Optimization of Helicobacter pylori Biofilm Formation in In Vitro Conditions Mimicking Stomach.

Helicobacter pylori is one of the most common bacterial pathogens worldwide and the main etiological agent of numerous gastric diseases. The frequency of multidrug resistance of H. pylori is growing and the leading factor related to this phenomenon is its ability to form biofilm. Therefore, the establishment of a proper model to study this structure is of critical need. In response to this, the aim of this original article is to validate conditions of the optimal biofilm development of H. pylori in monoculture and co-culture with a gastric cell line in media simulating human fluids. Using a set of culture-based and microscopic techniques, we proved that simulated transcellular fluid and simulated gastric fluid, when applied in appropriate concentrations, stimulate autoaggregation and biofilm formation of H. pylori. Additionally, using a co-culture system on semi-permeable membranes in media imitating the stomach environment, we were able to obtain a monolayer of a gastric cell line with H. pylori biofilm on its surface. We believe that the current model for H. pylori biofilm formation in monoculture and co-culture with gastric cells in media containing host-mimicking fluids will constitute a platform for the intensification of research on H. pylori biofilms in in vitro conditions that simulate the human body.

Phytochemical Profile, Plant Precursors and Some Properties of Georgian Propolis.

Propolis (bee glue) is a resinous substance produced by different species of bees i.a. from available plant resins, balsams, and exudates. It is characterized by significant biological activity (e.g., antimicrobial and antioxidant) and phytochemical diversity related to the available plant sources in specific geographical regions. The available scientific literature on propolis is quite extensive; however, there are only a few reports about propolis originating from Georgia. Therefore, our research was focused on the characterization of Georgian propolis in terms of phytochemical composition and antimicrobial/antioxidant activity. Performed research included UHPLC-DAD-MS/MS phytochemical profiling, determination of total phenolic and flavonoid content, antiradical and antioxidant activity (DPPH and FRAP assays) as well as antibacterial activity of propolis extracts obtained using 70% ethanol (70EE). Georgian propolis extracts exhibited strong activity against Gram-positive bacteria (22 mm-disc assay/64 µg/mL-MIC for S. aureus, sample from Imereti) and weaker against Gram-negative strains as well as strong antioxidant properties (up to 117.71 ± 1.04 mgGAE/g in DPPH assay, up to 16.83 ± 1.02 mmol Fe2+/g in FRAP assay for samples from Orgora and Qvakhreli, respectively). The phytochemical profile of Georgian propolis was characterized by the presence of flavonoids, free phenolic acids, and their esters. In most of the samples, flavonoids were the main chemical group (52 compounds), represented mainly by 3-O-pinobanksin acetate, pinocembrin, chrysin, galangin, and pinobanksin. The primary plant precursor of the Georgian bee glue is black poplar (Populus nigra L.) while the secondary is aspen poplar (P. tremula L.).

Nanoapatites Doped and Co-Doped with Noble Metal Ions as Modern Antibiofilm Materials for Biomedical Applications against Drug-Resistant Clinical Strains of Enterococcus faecalis VRE and Staphylococcus aureus MRSA.

The main aim of our research was to investigate antiadhesive and antibiofilm properties of nanocrystalline apatites doped and co-doped with noble metal ions (Ag+, Au+, and Pd2+) against selected drug-resistant strains of Enterococcus faecalis and Staphylococcus aureus. The materials with the structure of apatite (hydroxyapatite, nHAp; hydroxy-chlor-apatites, OH-Cl-Ap) containing 1 mol% and 2 mol% of dopants and co-dopants were successfully obtained by the wet chemistry method. The majority of them contained an additional phase of metallic nanoparticles, in particular, AuNPs and PdNPs, which was confirmed by the XRPD, FTIR, UV-Vis, and SEM-EDS techniques. Extensive microbiological tests of the nanoapatites were carried out determining their MIC, MBC value, and FICI. The antiadhesive and antibiofilm properties of the tested nanoapatites were determined in detail with the use of fluorescence microscopy and computer image analysis. The results showed that almost all tested nanoapatites strongly inhibit adhesion and biofilm production of the tested bacterial strains. Biomaterials have not shown any significant cytotoxic effect on fibroblasts and even increased their survival when co-incubated with bacterial biofilms. Performed analyses confirmed that the nanoapatites doped and co-doped with noble metal ions are safe and excellent antiadhesive and antibiofilm biomaterials with potential use in the future in medical sectors.

Antimicrobial activity of Apis mellifera L. and Trigona sp. propolis from Nepal and its phytochemical analysis.

In this study, we evaluated antimicrobial activity, antimicrobial activity in combination with antibiotics, and chemical composition of Nepalese propolis 70% ethanolic extracts. Propolis originated from two genera of bees - Apis mellifera L. and Trigona sp. HPLC-DAD-MS/MS analyses revealed that the composition of both extracts was almost the same and the main components were flavonoid aglycones (mainly neoflavonoids, isoflavonoids) and pterocarpans. The highest antibacterial activity (disc diffusion test) was observed against Helicobacter pylori, Staphylococcus aureus and Shigella flexneri. Antibiotics exhibited synergism with Apis mellifera L. and Trigona sp. propolis against S. aureus and the strongest effect was observed for the combination with amikacin and tetracycline. Moreover, Nepalase propolis inhibited filamentation of C. albicans and caused oxidative stress by production of the superoxide anion radical (O2-) and a lower concentration of the hydroxyl radical (OH). Propolis extracts are potent antibacterial agents and may be used in combination with antibiotics.

In Vitro Activity of Sertraline, an Antidepressant, Against Antibiotic-Susceptible and Antibiotic-Resistant Helicobacter pylori Strains.

Antibiotic resistance of Helicobacter pylori, a spiral bacterium associated with gastric diseases, is a topic that has been intensively discussed in last decades. Recent discoveries indicate promising antimicrobial and antibiotic-potentiating properties of sertraline (SER), an antidepressant substance. The aim of the study, therefore, was to determine the antibacterial activity of SER in relation to antibiotic-sensitive and antibiotic-resistant H. pylori strains. The antimicrobial tests were performed using a diffusion-disk method, microdilution method, and time-killing assay. The interaction between SER and antibiotics (amoxicillin, clarithromycin, tetracycline, and metronidazole) was determined by using a checkerboard method. In addition, the study was expanded to include observations by light, fluorescence, and scanning electron microscopy. The growth inhibition zones were in the range of 19-37 mm for discs impregnated with 2 mg of SER. The minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) counted for 2-8 µg/mL and 4-8 µg/mL, respectively. The time-killing assay showed the time-dependent and concentration-dependent bactericidal activity of SER. Bacteria exposed to MBCs (but not sub-MICs and MICs ≠ MBCs) underwent morphological transformation into coccoid forms. This mechanism, however, was not protective because these cells after a 24-h incubation had a several-fold reduced green/red fluorescence ratio compared to the control. Using the checkerboard assay, a synergistic/additive interaction of SER with all four antibiotics tested was demonstrated. These results indicate that SER may be a promising anti-H. pylori compound.

In Vitro Activity of 3-Bromopyruvate, an Anticancer Compound, Against Antibiotic-Susceptible and Antibiotic-Resistant Helicobacter pylori Strains.

Helicobacter pylori (H. pylori) is a bacterium capable of inducing chronic active gastritis, which in some people, develops into gastric cancers. One of the substances that may be useful in the eradication of this microorganism is 3-Bromopyruvate (3-BP), an anticancer compound with antimicrobial properties. The aim of this article was to determine the activity of 3-BP against antibiotic-susceptible and antibiotic-resistant H. pylori strains. The antimicrobial activity was determined using a disk-diffusion method, broth microdilution method, time-killing assay, and checkerboard assay. The research was extended by observations using light, fluorescence, and scanning electron microscopy. The growth inhibition zones produced by 2 mg/disk with 3-BP counted for 16⁻32.5 mm. The minimal inhibitory concentrations (MICs) ranged from 32 to 128 µg/mL, while the minimal bactericidal concentrations (MBCs) for all tested strains had values of 128 µg/mL. The time-killing assay demonstrated the concentration-dependent and time-dependent bactericidal activity of 3-BP. The decrease in culturability below the detection threshold (<100 CFU/mL) was demonstrated after 6 h, 4 h, and 2 h of incubation for MIC, 2× MIC, and 4× MIC, respectively. Bacteria treated with 3-BP had a several times reduced mean green/red fluorescence ratio compared to the control samples, suggesting bactericidal activity, which was independent from an induction of coccoid forms. The checkerboard assay showed the existence of a synergistic/additive interaction of 3-BP with amoxicillin, tetracycline, and clarithromycin. Based on the presented results, it is suggested that 3-BP may be an interesting anti-H. pylori compound.

High Antimicrobial Efficacy, Antioxidant Activity, and a Novel Approach to Phytochemical Analysis of Bioactive Polyphenols in Extracts from Leaves of Pyrus communis and Pyrus pyrifolia Collected During One Vegetative Season.

Dried leaf samples of Pyrus communis L. var. 'Conference' and Pyrus pyrifolia Burm. f. (Nakai) var. 'Shinseiki' were subjected to the successful extraction procedures using various solvents, followed by filtering and/or drying liquid plant preparations under reduced pressure. As a result of this, for each Pyrus leaf sample examined, four dried residues were obtained, including methanolic (EA), ethyl acetate (EC), water (EB), and the residue obtained from aqueous solution (ED). Antiradical activity of these preparations was measured using the ABTS+• assay, and antimicrobial activity was examined using various strains of bacteria and yeasts. The highest antiradical activity was observed for EC from leaves of P. communis var. 'Conference' collected in May, but the highest average antibacterial activity was noted for EC residues from P. pyrifolia var. 'Shinseiki' collected in May. Antibacterial activity positively correlated with concentration of hydroquinone in extracts. No antifungal activity was observed for any extract. In addition, qualitative and quantitative analyses of active polyphenolic components in extracts from Pyrus were performed. Hydroquinone and hydroxycinnamic acid derivatives were analyzed using a new optimized method comprising reversed-phase high-performance liquid chromatography (RP-LC) coupled with simultaneous photodiode-array and fluorescence detection.

Profile of Polyphenolic and Essential Oil Composition of Polish Propolis, Black Poplar and Aspens Buds.

In this work, we studied similarities and differences between 70% ethanol in water extract (70EE) and essential oils (EOs) obtained from propolis, black poplars (Populus nigra L.) and aspens (P. tremula L.) to ascertain which of these is a better indicator of the plant species used by bees to collect propolis precursors. Composition of 70EE was analyzed by UPLC-PDA-MS, while GC-MS was used to research the EOs. Principal component analyses (PCA) and calculations of Spearman's coefficient rank were used for statistical analysis. Statistical analysis exhibited correlation between chemical compositions of propolis and Populus buds' 70EE. In the case of EOs, results were less clear. Compositions of black poplars, aspens EOs and propolises have shown more variability than 70EE. Different factors such as higher instability of EOs compared to 70EE, different degradation pattern of benzyl esters to benzoic acid, differences in plant metabolism and bees' preferences may be responsible for these phenomena. Our research has therefore shown that 70EE of propolis reflected the composition of P. nigra or complex aspen⁻black poplar origin.

Antimicrobial and Antiradical Activity of Extracts Obtained from Leaves of Five Species of the Genus Bergenia: Identification of Antimicrobial Compounds.

An important focus of modern medicine is the search for new substances and strategies to combat infectious diseases, which present an increasing threat due to the growth of bacterial resistance to antibiotics. Another problem concerns free radicals, which in excess can cause several serious diseases. An alternative to chemical synthesis of antimicrobial and antiradical compounds is to find active substances in plant raw materials. We prepared extracts from leaves of five species of the genus Bergenia: B. purpurascens, B. cordifolia, B. ligulata, B. crassifolia, and B. ciliata. Antimicrobial and antiradical features of extracts and raw materials were assessed, and the quantities of phenolic compounds were determined. We also evaluated, using high-performance liquid chromatography, the amounts of arbutin and hydroquinone, compounds related to antimicrobial activity of these raw materials. The strongest antiradical properties were shown by leaves of B. crassifolia and B. cordifolia, the lowest by leaves of B. ciliata. The antiradical activity of extracts showed a strong positive correlation with the amount of phenols. All raw materials have significant antimicrobial properties. Among them, the ethyl acetate extracts were the most active. Antimicrobial activity very weakly correlated with the amount of arbutin, but correlated very strongly with the contents of both hydroquinone and phenolic compounds. Additional experiments using artificially prepared mixtures of phenolic compounds and hydroquinone allowed us to conclude that the most active antimicrobial substance is hydroquinone.

In vitro evaluation of the antioxidant and antimicrobial activity of DIMBOA [2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one].

The aim of this study was to evaluate the in vitro antioxidant and antimicrobial properties of the natural cyclic hydroxamic acid: 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA). Antioxidant activity of the isolated DIMBOA was examined using DPPH, FRAP and ABTS tests. It was found that DIMBOA exhibits a potent free-radical scavenging activity and a weaker iron (III) ions reducing activity. Antimicrobial activity against selected G(+), G(-) bacterial strains and against yeasts-like reference strains of fungi was investigated using disk-diffusion method. It has been shown that DIMBOA possess growth inhibitory properties against many strains of studied bacteria and fungi, such as Staphylococcus aureus, Escherichia coli as well as against Saccharomyces cerevisiae.

ß-Aescin at subinhibitory concentration (sub-MIC) enhances susceptibility of Candida glabrata clinical isolates to nystatin.

Aescin (escin) derived from the seeds of horse chestnut (Aesculus hippocastanum L.) is a natural mixture of triterpene saponins exhibiting a wide variety of pharmacological properties, including antiinflammatory, analgesic, and antipyretic activities. However, data concerning antifungal activities of these compounds are limited. This study aims to evaluate the in vitro antifungal susceptibility of Candida glabrata clinical isolates to α-aescin sodium, ß-aescin crystalline and ß-aescin sodium using the disk diffusion (DD) and broth microdilution (BMD) methods. Moreover, the influence of subinhibitory concentration (0.5×MIC) of ß-aescins on the nystatin MIC was also studied. In general, the results obtained by the DD assay correlated well with those obtained by the BMD method. Both ß-aescins effectively inhibited the growth of all 24 strains tested. The minimum inhibitory concentration (MIC) values ranging from 8 to 32 µg/ml for ß-aescin crystalline, whereas those of ß-aescin sodium were slightly lower and ranged from 4 to 16 µg/ml. In contrast, α-aescin sodium was found to be completely ineffective against the strains studied. MIC values of nystatin were reduced 2-16-fold and 2-4-fold in the presence of subinhibitory concentration of ß-aescin crystalline and ß-aescin sodium, respectively. Results of the present study may suggest the additive interaction between ß-aescin and nystatin.

ESBL-producing Escherichia coli isolated from bloodstream infections--antimicrobial susceptibility, conjugative transfer of resistance genes and phylogenetic origin.

BACKGROUND: The prevalence of bloodstream infections (BSIs) due to ESBL-producing Escherichia coli (ESBL-EC) strains has increased dramatically over the past years. OBJECTIVES: Characterization of ESBL-EC isolates collected from BSIs with regard to their antimicrobial susceptibility and phylogenetic background. The conjugative transfer of resistance determinants to the E. coli reference strain K12 C600 was also investigated. MATERIAL AND METHODS: A collection of forty-eight ESBL-EC strains recovered from BSIs was subjected to the study. These strains were obtained from the ICU (intensive care unit) of the Medical University Hospital, Wroclaw, Poland, during a four-year period (2009-2012). All the isolates were screened for ESBL production by the double disk synergy test (DDST). Transferability of plasmid-mediated resistance genes was performed by the conjugational broth method. Susceptibility to antibiotics and chemotherapeutics of clinical isolates and transconjugants was determined by the agar dilution method. PCR assay was used to detect the blaCTX-M gene in ESBL-EC tested and transconjugants. Affiliation to phylogenetic groups was done by the triplex PCR method. RESULTS: Conjugational transfer of plasmids responsible for ESBL to a recipient strain was successful for all the ESBL-EC analyzed (donors). The conjugation frequencies ranging from 2.3×10(-7) to 5.2×10(-1) per donor. In vitro susceptibility testing revealed that all the ESBL-EC isolates and their transconjugants were resistant to most of the antimicrobial agents tested with the exception of carbapenems, tigecycline, and ß-lactam-clavulanate combinations. Moreover, all the donor strains and their transconjugants were found to contain the blaCTX-M gene. The majority of the isolates analyzed belonged to phylogroups B2 (62.5%) and D (25%), whereas groups B1 and A were less frequently represented (8.3% and 4.2%, respectively). CONCLUSIONS: The results of the study confirm the need of antibiotic policies and effective infection control measures in hospital settings to minimize BSIs caused by multi-resistant ESBL-producing pathogens.

ESBL-producing Escherichia coli isolated from children with acute diarrhea - antimicrobial susceptibility, adherence patterns and phylogenetic background.

BACKGROUND: Escherichia coli remains the principal bacterial pathogen in childhood diarrhea and constitutes an important public health problem, especially in developing countries. Diarrheagenic E. coli strains often display resistance to beta-lactams due to the production of extended-spectrum beta-lactamases (ESBLs). OBJECTIVES: A total of thirty ESBL-producing E. coli strains colonizing the gastrointestinal tracts of children with acute diarrhea were studied in order to determine their antimicrobial susceptibility, adherence patterns to the HEp-2 cell line and phylogenetic background. MATERIAL AND METHODS: ESBL production was detected by the double disk synergy test (DDST). The minimal inhibitory concentrations (MICs) of antibacterial drugs were determined by an agar dilution technique on Mueller-Hinton agar. The presence of bla(TEM), bla(SHV) and bla(CTX-M) determinants in the strains studied was ascertained by polymerase chain reaction (PCR). RESULTS: The strains displayed the resistance pattern typical of ESBL producers. The majority of them (23 out of 30) were found to produce CTX-M-type ESBLs conferring a high level of resistance to oxyimino-beta-lactams, especially to cefotaxime and ceftriaxone. In many cases, the strains exhibited resistance to non-beta-lactam antimicrobials, such as gentamicin, amikacin, co-trimoxazole and tetracycline. On the other hand, these strains were uniformly susceptible to carbapenems, to oxyimino-beta-lactams combined with clavulanic acid and to tigecycline. The E. coli strains were distributed among the four main phylogenetic groups: A, B1, B2 and D. The in vitro adhesion assay revealed that all but two of the strains adhered to the HEp-2 epithelial cell line. Aggregative and diffuse adherence patterns were found to be the most prevalent. CONCLUSIONS: CTX-M-type enzymes were the most prevalent ESBLs among the strains studied. As many as 40% of the diarrheagenic E. coli isolates were found to belong to phylogenetic group D, which usually comprises E. coli strains associated with extra intestinal infections. The effectiveness of tigecycline against ESBL-producing E. coli strains was similar to that of imipenem and meropenem.

[Conjugative transfer frequency of resistance genes from ESBL-producing Enterobacteriaceae strains isolated from patients hospitalized in pediatric wards]. / Czestosc koniugacyjnego transferu genów lekoopornosci z ESBL-dodatnich paleczek Enterobacteriaceae izolowanych od chorych z oddzialow pediatrycznych.

The aim of this study was to evaluate the transfer frequency of plasmids encoding extended-spectrum beta-lactamases (ESBLs) from clinical isolates of Enterobacteriaceae to E. coli K12 C600 recipient strain. Additionally, resistance patterns to antimicrobial drugs of the isolates as well as transconjugants were analyzed. Fifty-four clinical strains belonging to the Enterobacteriaceae family were isolated from children hospitalized in Medical University Hospital in Wroclaw. All the strains studied were identified in automatic ATB system using ID32E tests. Besides, they were ESBL-positive as was confirmed by the double-disc synergy test (DDST). The minimal inhibitory concentration (MIC) was determined for twelve selected antibiotics and chemotherapeutics. The majority of the strains (87%) were able to transfer plasmid-mediated ESBL to E. coli K12 C600 recipient strain with a frequencies ranged from 10(-5) to 10(-1) per donor cell. All the isolates studied as well as their transconjugants were susceptible to imipenem, meropenem and norfloxacin (MIC <1mg/L). On the other hand, these strains displayed high level of resistance (MIC 512 - >1024 mg/L) to cefotaxime, ceftriaxone, gentamycin, amikacin and cotrimoxazole. Genetic markers conferring resistance to aminoglycosides and cotrimoxazole were often co-transferred to recipient strain in conjugation process.

[Biophysical parameters of early puberty in girls]. / Profil biofizyczny dziewczat oraz ocena pierwszo-, drugo-i trzeciorzedowych cech plciowych we wczesnej fazie dojrzewania.

OBJECTIVE: The appearance of breast development in girls characterizes an early period of puberty. Ultrasonographic examinations of the uterus and ovaries make possible the estimation of first pubertal changes in sexual organs. DESIGN: The aim of this work was to study the clinical and ultrasonographical features of early puberty in girls. MATERIAL AND METHODS: 33 healthy girls were observed quarterly in the course of prepuberty. Body mass, height, body mass index (BMI), quantity of adipose tissue were investigated. Stage of puberty was established according to Tanner. The uterus and ovaries were studied ultrasonographically, and the volume of the uterine body, length of the cervix and ovarian volume and size of ovarian follicles were scrutinised. RESULTS: Statistical differences were observed in weight, height, quantity of adipose tissue and the volume of body and that of the uterus, in length of the cervix between prepuberty and early puberty periods. Luminastity of mucus in the cervical canal in half of girls in the breast stage M1 was obtained. CONCLUSION: Ultrasonographic investigations of internal sexual organs with estimation of clinical sexual features are helpful in examination of early stage of puberty.