Screening for anthrax occurrence in soil of flooded rural areas in Poland after rainfalls in spring 2010.

INTRODUCTION AND OBJECTIVE: Anthrax spores remain viable and infectious in soil for decades. Flood water can percolate towards the surface the spores buried in soil. Moreover, the flood water might transport spores to areas previously unaffected. After the water recedes the spores located on the surface of the ground can be consumed by grazing animals and cause outbreaks of anthrax. MATERIALS AND METHOD: Soil samples were collected in areas of Poland most affected by floods in 2010 (Lubelskie, Swietokrzyskie, Podkarpackie and Mazowieckie provinces). After heating with the aim to kill vegetative forms of bacteria, the samples were cultured on PLET agar and the resulted colonies were investigated in terms of motility and presence of anthrax specific chromosomal (SG-749, plcR) and plasmid markers (capB, pagA). RESULTS: In total, 424 spore-forming, aerobically growing isolates were collected from the tested soil samples. Eighty-nine of them were non-motile. All the isolates were negative in PCR for anthrax specific chromosomal and plasmid markers. CONCLUSIONS: Spores of B. anthracis that could be related to risk of anthrax outbreaks were not detected in soil samples tested in this study. The negative results presented may not be proof that Poland is country free of anthrax. The results, however, may suggest a relatively low risk of anthrax outbreaks being triggered in the sampled areas.

Seroprevalence of diphtheria toxoid IgG antibodies in children, adolescents and adults in Poland.

BACKGROUND: Recommendations for diphtheria immunization are to apply an effective primary immunization in infancy and to maintain immunity throughout life. Immunity against diphtheria depends primarily on antibody to the diphtheria toxin. This study evaluated the seroprevalence of IgG diphtheria antitoxin in sera of healthy children, adolescents and adults in Poland. METHODS: A total of 1387 serum samples collected between 2010 and 2012 from individuals with ages ranging from 1 month to 85 years were investigated. Antibody concentrations were measured with an enzyme-linked immunosorbent assay (Anti-Diphtheria Toxoid ELISA IgG, Euroimmun, Germany). RESULTS: The results showed that among 1387 individuals examined, 547 (39.4%) had anti-diphtheria toxoid IgG antibody levels below 0.1 IU/ml (36.9% ≤ 18 years and 40.5% >18 years old, respectively). The 212 (50.8%) children and 542 (55.9%) adults showed only basic protection (0.1-1.0 IU/ml) and need immediate booster. High levels of anti-diphtheria toxoid IgG antibodies (>1.0 IU/ml) were found more often in children and adolescent (12.2%) than in adults (3.6%) and this was statistically significant (P < 0.05). The proportion of seronegatives (< 0.1 IU/ml) in children below 2 years old, adolescents and young adults to 25 years old decreased from 53.5% to 17.4%. However, in older individuals the seronegative proportion tended to increase with age, from 22.7% in adults (26-30 years old) to 67.1% in subjects > 60 years old. Characteristically, in individuals > 40 years old high levels of anti-diphtheria toxoid IgG antibodies (>1.0 IU/ml) were not seen. There were no statistically significant differences in results in relation to gender. CONCLUSIONS: The present study showed inadequate immunity levels to diphtheria amongst the Polish population, especially in adults > 40 years old and children ≤ 2 years old. To prevent reemergence of diphtheria an information campaign reminding people about recommendations concerning diphtheria booster vaccination in adults should be conducted. Moreover, the immunogenicity of the DTP vaccine used in Poland should be verified.

[Serum immunoglobulin IgG subclass distribution of antibody responses to pertussis toxin and filamentous hemagglutinin of Bordetella pertussis in patients with whooping cough]. / Wystepowanie przeciwcial dla toksyny krztuscowej i wlókienkowej hemaglutyniny Bordetella pertussis w poszczególnych podklasach IgG w przebiegu krztusca.

INTRODUCTION: The present study was aimed at determining the IgG subclass distribution against pertussis toxin (PT) and filamentous hemagglutinin (FHA) of Bordetella pertussis in patients with whooping cough. METHODS: The total number of 222 serum samples obtained from patients suspected in clinical investigation for pertussis were tested separately by in-house ELISA for the presence of IgG antibodies to pertussis toxin and filamentous hemagglutinin. The percentage distribution of specific anti-PT and anti-FHA IgG subclass response was calculated only on the basis of group of sera confirmed in the present study as positive for total IgG antibodies (183 sera to PT antigen and 129 to FHA antigen). Paired serum specimens were obtained from 36 patients. Based on the results of determining the level of antibodies in the sera of 40 blood donors, the cut-off limit of serum antibodies for each subclass was set at arithmetic mean plus two standard deviations. RESULTS: Antibodies of IgG1 to pertussis toxin and filamentous hemagglutinin were diagnosed in 151 (82.5%) and 99 (76.7%), IgG2 in 72 (39.0%) and 50 (38.8%), IgG3 in 17 (9.3%) and 43 (33.3%), IgG4 in 55 (30.1%) and 53 (41.1%) serum samples, respectively. There were no significant differences in percentage of sera with IgG1, IgG2 and IgG3 in relation to age of the patients. However, the frequency of occurrence of IgG4 antibodies was highest in the group of the youngest children to the age of 6 years old (61.8% for PT and 68.0% for FHA), and decrease with age, reaching the minimum in the group of patients above 40 years old (13.2% and 4.2% for PT and FHA, respectively). We also found significantly higher frequency of IgG4 to PT and FHA antigens in men than in women. Statistically significant, essential changes in the pattern of IgG subclass during the course of infection were not found. CONCLUSIONS: In conclusion, this study showed that all four subclasses of IgG antibodies to pertussis toxin and filamentous hemagglutinin are produced during whooping cough.

[Evaluation of multiplex PCR to identify the species of microorganisms from Bacillus cereus group]. / Ocena przydatnosci metody multiplex PCR do identyfikacji i róznicowania drobnoustrojów z grupy Bacillus cereus.

INTRODUCTION: Bacillus genus comprises about 215 species. The most closely related are B. cereus, B. thuringiensis, B. anthracis, B. mycoides, B. pseudomycoides and B. weihenstephanensis. These bacterial species belong to the Bacillus cereus group. Identification and differentiation of Bacillus cereus group bacteria is difficult because of genetic and phenotypic similarity. Many molecular methods have already been suggested to differentiate B. cereus group members. However easier and more convenient methods are still sought. The aim of this study was to evaluate of multiplex PCR method to identify and distinguish strains of Bacillus cereus group, as proposed by Park et al. (J Microbiol Biotechnol 2007; 17: 1177-82). MATERIALS AND METHOD: Twenty four strains of Bacillus cereus group included B. cereus, B. anthracis, B. thuringiensis and B. weihestephanensis was examined. A multiplex-PCR assay for the differentiation of the species has been applied by using three pairs specific oligonucleotide primers based on sequences of gyrB genes which identify species from Bacillus cereus group and one pair specific primers based on sequences of groEL gene, which are used to identify Bacillus cereus group. RESULTS: Using a specific primers complementary to fragment of groEL gene, we received all PCR products and thus we identified Bacillus cereus group. We have not recived a specific products characteristic for each of the species. Oligonucleotide primers recognized by Park et al. as specific for each species were complementary (often 100%) for the gyrB gene sequence in almost all species of the B. cereus group. CONCLUSIONS: The multiplex PCR method proposed by Park et al. multiplex PCR method for identification ofB. cereus group and individual bacterial species has been proved to be useful only for identification the entire group of B. cereus. This method does not provide specific identification of the individual species. Lack of specificity of the primers used in this study creates a risk of obtaining PCR product in more than one species of the entire examined group of bacteria and does not allow the precise identification to the species level.

[Effect of multiple freeze-thaw cycles on detection of IgA, IgG and IgM antibodies to selected bacterial antigens]. / Wplyw wielokrotnego, cyklicznego zamrazania i rozmrazania próbki surowicy na poziom przeciwcial klasy IgA, IgG i IUgM dla wybranych antygenów bakteryjnych.

INTRODUCTION: In presented study we investigated the effect of multiple freeze-thaw cycles of human sera on the determination of IgA, IgG and IgM antibodies to selected bacterial antigens. METHODS: A panel of 15 serum samples with elevated levels of antibodies to Mycoplasma peumoniae, Yersinia enterocolitica and Salmonella spp. were used (5 positive sera for each pathogen). One set of aliquots designed as the baseline, was taken and stored at 4-8o C for the remainder for the study. The remaining seven sets of aliquots were divided into two parts and repeatedly frozen respectively at two different temperatures: -65 degrees C and -25 degrees C. Once a day the aliquot sets were removed from the freezer and allowed to stand at room temperature for approximately 1 h until completely thawed. For the determination of the level of antibodies the sera after: 2, 5, 10, 15, 20, 25 and 30 freeze/cycle were used. The measurement of IgA, IgG and IgM antibodies was done using a home-made ELISA with four different antigens: whole-cell antigen of M. pneumoniae FH strain, LPS and Yop antigens of Y. enterocolitica serotype O:3 and LPS extracted by Westphal method from Salmonella serogroup B +D. The results were presented as the arithmetic mean of the antibody titre in five sera which were treated by the same number of freeze-thaw cycles. RESULTS: There was no significant statistic difference between levels of antibodies in unfrozen and frozen sera even after 30 freeze-thaw cycles. Depending of the antigen used in ELISA a slight varations in the level of antibodies were observed but the changes were small and not clinically significant. Examination of the ELISA values does not suggest any consistent nonlinear trend in levels of IgA, IgG and IgM antibodies in sera frozen at -65 degrees C as well at -25 degrees C. CONCLUSIONS: Our study demonstrates that the IgA, IgG and IgM antibody activity levels measured for M. pneumoniae, Y enterocolitica and Salmonella antigens are stable even after 30 freeze-thaw cycles.

[Seroprevalence of antibodies against lipopolysaccharides of Bordetella parapertussis in patients with prolonged caught in Poland]. / Czestosc wystepowania przeciwcial dla lipopolisacharydów paleczek Bordetella parapertussis u osób z przewleklym kaszlem w Polsce.

INTRODUCTION: Bordetella parapertussis is a bacterium closely related to Bordetella pertussis, also causes a pertussis - like symptoms in humans. Because of unsatisfactory level of routine microbiological diagnosis of B. parapertussis infections in Poland most of parapertussis cases are not reported. The aim of the presented study was to investigate incidence of B. parapertussis in patients with cough in Poland using serology method. METHODS: Serum IgA, IgG and IgM antibodies were determined in 1192 serum samples obtained from patients with respiratory infections and chronic cough and who were previously suspected of B. pertussis infection. As a control we used 258 sera from healthy people - blood donors. The LPS antigen was extracted by Westphal method from wild B. parapertussis strain isolated in Poland. For exclusion of possible false positive results with B. pertussis some of the sera were tested against the purified pertussis toxin (PT). RESULTS: The diagnostically significant level of IgA antibodies to LPS of B. parapertussis was detected in 11.9%, IgG in 12.2% and IgM in 9.6% serum samples. More often the antibodies were diagnosed in women than in men. In 63 serum samples, previously positive in NovaTec ELISA with mixed antigen of pertussis toxin and filamentous hemagglutinin we found also IgA and IgG antibodies to LPS of B. parapertussis. However, after use of purified pertussis toxin antigen in ELISA we confirmed the B. pertussis infections only in 28 cases. CONCLUSIONS: This study shows that B. parapertussis is a serious causative agent of infections in patients with prolonged caught in Poland. The serodiagnosis ofparapertussis should be conducted with sera obtained from patients suspected in clinical investigation for pertussis but negative in serological investigation with purified pertussis toxin antigen.

[Comparison of usefulness of lipopolysaccharides extracted by phenol and trichloroacetic acid from Salmonella Typhimurium and Enteritidis for serodiagnosis of salmonelosis]. / Przydatnosc wybranych lipopolisacharydowych preparatów antygenowych uzyskanych z paleczek Salmonella grupy serologicznej B i D do serodiagnostyki salmonelozy.

Salmonella Typhimurium and Salmonella Enteritidis are the two predominant serogroups, responsible for about 80% of all human cases of salmonelosis in Poland. Therefore we compared the usefulness of lipopolysaccharides antigens extracted by phenol (Westphal method) and trichloroacetic acid (Boivine method) from Salmonella Typhimurium and Enteritidis in ELISA method for the determination of antibodies. We used one home - made LPS antigen and two others commercially available antigens from SIGMA - Aldrich. Our study showed that the presence of antibodies was found in 35 (74.5%) sera from 47 samples from patients with suspected salmonelosis. There was no significant statistical differences of frequency of appearance of antibodies to all three Salmonella antigens in sera from patients with salmonelosis and in sera from control group. This study showed that all three antigens are useful for determination of IgA, IgG, IgM antibodies for Salmonella serogroup B and D in routine serological diagnosis of salmonelosis. However, it should be considered possibility of cross-reaction between LPS antigen of Salmonella and antibodies to Yersinia enterocolitica which could be correlated with similarity between somatic antigens of these two pathogens.

[The use of MLVA for Corynebacterium diphtheriae genotyping--preliminary studies]. / Zastosowanie metody MLVA do genotypowania corynebacterium diphtheriae. Badania wstepne.

The complete genome sequence of strain NCTC 13129 C. diphtheriae were investigated in order to identify tandem repeats (VNTR). From 75 VNTR loci identified in the genome 14 were selected. Primers were designed and PCR conditions were optimized for amplification of the selected VNTR markers. Preliminary studies of usefulness of selected VNTR markers were conducted using a group of 28 C. diphtheriae strains. From 14 markers 8 were regarded as potentially useful. The diversity of individual markers ranged from 1 to 6 alleles (Simpson index from 0 to 0,746). No diversity were observed for 3 VNTR markers but it could be a results of too small group of strains analyzed in the tests. Simpson diversity index calculated for all the markers tested on 28 strains was 0,87. Results of the preliminary studies showed usefulness of MLVA for C. diphtheriae genotyping. Nevertheless, confirmation of reliability of the method should be done using a large group of strains. Moreover, the method should be compared with other genotyping methods.

[Detection of antibodies to Campylobacter jejuni in pediatrics patients with Gullain-Barré syndrome using different antigen preparations]. / Wykrywanie przeciwcial dla paleczek Campylobacter jejuni u dzieci z zespolem Guillain-Barré przy zastosowaniu róznych preparatów antygenowych.

Diagnosis of previous C. jejuni infections in GBS patients are mostly based on serological findings. However, there are not consensus what kind of antigen should be used in the serological assays. In our study we used ELISA with four different antigen preparations for investigation of specific antibodies to C. jejuni in serum samples obtained from 6 children with GBS. In all patients the high level of IgA and IgG antibodies to LPS were diagnosed. The antibodies in particular classes of immunoglobulins to recombinant proteins (Mikrogen), termostabile antigen and whole-cell antigen (Virion/Serion) of C. jejuni were diagnosed only in some of the children with GBS. However, in comparison to the control groups, the ELISA with recombinant proteins was most specific. Moreover, in two of the patients a characteristic decline of the level of antibodies to recombinant proteins in sera obtained in acute and chronic phase of disease have been observed. The results of this study showed that serodiagnosis, specially based on recombinant antigens, may be a reliable marker of recent or previous infection caused by C. jejuni in patients with GBS.

Plasmid-borne 16S rRNA methylase ArmA in aminoglycoside-resistant Klebsiella pneumoniae in Poland.

We characterized 17 clinical isolates of Klebsiella pneumoniae producing 16S rRNA methylase ArmA. The isolates originated in Poland from 2002 to May 2010 and encompassed four XbaI-PFGE clusters. All the isolates were resistant to amikacin, gentamicin and kanamycin (MIC range: 256-1024 mg l(-1)) and carried the armA gene on a large plasmid of approximately 90 or 130 kb in 15 and 2 isolates, respectively. The armA gene was found in a ~10 kb ClaI restriction fragment of the large plasmid and was flanked by the same elements as in Tn1548. All the isolates carried the bla(CTX-M) gene for a CTX-M-type extended-spectrum ß-lactamase. Our results show that ArmA has disseminated horizontally among K. pneumoniae isolates in Poland on the ~90 kb plasmid of the pCTX-M3 family.

Emergence of Klebsiella pneumoniae coproducing KPC-2 and 16S rRNA methylase ArmA in Poland.

A Klebsiella pneumoniae epidemic strain that coproduced carbapenemase KPC-2 (K. pneumoniae carbapenemase 2) and 16S rRNA methylase ArmA has emerged in Poland. Four nonduplicate isolates from patients in a hospital in Warsaw, Poland, were found to carry the bla(KPC-2) and armA genes on ca. 50-kb and 90-kb plasmids, respectively. Tn4401 with a 100-bp deletion in the variable region was detected in all the isolates. XbaI pulsed-field gel electrophoresis (PFGE) revealed 93.2% similarity of the isolates. All the isolates were resistant to carbapenems and 4,6-disubstituted 2-deoxystreptamines.

[Evaluation of usefulness of the enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to lipopolysaccharides of VTEC strains in patients suspected for Escherichia coli O104:H4 infection in Poland]. / Wykorzystanie odczynu ELISA do poszukiwania przeciwcial dla lipopolisacharydów werotoksycznych paleczek okreznicy u osób podejrzanych o zakaienie szczepem Escherichia coli O104:H4 w Polsce.

The aim of the study was to investigate the presence of antibodies to lipopolysaccharides obtained by modified Boivin's method from E. coli serotype O104:H4 and O26, O103, O111, O121, O145, O157 in sera of 7 patients with acute diarrhea, suspected in clinical investigation for infection caused by E. coli O104:H4. Additionally, to determine the cut-off levels, the 75 sera from blood donors were tested. The high level of antibodies to LPS E. coli O104 was diagnosed in three patients from family outbreak caused by E. coli serotype O104:H4. In one of those patients, 7-years boy with HUS, we observed also a significant decrease of level of IgA, IgG and IgM antibodies in serum sample obtained in chronic phase of the disease. Furthermore, we showed that two others patients with clinical evidence of VTEC infection, not connected with this family outbreak, had a high level of antibodies to E. coli of other serotypes: one to O157 and one to O103. We did not observe presence of antibodies to LPS from E. coli O26, O111, O121 i O145 in the sera of tested patients. In conclusion, we confirmed that ELISA based on lipopolysaccharides obtained from different serotypes of E. coli may be helpful in laboratory diagnosis of infection caused by VTEC in humans.

[Comparison and evaluation of the Binax EIA and Biotest EIA urinary antigen kits for detection of Legionella pneumophila antigen in urine samples]. / Ocena przydatnosci komercyjnych testów immunoenzymatycznych biotest EIA i Binax EIA do wykrywania antygenu Legionella pneumophila w próbkach moczu osób z legioneloza.

The Binax and the Biotest urinary antigen kits for detection of L. pneumophila antigen were compared by testing of selected 67 urine samples obtained from EWGLI as reference samples in External Quality Assessment Scheme. Thirty nine were positive with the Binax kit (100% of sensitivity), and 33 were positive with the Biotest (84.6% of sensitivity). The test specificities were 100% for the both kits. It was concluded that the Binax kit was more suitable for the routine diagnosis of Legionella infections than the Biotest kit.

[Occurrence of 16s rRNA methylase ArmA producing Enterobacteriaceae in a general hospital in Warsaw, Poland]. / Wystepowanie w jednym z warszawskich szpitali paleczek Enterobacteriaceae wytwarzajacych 16s rRNA metylaze Arma.

Resistance to gentamicin, amikacin and kanamycin was screened in 270 clinical isolates of Enterobacteriaceae originated from April 19 to May 19, 2010 in a regular hospital in Warsaw, Poland. Most of the isolated bacteria were considered pathogenic. Nineteen isolates (7%) were simultaneously resistant to two or three of the tested aminoglycosides. MICs of the three aminoglycosides ranged form 128 to 1024 mcg/ml for six isolates. These isolates were suspected to produce 16S rRNA methylase. Genes encoding for three methylases reported in Europe: ArmA, RmtB and RmtC were searched by PCR. The armA gene was detected in all of the six isolates. This group encompassed Enterobacter cloacae (n=4), Klebsiella pneumoniae (n=1) and Proteus mirabilis (n=1). Five isolates of this group carried the bla(CAX-M) gene for CTX-M type ESBL. The remaining isolate E. cloacae DM0340 was ESBL negative and lacked bla(CRX-M) that may suggest an altered genetic environment of the armA gene in this isolate. Our results showed that 2.2% of the tested isolates produced 16S rRNA methylase ArmA. This finding may argue for a high incidence of ArmA producing Enterobacteriaceae in Poland when compared to reports from other European countries.

[Comparison of routine serological investigations performed in three different laboratories in Poland for the diagnosis of pertussis]. / Porównanie wyników serologicznych badan w kierunku krztusca rutynowo prowadzonych w trzech róznych laboratoriach w polsce.

We compared the 2.653 results of routine serological investigations performed in three different laboratories in Poland for the diagnosis of pertussis. One of the laboratories used the NovaLisa Bordetella pertussis kit produced by NovaTec GmbH and the two others used Bordetella pertussis ELISA kit produced by Genzyme Virotech GmbH. In first laboratory the diagnostic level of IgA antibodies to the pertussis toxin was observed in 11.0%, IgG in 52.7% and IgM in 27.4% serum samples. In the second and third laboratory the diagnostic level of IgA antibodies were found respectively in 22.2% and 40.1%, IgG in 46.8% and 66.4% and IgM only in 8.7% and 4.8% serum samples. In total, IgA antibodies were found in 28.0%, IgG antibodies in 56.0% and IgM antibodies in 14.3% serum samples obtained from patients suspected in clinical investigation for pertussis. We have observed that the frequency of detection of the antibodies in children and adolescents increased with age reaching its peak among individuals with age range between 11-20 years. We also found statistical significant higher frequency of IgA, IgG and IgM antibodies to B. pertussis in outpatients than in hospital patients.

[Aminoglycoside resistance in Enterobacteriaceae originated in a gynecology-obstetrical hospital: a survey for 16s rRNA methylases: ArmA, RmtB and RmtC]. / Opornosc paleczek Enterobacteriaceae wyosobnionych w szpitalu polozniczo- ginekologicznym na wybrane antybiotyki aminoglikozydowe ze szczególnym uwzgludnieniem zdolnosci do wytwarzania 16s rRNA metylaz ArmA, RmtB i RmtC.

We examined the resistance of 2359 clinical isolates of Enterobacteriaceae to gentamicin, amikacin, netilmicin and neomycin by the disc-diffusion assay. The isolates originated from female-patients and newborn infants in a gynecology-obstetrical hospital in Warsaw, Poland. Isolates from adults predominated. Most of the isolated bacteria were considered non-pathogenic, colonization flora. The majority (above 95%) of the isolates were sensitive to all of the tested aminoglycosides. Forty five (1,90%) isolates were resistant to one or more agents. In this group, E. coli was prevalent (73,33%). As little as 14 isolates had no growth inhibition around the gentamicin disc (10 mcg) (contact-growth). MICs of seven isolates ranged from 256 to 1024 (mcg/ml) of the tested agents. One isolate had MIC 1024 for amikacin and kanamycin. All the contact-growth isolates were examined for genes encoding for TEM, SHV and CTX-M beta-lactamases, and genes armA, rmtB and rmtC for 16S rRNA methylases reported in Europe. All of them produced TEM enzyme while SHV and CTX-M was expressed by one and two isolates respectively. None of the tested 16S rRNA methylases was detected. Our results show the low carriage of the aminoglycoside-resistant Enterobacteriaceae in healthy pregnant-women and most probably their sexual partners. Our findings may argue that production of the 16S rRNA methylases is still limited to patients with a long-term antibiotic-therapy in regular hospitals rather than to non-hospitalized population in Poland.

[Dissemination of the KPC carbapenemase producing Klebsiella pneumoniae in a hospital in Warsaw, Poland]. / Wystepowanie w jednym z warszawskich szpitali paleczek Klebsiella pneumoniae wytwarzajacych karbapenemaze typu KPC.

Within the last decade, human infections caused by enterobacteria which produce the Klebsiella pneumoniae carbapenemase (KPC) became a serious therapeutic and epidemiological problem worldwide. The KPC producing strains of K. pneumoniae broadly disseminated in the USA then spread to Europe. Recently, the KPC-2 was found in Poland. In the presented study we tested 11 ertapenem resistant isolates of K. pneumoniae. The isolates were obtained from 10 patients of a regular hospital (RH) and from one patient of a palliative care hospital (PH) in Warsaw, Poland. Expression of the KPC was confirmed in all the tested isolates by the positive result of phenotypic test with boronic acid. All the isolates were also shown to harbour the bla(KPC) gene by PCR with primers targeting the core 372 bp fragment of the gene, and all but two were resistant to imipenem and meropenem as determined by the disc-diffusion method. The DNA sequence analysis of the complete bla(KPC) gene from representative isolate DM0269 revealed variant 2 of KPC (KPC-2). Tested isolates were subjected to genotyping by the PFGE with XbaI. Dendrogram based on the PFGE profiles was composed of two main branches with 82,3% of similarity. Branch A encompassed 9 isolates (93,2%), including the one from the PH-patient, while the two remaining isolates (86,5%) were located in branch B. Five isolates of the branch A were indistinguishable by the PFGE. The high genetic similarity of the branch A isolates strongly suggests the intra-hospital dissemination of epidemic K. pneumoniae KPC+ sensu stricto strain. Most probably, the strain was also transferred to the palliative care hospital. In contrast, the branch B isolates appear to belong to the distinct sensu stricto strain, that has acquired the bla(KPC) gene via horizontal transfer. This is the first report on the intra-hospital dissemination of the KPC producing K. pneumoniae in Poland. It is noteworthy, all the tested strains were also resistant to cefotaxime, ceftazidime, aztreonam, ciprofloxacin and sulphonamides, but sensitive to colistin.

[Evaluation of the usefulness of selected antigens of Mycoplasma pneumoniae for the serodiagnosis of mycoplasmosis]. / Ocena przydatnosci wybranych antygenów Mycoplasma pneumoniae do serodiagnostyki mykoplazmozy.

A panel of 33 different antigens, among them lipoproteins, glicolipids and proteins, of Mycoplasma pneumoniae used in commercial western-blotting (Virotech) were assessed for reactivity with sera of patients with mycoplasmosis and other bacterial infections of variable etiology. In addition, commercial ELISA (Virotech) with recombinant proteins as antigen and complement fixation test (CFT) with in-house prepared glicolipid-protein antigen were also assessed for comparison. The proteins with molecular weight of 170 kDa (P1) and 90 kDa (P90) were most recognized by the serum samples of patients with mycoplasmosis. The reactions of proteins P50, P47 and Fts monomer with positive sera were not such often and the response was usually weak. The other proteins of M. pneumoniae, particularly P88, Repet. Prot. or P20, were recognized occasionally or at all. We observed also the often reactions ofglicolipids of M. pneumoniae with IgM antibodies. The result of the study showed that the commercial ELISA (Virotech) was equivalent in sensitivity and specificity to the CFT in the case of sera obtained in the acute phase of mycoplasmosis (90.7% of agreement of results in the class IgA, 85.6% in the class IgG and 100% in the class IgM). Analytical specificity studied by screening serum samples from patients with different bacterial infections and blood donors have shown lower specificity of ELISA in compared to western-blotting. The present study confirmed the earlier observations of the high usefulness of P1 protein and P90 protein for reliable serologic diagnosis of acute M. pneumoniae infection.

[Evaluation of genotypic and phenotypic methods for the differentiation of Campylobacter jejuni and Campylobacter coli clinical isolates from Poland. I. Biotyping, resistance pattern, plasmid analysis, restriction enzyme analysis of plasmid]. / Ocena przydatnosci wybranych metod genotypowych i fenotypowych do wewnatrzgatunkowego rznicowania paleczek Campylobacter jejuni oraz Campylobacter coli izolowanych od ludzi. I. Przydatnosc biotypowania, okreslania profilu lekoopornosci i profilu plazmidowego oraz plazmidowego wzoru restrykcyjnego.

A total of 128 clinical isolates of C. jejuni and 17 clinical isolates of C. coli were characterized by their plasmid profiles, susceptibility to six antimicrobial agents and the Lior biotypes. We also analyzed seven isolates from three C. jejuni family outbreaks and three isolates from one C. coli family outbreak. Plasmids were found in 47 C. jejuni (36.7%) and 3 C. coli (17.6%) isolates. The sizes of plasmids detected ranged from about 3.3 to 89 kb. The majority of Campylobacter isolates (n = 33) had a single plasmid, 11 and three isolates of C. jejuni had two and three plasmids, respectively. The most frequently isolated plasmids ranged from 37 to 48 kb (n = 26). The single plasmids were discriminated on the basis of their BglII patterns. The majority of 45 kb (n = 12) plasmids had the indistinguishable pattern. The significant association (p < 0.0001) between tetracycline resistance and plasmid carriage was observed. All except one (hippuric-negative C. jejuni isolate) C jejuni isolates were biotyped using the Lior biotyping scheme. Three biotypes of C. jejuni and 2 biotypes of C. coli were identified among the isolates; 60.2% were C. jejuni I, 38.2% C. jejuni II, 0.8% C. jejuni III, 70.6% C. coli I and 29.4% C. coli II. In this study the least discriminatory subtyping method was biotyping (D = 0.505). C. jejuni isolates were the most resistant to ciprofloxacin and nalidixic acid (64.1%) followed by tetracycline (23.4%) and ampicillin (13.3%). Fifteen isolates of C. coli were resistant to ciprofloxacin and nalidixic acid (88.2%), three to ampicillin and one to tetracycline. All of the C. jejuni and C. coli isolates were sensitive to erythromycin and gentamicin. Resistance to ciprofloxacin was found in all quinolone-resistant isolates. Thirty seven isolates (28.9%) were susceptible to all antimicrobial agents tested. Six C. jejuni (4.7%) and one C. coli isolates showed resistance to more than three drugs. The fact that the most of the C. jejuni and C. coli isolates tested were biotype I and possessed no plasmid limited the usefulness of biotyping and plasmid analysis as epidemiological markers. The results of susceptibility testing of C. jejuni and C. coli isolates showed high percentage of resistance to quinolones.

[Evaluation of genotypic and phenotypic methods for the differentiation of Campylobacter jejuni and Campylobacter coli clinical isolates from Poland. II. PFGE, ERIC-PCR, PCR-flaA-RFLP and MLST]. / Ocena przydatnosci wybranych metod genotypowych I fenotypowych do wewnatrzgatunkowego róznicowania paleczek Campylobacter jejuni oraz Campylobacter coli izolowanych od ludzi. II. przydatnosc metod PFGE, ERIC-PCR, PCR-fla-A-RFLP i MLST.

Campylobacter jejuni and Campylobacter coli are leading cause of bacterial gastroenteritis in many developed countries. We compared pulsed-field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus (ERIC-PCR) and restriction fragment length polymorphisms of the flagellin gene (PCR-flaA-RFLP) for distinguishing 128 C. jejuni isolates and 17 C. coli clinical isolates isolated between 2006 and 2007 in Poland. We also analysed seven isolates from three C. jejuni family outbreaks and three isolates from one C. coli family outbreak. These isolates were also analysed by multilocus sequence typing (MLST). PFGE and PCR-flaA-RFLP were performed as described by CAMPYNET. The methods were evaluated and compared on the basis of their abilities to identify outbreaks isolates and discriminate between unrelated isolates (with D index). PFGE was found to be the most discriminatory method (D = 0.999), followed by ERIC-PCR (D = 0.997) and PCR-flaA-RFLP (D = 0.912). PFGE and ERIC-PCR clearly divided C. jejuni and C. coli into two clusters. PFGE, ERIC-PCR and PCR-flaA-RFLP distinguished 117, 107 and 18 distinct profiles, respectively, among 128 C. jejuni isolates. Among 17 C. coli isolates, 15 PFGE and ERIC-PCR, and 7 PCR-flaA-RFLP profiles were found. All the methods identified the outbreaks strains. MLST analysis showed that C. jejuni isolates obtained from three outbreaks belonged to three new 3847, 3848 and 3849 STs. We found isolates with the indistinguishable patterns in each method which were obtained from humans from the same region and related in time that potentially represent common-source outbreaks. We also found isolates with the indistinguishable patterns in each method that were obtained from humans from different part of Poland. This is the first report of using MLST, ERIC-PCR and PCR-flaA-RFLP methods to distinguish C. jejuni and C. coli clinical isolates in Poland. Our results demonstrate that all typing methods evaluated in this study are highly discriminatory and useful to investigate Campylobacter outbreaks in Poland. Our results also show that the genetic diversity of polish C. jejuni and C. coli isolates is very high.