Development and evaluation of latex agglutination tests for the detection of human antibodies to the lipopolysaccharides of verocytotoxin-producing Escherichia coli (VTEC) serogroups O157 and non-O157.

Latex agglutination tests (LAT) were developed and evaluated for the rapid detection of LPS antibodies against E. coli serogroup O157, O26, O104, O111 and O145. The latex tests have been proved to be sensitive, fast, easy-to-perform and cost-efficient tools for the screening serodiagnosis of VTEC infections causing haemolytic-uraemic syndrome.

[The occurrence of infections caused by Francisella tularensis in humans in Poland and laboratory diagnosis of tularemia].

Tularemia is a serious infectious zoonotic disease, caused by Gram-negative bacterium Francisella tularensis. Natural reservoir of infection are small mammals such a mice, voles, squirrels and rabbits. Transmission to humans occurs through contact with infected animals or contaminated environments, or through arthropod vectors. Because of its extreme infectivity it is a dangerous biological agent to human health. Tularemia has a broad geographical distribution, however is mainly in the northern hemisphere, in areas with cooler climates particularly in North America, Europe, Russia and Japan. Most of the cases among European countries have been reported in the Scandinavian region. The prevalence rate of tularemia in Poland is small, although in recent years stable increase has been observed. According to official epidemiological data during the years 2010-2016 only 61 cases of tularemia were reported in Poland. A laboratory diagnosis of tularemia is based on serological investigation, classical microbiology and molecular biology. The aim of this study was to evaluate the prevalence of infections caused by Francisella tularensis in humans in Poland and present characteristics of laboratory diagnosis of tularemia.

Usefulness of in-house obtained recombinant proteins Yop of Yersinia enterocolitica as highly specific antigens in ELISA and recom-dot performed in the serodiagnosis of yersiniosis.

INTRODUCTION: Proper analysis of the human immune response is crucial in the laboratory diagnosis of many bacterial infections-The current serological diagnosis of yersiniosis often is carried out using ELISA with native antigens. However, recombinant proteins increase the specificity of the serological assays, particularly in patients with chronic, non- specific infections. The aim of the present study was to evaluate the usefulness of in-house obtained recombinant proteins Yop of Yersinia enterocolitica as highly specific antigens in ELISA and recom-dot performed in the serodiagnosis of yersiniosis. MATERIALS AND METHODS: Recombinant YopD, YopB, YopE and V-Ag proteins of Y enterocolitica were expressing in E. coli BL21 (DE3) using the pET-30 Ek/LIC expression vector (Novagen). Purification was accomplished by immobilized metal (Ni2) affinity column chromatography (His-trap). The proteins were used as antigens in standard ELISA and recom-dot assay, which was performed on nitrocellulose strips. The study population, used for characterization of the humoral immune response to the recombinant proteins, consisted of 74 patients suspected for Y enterocolitica infection and 41 clinically healthy blood donors. Some of the results obtained by ELISA and recom-dot were compared with results obtained by commercial western-blot Yersinia (Virotech). RESULTS: In the group of patients suspected for yersiniosis in clinical investigation the most positive results were obtained in ELISA with the recombinant protein YopD (IgA respectively 25 (42.4%), IgG 41 (69.5%), IgM 24 (40.7%). The percentage ofpositive results in the group of blood donors did not exceed 10.0% in IgG and 5.0% in IgA/IgM classes of immunoglobulin. The results obtained in the recom-dot assay showed that among 74 tested serum samples obtained from individuals suspected of yersiniosis the most common IgA, IgG and IgM antibodies were found for recombinant protein YopD (respectively IgG in 60.8%, IgA in 37.8% and IgM in 33.8% of serum samples). IgG antibodies to the protein V-Ag were observed in 32.4%, protein YopB in 27.0% and for the protein YopE in 18.9% serum samples. Immunoglobulin A, and M for the recombinant proteins were found much less frequently than IgG antibodies (respectively 12.2% and 10.8% for V protein-Ag in 10.8% and 14.9% protein and_YopB 2,7% and 10.8% for the protein YopE). Further studies showed that results obtained in recom-dot assay with recombinant protein YopD were comparable with the results of commercial western-blot Yersinia. CONCLUSIONS: The study showed that in-house obtained recombinant proteins can be used as highly specific antigens in ELISA and recom-dot performed in the serodiagnosis of yersiniosis. The most useful was the protein YopD.

[Health risks associated with the environmental presence of toxigenic moulds].

INTRODUCTION: This study reviews the occurrence and most common health effect of exposure to moulds in different environment. The short characteristic of chosen toxigenic fungi and the major mycotoxin classes was also presented. Exposure to allergens may cause human disease, including allergic rhinitis, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis asthma and sick building syndrome. Moulds also reveal carcinogenic, cytotoxic, and neurotoxic properties.

Development and evaluation of a latex agglutination test for the rapid serodiagnosis of tularemia.

A latex agglutination test (LAT) was developed for a rapid detection of antibodies against Francisella tularensis. The assay is performed by mixing serum with antigen-coated latex beads and read within 5 min. Developed LAT has been proved to be a specific, sensitive, fast, easy-to-perform and cost-efficient tool for the routine diagnosis of tularemia.

Molecular Methods for Identification of Monophasic Salmonella Typhimurium Strains.

Two molecular biology methods were used to differentiate Salmonella enterica 1,4,[5],12:i:- strains: "Salmonella Check&Trace microarray" (CT) and multiplex PCR (mPCR). For 92 strains in CT result "Salmonella 1,4,[5],12:i:-" were obtained. Those strains were confirmed in mPCR as monophasic fljB-lack Salmonella Typhimurium. For 17 strains, which in CT assay were recognized as Salmonella Typhimurium, the same identification was obtained in mPCR. Reference Salmonella strains: Lagos, Agama, Tsevie, Glocester and Tumodi in CT were recognized as Salmonella genovar, in mPCR--as Salmonella O:4, H:i other than Salmonella Typhimurium, the same like Salmonella Farsta, recognized incorrectly in CT as Salmonella Typhimurium.

Usefulness of the (GTG)4-PCR for typing of monophasic Salmonella enterica isolates with antigenic shame l,4,[5],12:i:-.

INTRODUCTION: Monophasic Salmonella enterica strains presenting the antigenic shame 1,4,[5],12:i:- are becoming more prevalent. Accurate identification of such strains is hard with routine using biochemical and serological tests. Such strains can be identified with molecular tests. In this study we have tested the usefulness of(GTG)4-PCR for the diagnostic of such monophasic strains. This usefulness of this method was previously confirmed for genoserotyping of S. Enterica, Typhimurium, Infantis, Virchow, Hadar, Newport and Anatum. MATERIALS AND METHODS: 76 strains with antigenic shame l,4,[5],12:i:-, isolated in Poland in years 2007-12 were tested. Additionally (GTG)4-PCR patterns were obtained for reference strains of serotypes S. Lagos, S. Agama, S. Farsta, S. Tsevie, S. Glocester and S. Tumodi. (GTG)4-PCR was performed with DreamTaq DNA polymerase. Obtained patterns were analysed with BioNumerics software. RESULTS: No pattern specific for monophasic pattern was identified. Additionally it was also impossible to differentiate patterns obtained for S. Typhimurium, S. Farsta, S. Tsevie and S. Glocester. Only reference strains of serotypes S. Tumodi, Farsta and Agama has the distinguishable patterns of (GTG)4-PCR. CONCLUSIONS: Analysed (GTG)4-PCR method do not show the ability to distinguish S. enterica serotypes from group 04, H:i, including monophasic strains with the antigenic shame 1,4,[5],12:i:-.

[Problems in the serological diagnosis of atypical pneumonia caused by Legionella pneumophila and Mycoplasma pneumoniae]. / Problemy w serologicznej diagnostyce atypowych zapalen pluc na przykladzie wyników badan w kierunku zakazen wywolywanych przez Legionella pneumophila oraz Mycoplasma pneumoniae.

INTRODUCTION: The clinical presentation of atypical pneumonia is often similar to the presentation of more typical bacterial pneumonias and the etiological agent must be confirmed by laboratory diagnosis. This article will discuss the problems in the serological diagnosis of atypical pneumonia caused by Legionella pneumophila and Mycoplasma pneumoniae which are the agents most commonly associated with atypical pneumonia. Specifically, seeking the possibility of non-specific response, we evaluated the prevalence of antibodies to M. pneumoniae in serum samples obtained from patients suspected in clinical investigation for legionellosis. METHODS: The total numbers of 261 serum obtained from patients suspected in clinical investigation for legionellosis, were tested by in-house ELISA with M. pneumoniae sonicated antigen. Some of the positive sera were also re-tested by western-blot with high specific recombinant M. pneumoniae P1 protein. RESULTS: The diagnostic significant level of IgA antibodies to M. pneumoniae were diagnosed by ELISA in 71 (27,2%) of tested serum samples. Some of the IgA-positive sera have also high level of IgG and IgM antibodies to M pneumoniae (respectively 4,2% and 6,5%). Most from the 18 selected positive results obtained by ELISA were also confirmed by western-blot. It was characteristic that IgA antibodies to M pneumoniae were detected more than three times often in serum samples with positive serological tests for Legionnaires' disease than in samples with negative results for L. pneumophila. CONCLUSIONS: This study showed the possibility of non-specific reactions in serological diagnosis of atypical pneumonia. However, according to the data of the literature, co-infections of L. pneumophila and M pneumoniae can not be excluded.

Co-existence of plasmid-mediated quinolone resistance determinants and mutations in gyrA and parC among fluoroquinolone-resistant clinical Enterobacteriaceae isolated in a tertiary hospital in Warsaw, Poland.

Plasmid-mediated quinolone resistance (PMQR) determinants and the distribution of mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC were investigated in 215 ciprofloxacin-resistant (MIC>1mg/L) clinical Enterobacteriaceae collected during a 6-month prospective study in a tertiary hospital in Warsaw, Poland. PMQR determinants were present in 49 isolates (22.8%), among which aac(6')-Ib-cr and qnrB1 predominated (85.7% and 26.5%, respectively). Mutations in gyrA and parC QRDRs were detected among 89.8% of isolates (MIC≥4mg/L). Changes in Ser-83, Ala-84 and Asp-87 in GyrA and Ser-80 and Glu-84 in ParC were detected. Five isolates with ciprofloxacin MICs in the range 1.5-16 mg/L were found to have unaltered QRDRs, with PMQR as the only fluoroquinolone (FQ) resistance trait detected. The remaining 44 PMQR-positive isolates were found to carry altered QRDRs. Three substitutions (two in GyrA and one in ParC) were detected in 23 isolates, whilst 8 isolates carried four mutations (two in GyrA and two in ParC). One isolate of Klebsiella pneumoniae with two amino acid substitutions in the ParC QRDR in the presence of aac(6')-Ib-cr and qnrB1 had a ciprofloxacin MIC of 16mg/L. The results presented here show that FQ resistance in these clinical Enterobacteriaceae is a complex interplay between PMQR determinants and mutations in gyrA and parC rather than a single stepwise accumulation of mutations in the gyrase and topoisomerase subunits. In addition, these results show the role of PMQR determinants in promoting QRDR mutations and the acquisition of high-level FQ resistance in clinical settings.

[Occurrence and characterization of monophasic Salmonella enterica subsp. enterica with antigenic formula 1,4, [5], 12: i:-isolated in Poland in 2007-2012]. / Wystepowanie i charakterystyka jednofazowych paleczek Salmonella enterica subsp. enterica o wzorze antygenowym 1,4,[5],12:i:-izolowanych w Polsce w latach 2007-20121.

INTRODUCTION: Salmonella is significant etiological agent of bacterial intestinal infections in Poland and other European Union countries. Since the 90's increasing incidence of monophasic Salmonella antigenic formula 1,4,[5],12:i:-has been observed, which are divided into two lineages: Spanish and European. More common European lineage are characterized by antimicrobial resistance ASSuT and DT193 phagetype. In many European countries, these organisms have become one of the most commonly isolated serovars. The aim of this study was to analyze strains of Salmonella enterica subsp. enterica strains with antigenic formula 1,4,[5],12: i:-, isolated in Poland in 2007-2012 years. MATERIAL AND METHODS: The material for the study was 146 Salmonella strains initially identified as Salmonella antigenic formula 1,4,[5],12:i,-, isolated in 2007-2012 from clinical, food and animal samples. All strains has been reidentified according to the methodology routinely used in the laboratory. Serovar has been identified using classical method--slide agglutination for somatic and flagellar antigens and using Check&Trace Salmonella microarray. The fljB gene,fliB-fliA intergenic region, selected Salmonella Pathogenicity Islands' (SPIs) genes and spvC has been detected using PCR. For all strains PFGE typing with HindIII enzyme has been performed and phagetyping also. Moreover antimicrobial resistance has been evaluated by establishing of MIC according EUCAST recommendation. RESULTS: 110 (75%) strains were S. enterica 1,4,[5],12:i,-. In this group for 17 strains in Check&Trace Salmonella microarray result of identification "Salmonella Typhimurium" have been obtained. All 110 strains have 1000 bp size DNA fragment with IS200 sequence, characteristic for S. Typhimurium. Only in case of 4 strains fljB gene has been detected. All strains harbor avrA, ssaQ, mgtC and siiD genes. For 6 strains spvC gene has been not detected. 92 strains (83.6%) have been typed as DT193, but in case of 40 strains additional reaction with phage no 18 has been observed. For 104 (94.5%) strains resistance for at least one antimicrobial have been detected. Most frequent (44 strains - 40%) resistance pattern was ASSuT. Among all strains 11 pulsotypes, which group two or more strains have been recognized, which contain 37 strains. The rest of strains have unique REA-PFGE patterns. CONCLUSIONS: To this time epidemiological situation of S. enterisa 1,4,[5],12:i:- isolated from human cases in Poland has been not recognized. Results of current studies show, that studied strains belong to european non-spanish lineage of monophasic S. Typhimurium and problem of infection caused by those strains is unrecognized and probably increase.

[Occurrence and characterisation of aac(6')-Ib-cr gene encoding fluoroquinolone-modifying enzyme in clinical ciprofloxacin-resistant Enterobacteriaceae strains isolated in Poland]. / Wystepowanie i charakterystyka genu aac(6')-Ib-cr kodujacego enzym modyfikujacy fluorochinolony u opornych na ciprofloksacyne szczepów Enterobacteriaceae wyizolowanych od ludzi.

INTRODUCTION: The aac(6')-Ib-cr gene encodes a variant of aminoglycoside acetyltransferase that confers reduced susceptibility to hydrophilic fluoroquinolones such as ciprofloxacin and norfloxacin. AAC(6')-Ib-cr has two amino acid changes, Trp 102Arg and Asp179Tyr, which together are necessery and sufficient for the enzyme's ability to reduce the activity of fluoroquinolones, including ciprofloxacin and norfloxacin. The aim of this study was to evaluate the prevelance of aac(6')-Ib-cr determinant among 15 Enterobacteriaceae isolates randomly chosen from 215 fluorochinolone resistant strains recovered during the 6 months of 2010. METHODS: The aac(6')-Ib was detected by PCR. The presence of aac(6')-Ib-cr gene variant was futher identified by digestion with BseGI (BtsCI) and sequencing. RESULTS: 11/15 of the resistant (MIC CIP 2-1024 microg/ml) Enterobacteriaceae strains carried aac(6')-Ib-cr variant. CONCLUSION: This is the first study identifying the variant of aminoglycoside acetyltransferase determinant in Poland. Our results demonstrate that this enzym may be even more widespread than Qnr determinants among fluoroquinolone resistant Enterobacteriaceae in Poland.

[Prevalence of qnr genes in clinical Enterobacteriaceae non-susceptible to fluoroquinolone in Poland]. / Wystepowanie genów qnr u niewrazliwych na fluorochinolony paleczek z rodziny Enterobacteriaceae izolowanych z materialu klinicznego.

INTRODUCTION: Fluoroquinolone are broad-spectrum antimicrobial agents extensively used by physicians. This widespread use has been associated with increased level ofquinolone resistance strains, particularly in Enterobacteriaceae. Plasmid-mediated quinolone resistance (PMQR) including Qnr determinants with the potential for horizontal transfer confer to quinolone resistance. Plasmid harboring qnr genes may also encode extended-spectrum beta-lactamases (ESBLs) such as CTX-M, SHV and TEM type. The prevalence ofplasmid-mediated quinolone resistance (PMQR) determinants like qnrA, qnrB and qnrS was investigated in a collection of 215 Enterobacteriaceae strains with reduced susceptibility to fluoroquinolone. METHODS: The isolates (n=215) were collected from 1 March to 31 September, 2010 in a regular hospital in Warsaw, Poland. The resistance to nalidixic acid, norfloxacin and ciprofloxacin was determinated by twofold agar dilution method, while MICs of moxifloxacin were examined by using E-test. The prevalence of qnrA, qnrB, qnrS, blaCTX-M, blaSHV and blaiTEM was evaluated by PCR. All PCR-products for qnr were sequenced. The epidemiological relationship between positive isolates was studied by PFGE method. RESULTS: Eighteen isolates (8,3%) carried the qnr gene encoding the QnrA, QnrB or QnrS. The coexistence of both qnrA and qnrS genes was noted in one isolate of E. coli. The qnrB gene was the most common qnr type found. All the Qnr-producing strains were simultaneously resistant to naldixic acid and different - level non-susceptible fluoroquinolone (MIC CIP 1.5-1024 microg/ml). Most of qnr-positive strains (88.9%) were extended-spectrum beta-lactamase (ESBL) producers of CTX-M and TEM types predominantly. CONCLUSIONS: The present study highlights the wide spread of Qnr-like determinants in clinical Enterobacteriaceae non-susceptible to fluoroquinolone in Poland, with an association with the ESBL.

Molecular epidemiology of a household outbreak of Shiga-toxin-producing Escherichia coli in Poland due to secondary transmission of STEC O104:H4 from Germany.

We characterized two STEC O104 : H4 clinical isolates collected in Poland from a 7-year-old boy with haemolytic uraemic syndrome (HUS) and his nanny. This household outbreak began on 29 May 2011. Because of its time-frame, the outbreak was assumed to be part of the international STEC O104 : H4 outbreak that arose in Germany in May 2011. The two Polish isolates were Shiga-toxin-producing Escherichia coli (stx2 lpf) with enteroaggregative E. coli pathotype (aggR aap aggA), thereby sharing the unique virulence properties of the epidemic STEC O104 : H4 strain from the international outbreak. The Polish isolates were multi-drug resistant and carried bla(TEM), strA, strB, tetA, sul1 and sul2 markers together with the bla(CTX-M-15) gene for CTX-M-15 extended-spectrum ß-lactamase. PFGE patterns and plasmid profiles of the Polish isolates and the epidemic STEC O104 : H4 strain corresponded closely. This finding suggested an epidemiological link between the Polish STEC O104 : H4 isolates and the international outbreak. Retrospective serological investigations proved person-to-person transmission of the epidemic STEC O104 : H4 strain from a father who had visited Dortmund, Germany, to his 7-year-old son in Gizycko, Poland. To the best of our knowledge, this is the first report of household transmission of Shiga-toxin-producing E. coli in Poland.

[Molecular investigation of enteroaggregative, shiga toxin-producing E. coli O104:H4 isolated in Poland during the recent international outbreak--characteristic of epidemic clone]. / Ognisko wywolane przez enteroagregacyjny i werotoksyczny szczep Escherichia coli O104:H4 w Europie--postepowanie diagnostyczne w Polsce oraz charakterystyka szczepu.

Since early May 2011 a large food-borne outbreak caused by E. coli O104:H4 affected Germany then spread over 13 European countries, U.S.A. and Canada. The outbreak strain was found to possess an unusual combination of enteroaggregative E. coli pathotype with StxII. In this report we described the molecular investigation of epidemic clone in Poland during the international outbreak. We confirmed three cases of E. coli O104:H4 infections. The molecular characteristics of the Polish E. coli O104:H4 isolates including virulence profile, antimicrobial resistance, PFGE and plasmids profiles were corresponded with Germany outbreak strains.