Showing papers in "Medycyna doświadczalna i mikrobiologia in 1998"

A new method for evaluation of the antagonistic action of bacterial lactic acid (LAB) on selected pathogenic indicator bacteria

Abstract: A comparison was made of three methods: paper disc, double layer and a newly proposed agar slab techniques for testing antibacterial activity of Lactobacillus strains. Strains of indicator bacteria were selected from important pathogens of the human alimentary and genitourinary tract. The agar slab method, which is based on applying of slabs of MRS agar with overnight growth of the antagonistic bacteria to the surface of appropriate agar plates seeded with indicator bacteria, appeared to be the only method suitable for testing of aerobic and anaerobic fastidious bacteria. After testing of 27 Lactobacillus strains against 3 selected indicator bacteria: E. coli, Salmonella enteritidis and Staphylococcus aureus, calculating mean values and standard deviations for diameters of inhibitory zones and making variance analysis it was shown that in comparison to other methods the agar slab technique, although less sensitive than others, gave most consistent and reproducible results.

Detection of bacterial biofilm on medical biomaterials

Abstract: This study was performed to assess the value TTC assay in the diagnosis of biomaterial-associated infections. In this assay, soluble colourless TTC is reduced to insoluble red formazan by electron transfer associated with active oxidative bacterial metabolism and is precipitated intracellularly. Microbial adhesion and biofilm formation on the surface of medical prosthetic devices (vesicular and urinary catheters) made of various polymers (PTFE, H-PE, PCW, SL), were determined. The microorganisms which are most often isolated in medical device-associated infections: S. aureus, S. epidermidis, E. faecalis, E. coli, P. vulgaris, P. aeruginosa, C. albicans, were included into the study. The obtained results indicate that the assay using TTC as a metabolic indicator of bacterial biofilm presence, is technically simple to conduct with minimal setup time. Even when classical cultures yielded no bacterial growth, TTC assessments demonstrated bacterial biofilms. TTC assay could be recommended as a quick routine method for confirmation of biomaterial device-associated infection.

Bacterial flora in pharyngitis and tonsillitis

Abstract: The aim of the study was a microbiological analysis of pharyngeal swabs obtained from 158 patients with the diagnosis of pharyngitis and purulent exudates from the tonsillar crypts of 10 patients treated for chronic purulent tonsillitis. Beta haemolytic streptococci groups A, B, C and G were isolated from 30% of the patients. The most frequently isolated were Streptococcus pyogenes--12% of patients and Streptococcus group C--10.7%. Other streptococci were isolated less frequently: Streptococcus group B--44%, group G--2.5%. The majority of isolated bacteria belonged to potential pathogenic flora (70% patients). Staphylococcus aureus (37%) and Haemophilus spp. (36%) were isolated most frequently. Other bacteria were isolated in the following sequence: Moraxella catarrhalis--22%, Streptococcus pneumoniae--17% and Gram-negative rods from the Enterobacteriaceae family--6%. One case of Plaut-Vincent tonsillitis was diagnosed. Aerobic and anaerobic bacteria were isolated from purulent exudates from the tonsillar crypts of 10 patients treated for chronic purulent tonsillitis. The isolated anaerobic bacteria belonged to genus of Prevotella, Fusobacterium, Peptostreptococcus and Gemella.

The presence of antibodies to Borrelia burgdorferi associated with immunologic complexes in sera of foresters

Abstract: Binding of antibodies specific to Borrelia burgdorferi in circulating immune complexes can lead to false negative results in serological tests The aim of our study was to determine the presence of IgM antibodies to Borrelia burgdorferi bound in immune complexes in 52 sera of foresters the National Park in Karkonosze Free and bound in immune complexes IgM antibodies present in 6 (115%) examined sera In 24 (462%) seronegative sera after dissociation of immune complexes IgM antibodies to spirochaeta were found The rest of the examined seronegative sera we failed to find IgM antibodies to Borrelia burgdorferi The diagnostic assay, such as antibody analysis of immune components is useful in establishing of the diagnosis of borreliosis in seronegative cases and monitoring of disease activity That method should be introduced for routine diagnosis of Lyme disease

Characteristics of E.coli O157 strains isolated in Poland from clinical material and food samples

Abstract: E. coli belonging to the O157 serological group are among the organisms isolated most frequently out of all the so called entero-hemorrhagic E. coli strains (EHEC). Since several years they have been isolated also in Poland. The purpose of the present study was determination on selected phenotypic and genotypic properties of E. coli O157 strains isolated in our country from clinical material samples and from food. The serotype of the strains was determined, together with the following properties regarded as pathogenicity markers of verotoxic E. coli strains such as absence of beta-glucuronidase activity and sorbitol fermentation ability, as well as production of verotoxins SLT I and/or SLT II and entero-hemolysin. Besides that, by the PCR method the fragments of the genes coding for verotoxins, intimin and enterohaemolysin were amplified. The products of PCR were analysed by the restriction enzyme analysis (RFLP). All verotoxic E. coli O157 strains isolated in Poland were analysed by the pulsed field gel electrophoresis of genomic DNA (PFGE). The studied group comprised E. coli O157 strains, among them 40 strains were isolated from human faeces and 5 from food. The remaining strains were the reference E. coli O157:H7 EDL 933 and G 5244 strains and strains from NIH collection. The obtained results showed that the tested strains were a very varying population. 21 of them (all isolated from food, 11 from faeces and 5 reference strains) belonged to serotype O157:H7, five were not peritrichous O157:NM and the remaining ones had other ciliary antigen than H7. All strains isolated from food, reference strains and only 3 O157:NM strains isolated from humans were verotoxic. The strains from food and two reference strains produced only SLT II, 2 of 3 strains isolated from humans and one reference strain also produced only SLT II and the other produced both verotoxins. Apart from these 13 verotoxic strains all remaining strains caused sorbitol fermentation.

Hemolytic activity of Klebsiella pneumoniae and Klebsiella oxytoca

Abstract: We demonstrated that Klebsiella pneumoniae and Klebsiella oxytoca possess a selective haemolytic activity on rabbit erythrocytes. Thirty one Klebsiella strains (18 strains of K. pneumoniae and 13 strains of K. oxytoca) were isolated from hospitalized patients. The liquid (Trypcase-soy broth--TSB) and solid (Trypcase-soy agar--TSA) medium, containing the red cells were used for the tests. All the screened strains showed a haemolytic effect on rabbit erythrocytes, provided that the supernatants of the cultures were preincubated with beta-mercaptoethanol or calcium chloride. There was no human and sheep erythrocyte lysis.

The role of rotaviruses in digestive tract infections of hospitalized children with diarrhoea at the Health Care Consortium in Sokoł Podlaski

Abstract: The aim of the study was a trial of establishing of the frequency of rotavirus infection and mixed bacterial-viral infections in children treated for diarrhoea in a hospital in the period from July 1 1996 to June 30 1997. Moreover, an attempt was made at establishing of the frequency of rotavirus colonization of the digestive tract in healthy newborns born in that hospital during the period of that study. The studies were done on feces samples from 169 children with diarrhoea from 4 days to 13 years, and from 30 healthy peers. In all samples rotaviruses were sought along with pathogenic Enterobacteriaceae Tests for rotaviruses were done with Slidex-Rota Kit 2 (Bio-Merieux) and for EPEC with the EPEC latex test (Biomex). In all, rotaviruses were found in 88 ill children (52.1%) including mixed bacterial-viral infections in 10 (5.1%) children. In 9 children beside rotaviruses enteropathogenic E. coli (EPEC) were disclosed, and in 1 child S. enteritidis. Moreover, in 1 child (0.6%) culture yielded simultaneously S. sonnei and E. coli 0127. No rotaviruses were found in any 30 healthy newborns, but from one of them E. coli 018 was cultured. The second most frequent aetiological factor in diarrhoea were EPEC organisms found in 17 (10.0%) children, the third factor were S. enteritidis strains in 6 children (3.6%). In one case S. sonnei and E. coli 0127 were obtained from faces. Rotavirus infection was most frequent in children aged from 2 months to 3 years, and EPEC infection in children up to 2 years. The incidence was highest in winter/spring, with peak in April when 27 cases of rotavirus diarrhoea were noted.

Evaluation of the usefulness of selected commercial systems for identification of Streptococcus species

Abstract: The usefulness was assessed of three commercially available systems for rapid identification of streptococcal strains. The studied material comprised 68 strains of streptococci and enterococci (including 24 standard strains) belonging to serological groups: A (14 strains), B (10), C (11), D (10), F (3) and G (10), as well as 10 S. pneumoniae strains. The strains were isolated from throat, nasal, wound swabs, blood, pus of inpatients and throat and nasal swabs of outpatients. For the identification of streptococci 3 commercially available systems were used: API 20 STREP (bioMerieux, France), rapid ID 32 Strep (bioMerieux, France), Streptoplast PPL 18 (HTL, Poland). The determinations were done according to producer's instructions. The highest percent of correctly identified strains was obtained with the rapid ID 32 Strep--80.9%, with the API 20 STREP--76.4% strains were identified correctly and with the PPL 18--61.8%. The study showed that the API 20 STREP and rapid ID 32 STREP are suitable for the identification of streptococcal strains from groups: A, B, C, D, F and enterococci--group D. The proportions of correctly identified strains from these groups with the Streptoplast PPL 18 were lower than those determined with the bioMerieux systems. Using of three identification systems streptococci from group G and S. pneumoniae strains cannot be identified.

[Sensitivity to selected beta-lactam antibiotics of clinical strains of Escherichia coli and Klebsiella pneumoniae].

Abstract: The studies aimed at analysing the resistance to some beta-lactam antibiotics among E. coli and K. pneumoniae clinical isolates and at evaluating. The extended spectrum of beta-lactamases (ESBL) production in the isolates. The analysis included 137 E. coli strains and 52 K. pneumoniae strains, isolated from hospitalized patients and out-patients treated in the first trimester of 1998. The strains were identified using the ATB computer system. Antibiotic sensitivity of the isolates was determined by disc-diffusion tests. ESBL production capacity of E. coli and K. pneumoniae strains was estimated by double-disc and ATB BLSA tests. Most of the analysed E. coli strains were found to exhibit significant sensitivity to compound penicillin preparations containing beta-lactam inhibitor (Augmentin, Tazocin) and to the third generation cefalosporins, in contrast, K. pneumoniae strains much more frequently were resistant to the drugs. Among the obtained isolates, 3 (2.2%) E. coli strains and 21 (40.4%) K. pneumoniae strains produced ESBL but all the isolates proved sensitive to imipenem. In evaluation of ESBL production-detecting tests, the double-disc test was found to be more reliable than ATB BLSA test.

Detection of toxin producing strains of Clostridium difficile using rapid diagnostic methods

Abstract: Feces of 53 patients from different hospital wards suffering from long term post-antibiotic therapy diarrhea were tested. For direct detection of C. difficile toxin A, in samples TCD (Becton-Dickinson), and C. difficile Toxin A Test (Oxoid) tests were used. Toxin A was detected in 16 samples (29.6% tested). C. difficile strains were isolated from 40% of the fecal samples. Toxin A was detected in 25 Clostridium difficile strains with commercial tests and toxin B was detected using McCoy cell line. Toxin A was not produced by 3 C. difficile strains in vitro, but toxin B was produced by all strains. The polymerase chain reaction (PCR) test showed that all isolated strains possess genes of toxins A and B.

Drug resistance in nosocomial strains of staphylococci to methicillin

Abstract: The aim of the present study was the analysis of drug susceptibility of MRSA and MRCNS strains isolated from patients hospitalized in 14 wards of the State Clinical Hospital No 1 in Warsaw. The strains were identified (ID 32 STAPH), and their susceptibility to antimicrobial agents (ATB STAPH) was determined in ATB system (bioMerieux, France). Four methods were applied to confirm the resistance to methicillin: ATB-plus system, disc-diffusion method (Oxa 1 microgram, Oxoid, U.K.), Crystal MRSA ID (Becton Dickinson-BBL, USA) and agar screen test in TSA medium (Difco, USA) with methicillin (25 mg/l, Sigma, USA). 108 Staphylococcus spp. strains were found in 300 clinical specimens. 56 strains were methicillin-resistant (52%). Among methicillin-resistant strains 13 MRSA, 28 MRSE and 15 of other species were found. All MRSA strains were susceptible to vancomycin, teicoplanin and fusidic acid. MRCNS were susceptible first of all to vancomycin (43/43), minocycline (42/43) and pristinamycin (42/43). On the basis of the obtained results it can be stated that methicillin-resistant staphylococci occur in hospital wards. The greatest number of methicillin-resistant strains was cultured from patients hospitalized in surgery wards (32), methicillin-resistant strains much more frequently occur among coagulase-negative staphylococci, especially in Staphylococcus epidermis. Glycopeptide antibiotics are most active against isolated MRSA strains. The most active therapeutic agent against MRCNS is vancomycin.

Use of PCR methods for identification of Listeria monocytogenes in milk

Abstract: The aim of this work was to estimate the limit of Listeria monocytogenes cfu in polymerase chain reaction (PCR) for a DNA fragment of listeriolysine O (hly A) gene. The PCR method, with used primers selected in areas of the listeriolysin O gene, allows to differentiate L. monocytogenes strains from other Listeria species. The amplified fragment (456 bp) of hly A gene was obtained for all strains L. monocytogenes and no other Listeria species. The PCR method with the selected primers allowed to detect 50-500 cfu L. monocytogenes/ml suspended in water or milk. Among 20 samples of raw milk from cows, 10 samples contained > 50 cfu L. monocytogenes/ml. Obtained results indicate that the PCR assay of L. monocytogenes identification is technically simple and may be conduct with minimal time. So, it could be recommended as quick diagnostic method in identification L. monocytogenes in milk.

Adhesins of Acinetobacter strains

Abstract: One of the important factors contributing to the pathogenicity of bacteria is the presence of adhesins on cell surface, which facilitate colonisation in the macroorganism. The presence and type of adhesins occurring in four species of the genus Acinetobacter: A. baumannii (184), A. junii (59), A. lwoffii (65) and A. haemolyticus (22) was determined by haemagglutination test with a 3% suspension of fresh, tannic acid-treated of guinea pig, cow and human group O and AB erythrocytes, with or without the addition of one of sugar inhibitors (D-mannose, alpha-methylmannopyranoside, D-galactose-N-acetyl-D-glucosamine, L-fucose and D-ribose). In strains from all species, adhesines of the mannose-resistant (MR) type dominated. The mannose-sensitive (MS) type was present solely on the surface of one A. lwoffii strains. A. baumannii (36), A. junii (8), A. lwoffii (11) and A. haemolyticus (4) exhibited mannose-resistant hemagglutination in relation to fresh erythrocytes and that reaction was restrained by D-galactose, D-galactose and L-fucose (no other inhibitor used restrained it). The results achieved prove that cell adhesines other than those of MR type must be present on the cell surface. Additional adhesines occurred mainly in strains isolated from the respiratory and urinary tract infection simples, but were not found in isolates from blood cultures.

Evaluation of the interactions between strains of S. aureus and peripheral blood phagocytic cells

Abstract: The aim of this study was to investigate the interactions occurring between peripheral blood phagocytes and strains of S. aureus isolated from different clinical specimens (blood, respiratory tract, pus). To evaluate the sensitivity of microorganisms to bactericidal activity of phagocytes, monocytes and granulocytes separated from peripheral blood by standard density gradient and by counter-current centrifugal evaluation these cells were incubated with suspensions of opsonized bacteria. In parallel, the viability of phagocytes was examined by flow cytometry, and the ability of bacteria to trigger reactive oxygen intermediates (ROI) production was evaluated by chemiluminescence measurement. To investigate the efficiency of phagocytosis, bacteria were labelled with fluorescein isothiocynate (FITC) and the percentage of cells containing FITC-labelled bacteria were analysed by flow cytometry. The data obtained show the strains of S. aureus derived from different clinical specimens, differ in their sensitivity to bactericidal activity of phagocytes--strains isolated from the blood show the highest, but strains isolated from the respiratory tract have the lowest sensitivity to killing. These strains differ too in their ability to trigger monocyte CL response. On the contrary, there was no difference in toxicity of bacteria against phagocytes. Strains isolated from peripheral blood showed a significant negative correlation between the ability to trigger CL response and toxicity against phagocytes.

Distribution of IgG subclasses and their biological activity in bioglobulin

Abstract: Preparations of human immunoglobulin for intravenous use (IVIG) should contain proper percentage of IgG subclasses, corresponding to physiological proportions with preserved activity of antibodies. The amounts of IgG subclasses were determined using the radial immunodiffusion technique against to the WHO reference serum 67/97. It appears that variations are observed among the different lots and manufacturers of the various formulations of IVIG available in Poland. Although some of these differences are not statistically significant, significant differences in subclass concentrations were found between formulation of IVIG, with Bioglobulina being lower in IgG1 and higher in IgG2 and Endobulin having a lower per cent of IgG3. The study was aimed at establishment of activity values against diphtheria and tetanus toxoids and Streptococcus group B (GBS). IgG antibodies to tetanus and diphtheria toxoid antigens were predominantly of the IgG1 and IgG2 isotype. IgG4 subclass, was present in smaller proportion and IgG3 fractions was absent. Antibodies to GBS were only IgG2 and IgG1 isotypes. Basing on obtained results it can be stated that in spite of the imbalance between the amounts of IgG1 and IgG2 in Bioglobulina, the activity which was determined in subclasses is comparable with that of other IVIGs.

[Phagocytosis and killing of Staphylococcus aureus by mouse granulocytes after interleukin-3 (IL-3) injection].

Abstract: Interleukin-3 is a multipotential hematopoietic growth factor, which like other colony stimulating factors (CSFs) is effective "in vitro" stimulation of the mature cells function. It was found that IL-3 synergistically with GM-CSF and G-CSF stimulated the proliferation of the granulocytes. Therefore the purpose of this investigation was the evaluation "in vivo" of the influence of IL-3 on the phagocytosis, bactericidal activity, and enzyme activities of granulocytes. IL-3 was injected into mice subcutaneously during 5 days in dose 1 microgram/kg/d. The examination of the percent of cells phagocytizing bacteria (Staphylococcus aureus), NBT test and bactericidal activity, were performed every day and evident increase of the tested parameters was found. Additionally the enzyme activities in primary granules were measured and showed on increase of acid phosphatase and peroxidase activity.

Electrophoretic and immunologic comparative analysis of Mycoplasma pneumoniae and Mycoplasma genitalium proteins

Abstract: The Mycoplasma pneumoniae FH strain routinely used in our laboratory for over 25 years as antigen in serological tests, 2 reference M. pneumoniae strains from ATCC (29342 and M129) and 3 isolates of M. pneumoniae obtained in 1995 from pneumonia patients were compared by SDS-PAGE, complement fixation test (CFT) and by Western-immunoblotting against human and rabbit serum samples with high level of mycoplasmal antibodies. On SDS-PAGE all M. pneumoniae strains showed the same number of 23 polypeptides on the gel with identical molecular weights. The same strains on immunoblotting against human and rabbit serum samples showed six bands: 170, 89, 75, 55, 38 and 33 kDa with the strongest antibody staining in 170-(P1 protein) and 89-kDa bands. Because of its known antigenic relationships Mycoplasma genitalium was used for comparison. The pattern of M. genitalium proteins on SDS-PAGE was similar to pattern of M. pneumoniae but distinguishable. On immunoblotting six proteins of M. genitalium (135, 127, 110, 95, 75 and 45 kDa) reacted with human and rabbits immunoglobulins for M. pneumoniae antigens. Furthermore in complement fixation test both antigens, prepared from M. pneumoniae and M. genitalium, reacted as well with human and rabbit immunoglobulins for M. pneumoniae and with rabbit immunoglobulins for M. genitalium. These cross-reactions observed in serological techniques could give false positive results in routine diagnosis of M. pneumoniae infections. In such situations showing on immunoblott of presence in tested serum sample of antibodies to 170- and 89 kDa proteins could confirm M. pneumoniae infection.