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Objective: Helicobacter pylori expresses an increased resistance in respect to antimicrobials currently used in therapy. The aim of this study was to evaluate the antimicrobial profiles of H. pylori isolates to nine conventional antibiotics used in a Central Region (Abruzzo) of Italy., Materials and Methods: Biopsies were taken from antrum and fundus of 112 adult and 3 children with Urea Breath Test positive with dyspeptic symptoms and analyzed for H. pylori culture and antibacterial activity. Antimicrobial susceptibility tests were performed for clarithromycin, metronidazole, levofloxacin, moxifloxacin, ciprofloxacin, tetracycline, amoxicillin, ampicillin, and rifabutin by a modified agar dilution susceptibility test., Results: Bacterial culture was successful in 100 out of 115 patients. Helicobacter pylori strains were isolated from 98 antrum and 83 fundus samples. The rate of recovery of H. pylori strains was 90.50% (181/200). The percentages of resistance were as follows: clarithromycin 72.44% antrum, 72.28% fundus; metronidazole 34.69% antrum, 42.16% fundus; levofloxacin 42.85% antrum, 53.01% fundus; moxifloxacin 37.35% antrum, 46.57% fundus; ciprofloxacin 39.47% antrum, 44.28% fundus; tetracycline 2.63% antrum, 2.85% fundus; amoxicillin 1.02% antrum, 1.20% fundus; ampicillin 0% antrum and fundus and rifabutin 0% antrum, 1.20% fundus. A total of 35 subjects harbored multi-resistant strains., Conclusions: This study underlines the high rate of resistance to clarithromycin, metronidazole and quinolones, which may reflect an overuse of them. Culture and susceptibility test, should be performed to prevent the emergence of multi-resistance and to assess an efficacious regimen.

We developed a new transport medium (GESA--Helicobacter pylori transport medium [publication no. WO/2014/019696, patent pending no. PCT/EP2013/002292; Liofilchem s.r.l., Roseto degli Abruzzi, Teramo, Italy]) for recovery of Helicobacter pylori from gastric biopsy samples. GESA transport medium, in a semisolid state, provides the optimal conditions for maintaining the viability of the microorganism over time. The efficacy of the transport medium was assessed through in vitro and ex vivo experiments. We were able to recover different suspensions of H. pylori ATCC 43629 and H. pylori 13 A in GESA transport medium stored at 4 °C for up to 10 days. In particular, with a starting inoculum of ∼ 10(5) CFU, after 7 days of storage, 150 ± 25 CFU and 40 ± 7 CFU of the reference and clinical strains were detected, respectively. H. pylori colonies were isolated from gastric specimens taken from both the antrum and the fundus in 68 (90.66%) of 75 urea breath test (UBT)-positive patients. Moreover, GESA transport medium allowed the recovery and isolation of H. pylori colonies from additional biopsy samples from 13 of the 75 detected subjects at up to 10 days of biopsy sample storage at 4 °C. Finally, GESA transport medium preserved its characteristics when stored at 4°C for 1 year from its preparation, thus allowing good recovery of H. pylori. GESA transport medium can be considered a standardized transport medium with high performance that optimizes the recovery rate of H. pylori grown by culture., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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., (© 2014 The Society for Applied Microbiology.)

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., (© 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.)

The aim of this work was to analyze several clones of Helicobacter pylori isolated from a patient with gastric cancer, to evaluate i) genetic variability ii) virulence factors profile and iii) antimicrobial susceptibility against the drugs commonly used in the H. pylori therapy. A total of 32 H. pylori clones isolated from a biopsy sample coming from a patient with gastric cancer previously treated for H. pylori infection, were analyzed for: the genetic variability by amplified fragment polymorphism analysis; the vacA, cagA virulence status by PCR; the antimicrobial susceptibility by minimum inhibitory concentrations with the agar dilution method towards amoxicillin, clarithromycin, levofloxacin and tinidazole. The patient showed a mixed infection with the presence of at least 3 different strains. The clones isolated possessed the vacA, cagA virulence factors with a different allelic combination (vacA s1/i1/m1; s1/i1i2/m1; s2/i2/m2; s2/i1i2/m2) together with repeated cagA EPIYA motif pattern P1P2P3P3P3. Moreover, a pattern of multi-drug resistance was disclosed in the different clones. The presence of multiple H. pylori strains colonizing the same patient, with the main virulence factors displaying a different allelic combination and a different multi-drug resistance among isolates, point out the role of genetic variability generating, in time, more virulent and adapted strains.

The aim of this work was to investigate the effects of exposure to extremely low-frequency electromagnetic fields (ELF-EMF) both on biofilm formation and on mature biofilm of Helicobacter pylori. Bacterial cultures and 2-day-old biofilm of H. pylori ATCC 43629 were exposed to ELF-EMF (50 Hz frequency-1 mT intensity) for 2 days to assess their effect on the cell adhesion and on the mature biofilm detachment, respectively. All the exposed cultures and the respective sham exposed controls were studied for: the cell viability status, the cell morphological analysis, the biofilm mass measurement, the genotypic profile, and the luxS and amiA gene expression. The ELF-EMF acted on the bacterial population during the biofilm formation displaying significant differences in cell viability, as well as, in morphotypes measured by the prevalence of spiral forms (58.41%) in respect to the controls (33.14%), whereas, on mature biofilm, no significant differences were found when compared to the controls. The measurement of biofilm cell mass was significantly reduced in exposed cultures in both examined experimental conditions. No changes in DNA patterns were recorded, whereas a modulation in amiA gene expression was detected. An exposure to ELF-EMF of H. pylori biofilm induces phenotypic changes on adhering bacteria and decreases the cell adhesion unbalancing the bacterial population therefore reducing the H. pylori capability to protect itself.

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.

Objective: To assess the relationship between the presence of mixed infection of Helicobacter pylori and both antimicrobial susceptibility and virulence markers., Material and Methods: Thirty-six patients with H. pylori infection were included in the study. Three colonies were selected from each positive biopsy sample collected from each host for a total of 108 H. pylori strains. The genetic variability was evaluated through the amplified fragment length polymorphism (AFLP) analysis; the antibiotic susceptibility to amoxicillin, clarithromycin, moxifloxacin, rifabutin and tinidazole was determined using the minimum inhibitory concentrations (MICs) with the agar dilution method. Moreover, the vacA, cagA, iceA and babA2 statuse were detected by polymerase chain reaction (PCR)., Results: There was a strong connection between mixed H. pylori infection and antimicrobial resistance. In particular, H. pylori strains with genetic variability, in the same host, expressed more resistance to clarithromycin, moxifloxacin and tinidazole than that expressed in strains with a unique genetic host pattern. VacA s1m1/s1m2 genotypes were found in 70% of strains isolated in mixed infection, whereas the same allelic combinations were found in 42% of strains, isolated in single infection. The cagA(+) status prevailed both in patients with mixed (97%) and in those with single infection (85%) without significant differences. The iceA1 status was more commonly found in patients with mixed infection, whereas the babA2 status was significantly prevalent in single H. pylori infection., Conclusions: Mixed H. pylori infection harbouring in one patient is significantly related to strains that are more resistant to antibiotics and with a more virulent genotype (vacA s1m1/s1m2, cagA, iceA1) than strains responsible for single infection.

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., (Copyright 2006 John Wiley & Sons, Ltd.)

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., (Copyright 2005 John Wiley & Sons, Ltd.)

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 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.

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.

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.