriae type 1.

We examined a virulent strain of Shigella dysenteriae type 1 after induction into the viable but nonculturable (VBNC) state for its ability to (i) maintain the Shiga toxin (stx) gene; (ii) maintain biologically active Shiga toxin (ShT); and (iii) adhere to intestinal epithelial cells (Henle 407 cell line). PCR was used to amplify the stx gene from VBNC cells of S. dysenteriae type 1, thereby establishing its presence even when cells are in the VBNC state. VBNC S. dysenteriae type 1 ShT was monitored by the enzyme-linked immunosorbent assay with mouse monoclonal antibodies against the B subunit of ShT and affinity-purified rabbit polyclonal antibodies against ShT. We used the Henle 407 cell line to study the adhesive property of VBNC S. dysenteriae type 1 cells in a series of tissue culture experiments. Results showed that VBNC S. dysenteriae type 1 not only maintained the stx gene and biologically active ShT but also remained capable of adhering to Henle 407 cells. However, S. dysenteriae type 1 cells lost the ability to invade Henle 407 cells after entering the VBNC state. From results of the study, we conclude that VBNC cells of S. dysenteriae type 1 retain several virulence factors and remain potentially virulent, posing a public health problem.

Methionine uptake and cytopathogenicity of viable but nonculturable Shigella dysenteriae type 1.

A pathogenic strain of Shigella dysenteriae type 1 was selected for study to elucidate the physiology and potential pathogenicity of organisms in the viable but nonculturable (VBNC) state in the environment. Studies in our laboratory have shown that S. dysenteriae type 1 survives in laboratory microcosms in the VBNC state for long periods of time, i.e., more than 6 months. VBNC cells of S. dysenteriae type 1 were found to retain cytopathogenicity for cultured HeLa cells. To determine whether VBNC S. dysenteriae type 1 expressed protein after loss of culturability, 35S-labelled methionine was added to suspensions of VBNC cells. Total cellular proteins were extracted and examined by autoradiography. Results indicate that VBNC S. dysenteriae type 1 is capable of both active uptake of methionine and incorporation of methionine into protein. Amino acid uptake and protein synthesis substantiate the viability of cells of S. dysenteriae type 1 in the VBNC state, i.e., although the cells are unable to be cultured on laboratory media by standard bacteriological methods, the cells remain metabolically active. Furthermore, VBNC cells of S. dysenteriae type 1 may pose a potential public health hazard that has not yet been recognized.

Effect of temperature on survival of Legionella pneumophila in the aquatic environment.

Although Legionella spp. are often isolated from natural aquatic habitats, outbreaks of legionellosis are rarely traced to these sources. To determine the fate of Legionella pneumophila in the environment, filtered and unfiltered river water and seawater microcosms, incubated at 4 degrees C and 26 degrees C, were inoculated with [3H]thymidine-labeled L. pneumophila cells. Survival in these microcosms was monitored using [3H]thymidine labeling and culture on buffered-charcoal yeast extract agar amended with alpha-ketoglutarate (BCYE alpha). Immunofluorescent microscopy, direct fluorescent antibody staining, and acridine orange direct counts were also employed. To assess effects of grazing on Legionella, a duplicate set of samples was filtered through 2.0-microns Nuclepore filters to trap large protozoa. Over the test period, in the microcosms incubated at 4 degrees C, the culturable counts decreased ca. 1 log on BCYE alpha agar, with no substantial decline in thymidine count. Autoclaved seawater and river water controls held at 15 degrees C also showed no change in thymidine count. At 26 degrees C, a 3-log decline was observed in culturable counts, with ca. 1-log decline in thymidine counts. These results indicate that, although culturability declined by one to three orders of magnitude, when L. pneumophila microcosms were incubated at 4 degrees C and 26 degrees C, the cells remained metabolically active for extended periods, especially at 4 degrees C.

Use of autoradiography to assess viability of Helicobacter pylori in water.

Autoradiographic methods have been developed to detect metabolic activity of viable but nonculturable cells of Helicobacter pylori in water. Four strains of H. pylori were studied by using microcosms containing suspensions of 72-h cultures in water. The suspensions of aged, nonculturable cells of H. pylori were incubated with [3H]thymidine for 24 to 72 h, after which the cell suspensions were exposed to Kodak NTB2 emulsion for 3 to 28 days. Each sample was processed with three separate controls to rule out false-positive reactions. The organism remains viable and culturable under these conditions for up to 48 h and, in some cases, 20 to 30 days, depending on physical conditions of the environment. We found that temperature was a significant (P < or equal to 0.01) environmental factor associated with the viability of H. pylori cells in water. Autoradiographs of tritium-labeled cells of H. pylori revealed aggregations of silver grains associated with uptake by H. pylori of radiolabelled substrate. Findings based on the autoradiographic approach give strong evidence supporting the hypothesis that there is a waterborne route of infection for H. pylori. The possibility that H. pylori may persist in water in a metabolically active stage but not actively growing and dividing is intriguing and relevant to public health concerns.

Colonization of broiler chickens by waterborne Campylobacter jejuni.

Chickens on a broiler farm in southern England were found to be colonized with Campylobacter jejuni of a single serotype, Lior 1 Penner 4. The farm was the sole supplier of a local slaughterhouse associated with a campylobacter outbreak in 1984 caused by this serotype. The serotype persisted on the farm for at least 18 months after the outbreak; its prevalence in the human population served by the farm remained high until it disappeared from the farm in 1986. The possible sources and routes of transmission of C. jejuni to the broilers on the farm were investigated. The results showed that vertical transmission, feed, litter, small mammals, and environmental or airborne cross-contamination between sheds or successive crops could be excluded as persistent sources of C. jejuni. The predominant source of C. jejuni on the farm was shown to be the water supply. Direct microscopy and fluorescent antibody methods revealed presumptive campylobacters throughout the farm's water system. Campylobacter-free chickens raised in an animal house and given water from the farm supply became colonized with the serotype of C. jejuni endemic on the farm (Lior 1 Penner 4). An intervention program based on water chlorination, shed drinking system cleaning and disinfection, and withdrawal of furazolidone from feed reduced the proportion of birds colonized with campylobacter from 81 to 7% and was associated with a 1,000- to 10,000-fold reduction in campylobacters recoverable from the carcasses. Two months after the end of the intervention program colonization of the birds returned to high levels (84%), indicating that there was a temporal association between intervention and reduced colonization with C. jejuni. Investigations continue to establish the general applicability of these findings.

Isolation and identification of threeCellulomonas spp. from forest soils.

Three stains of cellulose-degrading, aerobic, mesophilic bacteria were isolated from forest soils and, from their cultural, biochemical, and physiological characteristics, they were identified as members of the genusCellulomonas. Unusual biochemical characteristics, e.g. urea hydrolysis, were observed in two isolates. These characteristics have not previously been reported for cellulomonads and may prove to be significant for characterization ofCellulomonas spp. The isolates were able to use urea as a N source in cellulose fermentation. All three strains were motile, with one to four peritrichous flagella observed. Amino acid and polysaccharide composition of the cell walls of the three isolates were identical.

Selected cryopreservatives for long term storage of Helicobacter pylori at low temperatures.

To meet the need for information on cryopreservation, a study was done on 32 Helicobacter pylori strains, comparing different cryopreservative media. Sheep blood, horse blood, horse serum with and without glycerol, and mineral oil media were used for long term storage of H pylori at -70 degrees C or in liquid nitrogen. Procedures were developed which permitted recovery of 87.5% of the strains included in the study after they had been stored for 24 months. Of those strains stored for more than three years, 60% were recovered. It is concluded that most strains of H pylori can be stored for up to one year or longer, under refrigeration, at -70 degrees C or in liquid nitrogen.

Evaluation of liquid media for growth of Helicobacter pylori.

Helicobacter pylori has routinely been isolated and grown on solid media. Recently, we have succeeded in obtaining growth of this organism in several liquid media in large volumes, including tryptic soy broth, Mueller-Hinton broth, brucella broth, brain heart infusion broth, and Columbia broth. Growth was tested in the media with and without supplementation. Growth was obtained after incubation under microaerobic conditions and with CO2 enrichment. Growth in a stationary system versus that in an agitated system was evaluated. Results from these experiments show that H. pylori can be grown in any of the liquid media tested except buffered yeast extract-alpha-ketoglutarate if serum is added. No growth was observed on buffered yeast extract-alpha-ketoglutarate even with serum and other supplementation. Growth of H. pylori in most of the liquid media with supplements was improved if the culture was incubated in a CO2 atmosphere. The findings reported here may be useful in clinical, industrial, and research laboratories that require harvests of large quantities of H. pylori cells.

Sequential culturing method improves recovery of Legionella spp. from contaminated environmental samples.

Investigations were undertaken to improve detection and isolation of Legionella spp. from samples containing a large number of non-legionellae isolates. The direct fluorescent antibody staining technique was used in conjunction with a sequential culturing method which was developed to improve the recovery rate of Legionella spp. from such samples. The technique for enrichment and isolation of Legionella spp. from environmental samples includes storage at 4 degrees C and repeated culture on freshly prepared media. Heat and acid treatments were included when deemed appropriate. A DNA probe was used for confirmation of Legionella. Treatment of the water samples, as described, and co-cultivation with amoebae naturally present in the samples are concluded to be responsible for increased success in recovery of Legionella isolates.

Isolation of amoebae and Pseudomonas and Legionella spp. from eyewash stations.

Forty eyewash units were sampled for protozoa, bacteria, and fungi. Total heterotrophic bacterial counts on nutrient agar and R2A agar (Difco Laboratories, Detroit, Mich.) ranged from 0 to 10(5) CFU/ml, with Pseudomonas spp. being the most frequently isolated. Total counts of 10(4) and 10(8) cells per ml were obtained with the acridine orange staining procedure. All samples were examined for Legionella spp. by direct fluorescent-antibody staining and by culturing on buffered charcoal-yeast extract agar containing alpha-ketoglutarate and glycine and supplemented with cycloheximide, vancomycin, and polymyxin B. DNA-DNA hybridization was used to confirm identification of the Legionella isolates. Legionellae were detected in 35 of 40 (87.5%) samples by direct fluorescent-antibody staining, with 3 samples yielding both Legionella spp. and amoebae. Amoebae identified as Hartmannella, Vahlkampfia, Acanthamoeba, and Cochliopodium spp. were detected in 19 of 40 (47:5%) samples. Sabouraud dextrose agar was used to obtain a crude estimate of viable fungal populations, pH, hardness, and ammonia, alkalinity, chlorine, copper, and iron contents were recorded for all water samples collected from eyewash stations; 33% of the samples had greater than or equal to 10 mg of CO2 per liter. It is concluded that eyewash stations not regularly flushed and/or cleaned and used to flush traumatized eye tissue may be a source of infection and can contaminate laboratory environments via aerosol transmission.

Survival ofLegionella pneumophila in the aquatic environment.

Survival ofLegionella pneumophila SG 1 in seawater and river water was assessed using plate counts on buffered charcoal yeast extract agar amended with α-ketoglutarate (BCYEα) and [(3)H]thymidine-labeling. The [(3)H]thymidine-labeling method for assessing survival ofL. pneumophila in aquatic environments was compared with viable counts, direct fluorescent microscopy (DFA), and acridine orange direct counts (AODC). Protozoa were isolated from the samples employed in the study and identified by characteristic trophozite and cyst morphology. Selective filtration employing 2.0 µm Nucleopore filters was used to determine the effect of grazing on survival ofL. pneumophila in seawater and river water.Legionella viability as measured by plate counts (CFU/ml), declined to a greater extent than cell lysis, assessed by thymidine, DFA, and AODC counts, suggesting thatL. pneumophila survives in aquatic habitats to a greater extent than revealed through culturable counts.

The extent of surface contamination of retailed chickens with Campylobacter jejuni serogroups.

Eighty-two chickens purchased at 11 retailers (supplied by 12 wholesalers) in the south of England were cultured for Campylobacter jejuni by a method involving total immersion. The organism was isolated from 22 (48%) of 46 fresh birds, 12 of 12 uneviscerated (New York dressed) birds, but only 1 of 24 frozen birds. Viable counts of up to 1.5 x 10(6)/chicken were obtained from fresh birds and 2.4 x 10(7)/chicken from uneviscerated birds. Surface swabbing of breasts, thighs, wings and vents of fresh chickens showed that contamination was generally distributed over the carcasses. Salmonellas were found in only 2 of 69 of the fresh chickens. The prevalence of several Lior and Penner C. jejuni serogroups was similar in chickens and sporadic human cases of enteritis. Penner serogroup 4 (mostly of Lior serogroup 1) was found in 26% of human isolates and 14% of chicken isolates. The rising incidence of campylobacter enteritis during the last 6 years could well be a reflection of the increasing proportion of fresh chickens consumed over that period (32% higher in 1986 than in 1981).

Survival of Listeria monocytogenes in high salt concentrations.

The bactericidal effects of high concentrations of common salt has been determined in a laboratory medium for Listeria monocytogenes strain (1, 2a, No. 18). The survival time for the organism was followed at three different incubation temperatures (4, 22, 37 degrees C). The influence of temperature on the action of salt is discussed.