Bacteriocin-like inhibitory activities of seven Lactobacillus delbrueckii subsp. bulgaricus strains against antibiotic susceptible and resistant Helicobacter pylori strains.

The aim of the study was to detect anti-Helicobacter pylori activity of seven Lactobacillus delbrueckii subsp. bulgaricus (GLB) strains by four cell-free supernatant (CFS) types. Activity of non-neutralized and non-heat-treated (CFSs1), non-neutralized and heat-treated (CFSs2), pH neutralized, catalase-treated and non-heat-treated (CFSs3), or neutralized, catalase- and heat-treated (CFSs4) CFSs against 18 H. pylori strains (11 of which with antibiotic resistance) was evaluated. All GLB strains produced bacteriocin-like inhibitory substances (BLISs), the neutralized CFSs of two GLB strains inhibited >81% of test strains and those of four GLB strains were active against >71% of antibiotic resistant strains. Two H. pylori strains were BLIS resistant. The heating did not reduce the CFS activity. Briefly, all GLB strains evaluated produced heat-stable BLISs, although GLB and H. pylori strain susceptibility patterns exhibited differences. Bacteriocin-like inhibitory substance activity can be an advantage for the probiotic choice for H. pylori infection control. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, anti-Helicobacter pylori activity of seven Lactobacillus delbrueckii subsp. bulgaricus (GLB) strains was evaluated by four cell-free supernatant (CFS) types. The GLB strains produced heat-stable bacteriocin-like inhibitory substances (BLISs) with a strong anti-H. pylori activity and some neutralized, catalase- and heat-treated CFSs inhibited >83% of the test strains. Bacteriocin-like inhibitory substance production of GLB strains can render them valuable probiotics in the control of H. pylori infection.

Evaluation of Helicobacter pylori eradication by triple therapy plus Lactobacillus acidophilus compared to triple therapy alone.

The purpose of this study was to evaluate the influence of adding Lactobacillus acidophilus to a triple regimen for Helicobacter pylori eradication in untreated patients with peptic ulcers or ulcer-scars. This was a pre-randomized, single-blind, interventional, treatment-efficacy study with active controls and parallel-assignment, set in Coimbra, Portugal, on 62 consecutive H. pylori-positive untreated adults with peptic ulcers or ulcer-scars, diagnosed by gastroduodenoscopy, with pre-treatment direct Gram-staining and culture of gastric biopsies. The first 31 patients received esomeprazole 20 mg, amoxicillin 1000 mg and clarithromycin 500 mg (EAC), all b.i.d., for 8 days. The remaining 31 added L. acidophilus, 5 × 10(9) organisms per capsule, 3 + 2 i.d. for 8 days (EACL). The main outcome measure was (13)C urea breath test (UBT), ≥6 weeks after completion of therapy. Successful eradication (UBT-negativity after treatment), was similar in both groups (EAC = 80.6%; EACL = 83.9%, p = 0.740) by both intention-to-treat and per-protocol analysis. The non-eradicated strains were susceptible in vitro to both antibiotics. Adding L. acidophilus to EAC triple therapy did not increase H. pylori eradication rates. Considering the cost and the burden of ingesting five extra capsules daily, supplementing the EAC therapy with L. acidophilus, at this dose, shows no benefit. Further studies with different dosages and duration of treatment, and other probiotics or probiotic combinations are required to improve eradication.

Anti-Helicobacter pylori activity of Lactobacillus delbrueckii subsp. bulgaricus strains: preliminary report.

AIMS: To evaluate the activities of six Lactobacillus delbrueckii subsp. bulgaricus (LB) strains against 30 Helicobacter pylori strains by agar-well diffusion method. METHODS AND RESULTS: LB cultures [4 x 10(8)-4 x 10(9) CFU ml(-1)) either were prepared in milk at their native pH, 3.8-5.0, or were adjusted to pH 6.4-7.7. At low and neutralized pH, LB strains inhibited the growth by 40-86.7% and 16.7-66.7% of H. pylori strains, respectively. LB activity was strain-dependent. At low and neutralized pH, one and five H. pylori strains, respectively, were not inhibited by any LB strain. LB2 and LB3, taken together, were active against most metronidazole and clarithromycin resistant strains. CONCLUSIONS: All LB strains inhibited a number of H. pylori strains, including also antibiotic resistant strains. LB activity was strain-dependent and better at low pH. At low pH values, the most active LB strains were LB1, LB2 and LB3, inhibiting 86.7% of H. pylori strains, while at neutralized pH values, the most active LB strains were LB2 and LB3, inhibiting 53.3 and 66.7% of H. pylori strains, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: LB could be utilized in the treatment or prophylaxis of H. pylori infection and warrants clinical investigations.

How to isolate, identify and determine antimicrobial susceptibility of anaerobic bacteria in routine laboratories.

BACKGROUND: There has been increased interest in the study of anaerobic bacteria that cause human infection during the past decade. Many new genera and species have been described using 16S rRNA gene sequencing of clinical isolates obtained from different infection sites with commercially available special culture media to support the growth of anaerobes. Several systems, such as anaerobic pouches, boxes, jars and chambers provide suitable anaerobic culture conditions to isolate even strict anaerobic bacteria successfully from clinical specimens. Beside the classical, time-consuming identification methods and automated biochemical tests, the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has revolutionized identification of even unusual and slow-growing anaerobes directly from culture plates, providing the possibility of providing timely information about anaerobic infections. AIMS: The aim of this review article is to present methods for routine laboratories, which carry out anaerobic diagnostics on different levels. SOURCES: Relevant data from the literature mostly published during the last 7 years are encompassed and discussed. CONTENT: The review involves topics on the anaerobes that are members of the commensal microbiota and their role causing infection, the key requirements for collection and transport of specimens, processing of specimens in the laboratory, incubation techniques, identification and antimicrobial susceptibility testing of anaerobic bacteria. Advantages, drawbacks and specific benefits of the methods are highlighted. IMPLICATIONS: The present review aims to update and improve anaerobic microbiology in laboratories with optimal conditions as well as encourage its routine implementation in laboratories with restricted resources.

Detection of Helicobacter pylori infection in symptomatic Bulgarian adults.

This study assessed the prevalence of Helicobacter pylori in symptomatic Bulgarian adults by means of culture, Gram's stain and an in-house rapid urease test (RUT), and also assessed the H. pylori density by culture. In total, 1441 non-treated and 270 treated patients were evaluated. Most non-treated patients with ulcers (87.7%), gastric malignancy (79.2%) and other gastroduodenal diseases (73.4%) were H. pylori-positive. Among non-treated and treated patients, 75.3% and 54.8%, respectively, of elderly patients, and 78.3% and 56.1%, respectively, of other adults were H. pylori-positive. Two (0.1%) non-treated adults were Helicobacter heilmannii-positive. The accuracy of direct Gram's stain and the in-house RUT were 74.8% and 64.2% in non-treated patients, and 73.7% and 63.0% in treated patients, respectively. Culture was highly accurate (>95%) in both groups. Older age decreased the sensitivity of the RUT in non-treated patients by 10.7% and that of all tests in treated patients by 6.9-8.1%. Incubation for 11 days was required for the growth of 2% and 4% of the strains from treated patients on selective and non-selective medium, respectively. There were no differences in isolation rates between positive fresh (74.2%) and frozen (75.2%) specimens. In non-treated adults, a high H. pylori density (growth in all quadrants of the plates) was more common (43.1%) in ulcer patients than in other patients (25.4%). In conclusion, H. pylori infection was common in Bulgarian patients, and at a high density in >40% of ulcer patients, while H. heilmannii infection was uncommon. Culture provided a highly accurate diagnostic approach. Stomach biopsies from non-treated patients can be frozen for several days. The benefit of reporting H. pylori density, as determined by culture, requires further evaluation.

Comparative evaluation of two methods for testing metronidazole susceptibility of Helicobacter pylori in routine practice.

Applicabilities of modified disk diffusion method (MDDM) and screening agar method (SAM) were evaluated in order to detect metronidazole resistance in vitro among 96 Helicobacter pylori strains. SAM was based on 8-mg/liter metronidazole as breakpoint, and MDDM was performed using disks containing 5 micrograms of the drug. Of 96 strains tested, 48 (50%) exhibited inhibitory zones of 26 mm or less by MDDM, and 44 (45.8%) were found resistant to 8 mg/liter metronidazole. Colonies growing within the inhibitory zones by MDDM were detected in six (6.2%) of all strains tested, and the subcultures revealed resistance by both methods. Overall agreement between the results of both tests was found in 90 (93.8%) of the 96 isolates. There is a possibility of great discrepancies between the two methods for testing H. pylori against metronidazole, so the results must be interpreted with caution.

Characteristics and trends in macrolide resistance among Helicobacter pylori strains isolated in Bulgaria over four years.

Macrolide resistance trends were examined among Helicobacter pylori strains from 154 patients between 1994 and 1998. Applicabilities of screening agar method (SAM) and modified disk diffusion method (MDDM) were evaluated. Overall primary resistance rates to erythromycin and clarithromycin were 14.8% and 8.7%, respectively. No association was found with age, sex, and diseases. Clarithromycin-resistance rate reached 12.5% in the last 2 years. Secondary resistance to erythromycin occurred more often (in 62.5%) than to clarithromycin (in 42.9%). Therapy with spiramycin or erythromycin in four cases induced no clarithromycin resistance. These data show a considerable prevalence of H. pylori resistance to macrolides, which exhibited a tendency to increase and was often associated with metronidazole resistance. By comparing the MDDM with SAM, an overall agreement was obtained in 81 (94.2%) of 86 results. MDDM and SAM are reliable techniques for testing H. pylori susceptibility to macrolides.

The status of antimicrobial resistance of Helicobacter pylori in eastern Europe.

OBJECTIVE: To evaluate the primary, secondary and combined resistance to five antimicrobial agents of 2340 Helicobacter pylori isolates from 19 centers in 10 countries in eastern Europe. METHODS: Data were available for centers in Bulgaria, Croatia, the Czech Republic, Estonia, Greece, Lithuania, Poland, Russia, Slovenia and Turkey. Susceptibility was tested by agar dilution (seven countries), E test (five countries) and disk diffusion (three countries) methods. Resistance breakpoints (mg/L) were: metronidazole 8, clarithromycin 1, amoxicillin 0.5, tetracycline 4, and ciprofloxacin 1 or 4 in most centers. Primary and post-treatment resistance was assessed in 2003 and 337 isolates respectively. Results for 282 children and 201 adults were compared. RESULTS: Primary resistance rates since 1998 were: metronidazole 37.9%, clarithromycin 9.5%, amoxicillin 0.9%, tetracycline 1.9%, ciprofloxacin 3.9%, and both metronidazole and clarithromycin 6.1%. Isolates from centers in Slovenia and Lithuania exhibited low resistance rates. Since 1998, amoxicillin resistance has been detected in the southeastern region. From 1996, metronidazole resistance increased significantly from 30.5% to 36.4%, while clarithromycin resistance increased slightly from 8.9% to 10.6%. In centers in Greece, Poland, and Bulgaria, the mean metronidazole resistance was slightly higher in adults than in children (39% versus 31.2%, P > 0.05); this trend was not found for clarithromycin or amoxicillin (P > 0.20). Post-treatment resistance rates exhibited wide variations. CONCLUSIONS: In eastern Europe, primary H. pylori resistance to metronidazole is considerable, and that to clarithromycin is similar to or slightly higher than that in western Europe. Resistance to amoxicillin, ciprofloxacin and tetracycline was detected in several centers. Primary and post-treatment resistance rates vary greatly between centers.

Comparison of three urease tests for detection of Helicobacter pylori in gastric biopsy specimens.

OBJECTIVE: To propose two modified urease tests for the detection of Helicobacter pylori in gastric biopsy specimens. PATIENTS AND METHODS: The presence of H. pylori infection was determined in 237 patients undergoing upper gastrointestinal endoscopy. Three media were used for the urease tests: Christensen's 2% urea broth and two urea agar media, modified by increasing the concentration of urea (to 4% and 10%) and phenol red and omitting the nutrients. RESULTS: The modified tests had good sensitivity (> 78%), specificity (96%) and accuracy (> or = 86%) at 2 h using small amounts (15%) of biopsy homogenates. They were statistically more sensitive and accurate than Christensen's broth. CONCLUSION: Both the modified 4% and 10% urea agar tests are simple, sensitive and specific and can be performed with small amounts of sample.

Xanthoma of the stomach--some morphometrical peculiarities and scanning electron microscopy.

BACKGROUND/AIMS: "Lipid Iceland's" in gastric mucosa, a rare finding in gastroenterology, is described in this paper. The aim of our investigation was to evaluate the frequency of the so-called "gastric xanthoma" in biopsy specimens and to describe the picture elicited by scanning electron microscopy. METHODOLOGY: We investigated cell characteristics histochemically, morphometrically and with scanning electron microscopy. RESULTS: The frequency of xanthoma in our gastrobiopsy material was 0.018% (4 of 21,650 cases) over a 6-year period. The picture of stomach xanthoma given by scanning electron microscopy is very typical of the condition and is called "rolling stones". CONCLUSIONS: We suggest that this diagnostic method warrants further investigation.

Bactericidal effect of amoxicillin on Helicobacter pylori in an in vitro model using epithelial cells.

The treatment of Helicobacter pylori with antimicrobial agents has largely been ineffective, and susceptibility results are in disagreement with those obtained by standard in vitro testing. The bactericidal effect of amoxicillin was tested in an in vitro model by using sessile bacteria attached to HEp-2 cells; this bactericidal effect was compared with that against planktonic bacteria. Viable cell counts were performed by standard procedures after 1, 6, and 24 h of contact with the antibiotic at different concentrations. A bactericidal effect (99.9% killing) was observed against sessile bacteria after 24 h with concentrations of only 10, 1, and 0.1 mg/liter, while against planktonic bacteria it was also noted at concentrations of 0.01 and 0.001 mg/liter, and the effect was observed after 6 h with the three highest concentrations. When the results for five strains were studied by analysis of variance at 6 and 24 h, the main variable was the antibiotic concentration, followed by the culture conditions, e.g., planktonic or sessile bacteria, the strain tested, and the time of contact. A decreased pH of 5.4 did not affect the action of amoxicillin. The bactericidal effect of the combination of amoxicillin and metronidazole was additive against sessile H. pylori.