No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Grow of Helicobacter pylori in various liquid and plating media
Abstract
The objectives of this research were to compare commonly used liquid and plating media to elucidate whether one medium provided superior growth of Helicobacter pylori in vitro. The liquid media compared were Mueller-Hinton broth, brain heart infusion broth and H. pylori special peptone broth, formulated in this laboratory. No significant differences in growth rates were noted and shaking during the incubation of broths was not essential for good growth. The plating media compared included Columbia agar, Mueller-Hinton agar, modified Glupczynski's Brussels campylobacter charcoal agar, Johnson-Murano agar and H. pylori special peptone agar (HPSPA). None of the non-specific plating media that have been used historically to culture H. pylori exhibited any particular advantage. However, HPSPA provided an obvious advantage in colony size. Helicobacter pylori special peptone agar enhances the cultivation of H. pylori and could improve the recovery of the bacterium from clinical samples in vitro.
... Similarly, variations in the compositions of liquid media appear to have a significant influence on H. pylori biofilm formation (20). The fastidious nature of H. pylori is well known and was in fact a major hurdle during the initial isolation of the bacterium (46). H. pylori is microaerophilic and thrives under conditions that include a carbon dioxide-enriched (5 to 10%) atmosphere, high humidity, and a temperature of 35°C to 37°C (46). ...
... The fastidious nature of H. pylori is well known and was in fact a major hurdle during the initial isolation of the bacterium (46). H. pylori is microaerophilic and thrives under conditions that include a carbon dioxide-enriched (5 to 10%) atmosphere, high humidity, and a temperature of 35°C to 37°C (46). In addition, cultures require rich medium that is supplemented with specific factors to promote optimal growth. ...
... In addition, cultures require rich medium that is supplemented with specific factors to promote optimal growth. Common H. pylori liquid media begin with a complex base, such as BB, brain heart infusion (BHI) broth, or Ham's F-12 medium, which are then supplemented with serum and additional carbon sources (e.g., fetal bovine serum [FBS] or 2,6-dimethyl--cyclodextrin [46,47]). One study assessed the effects of four types of common liquid culture media on H. pylori biofilm formation: BB supplemented with 2% FBS, BHI broth supplemented with 2% FBS, and Ham's F-12 medium supplemented with or without 2% FBS. ...
Despite decades of effort, Helicobacter pylori infections remain difficult to treat. Over half of the world's population is infected by H. pylori , which is a major cause of duodenal and gastric ulcers as well as gastric cancer. During chronic infection, H. pylori localizes within the gastric mucosal layer, including deep within invaginations called glands; thanks to its impressive ability to survive despite the harsh acidic environment, it can persist for the host's lifetime. This ability to survive and persist in the stomach is associated with urease production, chemotactic motility, and the ability to adapt to the fluctuating environment. Additionally, biofilm formation has recently been suggested to play a role in colonization. Biofilms are surface-associated communities of bacteria that are embedded in a hydrated matrix of extracellular polymeric substances. Biofilms pose a substantial health risk and are key contributors to many chronic and recurrent infections. This link between biofilm-associated bacteria and chronic infections likely results from an increased tolerance to conventional antibiotic treatments as well as immune system action. The role of this biofilm mode in antimicrobial treatment failure and H. pylori survival has yet to be determined. Furthermore, relatively little is known about the H. pylori biofilm structure or the genes associated with this mode of growth. In this review, therefore, we aim to highlight recent findings concerning H. pylori biofilms and the molecular mechanism of their formation. Additionally, we discuss the potential roles of biofilms in the failure of antibiotic treatment and in infection recurrence.
... Strains of H. pylori (ATCC 43504, 43629, and 43579) were obtained from the American Type Culture Collection (Rockville, Md.). These strains were maintained on H. pylori Special Peptone agar (HPSPA) plates and streaked onto fresh plates every 3 to 4 days (17). Plates were incubated at 37ЊC in 5.5-liter boxes (AnaeroPack, Mitsubishi Gas Chemical Co., New York, N.Y.) flushed with a microaerophilic gas mixture (6% O 2 , 10% CO 2 , and 84% N 2 ). ...
... Standard and selective plating media. HPSPA was prepared by adding Special Peptone (10 g/liter, Oxoid Ltd., Basingstoke, UK), granulated agar (15 g/liter, Difco), sodium chloride (5 g/liter, EM Science, Gibbstown, N.J.), yeast extract (5 g/liter, Difco), beef extract (5 g/liter, Becton Dickinson and Co., Cockeysville, Md.), and pyruvic acid, sodium salt (0.5 g/liter, Sigma Chemical Co., St. Louis, Mo.) (17). The selective agents added to the standard medium were vancomycin (10 mg/liter), amphotericin B (5 mg/liter), cefsulodin (10 mg/liter), polymyxin B sulfate (31,000 IU/liter), trimethoprim (40 mg/liter), and sulfamethoxazole (20 mg/liter) (18). ...
... The inoculum had been prepared previously by harvesting cells from HPSPA plates with 4-day-old cultures of H. pylori (ATCC strains 43504, 43629, and 43579). Cells from each plate were then used to inoculate 250-ml Erlenmeyer flasks (four flasks for each strain) containing 96 ml of HPSPB and 4 ml of iron-supplemented calf serum (Biologos), and the flasks were incubated in a microaerophilic atmosphere at 37ЊC for 48 h (17). The broths, containing 5.4 to 5.8 log CFU/ml, were then added to the coarsely ground meat and mixed for 2 min using a commercial mixer with a U-shaped pastry knife (Univex, Salem, N.H.). ...
This study focused on important factors related to the potential of cattle and beef products to transmit Helicobacter pylori to humans. Mucosal samples were collected from the rumen and abomasum of 105 cattle and were plated on a selective medium to isolate Helicobacter spp.; none of the samples examined contained these bacteria. Studies were also conducted to determine how long H. pylori survives in refrigerated or frozen ground beef; results indicated that the microorganism dies rapidly in ground beef, whether refrigerated or frozen. Packaging in vacuum or air had little effect on survival of the organism. The number of H. pylori decreased in refrigerated samples from 3.3 log10 CFU/g on day 0 to 1.4 log10 CFU/g on day 6. H. pylori died even more rapidly when frozen, decreasing from 3.3 log10 CFU/g on day 0 to 0.5 log10 CFU/g on day 6. Retail beef cuts (n = 20) were also examined for the presence of H. pylori by direct plating on a selective medium and by incubation in an enriched broth followed by plating on a selective medium. None of the retail samples contained H. pylori. This research suggests that transmission of H. pylori from beef and beef products is not a primary factor in the high prevalence of this bacterium in humans.
... This lack of data could be accounted for the difficulty in H. pylori isolation from foods, particularly in presence of a high load of accompanying microflora. In fact it is exacting and time-consuming since it requires the employment of selective media supplemented with numerous antibiotics, microaerophilic conditions and a long incubation periods (seven days) (Stevenson, Castillo, Lucia, & Acuff, 2000 ). Furthermore , H. pylori may produce viable nonculturable forms (VNC) (Cellini, Del Vecchio et al., 2004; Dunn, Cohen, & Blaser, 1997) not detectable by means of conventional microbiological techniques; however, it has been hypothesized that VNC forms are still infective (Bode, Mauch, & Malfertheiner, 1993; Cao, Li, Borch, Petersson, & Mardh, 1997 ) thus representing a potential microbiological risk for consumers. ...
... Foodstuff has been considered as the most likely source of infection (Wesley, 1997; Meng & Doyle, 1997; Velàzquez & Feirtag, 1999 ) since raw sheep and cow milk have been found contaminated with H. pylori (Cohen, 1996; Dore et al., 2001; Dunn et al., 1997; Fujimura et al., 2002; Hultèn et al., 1996; van Duynhoven & de Jonge, 2001; Wesley, 1997). However, since isolation of H. pylori from foods is extremely difficult due to the presence of accompanying microflora and to the presumably very low H. pylori load contaminating foodstuff (Cellini, Del Vecchio et al., 2004; Dunn et al., 1997; Stevenson et al., 2000), it is important to evaluate specific, sensitive and rapid methods for the detection of this pathogen from food products, and in particular from milk. This article reports the results of the employment of a Nested- PCR assay for the detection of H. pylori in raw sheep, goat and cow milk samples. ...
Helicobacter pylori is an organism widespread in the human population and sometimes responsible for serious illnesses. Since H. pylori has been detected in Italy from an high percentage of sheep milk samples, it has been hypothesized that contaminated milk, may act as a vehicle of transmission of the microorganism to humans. In this work, a Nested Polymerase Chain Reaction approach has been used to detect H. pylori phosphoglucosamine mutase gene (glmM) from sheep, goat and cow milk artificially contaminated with wild H. pylori strains isolated from human gastric biopsies and the reference strain (H. pylori ATCC 43504). The technique showed a high sensitivity of 3 CFU/ml and proved to be both specific and rapid, The authors suggest that it could be used as a sensitive method for a rapid screening of sheep, goat and cow milk samples during the microbiological control of these large consumed foods.
... The culture was grown on Mueller-Hinton agar with the addition of 10% defibrinated mutton blood and a selective additive-Campylobacter Selective Supplement (HiMedia, Thane West, India) [57]. Cultivation was carried out in the ILM-170 CO 2 incubator (LAMSYSTEMS, Miass, Russia) at 37 • C in an atmosphere with 10% CO 2 [59]. In the experiment, they used an overnight culture grown under similar conditions on a heart-brain broth with the addition of 10% defibrinated mutton blood and Campylobacter Selective Supplement (HiMedia, India). ...
The antimicrobial properties of baicalin against H. pylori and several probiotic cultures were evaluated. Baicalin was isolated from a dry plant extract obtained by extraction with water at 70 °C. For isolation, extraction was carried out with n-butanol and purification on a chromatographic column. The antimicrobial potential was assessed by evaluating changes in the optical density of the bacterial suspension during cultivation; additionally, the disk diffusion method was used. During the study, the baicalin concentrations (0.25, 0.5, and 1 mg/mL) and the pH of the medium in the range of 1.5-8.0 were tested. The test objects were: suspensions of H. pylori, Lactobacillus casei, L. brevis, Bifidobacterium longum, and B. teenis. It was found that the greater the concentration of the substance in the solution, the greater the delay in the growth of the strain zone. Thus, the highest antimicrobial activity against H. pylori was observed at pH 1.5-2.0 and a baicalin concentration of 1.00 mg/mL. In relation to probiotic strains, a stimulating effect of baicalin (1.00 mg/mL) on the growth of L. casei biomass at pH 1.5-2.0 was observed. The results open up the prospects for the use of baicalin and probiotics for the treatment of diseases caused by H. pylori.
... Bacterial strains, plasmids and culture E. coli cells were grown in LB media and appropriate antibiotics were added when required. H. pylori strains (26695 and P12) were grown on Brain Heart Infusion (BHI) agar (Difco) as described elsewhere [25,26]. Supplementary Table S1 shows the E. coli strains harboring different constructs used in this study. ...
Helicase loaders are required for the loading of helicases at the vicinity of replication origins. In Helicobacter pylori , Hp0897 has been shown to be a potential helicase loader for replicative helicase (HpDnaB) although it does not show any sequence homology with conventional DnaC like helicase loader proteins. Therefore, it is important to investigate the in vivo role of Hp0897 and structure function analysis with respect to domain mapping of Hp0897 and HpDnaB. Although HporiC is divided into oriC1 and oriC2 , the latter has been assigned as functional origin based on loading of initiator protein HpDnaA. Using chromatin immunoprecipitation (ChIP) experiment, we show preferential binding of Hp0897 at oriC2 over oriC1 like HpDnaA highlighting its helicase loader function in vivo Further, we generated series of deletion mutants for HpDnaB and Hp0897 that enabled us to map the domains of interaction between these two proteins. Interestingly, the C-terminal domain of Hp0897 (Hp0897CTD) shows stronger interaction with HpDnaB over the N-terminal region of Hp0897 (Hp0897NTD). Similar to the full-length protein, Hp0897CTD also stimulates the DNA binding activity of HpDnaB. Further, overexpression of Hp0897 full length protein in H. pylori leads to an elongated cell phenotype. While the overexpression of Hp0897CTD does not show a phenotype of cell elongation, overexpression of Hp0897NTD shows extensive cell elongation. These results highlight the possible role of Hp0897 CTD in helicase loading and Hp0897NTD's unique function linked to cell division that make Hp0897 as a potential drug target against H. pylori.
... using Blood Agar Base no. 2 supplemented with horse blood has been well established in our laboratory. However, alternate agar bases such as Brucella agar and Columbia blood agar can also be used 22 . It is important to ensure that only sterile glassware that is free of detergent is used to prepare the growth medium. ...
Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing ≥105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.
... 3,4 However, there are no studies that compare the effectiveness of these media by repeated experiments. 3,7,10,11 Therefore, we aimed to compare and contrast four agar-based media (chocolate media, brucella media, BHI media and Thayer-Martin [TM] agar) for allowing efficient growth of H. pylori and to find the most suitable one for laboratory use. ...
Background/Aims: Optimal culture conditions for Helicobacter pylori have not been established. We compared the effectiveness of four different agar-based media for the growth of H. pylori. Materials and Methods: G27, ATCC #43504 and 60190, and primary cultured strains were used. H. pylori strains were cultured for four days under four culture conditions: chocolate agar, Thayer-Martin (TM) agar containing vancomycin-colistin-nystatin inhibitor (VCNI), Brucella agar, and brain heart infusion (BHI) agar containing 5% horse blood and IsoVitaleX (BBL™ BD, USA). Culture of cells in each medium was repeated fourteen times. The growth of H. pylori was measured by using a spectrophotometer. Results: TM, Brucella, and BHI agars showed mean absorbance values of 0.099, 0.059, 1.410, and 0.913, respectively. These values were significantly different (P=0.030). After post-adjustment by Bonferroni correction, similar growth was noted for in chocolate, Brucella, and BHI agars; however, TM agar significantly suppressed H. pylori growth compared with Brucella agar (P=0.031). Conclusions: Chocolate, Brucella, and BHI agars provided effective culture conditions for the growth of H. pylori. TM agar containing VCNI suppressed the growth of H. pylori and other organisms.
... The culture of H. pylori from the stomach tissue is considered as the most important proof of colonization. For this purpose, it is necessary to create optimal conditions for the growth of bacteria, taking into ac- count their nutritional requirements, the composition of the atmosphere, the humidity and temperature, as well as the elimination of foreign microflora (Stevenson et al., 2000). Regarding these factors, we prepared bacterial suspension for the inoculation of guinea pigs in complete Brucella broth supplemented with 10% FCS, and a Skirrow complex of antibiotics limiting the growth of bacteria other than Helicobacter. ...
... The difficulty we faced was how to mimic special conditions encountered by this organism either in environment or in gastric niche. Stevenson et al. [28] showed that most media used for culturing H. pylori provide similar level of growth. To study the biofilm between liquid and air interface, the bacteria were cultured on blood agar before transferring into the liquid media to accelerate the biofilm formation process. ...
... Moreover, the method employed for H. pylori isolation may lack sufficient sensitivity to recover very low numbers of H. pylori due to the presence of viable nonculturable form (10 CFU per ml of liquid samples to recover at least one colony per plate) [20][21][22][23][26][27][28][29]. Furthermore, isolation is extremely difficult due to the presence of accompanying microflora and very low numbers of H. pylori in foodstuffs [33]. ...
Aim:
The aim was to detect the glmM gene of Helicobacter pylori (H. pylori) in cow's milk from different dairy farms in Khartoum State using Nested polymerase chain reaction (PCR).
Materials and methods:
A total of 50 milk samples were collected from different dairy farms in Khartoum State (13 from Khartoum, 24 Khartoum North, and 13 from Omdurman Provinces).
Results:
The generated results showed that 11/50 (22%) were harboring the investigated H. pylori glmM gene in Khartoum State (1/13 [7.7%] Khartoum, 9/24 [37.5%] Khartoum North, and 1/13 [7.7%] Omdurman provinces, respectively).
Conclusion:
To the best of our knowledge, this was the first report on the detection of H. pylori glmM gene in cattle milk in Khartoum State. Nonetheless, the high percentages of H. pylori DNA detection in milk opened new avenues toward exploring the risk of human infection with H. pylori through the consumption of raw milk.
... Most media contain blood as oxygen-detoxifying agent, rather than FBP, following the observation of Goodwin et al. (1985) that some strains of H. pylori are inhibited by sodium metabisulphite in this mixture. Stevenson et al. (2000a) examined the productivity of a variety of basal liquid and solid media for two strains of H. pylori. They found that the bacteria grew similarly in brain heart infusion, Mueller Hinton and a modification of Johnson and Murano's (1999a) broth for arcobacters. ...
The history of the development of selective media for isolation of campylobacters, including the rationale for choice of selective agents is described. Developments have included modifications to allow incubation at 37°C instead of 42 or 43°C and changes in the types and concentrations of antibiotics in order not to inhibit organisms such as Campylobacter upsaliensis, C. jejuni subsp. doylei and some strains of C. coli and C. lari. When examining foods, plating media originally developed for isolation from faeces are normally used, sometimes after liquid enrichment. Most of the media include ingredients intended to protect campylobacters from the toxic effect of oxygen derivatives. Most commonly used are lysed or defibrinated blood, charcoal, a combination of ferrous sulphate, sodium metabisulphite and sodium pyruvate (FBP); and haemin or haematin. The manner in which liquid enrichment media are used has been modified for food samples to avoid inhibitory effects on sublethally damaged cells by toxic components in the formula. This is done by a preliminary period of incubation at reduced temperature and sometimes by delayed addition of antibiotics. Expensive and time-consuming methods have been proposed to achieve a microaerobic atmosphere while using liquid enrichment media. To date there is no generally accepted "standard" method of isolating campylobacters from food, although Bolton broth and modified charcoal, cefoperazone deoxycholate agar have been proposed in a number of standard methods. Media for isolating arcobacters are similar to those for campylobacters, except that lower temperatures and sometimes aerobic atmosphere are used for incubation. Some strains of Arcobacter cryaerophilus and A. skirrowii are sensitive to 32 mg/l cefoperazone and all Arcobacter sp. are sensitive to colistin used in some media. Media for the human pathogen Helicobacter pylori have been developed, although, with one exception, using immunomagnetic beads, they have not been successful in isolating the organism from foods or the environment. Many other Helicobacter-like organisms, seen in gastric or intestinal tissue samples from a variety of animals have not been successfully cultivated.
... The pathogen Helicobacter pylori is the etiologic agent of gastritis and peptic ulcer disease and is also associated with the development of gastric adenocarcinoma and MALTlymphoma (1,2). The fastidious nature of this Gram-negative microorganism has been widely recognized and numerous solid and liquid media have been developed for its isolation and culture (3). Despite all the difficulties to grow this microorganism in vitro, growth in liquid media is particularly more challenging than in solid media (4). ...
The physiological behavior of Helicobacter pylori in different growth conditions was investigated to approach its growth standardization. H. pylori free-living and biofilm modes of growth were assessed in four different liquid culture media (Brucella broth, brain heart infusion broth and Ham's F-12 supplemented with 2% fetal calf serum and Ham's F-12 without serum). Free-living growth was monitored during 72 h in each medium and characterized for bacterial density, culturability, viability and morphology. The biofilm formation in the same media was evaluated for biomass production, colony forming unit (CFU) counts and microscopic visualization. Afterward, using Ham's F-12, the effect of amoxicillin and clarithromycin at sub- minimum inhibitory concentrations (sub-MICs) was evaluated on H. pylori biofilm formation and luxS gene expression. Differences in the free-living growth were observed between the media supplemented with serum and Ham's F-12 without serum. Biofilm formation was significantly dependent on the growth media used. Ham's F-12 seems to be a good medium to support both growth phenotypes of H. pylori. Moreover, sub-MICs of antibiotics increased the biofilm formation and affected the luxS gene expression. Optimizing the growth conditions of H. pylori, especially in the biofilm mode, will be helpful to perform more accurate in-depth studies that will allow increasing the knowledge about H. pylori biofilm, which should be a target to eradicate resistant infection.
... The isolation of H. pylori from foods with a high background flora (such as raw milk) is time consuming and requires complex selective media, lengthy periods of incubation and microaerobic conditions (Stevenson et al., 2000). In addition, under various adverse environmental conditions, such as exposure to atmospheric oxygen or antimicrobial agents, H. pylori can produce viable but non-culturable (VNC) coccoid forms (Bode et al., 1993). ...
The transmission pathways of Helicobacter pylori in humans have not been fully elucidated. Research in the last decade has proposed that foodborne transmission, among others, may be a plausible route of human infection. Owing to the organism's fastidious growth characteristics and its ability to convert to viable, yet unculturable states upon exposure to stress conditions, the detection of H. pylori in foods via culture-dependent methods has been proven to be laborious, difficult and in most cases unsuccessful. Hence, nucleic acid-based methods have been proposed as alternative methods but, to date, only PCR-based methods have been reported in the literature. In the current study, fluorescence in situ hybridization (FISH) was used for the detection of H. pylori in raw, bulk-tank bovine milk. After repeated milk centrifugation and washing steps, the bacterial flora of raw milk was subjected to fixation and permeabilization and H. pylori detection was conducted by FISH after hybridization with an H. pylori-specific 16S rRNA-directed fluorescent oligonucleotide probe. Using this protocol, H. pylori was detected in four out of the twenty (20%) raw milk samples examined. The data presented in this manuscript indicate that FISH can serve as an alternative molecular method for screening raw bovine milk for the presence of H. pylori.
... Traditionally, H. pylori has been isolated and grown on agar plates containing brain heart infusion media, brucella media, Columbia blood agar base, or Mueller-Hinton media supplemented with 5-10% horse or bovine serum in 5-10% CO 2 air incubators [6,7,[11][12][13]. There have been several attempts to facilitate the growth of H. pylori in liquid media [9,[14][15][16][17]. However, improvements in the liquid cultivation of H. pylori are needed for broader investigations, such as biochemical, genomic, and physiologic studies. ...
Several attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the growth of H. pylori in liquid media in order to get affluent growth and a simple approach for examining bacterial properties. We introduce here a thin-layer liquid culture technique for the growth of H. pylori.
A thin-layer liquid culture system was established by adding liquid media to a 90-mm diameter Petri dish. Optimal conditions for bacterial growth were investigated and then viability, growth curve, and released proteins were examined.
Maximal growth of H. pylori was obtained by adding 3 mL of brucella broth supplemented with 10% horse to a Petri dish. H. pylori grew in both DMEM and RPMI-1640 supplemented with 10% fetal bovine serum and 0.5% yeast extract. Serum-free RPMI-1640 supported the growth of H. pylori when supplemented with dimethyl-beta-cyclodextrin (200 microg/mL) and 1% yeast extract. Under optimal growth, H. pylori grew exponentially for 28 hours, reaching a density of 3.4 OD(600) with a generation time of 3.3 hours. After 24 hours, cultures at a cell density of 1.0 OD(600) contained 1.3 +/- 0.1 x 10(9 )CFU/mL. gamma-Glutamyl transpeptidase, nuclease, superoxide dismutase, and urease were not detected in culture supernatants at 24 hours in thin-layer liquid culture, but were present at 48 hours, whereas alcohol dehydrogenase, alkylhydroperoxide reductase, catalase, and vacuolating cytotoxin were detected at 24 hours.
Thin-layer liquid culture technique is feasible, and can serve as a versatile liquid culture technique for investigating bacterial properties of H. pylori.
... A wide variety of solid media have been used by many investigators for the isolation of H. pylori [16][17][18][19]. In this study, different results were obtained by using three selective media for isolation of H. pylori. ...
Helicobacter pylori (H. pylori) is the etiologic agent of a variety of gastrointestinal disorders. The rationale of the current study is to evaluate six enzyme immunoassays for detection of anti-H. pylori immunoglobulin G (IgG) and IgA in Jordanian patients. Biopsy specimens and blood samples were obtained from patients underwent the endoscopy unit at Al-Bashir hospital in Jordan. The serum samples were investigated for the presence of anti-H. pylori IgG and IgA antibodies in patients with positive H. pylori biopsy samples. The results showed that IgG utilizing kits are more sensitive than of IgA kits and the IgA kits are more specific than of that IgG kits. Moreover, the biopsy is seemingly the gold standard for diagnosis of H. pylori is followed by H. pylori culture on brucella agar medium. An imperfect relation between the presence of H. pylori infection and the antibody response was existed that could be explained either because of the unsatisfactory sensitivities and specificities of the commercial kits used or because of weak immunological response in our patients to H. pylori antigens. Collectively, the H. pylori diagnosis that depends on the detection of anti-H. pylori antibodies in the hospital setting and in the screening programs should consider another test for confirmation the initial diagnosis.
... The therapeutic value of charcoal is gained through absorption of toxic agents from the gastrointestinal tract [13]. On the other hand, the application value of charcoal in the promotion of growth of microorganisms such as Coryneabacteria, Brucella, Bordetella species, improvement of the production of virulence factors of other pathogens such as Listeria monocytogenes in vitro, and recovery of fastidious microorganism Flavobacterium psychrophilum, has been demonstrated [1,3,5,7]. It seems that the effect of charcoal in promoting the bacterial growth occurs through its direct adsorption of toxic substances produced during bacterial metabolism [15]. ...
Helicobacter pylori, a microaerophilic fastidious bacterium, has been cultured on various plating and broth media since its discovery. Although the agar media can be sufficient for the identification, typing, and antibiotic resistance studies, no secretory antigen of H. pylori can be evaluated in such media. Thus, satisfactory growth of H. pylori in liquid culture which is needed for analysis of secretory proteins without the presence of interfering agents is in demand. We assessed the impact of beta-cyclodextrin, Fetal Bovine Serum (FBS), and charcoal as supplements for H. pylori growth. Furthermore, we aimed to identify the most favorable supplement that supports the secretion of the dominant secretory protein, vacuolating cytotoxin (VacA). Five clinical strains were cultured on broth media and the growth, viability, morphology, and protein content of each strain were determined. Our results revealed that beta-cyclodextrin supports the growth rate, viability, and cell lysate protein content to the extent similar to FBS. Application of beta-cyclodextrin is found to postpone spiral to coccoid conversion up to 72 h of incubation. Although FBS supports a higher VacA protein content, presence of interfering macromolecules in FBS questions its utility particularly for purposes of studying extra cellular proteins such as VacA. This study recommends further application of beta-cyclodextrin as a culture supplement with the potential capacity in neutralizing toxic compounds and flourishing the secretion of H. pylori proteins without addition of interfering proteins.
... H. pylori NCTC 11637 and C. albicans ACTC 90025 were used in the study. H. pylori was grown for 2 days on Columbia blood agar at (Oxoid, UK) 37 o C under microaerobic conditions (Stevenson et al., 2001). C. albicans was grown on Columbia blood agar for 24hours at 37 o C. The number of bacterial cells was determined by obtaining viable counts of serial dilutions and measurement of the optical absorbance at 600nm (Ultrospec II, LKB, UK) of a suspension of bacterial cells to prepare a standard curve for H. pylori. ...
We have compared current image analysis software packages in order to find the most useful one for assessing microbial adhesion and inhibition of adhesion to tissue sections. We have used organisms of different sizes, the bacterium Helicobacter pylori and the yeast Candida albicans. Adhesion of FITC-labelled H. pylori and C. albicans was assessed by confocal microscopy. Four different Image analysis software packages, NIH-Image, IP Lab, Image Pro+, and Metamorph, were compared for their ability to quantify adhesion of the two organisms and several quantification methods were devised for each package. For both organisms, the dynamic range that could be detected by the software packages was 1x10(6)-1x10(9) cells/ml. Of the four software packages tested, our results showed that Metamorph software, using our 'Region of Interest' method, with the software's 'Standard Area Method' of counting, was the most suitable for quantifying adhesion of both organisms because of its unique ability to separate clumps of microbial cells. Moreover, fewer steps were required. By pre-incubating H. pylori with the glycoconjugate Lewis b-HSA, an inhibition of binding of 48.8% was achieved using 250 mug/ml Lewis b-HSA. The method we have devised using Metamorph software, provides a simple, quick and accurate way of quantifying adhesion and inhibition of adhesion of microbial cells to the epithelial surface of tissue sections. The method can be applied to organisms ranging in size from small bacteria to larger yeast cells.
... jejuni, a typical emerging foodborne pathogen, it is hypothesized that both Hp and C. jejuni have a similar transmission pattern and are thus foodborne pathogens (28). The isolation of Hp from foods, particularly when they present high loads of accompanying microflora, is exacting and time-consuming, since it requires selective media containing several antibiotics, microaerophilic conditions, and long incubation periods (7 days) (23). Under adverse environmental conditions such as desiccation, lack of protection against oxygen, and exposure to antimicrobial agents, Hp may produce viable nonculturable forms (5). ...
Helicobacter pylori (Hp) is an organism commonly present worldwide in the human population, sometimes causing serious illnesses such as duodenal and gastric ulcers, adenocarcinoma of the stomach, and low-grade B-cell mucosa-associated lymphoid tissue lymphoma of the stomach. This article describes a multiplex-touchdown PCR method for the identification and genotyping (vacA-s1/m1, sl/m2, and s2/m2-and cagA genes) of Hp directly from sheep milk artificially contaminated with Hp strains from human gastric biopsies and with Hp ATCC 43504. The strains from humans carried sl/m2 cagA+ and s2/m2 cagA allelic combinations, while the ATCC strains carried an sl/ml cagA+ allelic combination. The technique showed a sensitivity of 15 CFU/ml for species identification and of 1,500 CFU/ml for the detection of genes encoding for VacA and CagA. It has proven to be specific and rapid, and the authors suggest that it be used as a rapid screening method to ensure that sheep milk is uncontaminated with this organism.
... Isolation of H. pylori from food samples, particularly when they present high loads of accompanying microflora, is exacting and time-consuming since it requires the use of selective media added with numerous antibiotics, microaerophilic conditions and long incubation periods (7 days) (Stevenson et al., 2000). Furthermore, under adverse environmental conditions such as desiccation, lack of protection against oxygen, exposure to antimicrobial agent, H. pylori may produce viable nonculturable form (VNC) (Dunn et al., 1997;Cellini et al., 2004b). ...
Helicobacter pylori is an organism widespread in humans and sometimes responsible for serious illnesses, such as gastric and duodenal ulcers, MALToma and even gastric cancer. It has been hypothesized that the infection route by H. pylori involves multiple pathways including food-borne transmission, as the microorganism has been detected from foods such as sheep and cow milk. This work reports the results of a survey conducted in order to investigate the presence of H. pylori in raw goat, sheep and cow milk produced in Southern Italy, employing a Nested Polymerase Chain Reaction (Nested-PCR) assay for the detection of the phosphoglucosamine mutase gene (glmM), as screening method followed by conventional bacteriological isolation. Out of the 400 raw milk samples examined, 139 (34.7%) resulted positive for the presence of glmM gene, but no strains were isolated. In this work H. pylori DNA has been firstly detected from 41 (25.6%) raw goat milk samples. The results deserve further investigations on the contamination source/s of the milk samples and on the major impact that it may have on consumers.
... Various non-specific liquid culture media (Brucella broth, Mueller-Hinton broth, brain heart infusion broth) have been used historically to culture H. pylori, but none of them exhibited any particular advantage [15][16][17]. Previous studies on H. pylori growth in complex and chemically defined liquid media demonstrated that this species is not as fastidious as it was formerly considered to be [18][19][20][21][22]. The addition of blood to the medium or the maintenance of a strict microaerophilic atmosphere was found to be not absolutely essential [23][24][25][26][27]. ...
The goal of this study was to develop a liquid culture medium for the rapid isolation, cultivation, identification and subsequent antibiotics susceptibility testing of Helicobacter pylori directly from biopsy specimens. Five liquid media were tested: Ham's F-12, Brucella broth, tryptic soybroth, brain heart infusion broth and Mueller-Hinton broth. After optimisation of the medium, it was applied in order to investigate biopsy samples from 150 patients with gastro-duodenal disorders and compared with traditional culture methods, microscopy and an H. pylori-specific TaqMan real-time polymerase chain reaction (PCR). The most reliable and rapid growth of H. pylori, even at a small inoculum size, was obtained in Ham's F-12 medium with 5% horse serum. The developed system allowed the primary isolation of H. pylori in clinical samples and provided 87% sensitivity and 100% specificity.
Helicobacter pylori is a gram-negative bacteria that infects a majority of the world's population. It causes various diseases such as chronic gastritis, peptic ulcer and gastric cancer. While a majority of the people infected with H.pylori is asymptomatic. The main factors, which determine the development of H. pylori-related diseases might be bacterial virulence, host genetic and environmental factors.
The present study was carried out to determine the frequency of H. pylori infection in patients attending the endoscopic unit at Al-Sadr Teaching Hospital in Basrah who are suffering from gastrointestinal symptoms and were diagnosed by a specialist doctor.
During November 2020 to May 2021, three types of clinical samples were collected from 120 patients ( 53 male and 67 female), the age range was ( 11-76 years), including tissue, saliva and stool. To investigate H.pylori three biopsies of gastric mucosal tissue were taken for rapid urease test (RUT), bacterial culture and polymerase chain reaction (PCR) technique. Stool antigen detection. saliva swabs were taken and cultured.
The incidence of H. pylori infection among these patients was 48 by using four diagnostic methods, including rapid urease test( RUT), stool antigen test and PCR. While 20 H.pylori were isolated by culturing method on selective and modified media ( Modified Columbia Urea Agar, Modified Brain Heart Infusion Agar and H. pylori special peptone medium (HPSP )agar, and identified through biochemical tests ( Oxidase, Catalase, and Urease ) as well as Gram stain.
There was no significant difference (P>0.05) between males and females in the rate of H. pylori infection, the percentage of males was 54.2% and 45.8% . The highest percentage of infection was in the age group ≥ 60, which amounted to 55.6%, and there were no significant differences (P>0.05) between age groups of H.pylori infection.
An antibiotic sensitivity test was conducted on the isolates using six types of antibiotics. Multiple resistance to most antibiotics was recorded. Amoxicillin 60%, Doxycycline 70%, Metronidazole 100%, Ciprofloxacin 55%, Clarithromycin is the most used antibiotic 70%, while the most effective antibiotic according to the result of this study was Levofloxacin, which recorded a higher sensitivity 85%.
The results of 16S rRNA ( PCR) showed that 48 of H. pylori infection by using 138 base pairs gene-specific primer. The virulence factors were evaluated by using a gene-specific primer. Cag A gene was detected in 27 and Vac A gene was detected in 29 of the samples from both isolates and biopsies directly.
Helicobacter pylori crude supernatant production were performed through inoculated brain heart infusion broth by the bacteria isolates and incubated in a rotary-shaker incubator, respective supernatants were then carefully separated and poured into new vials and stored at room temperature after which the supernatant was freeze-drying the effective were the appearance of 10 chemical compounds that at the same time in with different percentages, when analysis GC-MS device.
The toxicity of H.pylori was examined by using experimental animals ( Balb/c) mice. Two mice from six were injected intraperitoneally ( I.P.) with 155 mg/ml H.pylori crude supernatant died after 48 hours of injection, while six mice that were administered H.pylori crude supernatant orally were survived and compared to the control group. For the interpretation of the toxin effect, the abdomen was opened and the complete stomach, intestine, kidney, and liver for all groups were removed and promptly fixed in 10% formalin overnight. The macroscopic results revealed the differences between normal ( control ) and H. pylori crude supernatant injected mice, such as hypertrophied liver, degenerated stomach characteristics, and infiltration of inflammatory cells in the lamina propria, when compared to normal mice.
To study the effect of H. pylori crude supernatant, three types of cell lines were used HBL-100 (normal breast), SKG (esophageal cancer), and HCAM (liver cancer).
The results indicated that H. pylori crude supernatant has an effect on the viability of the HBL-100 cell line. The viability of cells is dependent on concentrations, from selected five isolates only one affected the viability of the HBL-100 cell line concentration 1000 ìg /ml, while the effect of H. pylori crude supernatant on Esophagus Cancer and Liver Cancer can be inhibited DNA synthesis and stop the proliferation of the cells. The viability decreased from 30 %,40 %, and 50% when the SKG (Esophagus Cancer) and HCAM(Liver Cancer) cells were treated with H. pylori crude supernatant.
This is the highly anticipated third edition of a book written by the Working Party on Culture Media of the International Committee on Food Microbiology and Hygiene. It is a handy reference for microbiologists wanting to know which media to use for the detection of various groups of microbes in foods and how to check the performance of the media. The book is divided into two parts and concentrates on media for water as well as food microbes - selecting those which have been evaluated and shown to function optimally. The first part consists of a series of chapters written by various experts from all over the world, reviewing the media designed to detect the major groups of microbes important in food spoilage, food fermentations and food-borne disease. The history and rationale of the selective agents and indicator systems used, as well as the relative merits of the various media are surveyed by reference to the scientific literature. The second part contains monographs on almost 100 of the media considered most useful. Each monograph, written in the style of a pharmacopoeia, includes: a short section on the history and selective principle of the medium; a method for its preparation from basic ingredients; its appearance and physical properties, including pH; its shelf-life; instructions concerning method of inoculation, incubation and interpretation; the recommended method(s) and a list of test strains suitable for assessing the quality (productivity and selectivity) of the medium and a description of the typical appearance of the target organism.
The essential oil isolated from the bark of Cinnamomum glanduliferum (Wall) Meissn that grown in Egypt was screened for the first time. The chemical composition was analyzed by GC and GC/Mass. The antimicrobial activity of the oil was assessed using agar-well diffusion method toward representatives for each of Gram-positive bacteria, Gram-negative bacteria, and fungi. The cytotoxic activity was checked using three human cancer cell lines. Twenty seven compounds were identified, representing 99.07% of the total detected components. The major constituents were eucalyptol (65.87%), terpinen-4-ol (7.57%), α-terpineol (7.39%). The essential oil possessed strong antimicrobial activities against Escherichia coli, as the activity index (AI) equal to one and MIC is 0.49 μg/mL. Good antimicrobial activities were against methicillin-resistant Staphylococcus aureus, Geotrichum candidum, Pseudomonas aeruginosa, Bacillus subtilis, Helicobacter pylori, Aspergillus fumigatus (MIC: 7.81, 1.95, 7.81, 0.98, 31.25, and 32.5 μg/mL respectively). A considerable activity was observed against Staphylococcus aureus and Mycobacterium tuberculosis (MIC; 32.5 and 31.25 μg/mL). It was toxic to colon (HCT-116), liver (HepG2), and breast (MCF-7) carcinoma cell lines at IC50 9.1, 42.4, and 57.3 μg/mL. These results revealed Egyptian Cinnamomum glanduliferum bark oil possess antimicrobial and cytotoxic activities mainly due to eucalyptol and other major compounds. This article is protected by copyright. All rights reserved.
Helkobacter pylori (H.p.) is a widespread microorganism in humans and is able to cause diseases, in same cases, serious. Among the route of transmission it is possible that contaminated foods are able to induce the infection to humans. In this paper the Authors report a PCR method for the identification and genotyping of the microorganism directly from artificially contaminated milk. The technique shows sensibility, specificity and rapidity of detection.
Objective: To establish a rapid, specific and sensitive method for the detection of Helicobacter pylori, and provide an effective detecting evidence for Helicobacter pylori. Methods: A pair of primers and probe corresponding to the urease gene for real time PCR were designed according to Primer Express 3.0 software, similar sequences were searched by Blast method, and the excellent primers and probe were selected. DNA from common bacterial pathogens such as Escherichia coli, Staphylococcus, histeria monocytogenes, Campylobacter and Salmonella in food was used for specific test; the counted Helicobacter pylori in bacterium suspension and the bacterium and milk mixture were serially diluted, the DNA was extracted for real time PCR amplification, the sensitivity of method was tested. The practicability of the method was demonstrated through the detection of the artificial contaminative samples by real time PCR. Results: The primers and probe according to urease gene could only amplify Helicobacter pylori DNA, but not other reference bacterium DNA. Helicobacter pylori from the artificial contaminative samples could be amplified by the method. It can detect 3 CFU · mL-1 of Helicobacter pylori. Its sensitivity was sufficient to detect 3 CFU · mL-1 of Helicobacter pylori. The method was enough rapid to finish detection in 4 d. Conclusion: Real time PCR can rapidly and accurately detect the Helicobacter pylori contamination in food with high specificity and sensitivity. © 1994-2012 China Academic Journal Electronic Publishing House. All rights reserved.
Introduction* Physiology and growth requirements * Disease associations and mechanisms of virulence * Epidemiology and routes of transmission * Detection methods and culture from clinical samples, food and water * Survival in food and water * Conclusions and future trends * Sources of further information * References.
Helicobacter pylori is an etiological agent of chronic gastritis, gastric and duodenal ulcers and gastric cancers. The use of an appropriate animal model for experimental studies on the pathogenesis of H. pylori infections is necessary due to the chronic character of such infections and difficulties in identifying their early stage in humans. The aim of this study was to develop a guinea pig model of H. pylori infection and identify its microbiological, histological, serological and molecular determinants. Guinea pigs were inoculated per os with H. pylori strains: CCUG 17874 or ATCC 700312, both producing vacuolating cytotoxin A (VacA) and cytotoxin associated gene A (CagA) protein, suspended in Brucella broth with fetal calf serum (FCS) and Skirrow supplement of antibiotics. To determine H. pylori colonization, 7 and 28 days after the challenge, a panel of diagnostic methods was used. It included culturing of microorganisms from the gastric tissue, histopathological analysis of gastric sections, stained by Mayer,s haematoxylin and eosin to assess inflammatory response, by Giemsa as well as Warthin-Starry silver staining to visualise Helicobacter-like organisms (HLO) and with anti-Ki-67 antigen to assess epithelial cell proliferation. H. pylori infection was also confirmed by polymerase chain reactions (PCR) for detection in gastric tissue of ureC and cagA genes and by serological assessment of H. pylori antigens in faeces. This study showed the usefulness of microbiological, histological, immunological and molecular methods for the detection of persistent H. pylori infections in guinea pigs, which could be an appropriate model for studying H. pylori pathogenesis and the related immune response against these microbes.
Im Rahmen der vorliegenden Arbeit konnte gezeigt werden, dass kariesassoziierte Erreger Einfluss auf die Überlebensfähigkeit von H. pylori KE haben. Hierzu wurden Wachstumsexperimente mit Co-Kulturen in humanem Speichel durchgeführt. Die Überlebensrate von H. pylori KE war in Co-Kultur mit Streptococcus mutans, Actinomyces naeslundii und Candida dubliniensis höher als in Poolspeichel-Monokultur, in der H. pylori KE für mindestens 48 h überdauern konnte. Auch in Co-Kultur mit Lactobacillus casei überlebte H. pylori KE, jedoch in etwas geringerer Keimzahl als mit den erstgenannten kariesassoziierten Erregern. Ein hemmender Effekt wurde in Co-Kultur mit Streptococcus oralis nachgewiesen. Insgesamt war H. pylori KE nach einer Woche Inkubation in Poolspeichel weder in Mono- noch in Co-Kultur mit den ausgewählten kariesassoziierten Erregern noch anzüchtbar. Zur Verifizierung der Ergebnisse wurden die Wachstumsexperimente mit dem klinischen H. pylori-Isolat SS1 wiederholt. Im Gegensatz zu H. pylori KE überlebte H. pylori SS1 sogar eine gesamte Woche in Poolspeichel-Co-Kultur mit S. mutans und C. dubliniensis.
Ein inhibitorischer Einfluss von Probiotika auf H. pylori konnte für L. casei nicht nachgewiesen werden.
Mittels Doppelimmunfluoreszenz wurde zudem untersucht, ob sich H. pylori SS1 intrazellulär in C. dubliniensis einnisten könnte, was sich als negativ herausstellte. H. pylori kann jedoch eventuell durch extrazelluläre Einlagerung in das Pilzgeflecht kurzfristig besser in der Mundhöhle überdauern.
Aus den Wachstumsergebnissen in Poolspeichel-Monokultur lässt sich schlussfolgern, dass H. pylori in der Mundhöhle für kurze Zeit überleben kann. Dieses Bakterium kann jedoch nicht als permanenter Keim der Mundhöhle betrachtet werden. Das Wachstumsverhalten in Co-Kultur mit den untersuchten Mikroorganismen bestätigt die kurze Überlebensdauer in der Mundhöhle, was sein intermittierendes orales Auftreten erklären könnte.
Helicobacter pylori is a gastroduodenal pathogen that colonizes the human stomach and is the causal agent of gastric diseases. From the clinical and epidemiological point of view, enhancing and improving the growth of this bacterium in liquid media is an important goal to achieve in order to allow the performance of accurate physiological studies. The aim of this work was to optimize three culture conditions that influence the growth of H. pylori in the defined medium Ham s F-12 supplemented with 5 percent fetal bovine serum by using response surface methodology as a statistical technique to obtain the optimal conditions. The factors studied in this experimental design (Box-Behnken design) were the pH of the medium, the shaking speed (rpm) and the percentage of atmospheric oxygen, in a total of 17 experiments. The biomass specific growth rate was the response measured. The model was validated for pH and shaking speed. The percentage of atmospheric oxygen did not influence the growth for the range of values studied. At the optimal values found for pH and shaking speed, 8 and 130 rpm, respectively, a specific growth rate value of 0.164 h-1, corresponding to a maximal concentration of approximately 1.5x108 CFU/ml, was reached after 8 h. The experimental design strategy allowed, for the first time, the optimization of H. pylori growth in a semi-synthetic medium, which may be important to improve physiological and metabolic studies of this fastidious bacterium.
Background: To asses the status of two representative genes of cag PAI i.e cagA and cagE of Helicobacter pylori strains infecting Iranian patients suffered from various clinical outcomes using one-step PCR. Methods: A total of 120 H. pylori infected patients including non–ulcer dyspepsia, NUD (n=81), peptic ulcer disease, PUD (n=17), and gastric carcinoma, GC (n= 22) referred for endoscopy or gastric resection to AmirAlam Hospital or Cancer Institute from 2005 to 2008 were assessed. The status of cagA and cagE genes was determined by gene specific PCR. Results: 84.2% and 90.8% of the tested strains were positive for cagA and cage, respectively. 81.7% strains were positive for both cagA and cagE genes, whereas 8 (6.7%) were found double negative. The prevalence of cagA in GC patients (100%) was slightly higher than PUD patients (94.1%). All of GC cases were infected with cagA-positive strains. The same distribution pattern was indicated for cagE gene in GC and PUD patients. The cagA-positive strains were significantly associated with GC as compared with NUD (P< 0.05) but this association did not gain statistical significance when cagE gene was assessed. Conclusion: The concurrent detection of cagA/cagE genes allowed rapid and specific clarification of cag PAI status. The strains with cagA/cagE genotype are predominant in Iran regardless of clinical outcome and create a distinct cluster pattern from those in the West and similar to those of East Asian countries. The current study also demonstrated that cagE gene can be explored as a better indication of cag-PAI in Iranian H. pylori strains.
Infection by Helicobacter pylori is recognized as the major cause of gastric disease and its prevalence is elevated worldwide. It is hypothesised that the transmission of H. pylori involves multiple pathways: iatrogenic, oral–oral and faecal–oral. Food and water are suspects of serving as vehicles in the faecal–oral route of H. pylori infection. However, the difficult cultivation of H. pylori from samples with high loads of accompanying microflora and its conversion to viable but nonculturable state (VNC) impair the elucidation of the real role of waterborne and foodborne infections by this pathogen. In this sense, it is crucial the development of methods for isolation of H. pylori from environmental samples. In this work, an overview of the present knowledge on epidemiology and transmission of H. pylori is presented, attempting to the possible role of water and foods in the spread of the infection.
Background: The human bacterial pathogen Helicobacter pylori forms biofilms. However, the constituents of the biofilm have not been extensively investigated. In this study, we analyzed the carbohydrate and protein components of biofilm formed by H. pylori strain ATCC 43504 (NCTC 11637).
Materials and Methods: Development of H. pylori biofilm was analyzed using scanning electron microscopy (SEM) and quantified using crystal violet staining. The extracted extracellular polysaccharide (EPS) matrix was analyzed using GC-MS and nuclear magnetic resonance (NMR) analyses. Proteomic profiles of biofilms were examined by SDS–PAGE while deletion mutants of upregulated biofilm proteins were constructed and characterized.
Results: Formation of H. pylori biofilm is time dependent as shown by crystal violet staining assay and SEM. NMR reveals the prevalence of 1,4-mannosyl linkages in both developing and mature biofilms. Proteomic analysis of the biofilm indicates the upregulation of neutrophil-activating protein A (NapA) and several stress-induced proteins. Interestingly, the isogenic mutant napA revealed a different biofilm phenotype that showed reduced aggregated colonial structure when compared to the wild type.
Conclusions: This in vitro study shows that mannose-related proteoglycans (proteomannans) are involved in the process of H. pylori biofilm formation while the presence of upregulated NapA in the biofilm implies the potency to increase adhesiveness of H. pylori biofilm. Being a complex matrix of proteins and carbohydrates, which are probably interdependent, the H. pylori biofilm could possibly offer a protective haven for the survival of this gastric bacterial pathogen in the extragastric environments.
In the attempt to enrich the local contemporary laboratory data regarding the group B streptococcus (GBS) colonization, isolates obtained from the vaginal swab cultures were characterized for their serotype distribution and antibiotic susceptibility. The 100 GBS isolates analyzed were collected during a four-month period of year 2009 from women screened in ambulatory for vaginal carriage of GBS. The GBS isolates were classified based on their capsular polysaccharide structures using commercially available antisera. Susceptibility to penicillin, ampicillin, erithromycin, clindamycin, tetracycline, ofloxacin, and chloramphenicol was initially tested using antibiotic disk diffusion technique according to CLSI guidelines. Minimum inhibitory concentrations of erythromycin and tetracycline for the isolates with reduced susceptibility were evaluated according to the CLSI criteria and macrolide-lincosamide-streptogramin B (MLSB) resistance was investigated by a double-disk test with erythromycin and clindamycin disks. All the GBS isolates were serotypeable. Their distribution comprised six different serotypes of which serotypes II (26%), III (26%), and Ia (19%) prevailed and no serotype VI, VII, and VIII isolates were found. Overall, the GBS isolates were fully susceptible to penicillin and ampicillin, but the rates of susceptibility to the other antimicrobial agents tested were decreased, ranging from 87% for chloramphenicol to 5% for tetracycline. Reduced susceptibility to clindamycin and erythromycin was detected in 18% and 19% of isolates, respectively. For the latter, 84% displayed a constitutive MLSB phenotype, 11% had an inducible MLSB phenotype, and M phenotype was expressed by 5% of them. Erythromycin-resistant GBS isolates displayed concurrently resistance to at least one more antibiotic. In conclusion, according to our study the most frequent GBS serotypes isolated from the vaginal microflora were II and III, followed by serotype Ia. While the GBS isolates remain susceptible to beta-lactams, resistance to alternative antimicrobial drugs such as erythromycin and clindamycin seems to be an increasing concern for our region. Further phenotypic and genotypic studies are required to identify specific aspects of GBS strains colonizing or infecting the local population.
Unlabelled:
The aim of this study was to investigate the antibiotic resistance profile of 58 Gram negative bacilli strains (GNB): 36 non-fermentative GNB (NGNB), including 19 strains of Acinetobacter spp., 11 of Pseudomonas aeruginosa, 6 of Stenotrophomonas maltophilia and 22 enterobacterial strains (14 strains of KEHSs, 6 belonging to the group Proteus-Providencia and 2 Escherichia coli) isolated from nasal, pharyngeal exudates and also from bronchial secretions, from immuno-depressed patients admitted in the Intensive Care Unit of Fundeni Clinical Institute. METHHODS: the antibiotic susceptibility testing was performed according to CLSI 2009 recommendations and the production of beta-lactamases was investigated by ESBL chromogenic media, double disc diffusion test, ESBL E-test, Amp C E-test and MBL E-test.
Results:
68% of the enterobacterial strains produced extended-spectrum beta- lactamases (ESBL), 13.63% of them expressing simultaneously the Amp C enzyme. All enterobacterial strains were susceptible to carbapenems (Imipenem and Ertapenem). Metallo-beta lactamases production among NGNB strains with resistance to Imipenem was high (80%), these strains being also multi-resistant to the majority of tested antibiotics with the exception of colistin.
Conclusions:
our results showed that the majority of the analyzed strains were multi-drug resistant. Antibiotic multi-resistance and the increasing number of severe infections caused by these strains are a major issue for ICU, where patients with severe diseases and destabilized physiological condition are often admitted.
Regulatory constraints and environmental and human health concerns have promoted the search for alternative bio-control strategies of fire blight, a destructive disease of rosaceous plants which produces serious losses in apple and pear orchards all over the world. The aim of this study was to establish the antimicrobial activity of Citrus maxima essential oil against Erwinia amylovora. An agar diffusion method was used for the screening of the inhibitory effect of Citrus maxima essential oil on bacterial strains growth. The quantitative inhibitory effect of pomelo oil on in vitro biofilm development was established by a microtiter colorimetric assay. In order to investigate the ability of pomelo oil to interfere with bacterial adherence and subsequent biofilm development on leaves obtained from different pomaceous fruit trees species and cultivars: Pyrus (Napoca, Williams), Malus (Golden Delicious) and Cydonia (Aromate), leaves were immersed in pomelo oil for 1, 2, 3, 5 and 10 minutes before exposing them to bacterial colonization. The architecture of bacterial biofilms developed on leaf surface was analyzed using Confocal Scanning Laser Microscopy (CSLM). Our results showed that Citrus maxima essential oil inhibited the development of bacterial biofilms on leaves, pomelo oil being more active on Cydonia (Aromate) leaves when the leaves were treated for 5 minutes. The results obtained from this study may contribute to the development of new bio-control agents as alternative strategies to protect fruit trees from fire blight disease.
The vegetal extracts are used as an ecological alternative to classical anti-infectious treatments based on antibiotics, exhibiting the advantage of reduced secondary effects. Most of these compounds are secondary metabolites, especially aromatic substances synthesized by plants in a reduced concentration. The aim of this study was to investigate the influence of usnic acid against quorum sensing and response mechanisms involved in the initiation and development of the dental plaque biofilm and its tolerance to antimicrobials. Three samples of super-gingival dental plaque were treated for different time intervals with usnic acid at 200 ig/ml in dimethyl-sulfoxide, representing the MIC value. Each dental plaque sample was inoculated in Brain Heart Infusion medium to establish the microbial growing curve by viable cells counts using the tenfold microdilutions method. For strains identification there were used the microtest galleries: API 20Strep, API Staph, API 20NE, API 20E. MIC value for usnic acid was determined by twofold microdilution technique. Usnic acid selectively inhibited the biofilm development by Gram positive bacteria and the expression of haemolytic properties of strains isolated from the dental plaque. The growth curve of the isolated strains was affected by usnic acid, the changes consisting of the lag phase extension to 6-10 h (this time interval being considered as the persistence time of antimicrobial activity) and the significant decrease of the viable cell number and consecutively, the prolongation of the generation time. These effects are demonstrating the interference of the usnic acid with the intra- and interspecies signalling mechanisms based on quorum sensing and response and dependent on cell density, giving the possibility to use them as an active principle in some new pharmaceutical formula intended for the prevention and treatment of gingival and periodontal pathologies.
Unlabelled:
The purpose of this study was the assessment of the influence of three probiotic supernatants (Bifidobacterium breve ATCC 15700, Enterococcus faecium ATCC 19434, Lactobacillus casei subsp. casei ATCC 393) on the growth (quantified by viable cell counts) and virulence features expression (adherence ability to HEp-2 cells and inert substratum- slime test) of several selected EAggEC diarrhoeagenic strains as well as the cytotoxicity of the respective supernatants on HEp-2 cells.
Results:
Our in vitro studies are demonstrating that the selected supernatants, when added simultaneously with the bacterial culture are generally opposing to the adherence to the cellular substratum by the EAggEC strains. When added after the pre-adherence period, the supernatants did not change the adherence indexes of the EAggEC strains, but induced slight changes in the adherence pattern, reducing the frequency and size of bacterial aggregates. Only in few cases, the bacterial growth rate was slightly increased or sustained by the probiotic supernatants, a possible explanation being that we used supernatants obtained from 24 hrs fresh cultures, which are probably still containing some nutrients and probably also other growth factors.
Conclusion:
Our results are demonstrating that soluble probiotic metabolites accumulated in culture supernatants may interfere with the first step of adherence and colonization of the cellular and inert substrata by EAggEC strains, probablyby the cross-talk between probiotic soluble molecules and quorum-sensing mediators of opportunistic strains; so, direct contact between the probiotic and pathogenic bacteria are not always necessary for the occurrence of a protective effect, that could be partly mediated by the soluble molecules secreted by specific probiotic strains.
The chemotherapy success to kill cancer cells depends on its ability to stop cell division. The faster the cells are dividing, the more likely it is that chemotherapy will kill the cells, causing the tumor to shrink. Taking into account the severe side effects of chemotherapy, drugs producers also focus on natural products obtained either from medicinal plants, or from microorganisms. The complex polysaccharides named beta-glucans are active compounds with immune activity. beta-glucan polymers belong to a class of drugs with effects on the immune system, such as: anti-tumoral, anti-infectious, protection against fungi, bacteria and viruses infections. The correct selection of beta-glucans is essential to identify compounds with favorable clinical effects. The aim of this study was to investigate the capacity of six Curdlan (beta-glucan) derivatives to up-regulate the Doxorubicin, Actinomycin D and Cyclophophamide cytostatic drug activity on tumor cells (murine B16 melanoma and human HEp-2 laryngeal carcinoma cell lines). Our results demonstrated that Palm SP derivative, as well as SP and Palm CM/SP derivatives were able to potentiate Doxorubicin action or Actinomycin D effect on B16 tumor cells. SP derivative significantly enhanced cytostatic activity of Cyclophosphamide on B16 cells. All the investigated Curdlan derivatives (SP, Palm CM/SP, CM/SP, Palm CM, Palm SP and CM) were able to inhibit HEp-2 tumor cell growth, by up-regulating Doxorubicin and Actinomycin D cytostatic activity.
Molecular and epidemiological data indicated that the presence of HPV virus is not sufficient to induce transformation, suggesting the implication of other several cellular factors. Constitutive activation of the Ras signaling pathway is an important component of malignant progression for a number of different cancers. In this context, the objectives of our study were: the quantitative assessment of the K-ras gene expression changes in the development of the HPV positive cervical cancers. We observed that the K-ras mRNA expression levels did not gradually increase with the severity of injury. The mRNA expression in the ASCUS increased 2.02 times as compared with the control group, while in LSIL group only 1.76 times. However ras expression was increased in the HSIL/cancer group by 2.27 times when was reported to the control group. The presence of low risk HPV infection (IrHPV) does not lead to increased ras expression, remaining at baseline, but K-ras expression was increased in the presence of high risk HPV infection (hrHPV). In addition, we noted that in hrHPV single infections ras expression is increased (0.96 +/- 0.48) comparing with hrHPV co-infections. Our findings indicate that high expression of ras among hrHPV infection can be a marker of cervical cancer development.
Helicobacter pylori cell surface is composed of lipopolysaccharides (LPSs) yielding structures homologous to mammalian Lewis O-chains blood group antigens. These structures are key mediators in the definition of host-microbial interactions and known to change their expression pattern in response to environmental pressure.
The present work is focused on the identification of new H. pylori cell-surface glycosides. Special attention is further devoted to provide insights on the impact of in vitro subcultivation on H. pylori cell-surface phenotypes.
Cell-surface glycans from H. pylori NCTC 11637 and two clinical isolates were recovered from the aqueous phase resulting from phenol:water extraction of intact bacteria. They were evaluated in relation to their sugars and glycosidic-linkages composition by CG-MS, size-exclusion chromatography, NMR, and Mass Spectrometry. H. pylori glycan profile was also monitored during subcultivation in vitro in agar and F12 liquid medium.
All three studied strains produce LPS expressing Lewis epitopes and express bioaccumulate amylose-like glycans. Bioaccumulation of amylose was found to be enhanced with the subcultivation of the bacterium on agar medium and accompanied by a decrease in the expression of LPS O-chains. In contrast, during exponential growth in F12 liquid medium, an opposite behavior is observed, that is, there is an increase in the overall amount of LPS and decrease in amylose content.
This work shows that under specific environmental conditions, H. pylori expresses a phase-variable cell-surface alpha-(1-->4)-glucose moiety.
Nickel-dependent urease activity and nickel supply are essential for successful colonization of Helicobacter pylori in the acidic environment of the human stomach. A comparison of media effects on these two activities have never been carried out. Additionally to H. pylori we cultivated an Escherichia coli strain expressing the urease and the nickel transporter NixA of H. pylori on the same four media and measured in all cases urease and nickel uptake activity.
To compare nickel uptake and urease activity on an inter- and intraspecies level.
In H. pylori nickel uptake (four to 200 times) and urease activities (400 to 30,000 times) were found to be much higher in comparison to the tested E. coli strain after growth on all media. These differences could not be explained by reduced protein amounts in the heterologous host E. coli. On which media the two bacteria extracted most of the nickel were organism-dependent: E. coli on Brucella Broth, H. pylori on Trypticase Soy Broth, and Minimal Media.
H. pylori took nickel much more efficiently up than E. coli. The observed differences in urease activity are most likely due to additional protein components absent in the recombinant E. coli strain. The observed variety in nickel uptake and urease activities on the different media in the same organism depended on the intrinsic nickel content and chelating capacities of media components. Different culture conditions may lead to varying results; generalizations should be concluded only after excluding their media dependence.
Helicobacter pylori has been isolated from the human stomach with media containing only minimal selective agents. However, current research on
the transmission and sources of infection requires more selective media due to the higher numbers of contaminants in environmental,
oral, and fecal samples. The objective of this study was to develop and evaluate detection techniques that are sufficiently
selective to isolate H. pylori from potential animal and food sources. Since H. pylori survives in the acidic environment of the stomach, low pH with added urea was studied as a potential selective combination.
H. pylori grew fairly well on H. pylori Special Peptone plating medium supplemented with 10 mM urea at pH 4.5, but this pH did not sufficiently inhibit the growth
of contaminants. Various antibiotic combinations were then compared, and a combination consisting of 10 mg of vancomycin per
liter, 5 mg of amphotericin B per liter, 10 mg of cefsulodin per liter, 62,000 IU of polymyxin B sulfate per liter, 40 mg
of trimethoprim per liter, and 20 mg of sulfamethoxazole per liter proved to be highly selective but still allowed robust
colonies of H. pylori to grow. This medium was highly selective for recovering H. pylori from cattle and beef samples, and it is possible that it could be used to enhance the recovery of this bacterium from human
and environmental samples, which may be contaminated with large numbers of competing microorganisms.
Currently, different approaches are employed to detect the presence of Helicobacter pylori (H. pylori): invasive, if based on biopsies performed during endoscopy (urease test, histology, culture, polymerase chain reaction), and non-invasive, if endoscopy is not carried out. Another option is offered by the invasive non-endoscopic methods (string test). An update on the diagnostic invasive approaches to patients in the clinical setting is presented in this review. Although non-invasive tests are preferred, important information can be gained from histology, the unique technique which permits the evaluation of the status of the mucosa, and from culture, that allows strain typing and tests for antibiotic susceptibility. Until today, polymerase chain reaction has had a limited application in the clinical practice and the role of non-endoscopic methods needs to be better clarified in the future.
Helicobacter pylori is an important global human pathogen and there is growing evidence from PCR assays that contaminated drinking water might be a possible source of infection in some circumstances. There are no validated protocols for direct isolation but various culture media have been developed for possible environmental sampling. Our aim here was to investigate how inter-strain variation might affect the interpretation of results with such media. Two laboratory adapted reference strains and four recent clinical isolates were tested on four solid media and in ten liquid media. Considerable variation was found between strains in their ability to recover on the different media after stress exposure (suspension in sterile tap water). Generally, clinical isolates were less robust than the laboratory-adapted strains and, overall, the former required longer recovery times. Our findings highlighted the importance of using a range of isolates for evaluations, as examination of laboratory-adapted strains alone did not provide an accurate representation of the utility of media that may be used to recover H. pylori from water.
In order to increase the database for in vitro growth and/or susceptibility testing in liquid media, we evaluated the growth of Helicobacter pylori in broth media containing 5% sheep blood. We also compared the effect of bismuth on the growth of H. pylori in broth media containing 10% fetal calf serum with the effect on growth in media containing 0.5% starch. In contrast to the result seen with agar, we found that sheep blood, whether whole or laked, inhibited the growth of H. pylori in broth media. In addition, we found that bismuth inhibited growth in media with starch but that this inhibition was negated in media with serum.
We developed procedures for large-scale cultivation of Helicobacter pylori in flasks and fermentors. Flasks incubated closed under a microaerophilic gas phase with a cotton plug covered by a plastic bag, followed by removal of the bag after 8 h, gave excellent growth. Growth in a 10-liter fermentor led to excessive foaming if the medium was sparged with gas; silicone- or polyglycol-based antifoaming agents were severely inhibitory. Use of fermentor surface gassing, first with a microaerophilic 6% oxygen gas mixture, then with air, and then with 95% oxygen, allowed the culture to grow to an A600 of 2.5 in < 24 h. This method was modified for scale-up to a 100-liter fermentor.
Cultures of Helicobacter pylori on chocolate agar slants in bijou bottles and on chocolate agar plates inside BBL Campy Pouches were mailed from Dublin to Galway, Ireland; Bordeaux, France; and Beijing, China. Both systems maintained viability of H. pylori for at least 4 days under mailing conditions. Ninety percent of the isolates on the slants survived for 6 days, but only 30% of the isolates in the pouches survived. When the slants were stored at 4 degrees C after arrival, 50% of the isolates were recoverable 10 days after mailing. Failure of recovery was due to coccoid formation by the organisms. Contamination was not a problem in either system. Chocolate agar slants are considered the more suitable system for transporting H. pylori cultures, especially when transport time longer than 4 days is expected.
To evaluate a technique for culture of Helicobacter pylori in large quantities of liquid media and to determine the factors that could influence the results.
Fifteen clinical isolates of H pylori and a reference strain of H pylori NCTC11637 were used to evaluate a method to cultivate the organism in 100 ml liquid medium comprising brain heart infusion broth with 5% horse serum and 0.25% yeast extract. Tissue culture flasks containing the inoculated liquid medium were placed in a CO2 incubator with 5% CO2 for 2 hours and then incubated in a shaking incubator at 120 rpm.
All the clinical isolates and the reference strain grew in the broth, although only a moderate growth of the reference strain occurred. Inoculum size significantly influenced the kinetics of growth of H pylori in the liquid medium. Vancomycin, nalidixic acid, and amphotericin B, used to suppress contamination, did not affect growth of H pylori in the medium. CO2 was essential for H pylori to grow or survive in the liquid medium. Incubation with CO2 in a CO2 incubator for 30 minutes or 2 hours did not affect the results.
H pylori can be cultivated in large quantities of brain heart infusion broth with 5% horse serum and 0.25% yeast extract. Initial inoculum concentrations influence the kinetics of H pylori growth in the liquid medium. Vancomycin, nalidixic acid, and amphotericin B can be used as selective antimicrobial agents. CO2 is essential for initial growth of H pylori in liquid media. The findings in this study may provide a useful, reproducible, and simple method for biochemical, molecular, and physiological studies of H pylori, when those require large quantities of the organism.
In vitro, Helicobacter pylori converts from a bacillary to a full coccoid form via an intermediate U-shaped form. Organisms with a full coccoid form keep a double membrane system, a polar membrane, and invagination structures. Western blots (immunoblots) of sera from colonized patients show that some high-molecular-mass antigenic fractions are expressed only in coccoids. Conversely, fractions of 30 and 94 kDa were more intensively detected in the bacillary forms. These results suggest that (i) coccoid conversion is not a degenerative transformation and (ii) antigens specific to the coccoid forms are expressed in vivo.
Arcobacter, the newly reclassified Campylobacter species, has been shown to cause diarrhea in both humans and animals. Few studies have been conducted regarding its occurrence in foods because of the lack of effective isolation and identification methods. The purpose of this study was to develop a plating medium that would be selective for the three most commonly found Arcobacter species. The effect of common components used in media intended for the isolation of Campylobacter, Helicobacter, and other gram-negative rods was examined. These components were divided into five distinct groups: (1) basic growth nutrients, (2) reducing and growth-promoting agents, (3) detoxifying agents, (4) antibiotics, and (5) color-enhancing compounds. Components from each of these groups were tested for their ability to recover Arcobacter on a solid medium when incubated aerobically at 30 degrees C for up to 72 h. Growth was evaluated by the ecometric technique, colony size, and differential colony morphology after incubation. After initial evaluations, five formulas showing the best results were selected and tested in detail and compared with brucella agar. A medium containing a basal nutrient mix along with 0.05% thioglycolic acid, 0.05% sodium pyruvate, and 5% sheep's blood (pH 6.9+/-0.2) was found to be the most effective for the growth of A. butzleri, A. cryaerophilus, and A. nitrofigilis. In addition to superior growth characteristics, a deep red color around the colonies also was observed with this formulation.
Different types of studies have shown that Campylobucter pylori does not belong in the genus Campylobucter. Ribonucleic acid sequencing has indicated that C. pylon' might belong in the genus Wolinellu, but we describe five major groups of taxonomic features of the genus Wolinellu that dBer markedly from those of C. pylori, including ultrastructure and morphology, cellular fatty acids, menaquinones, growth characteristics, and enzyme capabilities, indicating that C. pylori should not be included in the genus Wolinella. Thus, we propose the establishment of a new genus, Helicobacter; C. pylori should be transferred to this genus as Helicobacter pylon' comb. nov., and H. pylori NCTC 11637 (= ATCC 43504) is the type strain. The gastric spiral organism from ferrets has been elevated recently from Campylobucter pylori subsp. mustelae to Campylobacter mustelae. We describe the similarities and differences between C. mustelae and C. pylori compared with other campylobacters, and we propose that C. mustelae should be included in the new genus Helicobacter as Helicobacter mustelae comb. nov. (type strain, ATCC 43772).
The ability of six variants of a new charcoal medium and Skirrow's medium to growHelicobacter pylori in 3 and 5 days was studied using 20 different strains ofHelicobacter pylori. The main admixtures for the charcoal media were serum, whole blood, and egg yolk emulsion. For this purpose, serum was significantly better and egg yolk emulsion significantly worse than whole blood. The addition of IsoVitalex resulted in significantly improved growth on the charcoal media. Skirrow's medium showed very poor performance after three days of incubation and needed a long incubation time.
Plating on solid media is the standard technique used in most laboratories for the isolation of Helicobacter pylori from gastric biopsies. Recently, various selective media were developed for this purpose. We compared and evaluated three selective media, Skirrow's, Dent's CP, and modified Glupczynski's Brussels campylobacter charcoal media, and chocolate agar medium for the isolation of H. pylori. Gastric biopsies taken from a total of 203 patients were plated in parallel on all four media. An isolation rate of 51% (104 of 203) was obtained with a combination of all four media. Of the 104, 92 (88%) were positive with Dent's medium and with modified Glupczynski's medium. Skirrow's medium gave the highest isolation rate, 96% (100 of 104). However, growth of H. pylori was scant (only one to five colonies) when growth occurred on Skirrow's medium alone. Overall, modified Glupczynski's medium provided significantly heavier growth. Chocolate agar medium yielded a 76% (79 of 104) positivity rate. We recommend the use of a combination of two selective media for the maximum recovery of H. pylori from antral biopsies.
Growth studies of Helicobacter pylori were performed involving analysis of the bacterium and its microenvironment, to lend insight into the factors responsible for the morphologic conversion phenomenon. H. pylori converted from bacillary to coccoid forms in broth culture after incubation for 5 days under microaerobic conditions with agitation. This morphologic conversion was paralleled by a dramatic decrease in colony-forming units per milliliter (CFU/ml) and a significant endogenous increase in the pH of the broth culture. In addition, removal of broth cultures from microaerobic conditions after 3 days of incubation resulted in a rapid increase in culture pH, a morphologic conversion, and a concomitant decrease of CFU/ml. These observations suggest an inhibitory effect of basic pH, endogenously produced, on the growth of H. pylori in vitro. Experiments designed to identify the reason for the endogenous increase in culture pH demonstrated that the urease enzyme of H. pylori is not primarily responsible for this phenomenon. Rather, H. pylori appears to produce a deaminase enzyme that is likely responsible for the generation of ammonia, which results in the increase in culture pH, the morphologic conversion, and the loss of culturability observed in vitro. Also indicated is the need for a buffering component (for example, bicarbonate) to maintain pH conditions favorable to the growth of H. pylori.
The human gastric pathogen Campylobacter pylori has recently been reclassified as Helicobacter pylori, and a related spiral bacterium found in the stomach of ferrets has been designated Helicobacter mustelae. The general microbiological features of Helicobacter pylori are delineated here, with details of phenotypic differences between Helicobacter pylori and Helicobacter mustelae; comparisons are made with Wolinella succinogenes and Campylobacter jejuni. The Helicobacter organisms possess an external glycocalyx which can be visualised by electron microscopy, and which may be involved in bacterial adherence. The finding of soluble and cell-associated haemagglutinins of Helicobacter pylori is reported. Detection of Helicobacter pylori in clinical specimens, susceptibility of the organism to antibacterial agents, and other aspects of practical and clinical significance are briefly reviewed.
Contaminating bacteria from the oropharynx and bacteria that colonise the stomachs of patients with a high gastric pH impede the isolation of Campylobacter pylori from gastric biopsy specimens. Commercially available selective supplements are inhibitory to this organism and therefore a specific selective medium is needed for isolation. Potential selective agents were evaluated for their activity against 97 strains of Campylobacter pylori. A modification of Skirrow's medium was developed; cefsulodin (5 mg/l) was substituted for polymyxin, and amphotericin B (5 mg/l) was added to inhibit Candida spp., a common contaminant of the stomach. No strains of Campylobacter pylori were inhibited by the Campylobacter pylori selective medium and it supported the growth of all strains compared with the biopsy urease test and Gram stained smear. Colonies were slightly larger and more easily recognised on the new medium compared with those grown on chocolate blood agar. This medium greatly improves the isolation of Campylobacter pylori and could be used alone, without a non-selective medium.
Campylobacter pyloridis is a spiral bacterium which was seen by histopathologists several years before it was cultured in 1982 in Perth, Western Australia. It has unique cellular fatty acids, predominantly tetradecanoic acid and cis-11, 12 methylene octadecanoic acid. It also has a unique ultrastructure which is different from that of other campylobacters. C pyloridis possesses a powerful urease enzyme and produces large amounts of extracellular catalase. Both these features may be important virulence factors, allowing it to occupy a protected niche in the stomach below the mucus layer but above the gastric mucosa. Specific lesions are found in the gastric mucosa, and ultrastructural studies show the presence of adherence pedestals identical with those found with enteropathogenic Escherichia coli of the intestine. Histological examination of gastric biopsy tissue has shown that C pyloridis is strongly associated with active chronic gastritis, when polymorphonuclear leucocytes are present, and is not found on normal mucosa except when a biopsy specimen from elsewhere in the stomach shows active chronic gastritis. When patients with symptoms caused by gastritis are identified dual antibacterial treatment, combining the action of bismuth in the stomach with a systemic antibiotic, can eradicate C pyloridis, with remission of symptoms and restoration of normal epithelial morphology. Most peptic ulcers relapse after modern acid reducing treatment, and antibacterial treatment may be beneficial in preventing relapse.
Until recently, broth cultivation techniques for Campylobacter pylori were unavailable. We developed a method to cultivate bacterial cells within 24 h in liquid media. Cultivation in broth depended on the adequate dispersion of appropriate gases. A static broth at 37 degrees C in a GasPak jar (BBL Microbiology Systems, Cockeysville, Md.) with a CampyPak (BBL) envelope did not support growth after 5 days of incubation. A broth placed in a flask on a Gyrotory water bath shaker (150 rpm; New Brunswick Scientific Co., Inc., Edison, N.J.) fitted with a gassing hood connected to a gas mixture of 10% CO2, 5% O2, and 85% N2 supported good growth. An initial inoculum of 10(5), 10(3) to 10(4), or 10(2) CFU/ml resulted in greater than or equal to 10(8) CFU/ml after incubation for 24, 48, or 72 h, respectively. Under these conditions, the bacteria grew as motile, spiral bacilli rather than the oval and coccal bacilli occasionally reported. Several bases supported good growth when supplemented with serum. For the determination of basal growth conditions, brucella broth base was used. Fetal calf serum (1%) provided maximum growth. Vitox was not necessary for growth and did not augment growth. C. pylori grew over a wide optimal pH range of 5.5 to 8.5.
One hundred and three gastroscopic biopsies from 80 patients were cultured for Campylobacter pyloridis and studied histologically. Active chronic gastritis, as shown by the presence of polymorphonuclear leucocytes, was diagnosed in 51 biopsies and C pyloridis was found in 47. Sixteen gastric biopsies showed normal histology (no inflammation); C pyloridis was detected in only one of these, and a second biopsy taken from this patient at the same time showed active gastritis. Biopsies could be kept at 4 degrees C for five hours without loss of viability of C pyloridis. An inoculum made by grinding the biopsy in a ground glass grinder consistently gave a much heavier growth of C pyloridis than one made by mincing the specimen. The campylobacter supplement ferrous sulphate, sodium metabisulphite, sodium pyruvate (FBP) (Oxoid) was inhibitory for some isolates; the inhibitory component was found to be sodium metabisulphite. Contaminants, but not C pyloridis, were inhibited by the incorporation of vancomycin 6 mg/l, nalidixic acid 20 mg/l, and amphotericin 2 mg/l, but higher concentrations inhibited C pyloridis. Undried plates kept in a plastic container at room temperature for up to two weeks were as satisfactory as freshly poured plates for the isolation of C pyloridis.
Biopsy specimens were taken from intact areas of antral mucosa in 100 consecutive consenting patients presenting for gastroscopy. Spiral or curved bacilli were demonstrated in specimens from 58 patients. Bacilli cultured from 11 of these biopsies were gram-negative, flagellate, and microaerophilic and appeared to be a new species related to the genus Campylobacter. The bacteria were present in almost all patients with active chronic gastritis, duodenal ulcer, or gastric ulcer and thus may be an important factor in the aetiology of these diseases.
Enprostil, a synthetic dehydroprostaglandin E2 structural analogue primarily developed for treatment of gastritis, has been shown also to lower serum cholesterol.
We studied cholesterol metabolism in seven hypercholesterolemic subjects before, during, and after a low-dose enprostil (18 micrograms/day) treatment, measuring serum lipids, cholesterol absorption by an oral double-isotope method, fecal cholesterol elimination by the balance technique, and fecal fat. In addition, an oral fat load test with vitamin A was performed.
The drug treatment reduced serum concentrations of total and low-density lipoprotein (LDL) cholesterol by 8.2% and 7.9% (p < 0.05), respectively, and cholesterol absorption efficiency by 18% (p < 0.05), and increased fecal output of neutral sterols by 20% (p < 0.05), bile acids by 24% (NS), and cholesterol synthesis by 30% (p < 0.05). Postabsorptive concentrations of triglycerides and vitamin A in chylomicrons were reduced 3-4 h after the intake of the test meal. Fecal fat excretion was doubled during the enprostil treatment.
Enprostil reduces serum cholesterol concentrations, apparently by inhibiting cholesterol absorption so that fecal cholesterol elimination is increased in association with a mild fat malabsorption. Enhanced intestinal motility may contribute to these changes, frequently causing abdominal fullness or mild pain without diarrhea.
Helicobacter pylori is an important cause of chronic active gastritis and is strongly associated with peptic ulcer disease and gastric cancer. H. pylori colonizes the surface of the gastric epithelium with production of a number of factors, resulting in inflammation and an altered mucosa. H. pylori infection occurs world-wide and the mode of transmission most likely is from human to human via the fecal-oral and/or the oral-oral route. Treatment and, in the future, prevention of this infection may result in a marked diminution of upper gastrointestinal tract disease.
Helicobacter pylori is now accepted as the major cause of chronic gastritis. The initial response to infection is acute neutrophilic gastritis, which progresses to active chronic gastritis in most people. To confirm the pathogenic role of H. pylori, both the individual histological features of chronic gastritis and its topographical patterns must be shown to be caused by the infection. Surface epithelial degeneration is a probable result of direct tissue injury by bacterial products. Candidates are ammonia or ammonium products, cytotoxins, phospholipases and pro-inflammatory products such as lipopolysaccharide and platelet-activating factor. Neutrophil polymorph and chronic inflammatory cell infiltration are consequences of the mucosal immune response to bacterial antigens. Complement products and interleukin (IL)-8 are polymorph chemotaxins, and monocyte processing of antigens, followed by T helper cell and B lymphocyte responses, explain the presence of these cells in the mucosa. Atrophy may be a consequence of autodestructive products of neutrophil and monocyte activation, such as reactive oxygen metabolites and proteases. Intestinal metaplasia is most probably an adaptive response, possibly to H. pylori infection, exacerbated by other injurious agents such as bile reflux and dietary irritants. Pangastritis is the usual outcome after H. pylori infection. This is followed by multifocal atrophy and intestinal metaplasia. The latter changes weaken mucosal defences further and peptic ulceration may ensue. Patients with an increased parietal cell mass who become infected with H. pylori will exhibit antral restriction of the gastritis because the high acid output protects the corpus mucosa from bacterial adhesion and the inflammatory consequences. Such patients also have acid-induced gastric metaplasia in the proximal duodenum.(ABSTRACT TRUNCATED AT 250 WORDS)
Helicobacter pylori is part of a genus of specialized bacteria that have adapted to the ecological niche provided by gastric mucus. H. pylori has exploited the human niche, while further species of Helicobacter have inhabited the gastric mucosa of other animals. The preferred habitat of H. pylori is the gastric antrum. In humans with normal gastric function, the organism is mainly restricted to the antral surface, where a number of specialized traits allow it to flourish, while causing minimal harm to its host. These include a characteristic motility that allows it to swim rapidly through viscous mucus, and the ability to manufacture large amounts of the enzyme urease. This enzyme breaks down endogenous urea to form ammonia, which protects the bacterium from gastric acidity. Specific adhesions bind a number of the bacteria to the gastric surface, some swim freely in the mucus, and others possibly endocytose into the epithelial cells. It is probably these inaccessible colonization sites that make the organism so difficult to eradicate. In some patients, the normally harmless balance between host and bacterium is disturbed, resulting in peptic ulceration. Modifications to the mucus or epithelial surface in the proximal duodenum, towards the gastric phenotype, make the tissue more susceptible to H. pylori infection of the duodenum by spread of organisms from the antrum. Gastric acid output becomes further increased and the duodenal mucosa is rendered more susceptible to acid attack, leading to peptic ulceration. In other situations, the level of inflammation is enhanced and immunopathology results, followed in the longer term in some cases by atrophy and gastric cancer.(ABSTRACT TRUNCATED AT 250 WORDS)
Helicobacter pylori has been successfully maintained in a continuous culture system. The culture was grown in 0.6 litre of brain heart infusion broth with supplements in a 1 litre fermenter with a flow rate of 25 ml/h, an 80% dilution rate and a specific growth rate of 0.06/h. Unlike the typical growth curve which occurred in batch culture, the growth pattern in continuous culture showed a short lag phase following which growth was maintained in exponential phase with > or = 10-fold increase in viability and the pH was stabilised at 6.95 +/- 0.05. The urease activity remained constant in relation to viability while the urea was completely hydrolysed. The system provides cells with growth characteristics similar to the fresh isolate which are highly adaptable to the in vitro environment. This study shows that continuous culture is a simple and efficient growth system for the cultivation of a 'synchronous' spiral form of H. pylori.
The ability of six variants of a new charcoal medium and Skirrow's medium to grow Helicobacter pylori in 3 and 5 days was studied using 20 different strains of Helicobacter pylori. The main admixtures for the charcoal media were serum, whole blood, and egg yolk emulsion. For this purpose, serum was significantly better and egg yolk emulsion significantly worse than whole blood. The addition of Iso Vitalex resulted in significantly improved growth on the charcoal media. Skirrow's medium showed very poor performance after three days of incubation and needed a long incubation time.
The morphologic conversion of Helicobacter pylori from bacillary to coccoid form was studied by microscopy, viable count on agar plates, and bioluminescence assay of bacterial ATP. When morphologic conversion from bacillary to coccoid form was detected by microscopy, the viable counts and the bacterial ATP decreased. No viable count was found after nine days of incubation, but bacterial ATP was still present. In these cultures in which only the coccoid form of Helicobacter pylori was present, there was no accumulation of extracellular ATP, indicating no leaky cells. During the transition phase from the bacillary to the coccoid form of Helicobacter pylori, the addition of fresh medium increased the intracellular ATP 26-fold. The coccoid form of Helicobacter pylori had a 1000-fold lower ATP level per cell compared to the bacillary form, which indicates a decreased metabolic activity in the coccoid form. Addition of fresh medium to the coccoid cultures from days 9 and 10 increased the ATP level twofold. However, no conversion from coccoid to bacillary form was found in these cultures during prolonged incubation in fresh broth for four weeks. Such conversion needs to be demonstrated before it is proven that the coccoid form of Helicobacter pylori is responsible for transmission and relapse of infection.
This study investigated the growth of Helicobacter (H.) pylori in Brucella broth supplemented with either IsoVitaleX (1% vol/vol), hemin (.01% wt.vol), agar (0.3% wt/vol), or blood agar blocks (1.5% wt/vol agar). IsoVitaleX was found to significantly shorten the lag phase, while hemin inhibited the growth within the first 24 hours but later acted as a growth stimulant. There was a tendency toward stronger growth when blood agar blocks were added to the medium. Subsequent electron microscopic evaluation revealed that cells of H. pylori were attached to blood agar block surfaces. In contrast, the supplementation of Brucella broth with agar did not significantly increase the cell density. When H. pylori was grown in the presence of IsoVitaleX, strongly stainable electron-dense bodies (140-200 nm) were seen in the cytoplasms. Incubation of cultures on rotary shakers at 10 rpm significantly enhanced growth. The addition of glycerol (15% vol/vol) or fetal bovine serum (15% vol/vol) showed good ultrastructural preservation of bacteria with undamaged cell walls and cytoplasmic membranes, and cytoplasms were ribosome-dense. Cell counts revealed that cultures stored in glycerol or fetal bovine serum had a significantly lower loss in viability when compared with cultures stored without cryopreservatives. Unprotected cells of H. pylori showed on electron micrographs, clumping, cell lysis, and flagellar damage. Finally, the survival rates of H. pylori after multiple thawing from storage at -80 degrees C were best in Brucella broth/glycerol, Brucella broth/fetal bovine serum, and Brucella broth without cryopreservative (in descending order).
Modified brucella broth medium was used to study the growth of Helicobacter pylori at varied pHs and partial pressures of oxygen and to determine the effect of urea on culture pH. Our findings suggested that the pHs of the media remained stable with or without urea and that H. pylori showed facultative acidophilism and obligate microaerophilism.
Bacteriological Analytical Manual, Edition 8, Revision A
FDA (1998) Bacteriological Analytical Manual, Edition 8, Revision
A. Gaithersburg, MD, USA: AOAC International.