[Analysis of laboratory tests results for Salmonella infections performed since 2008 to 2014 in Poland]. / Analiza wyników badan laboratoryjnych w kierunku zakazen wywolanych przez paleczki Salmonella w latach 2008-2014 w Polsce.

INTRODUCTION: Next to official surveillance system in Poland, established by Public Statistic Act in 1995, other surveillance data collection system exists - historically connected with Ministry of Health Decree, which is not in force from 1995 y. Data in this system comes from National Sanitary Inspection laboratories and contains results of clinical samples examinations for Salmonella and Shigella presence. MATERIAL AND METHODS: Analysed data were collected since 2007 to 2014 y. Database has been created, which contains data - results of Salmonella / Shigella (SS) detection in faecal samples, rectal swabs, urine samples, blood samples collected from patients, people exposed in outbreaks, Salmonella carriers, food workers and other people. RESULTS: The number of performed tests for SS in 2014 is 447033. Systematic decrease of this number has been observed since 2008. Most common material tested for SS was stool samples, then rectal swabs. Number of Salmonella identification going down each year during analysed time. The highest decrease in group of sick people has been observed. Most common serotype in 2008-2014 years was S. Enteritidis, then S. Typhimurium. Only in 2010 y. second most common serotype was S. Mbandaka. In 2014 y. highest number of S. enterica 1,4,[5],12:i:- has been observed (37 cases). CONCLUSIONS: The reason of decreased number of performed SS test and positive Salmonella results is unclear. One of theories is decreased level of quality of SS diagnostic. From the other hand, decreased number of false-positive Salmonella identification outside of National Sanitary Inspection laboratories is observed. This demonstrates improving quality of Salmo nella diagnostic in laboratories performing such tests. High disproportion between number of Salmonella cases in described database and official notification system is observed. It comes from, that both systems collect different data. Both systems could exist independently and could supplement each-other.

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.

[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.

[Salmonella interactions with intestinal flora and antibiotics influence on these pathogens infections]. / Interakcje paleczek Salmonella z flora jelitowa oraz wplyw stosowania antybiotyków na przebieg zakazenia tymi drobnoustrojami.

Human digestive system is colonized by a large number of bacteria, estimated to 10(6) - 10(12) per one gram. Those bacteria through a network of interactions and interdependencies, are integrated superorganism. The intestinal flora is a very important element in host's defense against infections of the gastrointestinal tract, caused by for example Salmonella. Therefore, this bacteria have evolved a number of mechanisms, which adapt pathogen to the conditions of the gastrointestinal tract, and on the other hand to the change this environment, for easier colonization and internalization into host cells. One of elements of mentioned above interactions are antimicrobial peptides produced by host's Paneths cells, which have antimicrobial feature. Salmonella mostly are resistant for those peptides, moreover they can stimulate AMPs production for increasing their abilities in competition for ecological niche. In case of Salmonella quorum sensing mechanism was also identified. It allows for recognition of other bacteria presence, which stimulate Salmonella for higher expression of SPI-1, SPI-4 genes. These genes encoded proteins are involved in many host-pathogens interaction, inter alia inflammatory induction. Using of antibiotics in case of Salmonella infections always cause dramatic changes in intestinal flora compositions, which facilitate Salmonella internalizations to host's cells and sometimes could even stimulate to this process. Antibiotic treatment could also cause increase of antimicrobial resistance. Also antibiotics influence on Salmonella carriage was confirmed. Moreover antibiotics could cause super-shedder phenotype, what was detected on streptomycin-treated mice with Salmonella carriage.

Impact of antibiotics on the intestinal microbiota and on the treatment of Shiga-toxin-producing Escherichia coli and Salmonella infections.

This review evaluates the current literature based on the impact of antibiotics on the intestinal microbiota and the critical role of intestinal bacteria in controlling infection and subsequent clinical disease caused by STEC and Salmonella, and the transmissibility of these important pathogens.A number of studies have indicated that antibiotic therapy could result in unexpected changes in the clinical picture of disease. This is observed, for example, in the case of infections associated with Shiga-toxin-producing Escherichia coli (STEC), when antibiotics used in treatment of the disease may increase the risk of hemolytic uremic syndrome (HUS) and thus fatal outcomes. In the case of such infections, treatment with antibiotics is usually discouraged. The use of antibiotics could cause also undesirable changes in the intestinal microbial flora and prolonged pathogen shedding, which is observed in the case of Salmonella infections. Inappropriate antibiotic therapy can result in Salmonella remaining in the host's cells (intracellular) and thus resulting in further asymptomatic carriage and a further complication is the development of resistance.

[Salmonella multiphasic flagellar antigen]. / Wielofazowosc antygenów rzeskowych u paleczek Salmonella.

In the Salmonella antigenic pattern, more than one phase of flagellar antigen is observed. The phase of flagellar antigen depends of the gene which encodes the protein building the filament of flagella. The fliC gene encodes the 1st phase of flagellar antigen and the fljB gene encodes the 2nd phase of flagellar antigen. The third phase of flagellar antigen is encoded by one of the genes localized on the plasmid. Expression of the fljB gene (part of the hinfljBA operon) is regulated by a mechanism of DNA fragment sequence inversion. The hin gene, which encodes Hin invertase, flanked by two regions - hixL and hixR - is inverted by Hin invertase together with Fis protein. This process turns on or turns off of the hinfljBA operon. When this operon is turned on, FljB protein is produced (structural protein of flagella filament), and also FljA protein, which is a transcriptional repressor of the fliC gene. This means that one Salmonella cell could have only one phase flagellar antigen--1st or 2nd phase. Sometimes, due to mutation in one of the mentioned genes, naturally diphasic Salmonella strains have the ability to produce only one phase of flagellar antigen. Mostly monophasic Salmonella with an active fliC gene are observed. In recent years such a strain, Salmonella enterica with the antigenic formula 1,4,[5],12: i: -, is one of the most often isolated strains from human cases in many European countries.

Validation of direct plating of a stool sample as a method for Listeria monocytogenes detection.

The aim of current studies was to validate the direct plating of a stool sample for Listeria monocytogenes detection, using selective medium Palcam agar with Palcam selective supplement. Validation was performed using stool samples collected from healthy humans inoculated with Listeria sp. strains. Stool samples were frozen to determine the influence of freezing on method robustness. The presented research defines the Listeria monocytogenes limit of detection (LOD) as 10(3) cfu/g of stools for fresh and frozen samples. Repeatability and reproducibility of the method has been confirmed using statistical methods. We show the effectiveness of direct plating of stool samples on Palcam agar with Palcam selective supplement collected for Listeria monocytogenes detection. This method could be useful for this pathogen detection in stool samples collected from patients with diarrhoea.

[Genotyping by REA-PFGE of Listeria monocytogenes isolated from clinical, environmental and food samples]. / Genotypowanie metoda REA-PFGE paleczek Listeria monocytogenes izolowanych z próbek materialu klinicznego, próbek srodowiskowych i próbek zywnosci.

Listeria monocytogenes strains isolated from clinical food and environmental samples were genotyped by Restriction Enzyme Analysis with Pulsed Field Gel Electrophoresis (REA-PFGE) using ApaI and AscI enzymes according to PulseNet Europe procedure. Analysis of DNA fragments profiles obtained by AscI digestion demonstrated presence of 62 REA-PFGE profiles grouped in 2 lineages (FI, FII). Diversity of strains source among both lineages was observed. Statistical analysis showed, that strains isolated from clinical samples more frequently are included to lineage FI, then lineage FII. Non-clinical strains were more frequently included to lineage FII. Combined analysis of REA-PFGE profiles for ApaI and AscI enzymes showed 8 unique pulsotypes characteristic for two or more L. monocytogenes isolates. Moreover researched L. monocytogenes strains were analyzed by multiplex-PCR according Doumith et al methodology. PCR-group 4B was most frequent among strains isolated from clinical samples. Correlation between PCR-group and pulsotype was observed only in few cases.

[Genotyping methods of Listeria monocytogenes]. / Genetyczne metody typowania paleczek Listeria monocytogenes.

Listeriosis could occur as sporadic case as well as epidemic outbreak. For that reason it is important to subtype the Listeria monocytogenes strains using the fastest and most effective method. Genetic techniques are the most effective methods for differentiation of this species. Basing on latest available data eight selected subtyping genetic methods of Listeria monocytogenes strains were characterized: REA-PFGE, rybotyping, AFLP, PCR-RFLP, multiplex PCR, and methods from RAPD groups, such as AP-PCR, rep-PCR and ERIC -PCR. Analysis of available information allowed to assess that the most effective methods for L. monocytogenes typing are REA-PFGE and rybotyping. However, the rep-PCR and ERIC-PCR methods could be used in the routine Listeria monocytogenes diagnosis.

[Listeria monocytogenes in human infections]. / Paleczki Listeria monocytogenes w zakazeniach ludzi.

The Listeria genus is distinguished into six species from which just one--Listeria monocytogenes is pathogenic for humans. The main route of acquisition of Listeria is through the ingestion of contaminated food products. An important element of the L. monocytogenes pathogenesis infection is affiliation with high-risk group of immunocompromised patients, infants or pregnant women, who infected by this microorganism can lead to miscarriage. Listeriosis can appear in the form of sepsis, infection of the nervous system or local abscesses. Another form of listeriosis is gastrointestinal tract infection--noticed in case of food poisoning outbreak.

[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.

[Evaluation of PremiTest Salmonella kit for identification of not-typable by conventional methods Salmonella]. / Ocena przydatnosci testu PremiTest Salmonella do identyfikacji paleczek Salmonella nietypujacych sie metodami klasycznymi.

Despite the downward trend, Salmonella is still one of the most important bacterial intestinal infections agents. For example, in 2007 y. in Poland over 14 thousands human salmonelosis cases were notified, in 2008 y.--over 10 thousands. Among all Salmonella isolated from human source, most common (more then 80%) are two serotypes--S. Enteritidis and S. Typhimurium, but every year the number of non-typable (because of the lack I or II phase flagellar antigens, autoagglutinative or non-motility properties) Salmonella is growing. This work describes the results of the evaluation of commercially available kit PremiTest Salmonella (DSM, Netherlands), which uses the microarray hybridization in ArrayTube, for serological identification of non-typable Salmonella strains. All 37 researched strains were submitted to the Department of Bacteriology in 2007-2008 y, were reidentified according to standard operating procedures and serotyped by slide agglutination for somatic and flagellar antigens if it was possible. All strains were tested using the PremiTest Salmonella Kit, according to manufacturer instructions. In 21 cases (56%) full identification were achieved, in 5 cases (14%) additional tests were required for precise identification (Salmonella Choleraesuis or Paratyphi C, what was detailed using examination of additional biochemical features), 5 strains (14%) achieved an incomplete identification (three of them--S. diarizonae were confirmed in National Reference Laboratory for Salmonella) and 6 cases (16%) were not identified at all. The total number of recognized strains is 30 (81%). The results of present studies show, that PremiTest Salmonella Kit is useful for non-typable Salmonella identification.

Virulotyping and antimicrobial resistance typing of Salmonella enterica serovars relevant to human health in Europe.

The combination of virulence gene and antimicrobial resistance gene typing using DNA arrays is a recently developed genomics-based approach to bacterial molecular epidemiology. We have now applied this technology to 523 Salmonella enterica subsp. enterica strains collected from various host sources and public health and veterinary institutes across nine European countries. The strain set included the five predominant Salmonella serovars isolated in Europe (Enteritidis, Typhimurium, Infantis, Virchow, and Hadar). Initially, these strains were screened for 10 potential virulence factors (avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC) by polymerase chain reaction. The results indicated that only 14 profiles comprising these genes (virulotypes) were observed throughout Europe. Moreover, most of these virulotypes were restricted to only one (n = 9) or two (n = 4) serovars. The data also indicated that the virulotype did not vary significantly with host source or geographical location. Subsequently, a representative subset of 77 strains was investigated using a microarray designed to detect 102 virulence and 49 resistance determinants. The results confirmed and extended the previous observations using the virulo-polymerase chain reaction screen. Strains belonging to the same serovar grouped together, indicating that the broader virulence-associated gene complement corresponded with the serovar. There were, however, some differences in the virulence gene profiles between strains belonging to an individual serovar. This variation occurred primarily within those virulence genes that were prophage encoded, in fimbrial clusters or in the virulence plasmid. It seems likely that such changes enable Salmonella to adapt to different environmental conditions, which might be reflected in serovar-specific ecology. In this strain subset a number of resistance genes were detected and were serovar restricted to a varying degree. Once again the profiles of those genes encoding resistance were similar or the same for each serovar in all hosts and countries investigated.

[Genoserotyping of Listeria monocytogenes in multiplex PCR]. / Serologiczne typowanie i genoserotypowanie paleczek Listeria monocytogenes izolowanych z próbek materialu klinicznego, próbek zywnosci i próbek srodowiskowych.

Listeria monocytogenes infection affects people throughout the all world, most often causing gastrointestinal disturbances, less inflammation of the central nervous system (meningitis, meningoencephalitis, absceses), sepsis, endocarditis and dispersed focal inflammation. Listeria monocytogenes infection in pregnant woman can lead also to premature birth or abortion. Listeriosis is a serious problem because of the high mortality in case of generalized infection, which can reach up to 20%. Listeria strains could be identified by determination of biochemical features--characteristic for each Listeria species. Biochemically identified strains could be serotyped using Seeliger method. Many authors developed a molecular biology-based methods of Listeria serotype determination The Doumith's method divide all Listeria monocytogenes serotypes for five genoserogroups. The purpose of this study was to determine the genoserogroups of Listeria monocytogenes strains and compare those results with results obtained by classical serotyping by Seeliger method. To this work 90 Listeria strains were used: 75 Listeria monocytogenes strains from clinical, food and environmental samples and 15 reference strains from Pasteur Institute collection and National Institute of Public Health--National Institute of Hygiene collection. All strains were serotyped using liquid stable antisera for determination of O and H Listeria monocytogenes antigens (Mast Diagnostic, UK). Multiplex PCR for Listeria monocytogenes serovars differentiation were performed according to Doumith et al procedure. The genoserogroup obtained from multiplex PCR of 10 Listeria monocytogenes reference strains were compatible with real serotype. In case of non-Listeria monocytogenes reference strains in multiplex-PCR, they were identified as non-monocytogenes. In group of 75 non-reference strains isolated from human, food and environmental sources, 34 belonged to genoserogroup I.1, two strains to genoserogroup I.2, 11 strains to genoserogroup II.1, 21 strains belonged to genoserogroup II.2 and 9 strains to genoserogroup III.

[Antimicrobial susceptibility of Yersinia enterocolitica and Yersinia pseudotuberculosis strains isolated from humans in Poland during 2004-2009]. / Ocena wrazliwosci na wybrane antybiotyki paleczek Yersinia enterocolitica i Yersinia pseudotuberculosis izolowanych z próbek materialu klinicznego w Polsce w latach 2004-2009.

The number of yersiniosis has increased in the last few years in Poland, especially an increase of Yersinia enterocolitica bioserotype 1B/O:8 infections was observed. From 2004 to 2009 265 of Y. enterocolitica 4/O:3, 108 of Y. enterocolitica 1B/O:8, 8 of Y. enterocolitica 2/O:9 and 4 of Y. pseudotuberculosis clinical isolates were collected. To obtain basic data for resistance monitoring purpose 385 Yersinia strains were tested by standard disc diffusion method for their susceptibilities to 12 antimicrobial agents. In addition beta-lactamase (enzyme A) inhibition assays were undertaken with ticarcillin and clavulanic acid and beta-lactamase (enzyme B) induction tests were perfonned with imipenem as the inducer for 135 strains. The present study demonstrated a high susceptibility of clinical strains to most of the tested antibiotics with the exception of ampicillin, ticarcillin and streptomycin. No strains were resistant to third-generation cephalosporins, fluoroquinolones, gentamicin and tetracyclin. Less than 10% isolates were resistant to amoxicillin with clavulanic acid (except--all Y. enterocolitica 2/O:9 strains were resistant), sulfonamide, trimetoprim/sulfamethoxazole and chloramphenicol. Four isolates of Y. enterocolitica 4/O:3 and one Y. enterocolitica 2/O:9 was multidrug resistant (MDR). Detection of enzyme A by disc diffusion in all tested strains, with the exception of the three Y. pseudotubeculosis I isolates, was highly reliable but results of enzyme B detection by the disc diffusion test were, especially for Y. enterocolitica 1B/O:8, faced with the difficulties.

[Evaluation of selected Listeria monocytogenes genotyping methods]. / Ocena przydatnosci wybranych metod genotypowania paleczek Listeria monocytogenes.

Listeria monocytogenes infections are found in Poland and all world as sporadic episodes and large outbreaks of human illness as well, where bacteria-contaminated food is the source of infection. Genotyping of strains isolated from outbreak is one of many other stages of epidemiologic investigation. In case of L. monocytogenes pulse-field gel electrophoresis (PFGE) - "golden standard" of genotyping and also ERIC-PCR and rep-PCR could be used. The goal of this study was to evaluate the usefulness of methods: PFGE, ERIC-PCR and rep-PCR to Listeria monocytogenes strains genotyping. Moreover estimation of L. monocytogenes strains isolated in Poland during 2003 - 2006 years genetic diversity was performed. In performed studies, 44 strains of L. monocytogenes from PZH strains collection from 2003 - 2006 period were used. Standard strains of L. monocytogenes, L. ivanovii, L. welschimeri and L. innocua were used also. All those strains were genotyped using following methods: PFGE with ApaI and AscI restrictases and random amplification of polymorphic DNA fragments (ERIC-PCR, rep-PCR). Obtained results were analyzed using Tenover method and specialistic software - Gene Profiler (Scan Analytics, USA). In every analysis, clonal groups were obtained - using computer analysis as well as Tenover method. For PFGE with ApaI and AscI six clonal groups were obtained in each analysis. For ERIC-PCR and rep-PCR four clonal groups were obtained (every group was different except one, where four the same strains were included). Taking into account such factors, as efficiency of strains differentiation, work consumption and costs of procedure performing, for quick application, especially in local laboratories, the best genotyping technique is rep-PCR and ERIC-PCR a little less. On account of appreciable costs of PFGE analysis, this method should be used as reference technique.

[Influence of spv plasmid genes group in Salmonella Enteritidis virulence for chickens. I. Occurrence of spv plasmid genes group in Salmonella Enteritidis large virulence plasmid]. / Znaczenie plazmidowego zgrupowania genów spv w chorobotwórczosci paleczek Salmonella Enteritidis dla kur. I. Wystepowanie zgrupowania genów spv w duzych plazmidach zjadliwosci paleczek Salmonella Enteritidis.

Many Salmonella Enteritidis virulence factors are encoded by genes localized on plasmids, especially large virulence plasmid, in highly conserved fragment, they create spv plasmid gene group. The aims of realized researches were spv genes occurrence evaluation and composition analysis among Salmonella Enteritidis strains caused infection in chickens. Researches were realized on 107 isolates, where in every cases large virulence plasmid 59 kbp size were detected. Specific nucleotides sequences of spv genes (spvRABCD) were detected in 47.7% of isolates. In the rest of examined bacteria spv genes occurred variably. Most often extreme genes of spv group, like spvR and spvD were absent, what could indicate that factors encoded by them are not most important for Salmonella Enteritidis live and their expressed virulence.