Clinical Isolates Of Helicobacter Pylori Research Articles

In vitro efficacy of combinations of antibiotics used in clinical practice on clinical isolates of Helicobacter pylori.

The main antibiotics used against Helicobacter pylori have been chosen empirically over time, with few preclinical studies to provide support. The rise in resistance to some of these antibiotics is prompting a reassessment of their use. This work aimed to evaluate the invitro efficacy of 2 × 2 combinations of the most widely used antibiotics against H. pylori. J99 reference strains and 19 clinical isolates of H. pylori with various antibiotic resistance phenotypes were used. Minimum inhibitory concentrations were carried out using the microdilution method in 96-well plates. The activity of 15 possible combinations of two antibiotics including amoxicillin, clarithromycin (CLA), levofloxacin, rifampicin, tetracycline, and metronidazole was determined for all strains by the checkerboard method. A mean fractional inhibitory concentration index (FICmean) was calculated for each combination and strain and the type of pharmacodynamic interaction was considered as synergic if FICmean ≤ 0.5, additive if 0.5 < FICmean ≤ 1, indifferent if 1 < FICmean < 4 or antagonistic if FICmean ≥ 4. Most of the 285 pharmacodynamic interactions tested with clinical strains were close to additivity (average FICmean = 0.89 [0.38-1.28]). No interaction was found to be antagonistic. When two antibiotics to which a strain was resistant were combined, the concentrations required to inhibit bacterial growth were higher than their respective breakpoints. The present results have shown that invitro, the different antibiotics used in therapeutics have additive effects. The addition of the effects of two antibiotics to which a strain was resistant was not sufficient to inhibit bacterial growth. In probabilistic treatment, the choice of antibiotics to combine should therefore be based on the local epidemiology of resistance, and on susceptibility testing in the case of CLA therapy, so that at least one antibiotic to which the strain is susceptible is used.

CRISPR-like sequences association with antibiotic resistance and biofilm formation in Helicobacter pylori clinical isolates

Role of clustered regularly interspaced short palindromic repeats (CRISPR)-like sequences in antibiotic resistance and biofilm formation isn't clear. This study investigated association of CRISPR-like sequences with antibiotic resistance and biofilm formation in H. pylori isolates. Thirty-six of H. pylori isolates were studied for existence of CRISPR-like sequences using PCR method and their correlation with biofilm formation and antibiotic resistance. Microtiter-plate technique was utilized for investigating antibiotic resistance profile of isolates against amoxicillin, tetracycline, metronidazole and clarithromycin. Biofilm formation of isolates was analyzed by microtiter-plate-based-method. Out of 23 CRISPR-like positive isolates, 19 had ability of biofilm formation and 7 of 13 CRISPR-like negative isolates were able to form biofilm (Pvalue = 0.445). In CRISPR-like positive isolates, 11 (48%), 18 (78%), 18 (78%) and 23 (100%) were resistant to amoxicillin, tetracycline, metronidazole and clarithromycin, respectively. Since CRISPR-like sequences have role in antibiotic resistance, may be applied as genetic markers of antibiotic resistance. But there was no substantial correlation between biofilm formation and existence of CRISPR-like sequences. These results indicate possible importance of CRISPR-like sequences on acquisition of resistance to antibiotics in this bacterium.

Antibacterial, Antibiofilm and Anti-Inflammatory Activities of Eugenol Clove Essential Oil against Resistant Helicobacter pylori.

Eugenol essential oil (EEO) is the major component in aromatic extracts of Syzygium aromaticum (clove) and has several biological properties, such as antibacterial, antioxidant, and anti-inflammatory activities, as well as controlling vomiting, coughing, nausea, flatulence, diarrhea, dyspepsia, stomach distension, and gastrointestinal spasm pain. It also stimulates the nerves. Therefore, the aim of this study was to extract and purify EEO from clove buds and assess its ability to combat resistant Helicobacter pylori. Additionally, EEO's anti-inflammatory activity and its ability to suppress H. pylori biofilm formation, which is responsible for antibiotic resistance, was also investigated. Syzygium aromaticum buds were purchased from a local market, ground, and the EEO was extracted by using hydro-distillation and then purified and chemically characterized using gas chromatography-mass spectrometry (GC-MS). A disk-diffusion assay showed that Helicobacter pylori is sensitive to EEO, with an inhibition zone ranging from 10 ± 06 to 22 ± 04 mm. The minimum inhibition concentration (MIC) of EEO ranged from 23.0 to 51.0 μg/mL against both Helicobacter pylori clinical isolates and standard strains. In addition, EEO showed antibiofilm activity at 25 µg/mL and 50 µg/mL against various Helicobacter pylori strains, with suppression percentages of 49.32% and 73.21%, respectively. The results obtained from the anti-inflammatory assay revealed that EEO possesses strong anti-inflammatory activity, with human erythrocyte hemolysis inhibition percentages of 53.04, 58.74, 61.07, and 63.64% at concentrations of 4, 8, 16, and 32 μg/L, respectively. GC-MS analysis revealed that EEO is a major component of Syzygium aromaticum when extracted with a hydro-distillation technique, which was confirmed by its purification using a chemical separation process. EEO exhibited antibacterial action against resistant Helicobacter pylori strains, as well as antibiofilm and anti-inflammatory activities, and is a promising natural alternative in clinical therapy.

Detection of the babA2 adhesin protein gene in Helicobacter pylori clinical isolates.

The study compared the effectiveness of two different primer sets for detecting and evaluating the prevalence of the babA2 gene in 52 H. pylori clinical isolates from patients with chronic gastritis (n=32), duodenal ulcer (n=16) and stomach cancer (n=4) in St. Petersburg, Russia. The PCR was used for detection of the babA2 gene with 271 bp and 832 bp primer sets followed by sequencing of the PCR-amplicons. The largest proportion of babA2-positive strains - 90.4% (47/52) was detected using a 271 bp PCR primer set. Detection of the 832 bp PCR positive samples was observed only in 51.9% of cases (27/52). The largest proportion of babA2-positive strains - 90.4% (47/52) was detected using 271 bp PCR primer set; detection of 832 bp PCR product was observed only in 51.9% cases (27/52), however, there were no significant differences in the babA2 gene detection rates (p>0.05). Bioinformatic analysis revealed a homology of Sanger sequenced PCR products 271 bp and 832 bp of babA2 gene with regions of the babA2, babA1, and chimeric babA/B genes of H. pylori strains annotated in the NCBI database. Regardless of the primer set used, the presence of babA2 was not significantly associated with duodenal ulcer nor gastric cancer (p>0.05). The combination of the three babA2, cagA, and vacAs1 genes did not reveal any association between the presence of babA2 gene and cagA/vacAs1 genes in H. pylori strains (p>0.05). Thus, none of the two primer sets (271 bp and 832 bp) appears sufficiently informative for detecting the babA2 gene to assess virulence of H. pylori Russian strains.

Chemical composition, antioxidant, antimicrobial and antiproliferative activity of Laureliopsis philippiana essential oil of Chile, study in vitro and in silico

Chilean Laureliopsis philippiana has been used in traditional medicine by the Mapuche and their ancestors. To evaluate its pharmacological activity, Laureliopsis philippiana leaf essential oil extract (LP_EO) was chemically and biologically characterized in the present study. In vitro antioxidant potential was analyzed, and antitumor activity was evaluated in non-tumor and tumor cell culture lines. Caenorhabditis elegans was used as a model for evaluating toxicity, and the chemical composition of the essential oil was analyzed using gas chromatography–mass spectrometry. The oil contains six major monoterpenes: eucalyptol (27.7 %), linalool (27.6 %), isozaphrol (19.5 %), isohomogenol (12.6 %), α-terpineol (7.7 %), and eudesmol (4.8 %). Based on quantum mechanical calculations, isosafrole and isohomogenol conferred in vitro antioxidant and antimicrobial activity to LP_EO. In addition, LP_EO showed antimicrobial activity against clinical Helicobacter pylori isolates (MIC 64 and MBC > 128 μg·mL−1), Staphylococcus aureus (MIC 32 and MBC > 64 μg·mL−1), Escherichia coli (MIC 8 and MBC 16 μg·mL−1) and Candida albicans (MIC 64 and > 128 μg·mL−1). LP_EO could selectively inhibit the proliferation of epithelial tumor cell lines but showed low toxicity against Caenorhabditis elegans (0.39 to 1.56 μg·mL−1). Therefore, LP_EO may be used as a source of bioactive compounds in novel pharmacological treatments for veterinary and human application, cosmetics, or sanitation.

High level of resistance to metronidazole and clarithromycin among Helicobacter pylori clinical isolates in Qazvin province, Iran

The treatment of patients with Helicobacter pylori infection has many limitations, especially because of antibiotic resistance. We aimed to investigate the prevalence and mechanism of antibiotic resistance to metronidazole and clarithromycin in H. pylori isolates collected from patients with gastrointestinal symptoms in Qazvin, Iran. In this cross-sectional study, antibiotic susceptibility testing to clarithromycin and metronidazole was performed among 80 clinical strains isolated from H. pylori-positive dyspeptic patients referred to Qazvin hospital from July 2018 to November 2018. Polymerase chain reaction (PCR) and sequencing tests were performed to determine the type of mutations in the rdxA gene in metronidazole-resistant isolates, and the 23SrRNA gene in clarithromycin-resistant isolates. Thirteen (40.6%) and Twenty-one (65.6%) isolates were resistant to clarithromycin and metronidazole, respectively. 37.5% and 59.4% of clarithromycin and metronidazole resistant isolates had MIC>256. In clarithromycin-resistant isolates, mutations in the 23SrRNA gene was seen at A2143G (15.6%), A2142G (9.4%), C2195T (6.3%), C2244T (3.1%), and G2212A (3.1%) locations. In one isolate, three simultaneous mutations were recorded in locations A2143G, G2110A, and C2121T. Mutations in the rdxA gene in metronidazole-resistant isolates, were missense. High resistance to metronidazole and clarithromycin antibiotics were seen in H. pylori isolates in Qazvin province. This is the first report of new mutation sites G2212A, G2110A, and C2121T on the 23SrRNA gene in clarithromycin-resistant isolates. It is necessary to evaluate the current situation in terms of resistance and identify the mechanisms involved in its occurrence for the successful treatment of infections caused by this organism.

Characterization of Domain V Mutations in Clinical Isolates of Helicobacter pylori in Pakistan and Their Effect on Clarithromycin MIC.

PurposeClarithromycin is commonly prescribed for H. pylori infection. Domain V mutations are responsible for clarithromycin resistance. This study was aimed to characterize the clarithromycin resistance and its associated mutations in clinical isolates of H. pylori in Pakistan.Materials and MethodsInfection was diagnosed in 93 patients’ biopsies using culture, rapid urease test, 16S rRNA, and vacA gene multiplex PCR. Clarithromycin resistance was assessed by the agar dilution method. Mutations were detected by PCR-RFLP using 46 (1.4 kb) domain V fragments. Sequencing was executed for 13 domain V fragments, of which 12 showed unusual amplicon size (1.2 kb) and 01 had a new MboII RFLP pattern.ResultsA total of 48 (83%) strains were obtained from 58 (62.3%) PCR H. pylori-positive samples. Resistance (MIC ≥ 0.001 mg/mL) and intermediate resistance phenotype (MIC = 0.0005 mg/mL) was observed in 22 (46%), and 10 (21%) isolates, respectively. The primary resistance was found in 23 (39.6%) samples. PCR-RFLP detected A2142G, A2143G, and double mutations in 19, 04, and 01 resistant strain, respectively. Sequencing of 10 amplicons obtained from intermediated resistant strains and 03 amplicons from resistant strains showed 138 new mutations. Among them, T2182C was also seen in 04 intermediated resistant isolates, whereas A2142G, A2143G, and A2143C were observed in resistant isolates. The new MboII RFLP pattern in an intermediated resistant strains was due to A1761G mutation.ConclusionH. pylori domain V mutations showed extensive diversity. Multiple mutations in domain V may give endurance to H. pylori against clarithromycin. Further investigations on the molecular mechanism of antibiotic resistance in H. pylori seem crucial at this stage.

In Vitro Screening Potential Antibacterial Properties of the Greek Oregano Honey against Clinical Isolates of Helicobacter pylori.

Oregano honey is an exceedingly rare and distinct product, not commercially available, produced by bees bred in oregano fields of alpine altitudes at the mountainous area of Epirus, Greece. In ethnic popular medicine, this product is used as a therapeutic in various gastric diseases. To test this hypothesis, 14 strains of Helicobacter pylori (H. pylori), 6 isolated from gastric ulcers and 8 from cases of clinical gastritis, were employed in the present study. The above bacterial strains were exposed to various concentrations (75% v/v, 50% v/v, 25% v/v, 12.5% v/v, and 6% v/v) of 50 oregano honey samples by using the agar well method and the inhibition zones observed around each well were recorded. Although the inhibitory zones of the H. pylori isolated from the gastric ulcers were wide enough (0–34 mm), those strains, in general, appeared more resistant than the other eight (0–58 mm). The same result was observed when the same strains were tested against six antibiotics used in clinical practice. Extracts of oregano honey were prepared by extraction with four different organic solvents. N-hexane and chloroform extracts had the most potent antibacterial action. Finally, pure oregano honey and diethyl ether extracts of honey showed significant inhibitory activity against urease secreted by the pathogen. These results strongly indicate the susceptibility of H. pylori strains to the oregano honey by more than one mode of action. Consequently, this variety of honey seems to have potential therapeutic properties against gastric ulcers and gastritis, thus explaining the preference of the locals towards this traditional remedy.

Growth-Inhibiting, Bactericidal, Antibiofilm, and Urease Inhibitory Activities of Hibiscus rosa sinensis L. Flower Constituents toward Antibiotic Sensitive- and Resistant-Strains of Helicobacter pylori.

The aim of the present study was to assess antimicrobial effects of naringenin (NRG), luteolin (LUT), myricetin (MCT), and protocatechuic acid (PCA) identified in a Hibiscus rosa sinensis flower against two reference strains and five clinical isolates of Helicobacter pylori. NRG displayed the most growth inhibitory and bactericidal activities to seven bacterial strains including six strains resistant to one or several antibiotics, azithromycin (MIC, 16–32 mg/L), erythromycin (MIC, 32 mg/L), levofloxacin (MIC, 32 mg/L), and/or metronidazole (24–64 mg/L), followed by LUT and MCT, while PCA showed weak activities toward the strains. These constituents had similar antibacterial activities toward the seven tested strains suggesting that these constituents and the antibiotics do not have a common mechanism of anti-H. pylori activity. NRG, LUT, and MCT resulted in a high percentage of coccoid forms of H. pylori. NRG exhibited the highest anti-biofilm formation activity. MCT produced the strongest inhibition of urease activity followed by LUT and PCA, whereas the activity of NRG was similar to standard inhibitor thiourea. The four constituents had no significant toxicity to human cell lines. A global attempt to decrease utilization of antibiotics justifies the need for further research on H. rosa sinensis derived materials containing NRG, LUT, MCT, and PCA as potential products or lead compounds for the prevention or treatment of diseases caused by H. pylori infection.

In vitro activity of new tetracycline analogues omadacycline and eravacycline against clinical isolates of Helicobacter pylori collected in China

Omadacycline and eravacycline are newly approved tetracycline analogues with excellent activity against a broad spectrum of Gram-positive and Gram-negative microorganisms; however, no data are available regarding Helicobacter pylori. The susceptibility of 201 clinical isolates of H. pylori collected in China to omadacycline, eravacycline, and the comparator tetracycline was determined by an agar dilution method. They showed greater activity than tetracycline. The MIC50/90 values of omadacycline, eravacycline, and tetracycline were 0.125/0.25 μg/mL, 0.063/0.125 μg/mL, and 0.25/1 μg/mL, respectively. Omadacycline and eravacycline were potent in vitro against all the isolates tested, including tetracycline-resistant strains, and warrant further investigation as potential antibiotics for H. pylori treatment.

Efflux Pump Mediated Antibiotic Resistance in Clinical Isolates of Helicobacter Pylori From South West Nigeria

Introduction Helicobacter pylori is a spiral shaped gram negative bacterium majorly causing peptic ulcers and gastric cancer in humans. The combinations of two or more antibiotics such as amoxicillin, clarithromycin, metronidazole or tetracycline with anti-secretory agents or bismuth have been used successfully for the treatment of H. pylori . Aim Increasing antibiotic resistance has been a contributory factor to treatment failures; as such evaluation of resistance mechanism will improve the management of H. pylori infection. Methodology The antibiotic resistance in H. pylori isolates from Nigeria was determined using E-test strips from Biomerieux SA, France and PCR for the efflux pump gene detection. Results Resistance to metronidazole was found to be 93% (97/104), amoxicillin 42% (44/104), clarithromycin 39% (41/104) and tetracycline 27% (28/104). However 51% (53/104) of the isolates harboured multidrug efflux pump gene hefA (Hp605) and 34% (35/104) hefD (Hp 971). However, hefG (Hp 1327) was absent in all the isolates. There was significant association between clarithromycin resistance and hefA with p-value 0.014, there was also association between amoxicillin resistance and hefD with p-value 0.004. Conclusion The present study revealed that H. pylori antibiotic resistance is on the increase in Nigerian strains and the mechanism of resistance may possibly include the possession of multidrug efflux pump.

Mutant selection window of clarithromycin for clinical isolates of Helicobacter pylori

BackgroundClarithromycin-resistance is becoming a global health concern in the treatment of Helicobacter pylori (H. pylori). The mutant prevention concentration (MPC) represent the propensities of antimicrobial agents to select resistant mutants. The concentration range between the minimum inhibitory concentration (MIC) and the MPC is defined as mutant selection window (MSW). In this study, we aimed to determine the cause of increasing clarithromycin resistance by investigating the MSW for clinical isolates of H. pylori.ResultsA retrospective subgroup, which included 68 clarithromycin-sensitive H. pylori strains, was selected from a double-blind trial. The MICs and MPCs were determined using agar plate assays. Genotypic tests were performed using Sanger sequencing. All isolates were wild-type, and 33.82% (23/68) had a 0.016 mg/L MIC, 45.59% (31/68) had a 0.031 mg/L MIC, 16.18% (11/68) had a 0.062 ≤ MIC ≤ 0.125 mg/L, and 4.41% (3/68) had a 0.25 mg/L MIC. The MPC50/90 (mg/L) of the isolates were: 0.062/0.125, 0.125/0.5, 0.25/0.25 and 1/2, respectively. The MPCs showed a moderate correlation with the MICs (rs = 0.65, P < 0.0001). Using published data and MPC90, we calculated the time inside the MSW (TMSW) for low- and high-dose (200 or 500 mg bid) clarithromycin that were 6 and 0 h, 24 and 4 h, 15 and 2 h, 5 and 17 h for the strains with MICs (mg/L) of 0.016, 0.031, 0.062–0.125, and 0.25, respectively.ConclusionsThis study showed that in the clarithromycin-sensitive clinical isolates of H. pylori, low-dose clarithromycin may lead to decreased drug sensitivity or even clarithromycin resistance; strains with a 0.25 mg/L MIC display a high risk of treatment failure.

In-vitro characterisation of a novel antimicrobial agent, TNP-2092, against Helicobacter pylori clinical isolates.

TNP-2092 is a novel dual-action lead compound consisting of rifamycin SV and 4H-4-oxo-quinolizine pharmacophores, with a broad spectrum of antibacterial activities. This compound is currently in the early stage of clinical development for Helicobacter pylori infection. The aim of the present study was to determine the antibacterial activity of TNP-2092 against H. pylori isolated from primary patients. A total of 100 H. pylori clinical isolates from primary patients were selected. The minimum inhibitory concentrations (MICs) for clarithromycin, levofloxacin, rifampin and TNP-2092 were determined using an agar dilution method. A time-kill study was performed with different concentrations of TNP-2092 relevant to MIC against H. pylori ATCC strain 43504 for up to 24 hours. The time-kill study with drug concentrations of 0-4 × MIC was also used to determine the antibacterial activity of TNP-2092 against H. pylori under different pH conditions (pH 4-7). The primary resistance percentages to clarithromycin, levofloxacin, rifampin and TNP-2092 were 13, 18, 1 and 1%, respectively. TNP-2092 killing kinetics were both concentration and time dependent. The effectiveness of TNP-2092 against H. pylori was gradually reduced with a decrease in pH. TNP-2092 is highly active against H. pylori and against strains resistant to clarithromycin or levofloxacin. Its antibacterial activity is both concentration- and time-dependent .The antibacterial activity of TNP-2092 appears to be pH-dependent and is more active under neutral pH. TNP-2092 represents a promising new therapy for the treatment of H. pylori infection in primary patients.

Phylogenetic study of drug resistance genes from clinical isolates of Helicobacter pylori

Hp strains were isolated and cultured from saliva and gastric mucosal tissues derived from patients with gastric diseases. Nine strains (including 5 isolated from oral tissues, 1 isolated from gastric tissues, and 3 representative strains of SS international standard strains used for animal models) were tested for common antibiotic resistance. Together with an ACTT 11637 international standard strain, these were subjected to re-sequencing to obtain drug-resistance genes. Such genes from various sources were compared with the resistance genes of Hp strains recorded by the NCBI website. Combined with results of drug-resistance experiments, correlation between molecular evolution and drug-resistance was analyzed. Testing of gastric mucosal tissues and salivary samples from 217 patients has found 89 Hp strains, which yielded a total infection rate of 41.01%. The resistance rates of 9 representative Hp strains for clarithromycin, amoxicillin, metronidazole, levofloxacin and tetracycline were 77.8%, 77.8%, 44.4%, 77.8%, and 77.8%, respectively. Compared with the reference strain, the similarity between clarithromycin-resistance genes was 99%, and that between amoxicillin- and metronidazole-resistance genes was 96%-97%. A2143G mutation was also found in clarithromycin-resistant genes of three Hp strains. The sensitivity of Hp to metronidazole is much higher in patients from Tibet region, and the sensitivity of Hp to clarithromycin, amoxicillin, levofloxacin and tetracycline is poor. Resistance mutations are consistent with drug resistance.

In – Vitro activity of Desmostachya bipinnata (L.) Stapf successive extracts against Helicobacter pylori clinical isolates

Helicobacter pylori are well acknowledged as a major cause of gastrointestinal ailments and gastric cancers. Therefore, the present study aimed to investigate the potential in vitro activity of Desmostachya bipinnata against H. pylori, focusing on the determination of the most active extract responsible for the anti-helicobacter activity to produce new active drug from natural source.Desmostachya bipinnata total alcohol and successive extracts were in vitro tested against H. pylori. All extracts showed promising anti Helicobacter pylori activities. The most effective extract was diethyl ether extract, it showed 75% growth inhibition of the clinical Isolates bacterial Helicobacter pylori, in addition it showed high count reduction on the selected organisms in the different concentrations used (2xMIC, MIC & ½ MIC) compared with the untreated controls as well as the other extracts (chloroform, ethyl acetate and n-butanol). The oral median lethal dose (LD50) of the alcohol extract of the plant by doses up to 5000 mg/kg didn’t showed any mortality or morbidity, in addition no side effects were recorded on both liver and kidney functions this means that the extract was safe for use.

Molecular analysis of low-level tetracycline resistance in clinical isolates of Helicobacter pylori among dyspeptic patients in South West Nigeria.

The aim of this study was to determine the occurrence of 16S rRNA mutations associated with low-level tetracycline resistance in Helicobacter pylori isolates from adult dyspeptic patients in South West Nigeria. Susceptibility testing to tetracycline of 50 H. pylori isolates was performed by Etest. The 535-bp conserved region of the H. pylori tetracycline-binding site of 16S rRNA was amplified by PCR, followed by sequencing and multiple sequence alignment for all 50 clinical isolates. Of the 50 clinical isolates examined, DNA sequence analysis revealed nucleotide substitutions in 7 isolates at positions 926-928. Of the seven isolates, two demonstrated reduced susceptibility to tetracycline with Etest minimum inhibitory concentrations (MICs) of 0.75-1.0mg/L, whilst the other five isolates were resistant with MICs of 1.5-24mg/L (resistance breakpoint >1mg/L). The two isolates with reduced susceptibility had single nucleotide substitution of A926G, whilst the five resistant isolates demonstrated double base pair substitutions of G927T/A928C and A926G/A928C and a single nucleotide substitution of A926G. This study shows that low-level tetracycline resistance amongst H. pylori-positive dyspeptic patients is associated with reduced susceptibility and resistance to tetracycline. This is the result of 1-bp and 2-bp differences in positions 926 and 926-928, respectively, in the 16S rRNA of H. pylori.

Characterization and Antibacterial Activity Against Helicobacter pylori of Lactic Acid Bacteria Isolated from Thai Fermented Rice Noodle.

A total of 32 lactic acid bacteria (LAB) were isolated from Khanom-jeen, a Thai traditional fermented rice noodle. They belonged to the genus Leuconostoc (Ln), Lactobacillus (Lb), Enterococcus (E), Lactococcus (Lc), and Weissella (W), based on their phenotypic characteristics and 16S rRNA gene sequence analyses. The strains were identified as Ln. pseudomesenteroides (group 1, two strains), Ln. citreum (group 2, three strains), Ln. lactis (group 3, three strains), Lb. paracasei subsp. tolerans (group 4, two strains), E. faecium (group 5, three strains), Lc. lactis subsp. lactis (group 6, one strain), W. confusa (group 7, six strains), Lb. fermentum (group 8, seven strains), and Lb. plantarum subsp. plantarum and Lb. pentosus (group 9, five strains). Fifteen strains exhibited the inhibitory activity against Helicobacter pylori clinical isolates by spot-on-lawn method. Lb. fermentum P43-01 resisted to bile acids showed the broad spectrum of antimicrobial activity against H. pylori strains MS83 and BK364. These antagonistic effects were associated with proteinaceous compounds which are sensitive to α-chymotrypsin and pepsin. Results indicated that production of bacteriocin-like substances of selected strain might be the significant mechanism that exerted the inhibition on H. pylori. A potential strain could be used as probiotics in alternative or adjunctive therapy for a patient suffering from H. pylori infection.

Comment on “Effect of biofilm formation by clinical isolates of Helicobacter pylori on the efflux-mediated resistance to commonly used antibiotics”

Attaran et al[1] have recently shown that decreased susceptibility of established Helicobacter pylori (H. pylori) biofilms to specific antibiotics, was associated with the overtly enhanced transcription of two efflux pump genes, hp1165 and hefA, involved in specific resistance to tetracycline and multiple antibiotics, respectively. Apart from antibiotic exposure, secretion of multiple antimicrobial peptides, such as human β-defensins (hβDs), by the gastric epithelium upon Hp challenge, may act as early triggering events that positively impact biofilm formation and thus, antibiotic resistance. In this regard, we undertook genomic transcriptional studies using Hp 26695 strain following exposure to sublethal, similar to those present in the gastric niche, concentrations of hβDs in an attempt to provide preliminary data regarding possible mechanisms of immune evasion and selective sensitivity of Hp. Our preliminary results indicate that hβD exposure ignites a rapid response that is largely due to the activation of several, possibly interconnected transcriptional regulatory networks – origons - that ultimately coordinate cellular processes needed to maintain homeostasis and successful adaptation of the bacterium in the gastric environment. In addition, we have shown that both antibiotic and hβD resistance are mediated by dedicated periplasmic transporters, including the aforementioned efflux pump genes hp1165 and hefA, involved in active export of antibiotics from the cell membrane and/or, as recently suggested, substrate sensing and signalling. Furthermore, it appears that sublethal doses of hβDs may enhance biofilm formation by the sustained expression of, mainly, quorum sensing-related genes. In conclusion, we provide additional data regarding the role of specific innate immune molecules in antibiotic cross-resistance mechanisms that may deepen our understanding in the context of the development of novel eradication regimens.

Antimicrobial susceptibility of 6 antimicrobial agents in Helicobacter pylori clinical isolates by using EUCAST breakpoints compared with previously used breakpoints

IntroductionThe aim of this study was to determine the differences in percentage resistance in H. pylori clinical isolates using EUCAST breakpoints compared with previously used breakpoints. MIC value distribution in H. pylori clinical isolates was also studied. MethodsSusceptibility to amoxicillin, tetracycline, metronidazole, clarithromycin, rifampicin and levofloxacin was performed by E-test in 824 H. pylori clinical isolates. EUCAST and previous breakpoints defined resistance as follows: MIC >0.12mg/L and ≥2mg/L for amoxicillin, >8mg/L and ≥8mg/L for metronidazole, >0.5mg/L and ≥1mg/L for clarithromycin, >1mg/L and ≥32mg/L for rifampicin, and >1mg/L and ≥4mg/L for tetracycline and >1mg/L levofloxacin. ResultsOverall resistance rate by EUCAST and by previous breakpoints was 8.5% and 3.2% for amoxicillin, 0.6% and 0.1% for tetracycline, 39.2% and 39.7% for metronidazole, 51.2% and 51.2% for clarithromycin, 32% and 3.1% for rifampicin, and 6.7% and 6.7% for levofloxacin. ConclusionsWhen using the different breakpoints for antimicrobial susceptibility testing, similar results were found with most antibiotics tested (tetracycline, metronidazole, clarithromycin, and levofloxacin), except for amoxicillin and rifampicin.

Identification of Factors Associated with Biofilm Formation Ability in the Clinical Isolates of Helicobacter pylori.

Background A few reports confirm the ability of Helicobacter pylori to form biofilm. However, conclusive data do not exist concerning the factors that favor this ability. Objectives Evaluation of the factors associated with the biofilm formation ability of H. pylori including bacterial, physical and chemical, and environmental factors was the research’s aim. Materials and Methods H. pylori isolates from gastric biopsy specimens of patients infected chronically were screened for biofilm formation ability. Association of bacterial properties such as motility, auto-aggregation, cell hydrophobicity, and extracellular polymeric substances (EPS) with in vitro biofilm formation ability of H. pylori was evaluated. The effects of environmental factors such as growth-medium, temperature, oxygen-tension, pH, β-cyclodextrin, gastric secreted mucins, and sub-inhibitory concentration of amoxicillin were also evaluated. Results Ability of clinical H. pylori isolates to form biofilm in was quantitatively compared. The coccoid shape H. pylori cells were observed by scanning electron microscopy, the images were illustrative of the attachment of cells to form microcolony. The levels of hydrophobicity, motility and auto aggregation of two isolates with highest and lowest biofilm formation ability were the same. However, the signifi cant role of mucins (P < 0.05) in elevating the biofilm formation was observed. Other factors influencing biofilm formation were: pH, atmosphere and sub-MIC of antibiotics. Conclusion Mucins have a signifi cant role in elevating the biofilm formation, also pH, atmosphere and sub-MIC of antibiotics influence biofilm formation.