Prevalence of ESBL-producing Pseudomonas aeruginosa isolates in Warsaw, Poland, detected by various phenotypic and genotypic methods.

Knowledge of the prevalence of ESBL enzymes among P. aeruginosa strains compared to the Enterobacteraiceae family is limited. The phenotypic tests recommended by EUCAST for the detection of ESBL-producing Enterobacteriaceae are not always suited for P. aeruginosa strains. This is mainly due to the presence of other families of ESBLs in P. aeruginosa isolates more often than in Enterobacteriaceae, production of natural AmpC cephalosporinase and its overexpression, and co-production of metallo-ß-lactamases. The aim of this study was to determine the occurrence of ESBLs in P. aeruginosa isolated from patients from hospitals in Warsaw, to evaluate the ESBL production of these isolates using currently available phenotypic tests, their modifications, multiplex PCR and molecular typing of ESBL-positive isolates by PFGE. Clinical isolates of P. aeruginosa were collected in 2000-2014 from four Warsaw hospitals. Based on the data obtained in this study, we suggest using three DDST methods with inhibitors, such as clavulanic acid, sulbactam and imipenem, to detect ESBL-producing P. aeruginosa strains. Depending on the appearance of the plates, we suggest a reduction in the distance between discs with antibiotics to 15 mm and the addition of boronic acid at 0.4 mg per disc. The analysed isolates carried genes encoding ESBL from the families VEB (69 isolates with VEB-9), GES (6 with GES-1, 1 GES-5, 5 GES-13 and 2 with GES-15), OXA-2 (12 with OXA-15, 1 OXA-141, 1 OXA-210, 1 OXA-543 and 1 with OXA-544) and OXA-10 (5 isolates with OXA-74 and one with OXA-142). The most important result of this study was the discovery of three new genes, blaGES-15, blaOXA-141 and blaOXA-142; their nucleotide sequences have been submitted to the NCBI GenBank. It is also very important to note that this is the first report on the epidemiological problem of VEB-9-producing bacterial strains, not only in Poland but also worldwide.

Identification of infectious microbiota from oral cavity environment of various population group patients as a preventive approach to human health risk factors.

INTRODUCTION AND OBJECTIVE: This study presents the results of comparative investigations aimed to determine microbiota that can occur in the oral environment in different human populations. The objective of the research was to identify pathogenic oral microbiota, the potential cause of health complications in patients of different population groups. MATERIALS AND METHOD: The study included 95 patients requiring dental or surgical treatment; their oral cavity environment microbiota as risk factors of local and general infections were assessed. RESULTS: In clinical assessment, differences occurred in oral cavity conditions between patients with malformations of the masticatory system, kidney allograft recipients and individuals without indications for surgical procedures. The presence of various pathogenic and opportunistic bacterial strains in oral cavities were revealed by direct microscopic and in vitro culture techniques. CONCLUSIONS: Colonization of oral cavities of patients requiring surgical treatment by the potentially pathogenic bacteria constitutes the threat of their spread, and development of general infections. Assessment of oral cavity infectious microbiota should be performed as a preventive measure against peri-surgical complications.

Comparative assessment of selected intraoral microorganisms ­ potential factors for peri-surgical management complications

In this research, a comparative analysis of results of investigations involving different human populations, in terms of a relation between the oral cavity health and the species composition of mouth microbiota is reported. The purpose of this analysis was to identify and assess microorganisms that could cause health complications in patients with neoplasm requiring dental problem-related surgical management. The patients with the oral cancer surgically treated and those without neoplasm were assessed for their oral health: status of teeth, gingiva, periodontium, and occurrence of inflammatory processes. From each patient, microorganisms isolated of periodontium, dental plaque, and dental pocket swabs were identified in wet and stained microscopic preparations; standard microbiology in vitro techniques were also applied to determine the fungal and bacterial strains. The comparative analysis of results of direct microscopic examinations and in vitro cultures assessment indicated significant differences in prevalences of fungi, parasitic oral protozoans and bacteriae in particular patient's groups. Yeast-like fungi belonging to Candida genus, mostly of C. albicans group, were identified in 93.75% patients with the oral cancer, while in 25% of individuals assessed without neoplasm. E. gingivalis amoebae were only found in 12.5% patients with the serious disease; no trichomonads were detected in all patients analyzed. Among bacteria species, potentially pathogenic Enterobacteriaceae were found in the patients with oral cancer. The pronounced shift in the microbiota species composition in the patients who needed prolonged treatment due to oral cavity cancer, compared to other generally healthy persons has been showed in this analysis.

Evaluation of selected oral cavity microbiota--risk factors of management complications in patients with masticatory system disorders.

The retrospective analysis of data on oral cavity clinical status in relation to microbiota species composition is presented. The research regards patients of different age, with and without congenital malformation, pretreatment assessed for occurrence of pathological changes in the masticatory system. Samples of the swabs collected from each patient (from dental plaque, periodontium and dental pockets) were used for identification of oral protozoans in wet slides and stained preparations; additionally, transmission electron microscope examination was performed. The material was used for in vitro cultures to identify bacteria strains. Clinically, intensity of tissue deteriorations was higher in patients with a congenital disease. Alive Trichomonas tenax and Entamoeba gingivalis, species with confirmed pathogenic impact on oral cavity and neighboring structures, were detected with higher prevalence in older patients. Enterococci, Staphylococcus aureus, various Enterobacteriaceae were more frequently detected in patients with somatic and mental retardations; in mouths of those patients, Klebsiella pneumonia and Pseudomonas aeruginosa opportunistic strains occurred. Masticatory system abnormalities favor colonization of oral cavity by exogenous species and dissemination of infections, especially dangerous for patients with congenital diseases. Oral microbiota assessment and preventive measures may be helpful to avoid subsequent peri-surgery complications.

Assessment of in vitro dynamics of pathogenic Acanthamoeba strains originating from contact lens wearers with infectious keratitis

Recently, incidents of Acanthamoeba keratitis, the vision-threatening eye disease, are reported with increasing frequency worldwide, particularly in contact lens wearers. In our study, the retrospective assessment of in vitro dynamics of subsequent pathogenic Acanthamoeba isolates cultured at 24°C, detected in Polish contact lens wearers with keratitis is presented and results compared with those of environmental A. castellanii Neff strain. There were delayed the proper diagnosis that influenced prolonged and severe course of this eye disease and treatment difficulties. The corneal material was examined directly to visualize developmental amoeba stages for diagnose verification, microbiologically tested for the specific identification of bacteriae and fungi, and in vitro grown in culture medium in temperature 24°C. Among twenty-six keratitis incidents analyzed, eleven were cases of Acanthamoeba keratitis; in the six of them, Acanthamoeba strains and concomitant bacterial and/or fungal infectious agents were detected. In vitro assays showed variability in population density of several clinical strains in the exponential growth phase expressed in various range of overall amoeba number and different proportion between trophozoites and cysts. The clear influence of temperature on the in vitro cultivation of the amoebae was observed: statistically significant lower population dynamics was revealed by most of pathogenic clinical isolates in comparison with those showed by environmental strain. The in vitro monitoring of dynamics of Acanthamoeba strains isolated from infected eyes may be helpful for diagnostics verification, especially in mixed infectious keratitis.

The threats for human health induced by food pests of Plodia interpunctella as reservoirs of infectious microbiota / Zagrozenia zdrowia czlowieka indukowane przez szkodniki zywnosci Plodia interpunctella jako rezerwuary infekcyjnych microbiota

INTRODUCTION: World-wide distributed pests of Plodia interpunctella occur with increasing frequency also in Poland, in areas where food is prepared and stored, in dwellings, buildings of public use, hospitals. Larvae damage various products causing economic losses. There were no data about microbiota transmission by pests. The aim of our systematic studies firstly conducted in Poland was to explain a role of pests as reservoirs of microbiota and assess health risk induced by them in human environments. MATERIAL AND METHODS: 300 adults and 200 larvae, collected in households and health facilities by traps and directly from products, were examined by light microscopy, in vitro cultivations, molecular techniques; the susceptibility /resistance of microbiota to chemicals was also assessed. RESULTS: Gram+ bacteriae of genera Enterococcus, Micrococcus, Bacillus, Gram-: Klebsiella, Escherichia, mold fungi: Aspergillus, Penicillium and yeast-like fungi were identified, including strains potentially pathogenic for humans. CONCLUSIONS: In the European Union countries, the food circulation is audited by the law; chemicals are applied to eliminate P.interpunctella pests causing economic losses. Our successive studies showed that pyralids may generate health problems as food pests and as reservoirs of microbiota. Sources of the pathogenic, drug-resistant strains revealed by us, not identified earlier, may be particularly dangerous for elder persons, with weakened immune system, persons from groups of high risk of infections. The increased awareness of the problem is necessary for more efficacy of preventive measures. A monitoring of consequences of the health risk induced by the pests may supply data useful for adequate practical approach.

Synthesis and antimicrobial and nitric oxide synthase inhibitory activities of novel isothiourea derivatives.

The reaction of substituted benzylhalides, or of halomethyl derivatives of thiophene or furane, with thiourea or its derivatives yielded the respective isothioureas as hydrohalide salts. The products (a total of 17, including 16 novel compounds) were tested for activity against five Gram-positive and nine Gram-negative bacterial strains, six yeast species and two protozoan species. The most active against Gram-positive bacteria were S-(2,4-dinitrobenzyl)isothiourea hydrochloride (MIC range for four out of five strains tested: 12.5-25 microg/mL) and S-(2,3,4,5,6-pentabromobenzyl)isothiourea hydrobromide (MIC range: 12.5-50 microg/mL). The lowest MICs of novel isothioureas for yeast and Gram-negative bacteria ranged between 50 and 100 microg/mL. Nine novel isothioureas showed appreciable genotoxicity in the Bacillus subtilis 'rec-assay' test, the most potent being S-2-(5-nitrofuran-2-ylmethyl)isothiourea and S-(2-nitrobenzyl) isothiourea. At 10 muM concentration, S-(3,4-dichlorobenzyl)isothiourea hydrochloride and S-(2,3,4,5,6-pentabromobenzyl)isothiourea hydrobromide inhibited Ca(2+)/calmodulin-dependent (non-inducible) nitric oxide synthase activity in normal rat brain homogenates stronger (p < 0.05) than the reference drug 7-nitroindazole (by 78, 76 and 60%, respectively); ten other new isothiourea derivatives significantly inhibited the activity to a lower extent (by 28-60%). These results extend the list of promising isothioureas with substantial activity in vitro and suggest that an in-depth study of toxicity, antimicrobial properties in vivo and nitric oxide synthase isoform selectivity of selected novel compounds is warranted.

Acanthamoeba castellanii: in vitro effects of selected biological, physical and chemical factors.

Trophozoites and cysts of free-living Acanthamoeba castellanii present a serious risk to human health as causative agents of human diseases such as fatal granulomatous amoebic encephalitis and Acanthamoeba keratitis that is reported from various part of the world, also in Poland, with increasing frequency, particularly in the contact lens wearers. The amphizoic amoebae are generally extremely resistant to different chemical agents, however, several strains/isolates within A. castellanii may differ in virulence. Among the features considered as associated with the amoeba pathogenicity, temperature tolerance and resistance to different environmental conditions are reported. In the present study, A. castellanii strain cultured in 26 degrees C after several year passages were tested for sensibility/tolerance to instant temperature changes as well as exposition to deuterium oxide, D2O. Significant decrease of number of viable amoebae during in vitro exposition to D2O occurred, but no changes in trophozoites/cysts ratio. The ability of the strain examined to develop in higher temperature may indicate a wide adaptation reserve and its pathogenic potential.

Synthesis of 1-{4-[4-(adamant-1-yl)phenoxymethyl]-2-(4-bromophenyl)- 1,3-dioxolan-2-ylmethyl}imidazole with expected antifungal and antibacterial activity.

A multistep synthesis of a new analogue of ketoconazole, which was expected to show antifungal and antibacterial activity, has been described.

Synthesis and antibacterial activity of 5-adamantan-1-yl-methyl analogues of trimethoprim.

A series of new trimethoprim [5-(3,4,5-trimethoxy-benzyl)-pyrimidine-2,4- diamine] analogues were prepared by condensation of adamantane-1-carbaldehyde with 3-methoxypropionitrile, followed by reaction of resulting mixture of 2-adamantan-1-ylmethyl-3-methoxy-acrylonitrile and 3-adamantan-1-yl-2-methoxymethyl-acrylonitrile with guanidine, acetamidine and thiourea, respectively. The activity of compounds obtained and sulfamethoxazole, alone and in combination, against several bacterial strains, as well as fungi was investigated.

Novel adamantylated pyrimidines and their preliminary biological evaluations.

The synthesis of adamantylated pyrimidines was based on the reaction of 3-(adamantan-1-yl)-3-oxopropionic acid ethyl ester with urea, thiourea, guanidine as well as acetamidine, respectively. Then the compounds obtained were converted into respective bromo-, thio- and S-alkyl derivatives. The molecular structures for some compounds were studied by X-ray methods. The significant anticancer and antimicrobial properties of [2-(6-adamantan-1-yl-2-methylpyrimidin-4-ylthio)ethyl]dimethylamine were found.

Synthesis, and antiprotozoal and antibacterial activities of S-substituted 4,6-dibromo- and 4,6-dichloro-2-mercaptobenzimidazoles.

The synthesis and some germicidal activities in vitro of two congener series of S-substituted 4,6-dihalogeno-2-mercapto-1H-benzimidazoles are reported. There was no substantial difference between antibacterial activities of corresponding 4,6-dichloro- and 4,6-dibromo-derivatives. The present results confirm lower susceptibility to substituted benzimidazoles of Gram-negative compared to Gram-positive bacteria. Minimum inhibitory concentrations (MICs) of a majority of the novel derivatives ranged between 25 and 100microg/ml for Gram-positive bacteria. The most active compounds (MICs for Gram-positive bacteria: 0.78-50microg/ml) were 4,6-dichloro-2-(4-nitrobenzylthio)-1H-benzimidazole and 4,6-dibromo-2-(4-nitrobenzylthio)-1H-benzimidazole that were 4-32 times more potent than nitrofurantoin against all Gram-positive bacteria utilized but Escherichia faecalis, against which they were, respectively, 2 and 4 times less potent than nitrofurantoin. Among Gram-negative bacteria used, Stenotrophomonas maltophilia and Bordetella bronchiseptica were most sensitive (as evidenced by a number of MICs 400microg/ml). All the new compounds were at least several times more active against Giardia intestinalis (IC(50): 0.006-0.053microg/ml), and a half of them were at least several times more active against Trichomonas vaginalis (IC(50): 0.0015-0.182microg/ml) than metronidazole (IC(50): 0.210 and 0.037microg/ml, respectively), the drug of choice in the treatment of G. intestinalis and T. vaginalis infections.

Research on the connections of isoindole derivatives and ethyl esters of formic, acetic and propionic acids.

A series of new derivatives of isoindole in the reaction of nucleophilic substitution with ethyl chloroformate, ethyl bromoacetate and ethyl 3-bromopropanoate were synthesized.

Synthesis and antibacterial activity of adamantyl substituted pyrimidines.

A series of new S-substituted 6-adamantylpyrimidines were prepared by alkylation on the thiol groups in 6-adamant-1-yl-2-methyl-3H-pyrimidine-4-thione and 6-adamant-1-yl-2-thioxo-2,3-dihydro-1H-pirymidin-4-one, respectively. Then, these compounds were tested against several bacterial strains as well as fungi.

[Occurrence of oral subclinical infections in insulin treated diabetics]. / Wystepowanie subklinicznych zakazen jamy ustnej u pacjentów z cukrzyca insulinozaleina.

The purpose of this study was to examine oral cavity condition, identify microorganisms and evaluate interrelation between protozoans, bacteria and fungi occurring in mouths of insuline treated diabetics. 30 men and women, 25-60 years old, were clinically assessed for their oral cavity condition including pH range. Fifteen of them were the insuline treated diabetics. Swabs taken of different sites of periodontium, dental plaques and dental pockets collected from each patients were used for detection and identification of protozoans, bacteria and fungi. Mucosal inflammation, dental caries, loose teeth, periodontitis were observed in the diabetic patients, whereas the oral cavities were generally in better condition in the control patients. Prevalence of the protozoan parasites Entamoeba gingivalis and Trichomonas tenax was higher in the control patients than in the diabetics (26.6% and 13.3%, respectively). Fecal bacteria Enterococcus fecalis, E. faecium, Klebsiella pneumoniae occurred with significantly higher frequency in the insuline treated patients (60%) than in the control (6.6%). Various Candida albicans strains were more often found in the diabetic (53.2%) than in the control patients (13.3%). The results of our studies show a clear interrelation between alteration of oral health and the occurrence of microorganisms in the insuline treated diabetics. Metabolic disease that causes serious multi-organ disturbances as well as insulinotherapy are the very important factors changing oral cavity ecology of the persons with diabetes mellitus; decresed pH (the average 5.5) may inhibit development of infection with protozoans in the oral cavity of these patients and favour subclinical infections with other microorganisms. Thus, in the insuline treated diabetics, examination of mouths with regard to the occurrence of opportunistic fecal bacteria and fungi, particularly before a therapy with antibiotics is strongly recommended.

Synthesis and antimicrobial activity of new adamantane derivatives III.

Some novel ester imides synthesised from trimellitic acid anhydride and 1-adamantanol or 2-adamantanol, were tested as antimicrobial compounds. Unfortunately, these agents showed a modest antibacterial activity (MIC > 6 microg/ml). However, a comparison of these N-substituted adamantylester imides with the series published previously, indicated that the incorporation of L-alanine and L-phenylalanine into the phthalimide moiety was the best choice regardless of the series and leads to antimicrobial activity against Staphylococcus aureus strains.

Synthesis, antiprotozoal and antibacterial activity of nitro- and halogeno-substituted benzimidazole derivatives.

Two series of benzimidazole derivatives were sythesised. The first one was based on 5,6-dinitrobenzimidazole, the second one comprises 2-thioalkyl- and thioaryl-substituted modified benzimidazoles. Antibacterial and antiprotozoal activity of the newly obtained compounds was studied. Some thioalkyl derivatives showed remarkable activity against nosocomial strains of Stenotrophomonas malthophilia, and an activity comparable to that of metronidazole against gram-positive and gram-negative bacteria. Of the tested compounds, 5,6-dichloro-2-(4-nitrobenzylthio)-benzimidazole showed the most distinct antiprotozoal activity.

Antimicrobial activity of substituted 2-trifluoromethyl- and 2-pentafluoroethylbenzimidazoles.

Antibacterial and antifungal activity of 2-trifluoromethyl- and 2-pentafluoroethylbenzimidazoles, including a number of newly obtained derivatives, were examined by diffusion method (inhibition area diameter in solid agar medium) and minimum inhibitory concentration (MIC, in liquid and agar medium). Some of the derivatives tested affected fungal colony morphology and exerted genotoxic effects in bacteria. Of the tested compounds, 5,6-dichlorosubstituted derivatives appeared the most active against the majority of microorganisms used.

[Detection of smr and qacA genes in Staphylococcus aureus strains using PCR]. / Wykrywanie genów smr I qacA u szczepów Staphylococcus aureus z zastosowaniem metody PCR.

The 31 strains of Staphylococcus aureus were examined for the presence of smr and qacA determinants. The smr gene was found in 15 strains. Fourteen of them were MRSA resistant to quaternary ammonium compounds, ethidium bromide, and acriflavine. One was MSSA strain resistant to ethidium bromide and acriflavine. The qacA gene was found in two MRSA strains resistant to quaternary ammonium compounds, ethidium bromide, chlorhexidine and acriflavine. One of these two strains possessed both smr and qacA genes.

[Resistance of Staphylococcus aureus strains to quaternary ammonium compounds and chlorhexidine]. / Opornosc szczepów Staphylococcus aureus na czwartorzedowe sole amoniowe i chlorheksydyne.

The level of susceptibility of 90 different Staphylococcus aureus strains to chosen quaternary ammonium compounds: cetyltrimethyl ammonium bromide, benzalkonium chloride and benzethonium chloride as well as to chlorhexidine digluconate were examined. The examined strains consist of three groups: hospital originated MRSA, hospital originated MSSA and non-hospital MSSA. The significant differences between these groups were observed in they susceptibility to the investigated disinfectants. The obtained MIC values showed that the most resistant were hospital MRSA strains, where 55% was estimated as resistant to cetyltrimethyl ammonium bromide, 72% were resistant to benzalkonium chloride and benzethonium chloride and 7% were resistant to chlorhexidine digluconate. Among hospital originated MSSA 3% of strains were resistant to cetyltrimethyl ammonium bromide and 6% were resistant to benzalkonium chloride and benzethonium chloride. 14% non-hospital S. aureus strains were resistant to benzalkonium chloride and benzethonium chloride. None were resistant to chlorhexidine digluconate or cetyltrimethyl ammonium bromide.