Medycyna doswiadczalna i mikrobiologia最新文献

Introduction: Campylobacterjejuni is has been found to be the leading cause of bacterial gastroenteritis worldwide. Clinical manifestation on enterocolitis caused by C. jejuni are diarrhea, fever, abdominal pain and in some patients, fecal blood. After C. jejuni infection, squeals may occur such as reactive arthritis. The aim of the study was to investigate the frequency of antibodies to the recombinant protein P39 sticks C. jejuni in patients with gastrointestinal disorders and reactive arthritis in Poland.

Material and methods: Serum samples collected from 46 patients with bacteriology confir- med infection caused by Campylobacter jejuni, 472 sera from patients with gastrointestinal disorders, 97 serum samples obtained from patients with reactive arthritis and 84 sera from healthy adults and children. Sera were screened for anti-P39 C. jejuni recombinant protein IgA, IgG andIgM antibodies by using the home-made ELISA. Protein P39 C. jejuni was expressing in E. coli BL21 (DE3) using the pET-30 Ek/LIC expression vector. Purification was accomplished by immobilized metal (Ni2+) affinity column chromatography (His Bind Resign, Novagen).

Results: SDS-PAGE and Coomassie brilliant blue staining confirmed a high purity of the recombinant P39 protein preparation with an expected molecular mass of 39 kDa. The results of ELISA with the P39 recombinant protein revealed that IgA antibodies in diagnostically significant level (x + 2SD) were found in 18.8%, IgM in 14.8% and IgG in 7.8% of sera obtained from patients with of gastrointestinal disorders. On the other hand, antibodies to recombinant P39 protein in sera obtained from patients with reactive arthritis were found in more than twice the percentage than in patients with gastrointestinal disorders (IgA in 34.0%, IgG in 26.8% and IgM in 19.6%).

Conclusions: In conclusion, based on the data obtained, C. jejuni may be important factor in triggering the gastrointestinal disorder and reactive arthritis in humans in Poland.

Introduction: Azithromycin is one of the most commonly used macrolide antibiotics. As other macrolides it inhibits bacterial proteins synthesis by binding with V domene of bacterial 23S rRNA. Resistance to azithromycin can be related to: 1. Mutations in gene encoding 23S rRNA. Significant effect on azithromycin MIC (minimal inhibitory concentration) is not observed when the mutation occurs only in 1 allele. In case of mutations occurring in 4 alleles, more common mutation C2611T is associated with MIC 2-16 mg/L and the second mutation A2059T results in high level resistance to azithromycin MIC > 256 mg/l 2. Over- production of membrane pumps proteins MtrCDE and MacAB, that remove antibiotics from bacterial cells. The mechanism is not able to cause azithromycin resistance itself but coexisting with other mechanisms of resistance can additionally increase MIC. 3. Synthesis of 23S rRNA methylases ErmB, ErmF, ErmC, ErmA. These enzymes cause demethylation of adenine (A2058) in V domain of 23S rRNA. The mechanism was common in the past, but it has been replaced by mutations in in V domain of 23S rRNA. Nowadays 23S rRNA methylases are very rare in N. gonorhoeae.

Methods: Sixty five Neisseria gonorrhoeae strains isolated from patients of the Department of Dermatology and Venereology in Warsaw in the second half of 2012 and first of 2013 were investigated. The strains were cultured on chocolate agar plates in a 5% CO2 atmosphere at 37 °C and identified by colony morphology, Gram staining and oxidase reaction, followed by carbohydrate utilization test. Azithromycin susceptibility was determined by E-Tests (bioMerieux). Bacteria were incubated at 37°C in 5% CO2 for 24 h on chocolate agar plates. Tests were performed according to producers recommendations. The results (sensitive or resistant) were interpreted according to EUCAST recommendations.

Results: The MIC of azithromycin in investigated strains ranged from 0,064 to 4 mg/L, MIC50 = 0.5 mg/L, MIC90 = 2 mg/L. It was shown that only 38.5% of the strains were sensitive to azithromycin according to EUCAST criteria from 2014 year and 89.3% of the strains were sensitive to azithromycin according to CDC criteria from 2014 year.

Conclusions: The percentage of azithromycin resistant Neisseria gonorrhoeae strains is increasing in Poland and the antibiotic should not be used in monotherapy as gonorrhoea patients. It should only be used in combination with ceftriaxone or cefixime.

The prevalence of anti-HCV antibodies in pregnant women ranges from 0.1% to 3.6% worldwide. In Poland, one work was published on the prevalence of HCV antibodies in pregnant women. Based on studies conducted by Aniszewska et al. in 544 women, the percentage of anti-HCV antibodies was estimated at 2.02%. Since 2011, the NIPH-NIH performs "Preliminary programme of routine HCV testing among pregnant women" within the Swiss-Polish Cooperation Programme, co-financed by the Ministry of Health, with the aim to, i.a. estimate the prevalence of HCV infection in the population of pregnant women. The transmission of the virus from mother to fetus is now considered to be the most common route leading to infections in children and infants. According to available data, the risk of vertical transmission from infected mother is relatively low and ranges from 1.8% to 5%. Transmission of HCV can occur both in the prenatal period as well as during the labor. Irrespective of the numerous studies on the transmission of the virus from mother to child, its mechanism has not been completely understood. Exclusively the factors favoring this route of infection are known. The main risk factor for vertical transmission is the presence of viral RNA in maternal peripheral blood. Other risk factors include: the presence of viral RNA in PBMC, HIV coinfection, significant increase in ALT in a year preceding pregnan- cy and during labor in women infected with HCV, extended time between the rupture of membranes and delivery as well as female gender of the baby. The impact of amniocentesis and cesarean delivery as risk factors for vertical transmission of HCV are still discussed. Breastfeeding by mothers infected with HCV is safe and does not lead to transmission of infection to the baby. As ribavirin and interferon, which are used in therapeutic regimens, cannot be administered during pregnancy, it is important to perform testing for HCV prior to a planned pregnancy. This gives the opportunity to cure the infection and eliminate the vertical route of HCV transmission.

Introduction: Mycoplasma pneumoniae is a common causative agent of tracheobronchitis and atypical pneumonia, mainly in children and adolescents. The infections are often seen as epidemics occurring in autumn-winter seasons at intervals of 4-7 years. Epidemiological studies showed that M. pneumoniae is responsible for 30% to 40% of all cases of bacterial respiratory infections in Poland. The aim of the study was estimate the seroprevalence of M. pneumoniae in Poland in 2008-2013 in comparing to results obtained in other European countries.

Material and methods: The results of diagnostic serological tests (ELISA) in particular immunoglobulin classes for infection with M. pneumoniae performed in 16.825 persons were retrospectively analyzed. The patients were mostly children at the preschool and school age with clinical symptoms of respiratory tract infection. The data were obtained from Bacteriology Department of National Institute of Public Health-National Institute of Hygiene in Warsaw and from 13 Sanitary and Epidemiological Stations through the country which send quarterly or monthly reports.

Results: The serological results showed that in autumn-winter seasons of 2011-2012 the "early antibodies" (IgA and/or IgM) for M. pneumoniae were twice more often diagnosed in sera of patients with respiratory tract infection than in analogous seasons of 2008-2010. The antibodies were detected in 34% and 42% of patients, respectively in third quarter of 2011 and 2012.

Conclusions: Epidemic increase of M. pneumoniae infections in Poland in autumn-winter seasons of 2011-2012 was mainly observed due to diagnosis of the IgA and/or IgM antibodies in serological tests.

Human organism consists of not only from numerous of eukaryotic cells but also from thousands of microorganisms. The most complicated is the microflora of gastrointestinal tract. Numerous studies indicates that the complex network of interactions between the host organism and its microbiome can have a very significant impact on the health condition of the host. These interactions can affect not only to gastrointestinal tract but can be related to different processes and organs. Disturbance of the homeostasis, e.g. after antibiotic course, can therefore have significant health implications Therefore, very important is the deepest exploring of the network of these interactions and dependencies.

Introduction: Infections caused with a variety of bacteria, fungi and viruses are still responsible for high level of mortality and morbidity in immunosupressed individuals. A case of fatal post-transplant reactivation with four herpesviruses in 49-year-old immunocompromised male with MDS-RAEB2, subjected to allogeneic haematopoietic stem cell transplantation was described.

Methods: Full microbiological examination of was performed in different types of clinical samples (whole blood, stool). Sera specimens were tested for the presence of different viral DNA using the real-time PCR assays.

Results and conclusions: DNA of HSV-1, VZV, HHV-6 and EBV in serum samples was detected using molecular biology techniques. Viral level of HSV-1 and VZV was constantly increasing despite routine applied oral acyclovir therapy. These findings underline the value of real-time PCR technique used in current therapeutic procedures and for monitoring of antiviral therapy with nucleoside analogs. We found that real-time PCR is a useful tool in detection and monitoring of disseminated herpesviral infection, especially for the detection of low-copy viraemia in clinical specimens.

Introduction: The present study was aimed at determining the IgG subclass distribution against F. tularensis in patients with tularemia.

Methods: The total number of 56 serum samples obtained from patients with serologically confirmed tularemia were tested by in-house ELISA with bacterial sonicate as the antigen for the presence of IgG1, IgG2, IgG3 and IgG4 antibodies to F. tularensis. Based on the results of determining the level of antibodies in the sera of 30 blood donors, the cut-off limit of serum antibodies for each subclass was set at arithmetic mean plus three standard deviations.

Results: Antibodies of subclass IgG1 to F. tularensis were diagnosed in 41 (73.2%), IgG2 in 52 (92.9%) and IgG3 in 13 (23.2%) serum samples. The arithmetic mean of OD450 of antibodies IgG2 was over three-times higher than antibodies IgG1 and IgG3 measured in all of tested serum samples. The concentration of IgG4 was below the detection level.

Conclusion: In conclusion, IgG2 antibodies to F. tularensis are predominating IgG subclass in tularemia. This study showed also that subclasses of IgG1 and IgG3 but not IgG4 antibodies to F. tularensis are produced during natural infection in humans.

Introduction: Bacterial resistance is growing because of treatment a broad spectrum antibio- tics. Gram-negative pathogens which producing carbapenemase are a one of major problem in many hospitals. Rapid detection those strains provide an early inhibition of infection and control the expansion of microorganisms. The aim of work was to characterize the frequency of appearance MBLs in specific groups of Gram-negative bacilli which are resistant or intermediate to at least one of carbapenems.

Methods: Bacterial isolates were collected from Baby Jesus Clinical Hospital from 2003 to 2009. Pathogens were isolated from urine, blood, fluids, swab of the wound, pharyngeal swab. They were identified by the ID 32 E (bioMérieux, France) and Vitek2. Antimicrobial resistance was marked by the ATB G-5 and ATB UR (bioMérieux, France). Detection of metalo-beta-lactamases was tested by disk diffusion test recommended by the EUCAST. The DDS test using imipenem, ceftazidime, ethylenediaminetetraacetic acid (EDTA) and 2-mercaptopropionic acid (2-MPA). Positive test was reading as enlargement of inhibition zone about imipenem- or ceftazidime-impregnated disk.

Results: Of the 88 isolates, 32 come fromEnterobacteriaceae and 56 from non-fermentative bacilli. All strains were tested of production of MBL by disk diffusion test. This method used two inhibitors: ethylenediaminetetraacetic acid and 2-mercaptopropionic acid. As a result of EDTA there was 45 MBL positive strains. In apply 2-MPA there was 55 MBL positive strains. Both the EDTA and 2-MPA disk test showing the highest percentage of positive result in Enterobacter cloacae, Serratia marcescens, Pseudomonas aeruginosa and Pseudomonas putida.

Conclusions: Resistance to carbapenems in the non-fermentative bacilli occurs more often than in the Enterobacteriaceae. Method with 2-mercaptopropionic acid was more effective to detect metallo-beta-lactamases than EDTA. Concerns especially bacilli from Enterobacteriaceae.

Introduction: The aim of this study was to evaluate the usefulness of ITS-PCR and PCR MP methods for genotyping of S. agalactiae strains isolated from women in reproductive age.

Methods: In the course of the study 250 strains of S. agalactiae were isolated and their serotype was identified. The ITS-PCR and PCR MP methods allowed to differentiate 20 strains and then the correlation between the serotypes of the tested isolates and their geno- type was evaluated.

Results: Among 250 strains of S. agalactiae the following serotypes were identified as the most common: III (54%), Ia (17%) and V (12%). Also other serotypes have been found: IV (8%), Ib (5%) and II (4%). PCR MP has a higher discriminative power than the ITS-PCR and it allowed for the efficient differentiation of strains. There is no direct relationship between genotypes and serotypes ofS. agalactiae.

Conclusions: PCR MP is useful in the differentiation of S. agalactiae. Exactly as in the case of RAPD and PFGE methods, PCR MP can be used for thediagnosis and analysis of GBS isolates colonization. This method is also used for successful genotyping of other microbial species, often closely related ones, as well as in epidemiological studies.

Introduction: Enterohemorrhagic Escherichia coli (EHEC) strains are an important zoonotic food-borne and waterborne pathogens causing diarrhea and the severe hemolytic uremic syndrome (HUS) in humans. The aim of the study was to evaluate the usefulness of enzyme immunoassay ELISA for detection of antibodies to the lipopolysaccharides (LPS) of EHEC in patients with gastrointestinal disorders and patients with hemolytic-uremic syndrome.

Material and methods: Sera obtained from 526 patients with gastrointestinal disorders, 26 patients with HUS and 74 patients with different bacterial gastroenteritis infections were screened by an LPS-based ELISA. The LPS antigens of EHEC belonging to serogroups O26, O103, O104, O111, O121, O145, and O157 were obtained by modified Boivin's method. Additionally, to determine the cut-off level, the 122 sera from healthy people were tested. Cellular extract from E. coli O14 were used to remove by absorption antibodies to the Enterobacteriaceae Common Antigen (ECA).

Results: Generally, seroprevalence of antibodies to the LPS of different EHEC serogroups in patients with gastrointestinal disorders was low. Additionally, interpretation of the some positive results was difficult to the fact of many serological mutual interactions. Particularly a lot of cross-reactions were seen in the group of sera obtained from patients with different bacterial gastroenteritis infections. The study showed also that in most cases the absorption of antibodies to the ECA had no significant effect on the cross-reactions observed in ELISA. On the other hand, the very high level of antibodies to the LPS antigen of E. coli O26 was found in 5 patients, to E. coli O157 in 4 patients, to E. coli O104 and O145 in 3 patients and E. coli O111 in 2 patients with HUS. Analysis of antibody levels in paired sera taken 2-3 weeks apart obtained from six HUS patients showed a rapid decline of antibody levels to the LPS antigens.

Conclusions: The results showed the usefulness of the ELISA with lipopolysaccharides antigens to serodiagnosis of infection caused by EHEC. Due to the possibility of cross- -reaction there is a need to develop more specific antigens, based on the recombinant proteins of verotoxin-producing Escherichia coli.