In vitro activity of Aloe vera inner gel against microorganisms grown in planktonic and sessile phases.

The failure of traditional antimicrobial treatments is becoming a worldwide problem. The use of Aloe vera is of particular interest for its role as curative agent and its efficacy in complementary therapies for a variety of illnesses. This study evaluated the antimicrobial activity of A. vera inner gel against a panel of microorganisms, Gram-positive and -negative bacteria, and Candida albicans. In addition to A. vera inner gel being used in the treatment of peptic ulcers, in dermatological treatments, and wound healing, it was also tested on the sessile phase of clinical Helicobacter pylori strains (including multi-drug-resistant strains) and on planktonic and sessile phase of Staphylococcus aureus/Pseudomonas aeruginosa clinical isolates from venous leg ulcers.A. vera inner gel expresses its prevalent activity against Gram-negative bacteria and C. albicans in respect to Gram-positive bacteria. The results of the A. vera antibiofilm activity showed a decrease of the produced biomass in a concentration-dependent-way, in each analyzed microorganism. The data obtained show that A. vera inner gel has both an antimicrobial and antibiofilm activity suggesting its potential use for the treatment of microbial infections, in particular for H. pylori gastric infection, especially in case of multi-drug-resistance, as well as for an effective wound dressing.

In vitro activity of Aloe vera inner gel against Helicobacter pylori strains.

UNLABELLED: Aloe barbadensis Miller (Aloe vera) is a herbal remedy widely used for a variety of illnesses; A. vera leaf extracts have been promoted for detoxification, cure constipation, help flush out toxins and wastes from the body, promote digestion and are used in the treatment of peptic ulcer for cytoprotective action. The aim of this study was to evaluate the antibacterial activity of A. vera inner gel against both susceptible and resistant Helicobacter pylori strains isolated in Abruzzo region, Italy. The inner gel of leaves of a 5-year-old plant of A. vera was extracted, homogenized and tested from 800 to 1.56 mg ml(-1) against 14 clinical strains and one reference strain of H. pylori using the broth microdilution methodology. Furthermore, the sample of A. vera was investigated for the chemical fingerprint of anthraquinones. The inhibitory concentrations of A. vera inner gel were similar to the bactericidal ones, with values ranging from 6.25 to 800 mg ml(-1) . Fifty per cent of the detected strains, independently of their susceptibility profile, were inhibited in their growth at 100 mg ml(-1) . Aloe vera inner gel expresses antibacterial properties against H. pylori and, therefore, in combination with antibiotics, could represent a novel strategy for the treatment of the infection of H. pylori, especially in cases of multiresistance. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates that the Aloe vera inner gel expresses antibacterial properties against both susceptible and resistant Helicobacter pylori strains. These findings may impact on the antimicrobial resistance phenomenon of H. pylori, proposing the A. vera inner gel as a novel effective natural agent for combination with antibiotics for the treatment of H. pylori gastric infection.

Helicobacter pylori biofilm: a protective environment for bacterial recombination.

AIMS: The aim of this work was to investigate the interaction between two Helicobacter pylori strains in promoting genetic transfer, when grown in the biofilm mode. METHODS AND RESULTS: Biofilms produced by H. pylori 9/10 (A), H. pylori 15/4 (B) and their mixture (C) were studied for biomass production and cell viability. The genetic heterogeneity of 45 clones, coming from mature biofilm of co-cultured H. pylori strains was studied by both RAPD and cagA (EPIYA motifs)/vacA virulence genes analysis. Helicobacter pylori A, B and C developed a well-structured biofilm without significant differences in viability. No significant differences were recorded between A and B biomass measurement, whereas C biofilm expressed a significant (P < 0.001) higher adhesive capability when compared with A and B biofilms. C-clones DNA-fingerprintings showed an high genetic heterogeneity (mean similarity value = 0.528). The 60% of C-clones displayed vacA allelic combination s1i1m1m2 associated with cagA EPIYA motif pattern P1P2P3P3P3. CONCLUSIONS: Biofilms developed by multiple H. pylori strains are more complex than those associated with single strains. Such condition might promote the genetic exchange favouring the generation of more virulent strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The 'biofilm niche' represents a successful strategy and a suitable environment for promoting bacterial population persistence by recombination events.

Effect of 2-hydroxyethyl methacrylate on Streptococcus spp. biofilms.

AIMS: The effect of different concentrations of 2-hydroxyethyl methacrylate (HEMA) was evaluated on biofilm formation and preformed biofilm of Streptococcus mitis, Streptococcus mutans and Streptococcus oralis, alone or combined to each other. METHODS AND RESULTS: Twofold serial dilution of HEMA ranged from 12 to 0·75 mmol l(-1) was added to Streptococcal broth cultures and mature biofilms in 96-well-microtitre plates to evaluate bacterial biomass and cell viability. HEMA affected the Streptococcal population in a strain-specific way producing few significant effects. A reduction on biofilm formation and a detachment of preformed biofilm was recorded in Strep. mitis ATCC 6249, whereas in mixed cultures, the monomer expressed a general aggregative effect on mature biofilms. A reduction in cell viability was also recorded in an HEMA-concentration-dependent way in each experimental condition studied. CONCLUSIONS: These results suggest that the HEMA prevalent effects are both the reduction of bacterial adhesion to a polystyrene surface and the increase in dead cells also characterized by an aggregative status. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the potential effect of HEMA, released from resin-based materials, on oral bacteria may furnish information for surveillance of the risk reduction in secondary caries via hindering biofilm generation.

Extracellular DNA in Helicobacter pylori biofilm: a backstairs rumour.

AIMS: This study detected and characterized the extracellular DNA (eDNA) in the biofilm extracellular polymeric substance (EPS) matrix of Helicobacter pylori and investigated the role of such component in the biofilm development. METHODS AND RESULTS: Extracellular DNA was purified and characterized in a 2-day-old mature biofilm developed by the reference strain H. pylori ATCC 43629, the clinical isolate H. pylori SDB60 and the environmental strain H. pylori MDC1. Subsequently, the role of eDNA in the H. pylori biofilm was evaluated by adding DNase I during biofilm formation and on mature biofilms. Extracellular DNA was detected in the 2-day-old EPS biofilm matrix of all analysed H. pylori strains. The DNA fingerprintings, performed by RAPD analysis, on eDNA and intracellular DNA (iDNA), showed some remarkable differences. The data obtained by microtitre biofilm assay as well as colony forming unit count and CLSM (confocal laser scanning microscopy) qualitative analysis did not show any significant differences between the DNase I-treated biofilms and the corresponding not treated controls both in formation and on mature biofilms. CONCLUSIONS: In this study, we provide evidence that eDNA is a component of the EPS matrix of H. pylori biofilm. The different profiles of eDNA and iDNA indicate that lysed cells are not the primary source of eDNA release, suggesting that other active mechanisms might be involved in this process. Moreover, the biomass assay suggests that eDNA may not be the main component of biofilm matrix, suggesting that it could be primarily involved in other mechanisms such as recombination processes, via transformation, contributing to the wide genomic variability of this micro-organism defined as a 'quasi-species'. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of eDNA in H. pylori biofilm can contribute to the active dynamic exchange of information aimed to reach the best condition for the bacterial survival in the host and in the environment.

Characterization of an Helicobacter pylori environmental strain.

AIMS: To investigate the main genotypic virulence markers and the phenotypic features of an environmental Helicobacter pylori strain, named MDC1. METHODS AND RESULTS: The H. pylori MDC1 genotypic status was evaluated by PCR amplification. The mosaicism in vacA alleles was expressed by the s1m1 allelic combination, as found in strains which are strong vacuolating cytotoxin producers; the number of cagA variable EPIYA motifs displayed P1P2P3P3 pattern and the iceA1 was recorded between the iceA allelic types and the babA2 gene found in strains causing more severe disease. The biofilm formation was evaluated on a polystyrene surface in static conditions by scanning electron microscopy and confocal scanning laser microscopy. Helicobacter pylori MDC1 displayed a dense mature biofilm with cells in a coccoid morphology persistent in time in which the expression of the luxS gene, related to the quorum-sensing signalling, was always detected. CONCLUSIONS: Helicobacter pylori MDC1 strain had the main virulence markers closely related to gastric pathogenesis and displayed a well-structured biofilm which allowed this bacterium to be more protected in the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The persistence of the environmental virulent H. pylori strain in a clustered state suggests a long-term survival of this bacterial community outside of the host, enabling the bacterial transmission with important clinical repercussions.

Effects of combining extracts (from propolis or Zingiber officinale) with clarithromycin on Helicobacter pylori.

Propolis and Zingiber officinale have been shown to be specifically targeted against Helicobacter pylori strains, to possess antiinflammatory, antioxidant and antitumoral activity and to be used in traditional medicine for the treatment of gastrointestinal ailments. Considering that these natural products could potentially serve as novel therapeutic tools also in combination with an antibiotic, the aim of this work was to evaluate their effect when combined with clarithromycin on clinical H. pylori isolates (n = 25), characterized in respect to both clarithromycin susceptibility and the presence of the cagA gene. The results showed that the combinations of propolis extract + clarithromycin and Z. officinale extract + clarithromycin exhibited improved inhibition of H. pylori with synergistic or additive activity. Interestingly, the susceptibility to combinations was significantly independent of the microbial clarithromycin susceptibility status. Only one H. pylori strain showed antagonism towards the Z. officinale extract + clarithromycin combination. The data demonstrate that combinations of propolis extract + clarithromycin and Z. officinale extract + clarithromycin have the potential to help control H. pylori-associated gastroduodenal disease.

Antibacterial effect of plant extracts against Helicobacter pylori.

The aim of this work was to evaluate the antibacterial effect of plant extracts as alternative and[sol ]or as active agents supporting antibiotics for treating Helicobacter pylori infection. The effect of either, ethanolic or aqueous extracts from 17 plant materials were studied against one H. pylori standard strain and 11 clinical isolates using a disc diffusion test and by evaluating the minimum inhibitory concentration (MIC) on solid media. An inhibitory activity against H. pylori strains was recorded in a large percentage of tested plants. MIC values of ethanolic extracts were from two to four concentration steps lower than the aqueous ones. In particular, ethanolic extracts of Cuminum cyminum L. and Propolis expressed MIC90 values of 0.075 mg/mL. The results show a significant in vitro effect of plant extracts against H. pylori that could be considered a valuable support in the treatment of the infection and may contribute to the development of new and safe agents for inclusion in anti-H. pylori regimens.

Detection of free and plankton-associated Helicobacter pylori in seawater.

AIMS: To detect both free and plankton-associated Helicobacter pylori in seawater samples collected on the Italian coast of the Adriatic Sea using a nested-PCR. METHODS AND RESULTS: Dissolved oxygen, pH, salinity and chlorophyll 'a' were the parameters recorded together with the characterization of zooplanktonic organisms. Plankton-associated H. pylori DNA was searched for in water samples filtered through 200 and 64 microm nylon nets whereas free bacteria were retained with the subsequent filtration through 0.22 microm pore-size membranes. Nested-PCR using primers for the glmM (ureC) gene was performed to reveal the presence of H. pylori. The DNA sequencing of amplified products confirmed the specificity of the assay. The sensitivity of the nested-PCR assay for H. pylori detection was 62 CFU per 100 ml in spiked water samples. Helicobacter pylori either free or bound to planktonic organisms was found in seven of 12 monthly samples. In particular, free bacteria were detected during the summer sampling and in November, December and March associated to planktonic cells. CONCLUSIONS: The presence of free and plankton-associated H. pylori in seawater suggests that it can be a significant reservoir and a potential route of transmission for the microorganism. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study seems to provide a promising background to define new and effective strategies for surveillance of this human pathogen.

Population dynamics in ageing Helicobacter pylori.

The aim of this work was to characterize population changes occurring in aged broth cultures of Helicobacter pylori. Experiments were performed using clinical strains cultured immediately after isolation and after multiple subcultures in solid medium. Morphological changes in the ageing bacteria during a 7-day broth culture were analysed by optical and electron microscopy. The expression of the virulence factor, CagA, together with the presence of the cell cycle regulator, cGMP, were also assessed. The transition from bacillary to coccoid forms was the main morphological change observed in freshly isolated bacteria, together with the increase in cGMP from 1 to 2.25 nmoles/mg of proteins within the first 7 days of broth culture. A similar trend of morphological and physiological changes was observed in cells after multiple subcultures in solid medium with a major presence of large cell clusters. The cagA gene product was always expressed in all experimental conditions evaluated. These data show a significant morphological and physiological diversity in fresh, ageing and aged cultures of H. pylori.

Molecular fingerprinting of Helicobacter pylori strains from duodenal ulcer patients.

AIMS: To characterize the molecular fingerprinting of Helicobacter pylori population isolated in duodenal ulcer patients treated with triple therapy. METHODS AND RESULTS: Gastric biopsy specimens from corpus and antrum, were cultured for H. pylori isolation. Helicobacter pylori eradication was evaluated after 4 and 16 weeks. DNAs of all isolates were characterized by random amplified polymorphic DNA typing and cagA gene was also detected. After the therapy, five patients harboured the microorganism at 4 weeks and two of them remained H. pylori positive at 16 weeks. The analysis of DNA fingerprinting of strains isolated from antrum and corpus of patients susceptible to treatment, showed similar patterns. Instead, when the therapy was not effective, strains isolated from sequential biopsies from initial and after 4 and 16 weeks, showed distinct fingerprintings and retained the cagA status, over time. CONCLUSIONS: The drugs used for therapy could exercise an effect in genotypical rearrangement among H. pylori cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The variableness among H. pylori strains represents a way to challenge environmental stress.

Quantitative microbial monitoring in a dental office.

The aim of this study was to evaluate the environmental pollution before and after dental procedures (during one year) in a dental office in which a system of air filtration was effective and suitable procedures of microbial controls were routinely applied for instruments and small surfaces. The air contamination was evaluated during one year by the 'plate' method (Air Microbial Index, AMI) in each room of the dental office following a bimonthly monitoring program. Nutrient agar plates were exposed, in monitored areas for 1 h for each control time and incubated at 37 degrees C for 2 days. The number of viable cells was expressed as colony forming units per plate per hour (CFU/plate/h). During the observation year, the quantitative analysis of the microbiological levels in the operative areas was always within acceptable values. In fact, a range from 4-18 CFU/plate/h was found as the mean of AMI in each controlled room. In particular, the aerosol pollution following dental procedures did not significantly modify AMI values compared with AMI values recorded before dental procedures. Data presented here demonstrate that the combined use of effective infection control procedures and a system of air filtration can be efficacious in reducing airborne environmental contamination in a dental office and emphasise the use of an inexpensive method such as AMI to verify the environmental bacterial pollution.

Transmission of Helicobacter pyori in an animal model.

An experimental murine model was studied to evaluate the orogastrointestinal colonization of Helicobacter pylori and the animal-to-animal transmission. Balb/C mice were infected with H. pylori and housed with uninoculated mice in cages with and without a grate on the floor. Mice were killed after 7, 14, 30, and 45 days, and samples from the esophagus, stomach, small intestine, colon, and rectum were analyzed for H. pylori by PCR and immunohistochemistry and for histological changes. Bacterial colonization was assessed also by culture from stomach samples. H. pylori was cultured by stomach samples of infected mice at 7, 14, and 30 days. Using PCR and immunohistochemistry, H. pylori was detected in inoculated and uninoculated mice in all areas examined, with an high percentage of positive samples in the esophagus and stomach. Moreover transmission was detected, without differences, regardless of whether mice were housed with or without a grate on the floor, supporting an orooral animal transmission.

Role of antimicrobial susceptibility testing on efficacy of triple therapy in Helicobacter pylori eradication.

BACKGROUND: Helicobacter pylori treatment failure may be due to resistance to macrolides and 5-nitroimidazoles. AIM: To test whether a preliminary in vitro susceptibility test of H. pylori to tinidazole and clarithromycin and a consequent specific regimen could improve the eradication rate. METHODS: A total of 109 consecutive H. pylori-positive patients with dyspeptic symptoms were included. At endoscopy, biopsy from the antrum was obtained for H. pylori culture and antimicrobial susceptibility testing. Fifty-six patients were treated with omeprazole, tinidazole and clarithromycin for 10 days (group OTC) and 53 patients received therapy on the basis of the susceptibility test (group SUSC). Treatment success was evaluated by the 13C-urea breath test 1 month after the end of therapy. RESULTS: Eight patients dropped out. Overall primary resistance to clarithromycin, tinidazole and both antibiotics was 13%, 33% and 4%, respectively. In group OTC, H. pylori was eradicated in 81% and 75% of patients by per protocol and intention-to-treat analysis, respectively. Per protocol and intention-to-treat eradication rates for group SUSC were 98% and 91% (P < 0.05 vs. group OTC). CONCLUSIONS: These data show that in H. pylori infection, antibiotic therapy based on the results of culture and susceptibility testing gives, in comparison to standard therapy, a significant improvement in eradication rate.

Evidence for an oral-faecal transmission of Helicobacter pylori infection in an experimental murine model.

An experimental murine model was used to evaluate the possible animal-to-animal transmission of Helicobacter pylori and the mechanism involved. Twenty-four Balb/C mice were infected with H. pylori type I strain culture and kept with 24 noninoculated mice to evaluate the possible transmission of the microorganism. Twelve inoculated mice were housed with 12 noninoculated mice in a grated cage (supporting an oral-oral transmission); the remaining inoculated and noninoculated mice were housed in another cage without grating on the floor (supporting a faecal-oral transmission). The bacterial colonization was assessed by culture and immunohistochemistry. The systemic antibody response to H. pylori and the histopathological changes were evaluated; controls were examined at 2, 4, 8, 12 weeks after the start of the experiment. Faecal samples were also collected from each mouse on the day before sacrifice, to assess the presence of H. pylori by culture and by immunohistochemistry. In the gastric mucosa of inoculated mice, histopathological changes were recorded at each control time and H. pylori was detected both by immunohistochemistry and by a systemic antibody response; the microorganism was also cultured at 2, 4, 8 weeks postinoculation. H. pylori was detected in noninoculated mice, housed in the cage without grating, using an immunoperoxidase technique at 2, 4, 8 weeks after starting the experiment, and these positive values were supported by histopathological changes, and, in one case, at 8 weeks, also by the serum immune response. No colonies of H. pylori were detected by culturing faecal samples from either noninoculated or inoculated mice. The results obtained in this study seem to support an oral-faecal route as the mode of transmission of H. pylori infection in this animal model.

The effect of oxygen on the growth and cell morphology of Helicobacter pylori.

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

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

Studies were conducted following the formation and characterization of the coccoid morphology of Helicobacter pylori. H. pylori ATCC43504 was incubated in brucella broth plus 2% fetal calf serum at three different temperatures: 37 degrees C, room temperature and 4 degrees C in a microaerophilic environment, and readings were taken at 2, 7, 15, 30 and 45 days. At control times, the total and the viable count, viability tests with tetrazolium salts, and ultrastructural studies were carried out. On solid media, H. pylori became nonculturable after 7 days of incubation at room temperature and 4 degrees C, and after 15 days of incubation at 37 degrees C. At these times of incubation, after subculturing in liquid medium under the same conditions, the growth of H. pylori was detected until the 15th day from cultures incubated at 4 degrees C and until the 30th day from cultures stored at 37 degrees C, and at room temperature. Ultrastructural studies showed a gradual reduction of integrity of bacterial cells that remained stable at 30 and 45 days of incubation: 30% of whole cells of bacteria incubated at 37 degrees C and room temperature and 50% in bacteria incubated at 4 degrees C. The viability of the VNC (viable nonculturable) state was assessed by studying the reduction of tetrazolium salts INT (p-iodonitrophenyl tetrazolium violet) and CTC (cyanoditolyl tetrazolium chloride) to their respective formazans and this was linked to the cellular respiration. At 45 days of incubation, when bacterial regrowth was not observed in solid or in liquid medium, different resuscitation methods were applied to evaluate a possible resuscitation of VNC H. pylori. No significant growth on solid medium was observed.

Anti-Helicobacter pylori specific antibody immunohistochemistry improves the diagnostic accuracy of Helicobacter pylori in biopsy specimen from patients treated with triple therapy.

OBJECTIVE: To investigate the effectiveness of immunohistochemical technique to detect Helicobacter pylori (H. pylori) in patients treated with triple therapy. METHODS: Forty patients (18 men, 22 women, mean age 43 years) with active antral gastritis, H. pylori positive at urease test, culture, and histology, were treated for 1 wk with omeprazole, amoxicillin, and metronidazole. Gastritis was scored according to Sydney criteria. Two months after the end of therapy, endoscopy, urease test, culture, and histology were repeated. RESULTS: Culture and histology were negative in 32 (80%) of treated cases. Biopsy specimens of the eradicated group were stained with immunohistochemical technique using an anti-H. pylori specific polyclonal antibody. In 12 of 32 (37.5%) patients, clusters of round or vibrio-shaped bacteria, unidentified at histology, were stained by the specific anti-H. pylori antibody. After triple therapy, at histology all patients were found with improved gastritis. In six patients however, mucosal-associated lymphoid tissue (MALT) appearance, present before therapy, persisted after therapy. In five of six patients with MALT, immunostaining with anti-H. pylori antibody was positive. CONCLUSIONS: The immunohistochemical technique is more accurate than classical methods in identifying H. pylori after specific therapy. This method should, therefore, be used in all studies that aim to achieve eradication. Whether the H. pylori identified at immunohistochemistry is able to reactivate and induce recrudescence of infection remains to be clarified.

Inhibition of Helicobacter pylori by garlic extract (Allium sativum).

The antibacterial effect of aqueous garlic extract (AGE) was investigated against Helicobacter pylori. Sixteen clinical isolates and three reference strains of H. pylori were studied. Two different varieties of garlic were used. The concentration of AGE required to inhibit the bacterial growth was between 2-5 mg ml-1. The concentration, for both AGE types, to inhibit 90% (MIC90) of isolates was 5 mg ml-1. The minimum bactericidal concentration (MBC) was usually equal to, or two-fold higher than, minimum inhibitory concentration (MIC). Heat treatment of extracts reduced the inhibitory or bactericidal activity against H. pylori; the boiled garlic extract showed a loss of efficacy from two- to four-fold the values of MIC and the MBC obtained with fresh AGE. The antibacterial activity of garlic was also studied after combination with a proton pump-inhibitor (omeprazole) in a ratio of 250:1. A synergistic effect was found in 47% of strains studied; an antagonistic effect was not observed.

Helicobacter pylori: a fickle germ.

The morphologic changes from bacillary to coccoid forms of Helicobacter pylori were studied. These form changes were analyzed by bacterial growth in Brucella broth plus 2% fetal calf serum. The coccoid forms were observed at five days of incubation and a rapid decrease of CFU/ml was recorded. At two weeks of microaerophilic incubation, all coccoid forms observed were not culturable in vitro. The coccoid morphology was observed earlier when the culture of H. pylori was incubated in aerobic conditions and with subinhibitory concentrations of omeprazole and roxithromycin. To evaluate the possibility of resistance of coccal forms, before plating, the cultures were heated to 80 C for 10 min and sonicated. In the absence of these treatments the cultures did not show growth in vitro. The proteic patterns of the same strains of two different morphologies were studied revealing significant differences.