Elżbieta Karczewska

Oral cavity as a potential source of gastric reinfection by Helicobacter pylori

Helicobacter pylori (Hp) is a common pathogen colonizing the a gastric mucosa, but some reports indicated that it may also be found in the oral cavity, which could serve as a reservoir of the bacteria and a source of gastric reinfection. Accordingly, we aimed to study whether the oral cavity, particularly gingival pockets, are colonized by Hp and whether it could be the source of gastric reinfection. We studied 329 patients with dyspeptic symptoms (257 with chronic gastritis, 15 with gastric ulcer, and 57 with duodenal ulcer). The [13C]urea breath test (UBT), gastroscopy, and Hp culture from gastric biopsies were carried out, and material was collected from the oral cavity (gingival pocket) for bacteriological culture and genomic DNA studies. The serum was obtained for anti-Hp IgG and anti-CagA assays and saliva for anti-Hp IgA determination using the ELISA technique. Bacteria in material from gingival pockets and biopsies from the corpus and antrum of stomach of 30 DU patients before and after Hp eradication were also examined by PCR technique, using primers specific for 16S rRNA. All Hp-positive patients (276) were subjected to one week of triple therapy (omeprazole 2 × 20 mg twice a day, clarithromycin 2 × 500 mg twice a day, and metronidazole 2 × 500 mg twice a day). The measurements described above were then repeated at four weeks and six months. Bacteriological culture showed the presence of Hp in the material from oral cavity in about 50% of patients, whereas UBT, used as a gold standard, revealed gastric Hp infection in about 84% of these patients. The eradication was successful in the majority of patients (87%), but about 13% of them were still Hp positive after four weeks and about 21% after six months. Four weeks after Hp therapy, Hp was found in culture from oral samples in 23% (P < 0.05 vs initial) and after six months in 35.1%. The IgA levels recorded in saliva were in a close agreement with UBT results. Hp DNA assessed by PCR in 30 DUs before eradication of Hp was detected in 95% of antral mucosa, 90% in corpus mucosa, and in 35% of gingival pocket material, and after eradication therapy Hp DNA values fell to 25%, 20%, and 10%, respectively. In conclusion, Hp is commonly detected in the oral cavity of patients with dyspeptic symptoms, but the gastric reinfection does not appear to occur in the patients despite oral Hp colonization.

Antifungal and Antibacterial Activity of the Newly Synthesized 2-Xanthone Derivatives

A series of 2‐substituted xanthone derivatives 8–20 containing selected allyl, cinnamyl, morpholine, and imidazole moieties were synthesized and tested for their antifungal and antibacterial in‐vitro properties. Of the newly synthesized derivatives, ten revealed antifungal activity especially against Trichophyton mentagrophytes (the biggest inhibition zones ranged 35 mm for 11 and 13). 2‐(3‐(Allylamino)propoxy)‐9H‐xanthen‐9‐one hydrochloride 9 inhibited growth of all of the examined fungal species. Significant efficacy against evaluated yeasts and dermatophytes was also observed for 6‐chloro‐2‐methyl‐9H‐xanthen‐9‐one derivatives 11–13 containing encyclic amine moieties. Additionally, compounds 9, 11, and 12 hindered development of bacteria species but in a lesser degree. They were efficacious against Staphylococcus aureus, Escherichia coli, and Enterococcus faecalis.

Functional and morphological aspects of Helicobacter pylori-induced gastric cancer in Mongolian gerbils

Background Helicobacter pylori infection of Mongolian gerbils is an established model of gastric carcinogenesis, but gastric secretory aspects of this carcinogenesis have not been studied. Methods The effects of single intragastric inoculation of gerbils with H. pylori strain (cagA+ vacA+, 5 × 106 CFU/ml) or vehicle (saline) were examined at 1, 2, 4, 6, 9, 12 and 30 weeks from inoculation. Gastric morphology, the presence of H. pylori using the rapid urease test, the density of H. pylori and 16S rRNA and the plasma gastrin and somatostatin were determined. Results H. pylori was detected in gastric mucosa in all infected animals. Basal gastric acid in gerbils was reduced by about 50% after H. pylori inoculation. Early lesions seen at 4 weeks after H. pylori inoculation consisted of chronic gastritis with thickened mucosal folds, oedema, congestion and mucosal lymphocytic infiltration. Adenomatous hyperplasia with cellular atypia with increased mitotic activity and the formation of apoptotic bodies and visible erosions and ulcerations were observed at 12‐30 weeks after inoculation. The atypical gastric glands were situated ‘back‐to‐back’, suggesting gastric pre‐cancer. The gastric blood flow in H. pylori‐infected gerbils was significantly lower than that in the controls. Six‐ to seven‐fold increase in plasma gastrin levels combined with significant fall in gastric somatostatin contents and the intraepithelial neoplasia were noticed in gerbils at all tested periods. Conclusion H. pylori‐infection in gerbils resulted in gastric pre‐cancer associated with functional changes, such as suppression of gastric secretion and impairment of both gastric mucosal microcirculation and the gastrin‐somatostatin link.

Variability in Prevalence of Helicobacter pylori Strains Resistant to Clarithromycin and Levofloxacin in Southern Poland

Background. An increasing resistance of Helicobacter pylori strains to antimicrobial agents is the serious therapeutic problem. The aim of this study was to compare the primary and secondary resistance of H. pylori strains isolated between 2006–2008 (data published) and 2009–2011 to clarithromycin and levofloxacin. Material and Methods. 220 dyspeptic patients (153 before treatment, 67 after), were enrolled in the study. 51 H. pylori strains were isolated. MIC values of clarithromycin and levofloxacin were determined by the E-test method. The statistical analysis was conducted with the χ 2 test with Yates correction at the 0.05 significance level (P ≤ 0.05). Results. Between 2006 and 2008, 34% (39/115) of H. pylori strains were resistant to clarithromycin (primary 21% (19/90), secondary 80% (20/25)). 5% (6/115) of strains were resistant to levofloxacin (primary 2% (2/90), secondary 16% ((4/25); data published) Between 2009–2011, 22% (11/51) of H. pylori strains were resistant to clarithromycin (primary 19% (8/43), secondary 38% (3/8)). 16% (8/51) of strains were resistant to levofloxacin (primary 12% (5/43), secondary 38% (3/8)). Conclusion. The present study has shown the increasing amount of resistant H. pylori strains isolated from patients in Southern Poland to levofloxacin and decreasing number of resistant strains to clarithromycin.

Serum progastrin and its products, gastric acid secretion and serum pepsinogen I in gastric cancer.

Background: Numerous studies have shown an association between Helicobacter pylori (Hp) infection and gastric cancer (GC). Study: This study was designed to determine the role of cytotoxin-associated gene A (CagA)-positive Hp infection, serum amidated gastrins and their precursor, progastrin, gastric acidity and serum pepsinogen I (PG-I) levels in gastric cancerogenesis in 74 cancer patients and in 77 age- and gender-matched controls. Serum IgG antibodies to Hp and CagA and levels of IL-8 and PG-I were measured by ELISA, while progastrin and amidated gastrin by specific radioimmunoassay. Results: The overall Hp and CagA seropositivity in GC patients were significantly higher (82 and 60%) than in matched controls (61 and 27%, respectively). Progastrin and amidated gastrin levels over their cutoff points (122 and 32 pM, respectively) were found in a significantly larger number of GC (59.4 and 44.5%) than in controls (9.0 and 16.8%, respectively). Histologically, all these GCs with increased serum progastrin and amidated gastrins were of intestinal type and showed CagA and Hp seropositivity. Serum IL-8 and gastric pH, above their cutoff points (pH >4.5), and serum PG-I level below its cutoff point (44.2 µg/l) were observed in a significantly higher number of GC patients as compared to controls. Conclusions: (1) GC patients have higher Hp and CagA seroprevalence than matched controls, confirming that CagA-positive Hp infection is associated with higher risk of GC; (2) serum levels of amidated gastrins and their precursor, progastrin, as well as IL-8 are significantly higher, while serum PG-I levels are reduced in intestinal type GC compared to controls, and (3) determination of high serum progastrin, amidated gastrins and IL-8 combined with low serum PG-I may be useful biomarkers of GC.

Anti-Helicobacter pylori activity of some newly synthesized derivatives of xanthone

A series of 20 xanthone derivatives was synthesized and evaluated for anti-Helicobacter pylori (H. pylori) activity. Qualitative and quantitative in vitro tests using the Kirby–Bauer method (agar disc-diffusion method) were performed. The tested compounds were screened against clarithromycin- and/or metronidazole-resistant strains of H. pylori. As a reference, Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacterial strains were examined. On the basis of microbiological assays, xanthones can be considered as potential anti-H. pylori agents. They displayed significant activity against the examined strains, which was higher against the bacteria resistant to metronidazole than clarithromycin. The lowest MIC values ranging up to 20 mg l−1 were observed for the following compounds: 3, 4, 8, 9, 12, 19 (against the metronidazole-resistant strains) and the compound 10 (against the clarithromycin-resistant strain). These preliminary results for screening of xanthone derivatives form a part of an ongoing study of the structure–activity relationships of a large group of compounds. Microbiological assays will be conducted afterwards to determine the mechanism of xanthones’ action against H. pylori.

Anti-Helicobacter pylori activities of selected N-substituted cinnamamide derivatives evaluated on reference and clinical bacterial strains

In this study, thirty-five N-substituted derivatives of cinnamic acid amide (cinnamamide) were evaluated for anti-Helicobacter pylori activity using an agar disc-diffusion method. Qualitative screening was performed on a reference H. pylori strain (ATCC 43504), resulting in the identification of the three most active compounds, 8 (R,S-(2E)-3-(4-chlorophenyl)-N-(2-hydroxypropyl)prop-2-enamide, minimal inhibitory concentration, MIC = 7.5 µg/mL), 23 ((2E)-3-(4-chlorophenyl)-N-(2-hydroxycyclohexyl)prop-2-enamide, MIC = 10 µg/mL), and 28 ((2E)-3-(4-chlorophenyl)-N-(4-oxocyclohexyl)prop-2-enamide, MIC = 10 µg/mL). These compounds were further tested on twelve well-characterized clinical strains, yielding MIC values that ranged from 10 to 1000 µg/mL. Preliminary safety assessments of the compounds were made using the MTT viability test for cytotoxicity and Ames test for mutagenicity, which showed them to be generally safe, although compounds 8 and 28 showed mutagenic activity at some of the tested concentrations. The results of this study showed the anti-H. pylori potential of cinnamamide derivatives.

Nested PCR for the detection of Aspergillus species in maxillary sinus samples of patients with chronic sinusitis

BACKGROUND Fungal rhinosinusitis has become an increasingly recognized disease, being Aspergillus species responsible for most of the cases. Its diagnosis is quite difficult because of the non-specific symptoms and low sensitivity of the current diagnostic methods. AIMS An Aspergillus-specific nested polymerase chain reaction (PCR) assay using biopsy specimens taken from the maxillary sinuses was performed in order to assess its usefulness. Conventional diagnostic methods (histology and culture) were also carried out. METHODS A case-control study was performed in the Institute of Stomatology, Jagiellonian University in Kraków, between 2011 and 2014. The case group consisted of 21 patients with suspected rhinosinusal mycetoma while the control group included 46 patients with no suspicion of fungal rhinosinusitis. The two-step PCR assay amplified an Aspergillus specific portion of the 18S rRNA gene. Interval estimation of sensitivity, specificity, positive (PPV) and negative (NPV) predictive values were calculated to assess the diagnostic test performance. The agreement between the PCR and the other tests was evaluated using the Kappa coefficient (k). RESULTS Ninety percent of the samples obtained from patients diagnosed with mycetoma yielded positive PCR results. The PCR showed almost perfect concordance with histology (k=0.88). Sensitivity, specificity, PPV and NPV estimates were 90%; 95% CI: (55.5-99.7%), 98.3%; 95% CI: (90.9-100%), 90%; 95% CI: (55.5-99.7%) and 98.3%; 95% CI: (90.9-100%), respectively. One clinical sample showed growth of Aspergillus fumigatus and positive PCR despite the negative histological examination. CONCLUSIONS Nested PCR assay is a promising diagnostic tool to evaluate the presence of Aspergillus in the tissue of maxillary sinus from patients with suspicion of sinus aspergillosis.

Prevalence of human pathogens of the clade Nakaseomyces in a culture collection—the first report on Candida bracarensis in Poland

Human pathogens belonging to the Nakaseomyces clade include Candida glabrata sensu stricto, Candida nivariensis and Candida bracarensis. Their highly similar phenotypic characteristics often lead to misidentification by conventional laboratory methods. Therefore, limited information on the true epidemiology of the Candida glabrata species complex is available. Due to life-threatening infections caused by these species, it is crucial to supplement this knowledge. The aim of the study was to estimate the prevalence of C. bracarensis and C. nivariensis in a culture collection of C. glabrata complex isolates. The study covered 353 isolates identified by biochemical methods as C. glabrata, collected from paediatric and adult patients hospitalised at four medical centres in Southern Poland. The multiplex PCR was used to identify the strains. Further species confirmation was performed via sequencing and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis. One isolate was recognised as C. bracarensis (0.28%). To our knowledge, it is the first isolate in Poland. C. glabrata sensu stricto species has been confirmed for all the remaining isolates. No C. nivariensis was found. Our study has shown that the prevalence of C. nivariensis and C. bracarensis strains is infrequent. However, it should be emphasised that the incidence of these strains may differ locally and depend on environmental factors and the population.

Preliminary antifungal activity assay of selected chlorine-containing derivatives of xanthone and phenoxyethyl amines

The aim of this study was to evaluate antifungal activity in a diverse group of chlorine‐containing xanthone and phenoxyethyl amine derivatives – and to select the most promising compounds for further studies. The antifungal efficacy of 16 compounds was tested with qualitative and quantitative methods against both reference and clinical strains of dermatophytes, moulds and yeasts. The disc‐diffusion method has demonstrated that from 16 tested compounds, 7 possess good antifungal activity against dermatophytes and/or moulds while none of them has shown good efficacy against yeasts or bacterial strains. The most active compounds (2, 4, 10, 11, 12, 15, 16) were tested quantitatively by broth dilution method to obtain MIC values. The MIC values against dermatophytes ranged from 8 to 64 μg/ml. Compound 2 was the most active one against dermatophytes (MIC 50 and MIC 90 were 8 μg/ml). The MIC values for moulds ranged from 16 to 256 μg/ml. Compound 4 was the most active one against moulds, with MIC 50 and MIC 90 values amounting to 32 μg/ml. Among the tested compounds, compound 4 (derivative of xanthone) was the most active one and expressed good antifungal efficacy against clinical strains of dermatophytes and moulds. However, another xanthone derivative (compound 2) was the most active and selective against dermatophytes.