Genetic diversity of human strains of Listeria monocytogenes in the Czech Republic in 2016-2020.

STUDY AIM: To determine the genetic diversity of human isolates of Listeria monocytogenes obtained in 2016-2020 from clinical laboratories in various locations of the Czech Republic with a focus on their possible epidemic links and virulence using whole genome sequencing data. METHODS: A total of 102 human L. monocytogenes isolates, serotyped by slide agglutination in combination with multiplex PCR serotyping, were used in this study. Whole genome sequencing was performed retrospectively, and based on the obtained data, the clonal relatedness of the tested strains and the presence of virulence genes were assessed using the Ridom SeqSphere+ software. RESULTS: In 2016-2020, 102 human isolates of L. monocytogenes were characterized, which represented 65% of all cases of listeriosis reported to the ISIN/EPIDAT systems in the Czech Republic in the monitored period. Serotype 1/2a (57%) was dominant, followed by serotype 4b (30%). Strains of serotype 1/2b (12%) and 1/2c (1%) were rarely detected. Based on the analysis of whole genome sequencing data, the strains were assigned to 26 clonal complexes and 27 sequence types. The cgMLST (core genome Multi-Locus Sequence Typing) analysis revealed four clusters of more than three strains, showing high relatedness (differences up to 10 alleles) with a possible epidemic link. The presence of all key virulence genes was confirmed in all strains. Only three strains (of serotypes 1/2a, 1/2b, and 1/2c) carried a point mutation in the inlA gene responsible for the expression of truncated internalin A protein, which is involved in the mechanism of intestinal barrier crossing by L. monocytogenes. CONCLUSION: Molecular epidemiology based on whole genome sequencing is an effective tool to study the population structure of L. monocytogenes strains. This study found high heterogeneity of human L. monocytogenes strains, especially for serotype 1/2a, dominant in the Czech Republic. Several clusters with a possible epidemic link have been identified, and their occurrence will be further monitored.

An outbreak of listeriosis linked to turkey meat products in the Czech Republic, 2012-2016.

Since 2012-2016 an increased number of listeriosis cases, especially from one region of the Czech Republic, were observed. Most of them were caused by strains of serotype 1/2a, clonal complex 8, indistinguishable by pulsed-field gel electrophoresis. Twenty-six human cases were reported, including two neonatal cases in twins. Three cases were fatal. The typing of Listeria monocytogenes isolates from food enabled to confirm a turkey meat delicatessen as the vehicle of infection for this local outbreak in the Moravian-Silesian Region. The food strains belonging to identical pulsotype were isolated from ready-to-eat turkey meat products packaged by the same producer between 2012 and 2016. This fact confirms that the described L. monocytogenes outbreak strain probably persisted in the environment of the aforementioned food-processing plant over several years. Whole-genome sequencing confirmed a very close relationship (zero to seven different alleles) between isolates from humans, foods and swabs from the environment of the food-processing plant under investigation.

Properties of Staphylococcus aureus strains from food processing staff.

AIM: The food processing staff may act as a reservoir of virulent strains of Staphylococcus aureus and contribute to their transmission to foods. The aim of this study was to assess the occurrence and properties of S. aureus in cheese factory staff. MATERIAL AND METHODS: Throat and hand swabs collected from the staff of three different cheese factories were tested. The obtained isolates were characterized on the basis of detection of virulence factors, antimicrobial resistance testing, spa typing, and macrorestriction analysis. RESULTS: S. aureus was detected in 58% of the hand swab samples (7/12) and in 47% (17/36) of the throat swab samples. No methicillin-resistant S. aureus was detected in the throat or on the hands of the food processing staff. Strains carrying genes responsible for the production of enterotoxins (58%) and/or toxic shock syndrome toxin (25%) were recovered from employees of all three premises. One throat swab isolate was positive for the gene encoding production of exfoliatin A. There was no clonal relationship between S. aureus strains isolated from the throat and hands, which suggests possible contamination of the employees' hands arising from the manufacturing environment. CONCLUSION: Good compliance with hygiene guidelines (washing and disinfecting hands and the environment regularly, using gloves and masks, etc.) is a necessary tool for reducing the risk of S. aureus spread by the employees working in the food industry.

[Molecular-epidemiological characteristics and diversity of Listeria monocytogenes in the human population in the Czech Republic in 2013-2016]. / Molekulárne epidemiologická charakteristika a diverzita Listeria monocytogenes v humánní populaci Ceské republiky v letech 2013-2016.

Listeria monocytogenes is the cause of a serious foodborne infection with a high fatality rate. The aim of this study was to assess the diversity of human isolates of L. monocytogenes recovered in the Czech Republic from 2013 to 2016 by molecular biological methods. Most cases of listeriosis were caused by strains of serotype 1/2a (58 %) from clonal complex CC8 (28 %) and serotype 4b (28 %) from CC6 (16 %). The results of macrorestriction analysis confirmed primarily the occurrence of sporadic cases of listeriosis. A local outbreak of listeriosis was reported in the Moravian-Silesian Region. Real-time molecular subtyping methods have proved helpful in the investigation of Listeria outbreaks.

Virulence and antibiotic resistance genes in Campylobacter spp. in the Czech Republic.

OBJECTIVE: Thermotolerant species of the genus Campy-lobacter are the important agents causing human foodborne infections throughout the world. The aims of this study were to evaluate the presence of nine putative virulence genes in Campylobacter spp. isolated from patients and from foods (poultry meat, pork liver), to determine the resistance of Campylobacter isolates to eight antibiotic agents and to detect four resistance genes.Matherial and methods: The presence of the virulence genes cdtA, cdtB, cdtC, virB11, ciaB, wlaN, iam, dnaJ and racR was detected by polymerase chain reaction (PCR) in 94 Campylobacter spp. isolates from humans and 123 campylobacters from foods. The phenotypic resistance to selected antimicrobial agents was tested with microdilution method in 82 human isolates and 91 food isolates. The isolates with antibiograms were tested for the presence of blaOXA-61, tet(O), aph-3-1 and cmeB genes by PCR with specific primers. RESULTS: In both human and food C. jejuni isolates the preva-lence of the studied virulence genes, especially dnaJ, racR, ciaB genes and the toxigenic genes cdtA, cdtB, cdtC, was considerably higher than in C. coli isolates. The only exception was the iam gene identified in only C. coli. The tested isolates of both C. jejuni and C. coli were highly resistant to quinolone antibiotics. Additionally, C. coli was also more resistant to erythromycin, streptomycin and, in case of isolates from pork liver, to tetracycline. High prevalence rates of genes encoding antibiotic resistance was noted for the blaOXA-61 and tet(O) genes in both Campylobacter species. CONCLUSIONS: The presented study is the first to assess the presence of genes for virulence and resistance to antibiotics in thermotolerant Campylobacter spp. isolated from humans and foods in the Czech Republic. The resistance of Campylobacter isolates to eight antibiotic agents was also assessed. The prevalence of genes responsible for virulence and resistance is rather varied in thermotolerant Campylobacter spp.

Virulence factors and resistance to antimicrobials in Listeria monocytogenes serotype 1/2c isolated from food.

AIMS: The aim of this study was to assess the potential risk posed to the human population by the presence of Listeria monocytogenes serotype 1/2c in food based on the characterization of virulence factors of Listeria involved in the invasion of host cells and sensitivity to antimicrobial agents. METHODS AND RESULTS: In addition to sequencing of the inlA and inlB genes, the presence of genes lapB, aut, fbpA, ami, vip and llsX was tested. A premature stop codon (PMSC) in the inlA gene was detected in all tested strains of serotype 1/2c and, concurrently, two novel PMSC mutation types were identified. However, neither PMSC in the inlB gene nor deletion of the lapB, aut, fbpA, ami and vip genes were found in any of the strains. The presence of the llsX gene was not confirmed. Even though all L. monocytogenes strains showed sensitivity to the tested antimicrobials on the basis of their phenotype, sequencing revealed the presence of IS1542 insertion in the inlA gene, indicating the possibility of sharing of mobile genetic elements associated with antimicrobial resistance among strains. CONCLUSIONS: Other than the presence of PMSCs in the inlA gene, no PMSC in inlB or deletion of other factors linked to the invasiveness of listeria were detected. Tested strains showed sensitivity to antibiotics used in the therapy of listeriosis. SIGNIFICANCE AND IMPACT OF THE STUDY: Strains of L. monocytogenes serotype 1/2c typically carry a PMSC in the inlA gene, but these strains still represent a potential threat to public health. The possibility of transfer of IS1542, associated with resistance to vancomycin, between enterococci and Listeria spp. was revealed.

[Diversity of human Salmonella isolates in the South Moravian Region in 2009-2012]. / Diverzita humánních izolátu salmonel v Jihomoravském kraji v letech 2009-2012.

STUDY OBJECTIVE: To characterize the diversity of human Salmonella isolates from one of the administrative regions of the Czech Republic in a time horizon of four years using different typing methods. MATERIAL AND METHODS: The following phenotyping methods were used: serotyping, phage typing, and the disk diffusion method for antimicrobial susceptibility testing. Genotyping was performed using DNA macrorestriction analysis resolved by pulsed-field gel electrophoresis (PFGE). RESULTS: The most common serotypes involved in human cases of salmonellosis in the South Moravian Region were Salmonella Enteritidis, Salmonella Typhimurium, and monophasic Salmonella 4,[5],12:i:-. Phage typing revealed the predominance of phage type 8 in S. Enteritidis and changes in phage types of S. Typhimurium over years. The resistance levels in S. Typhimurium, monophasic Salmonella, and some other serotypes give increasing cause for concern about multidrug resistant strains as human pathogens. The study isolates were assigned to the complexes implicated in both local and international outbreaks. Isolates of serotype 9,12:l,v:- were identified for the first time in the Czech Republic. CONCLUSIONS: The diversity of the study isolates from 2009-2012 collected in the South Moravian Region suggests a high heterogeneity of the sources of human salmonellosis. However, the detection of several cases likely to be epidemiologically linked points out the need for careful typing of human isolates and their comparison with the strains of non-human origin. Higher resistance levels in some serotypes is behind the need for the surveillance of multidrug resistant strains.

[Occurrence and characteristic of Staphylococcus aureus in pork meat]. / Výskyt a vlastnosti baktérií Staphylococcus aureus ve veprovém mase.

This study was conducted to evaluate the contamination of raw pork meat with Staphylococcus aureus in retail market and their ability to participate in the creation of foodborne intoxication. Strains were characterized by genotypic traits. Of the 197 samples examined 43 (21.8%) were found to be positive for the presence of S. aureus. Toxigennic properties were found in 21 (48.8%) isolates, the major enterotoxigennic gene found was seh (81%). All the S. aureus isolates were screened for resistance to antimicrobial agents by disk diffusion method and for mecA gene encoding resistance to meticillin. Two of the strains isolated in this study harboured the mecA gene (MRSA). The determination of sequence type 398 (ST398) has been confirmed in both MRSA isolates.

[Nosocomial transmission of listeriosis]. / Nozokomiální prenos listeriózy.

Listeriosis is a severe and possibly fatal disease. Its incidence in the Czech Republic is low, with the most common vehicles of transmission being cheese, pastes, meat products, and salads. Nosocomial transmission is rare in the Czech Republic. Phylogenetic analysis and genotyping of isolated Listeria strains are very helpful in identifying the source of infection. A careful adherence to the preventive anti-epidemic measures in the health care institutions is still critically important.

[Prevalence and characteristics of thermotolerant Campylobacter spp. in the human food chain]. / Výskyt a charakteristika termotolerantních kampylobakteru v potravinovém retezci cloveka.

OBJECTIVES: To monitor the prevalence of Campylobacter spp. in poultry in slaughterhouses, poultry and pork liver at retail, and cows milk in Moravia. To determine the resistance of animal isolates to selected antibiotics; and to compare it with an antibiogram of human strains. MATERIAL AND METHODS: Throughout the year 2013, the following samples were collected in the South Moravian and Olomouc Regions: mixed samples of broiler cecal contents in slaughterhouses, fresh and frozen chickens and pork liver at retail, and raw cows milk from vending machines. The samples were both qualitatively and quantitatively analyzed for the presence of Campylobacter spp. The isolates recovered were tested for resistance to antibiotics. For comparison, antimicrobial resistance was also studied in human isolates from the same regions. RESULTS: A total of 41.8% of the tested food samples were found to contain Campylobacter spp.. The most contaminated (73.2%) were fresh chickens. Campylobacter spp. were not detected in raw cows milk samples. The isolates showed high levels of resistance to quinolone antibiotics and, in the case of C. coli, also to tetracycline and streptomycin. CONCLUSION: The studied commodities were frequently contaminated with Campylobacter spp. The levels of contamination (in CFU/g) varied between commodities and so, evidently, did the real risk for human infections. When antibiotic therapy is needed, quinolone antibiotics cannot be used. Adherence to high standards of consumer safe food handling is crucial for the prevention of diseases.

Salt stress-induced invasiveness of major Listeria monocytogenes serotypes.

UNLABELLED: Of the 13 serotypes, 4b serotype strains are responsible for the majority of recorded invasive listeriosis outbreaks, although some recent listeriosis outbreaks have been attributed to strains of serotypes 1/2a and 1/2b. Virulence and response to osmotic stress in 41 Listeria monocytogenes strains representing serotypes 1/2a, 1/2b and 4b was investigated. It was found that serotype 4b and 1/2b strains exhibited highest invasion efficiency and formed largest plaques in HT-29 cell monolayer. Invasiveness in response to 10-min exposure to 0·3 mol l⁻¹ NaCl was the highest in serotype 4b strains. We demonstrated that 4b serotype L. monocytogenes strains not only have the greatest pathogenic potential but also are the most invasive in response to salt stress. SIGNIFICANCE AND IMPACT OF THE STUDY: Listeria monocytogenes 4b serotype strains are responsible for the majority of recorded invasive listeriosis outbreaks. We showed that strains of serotype 4b are not only the most virulent L. monocytogenes strains but also have the best capacity to enhance their invasiveness in response to salt stress. Our results suggest possession of effective stress response mechanisms of 4b serotype strains, which may contribute to the high infection potential of this subpopulation.

[Occurrence and typing of Listeria monocytogenes isolated from raw cow's milk collected on farms and from vending machines]. / Výskyt a typizace izolátu Listeria monocytogenes v syrovém kravském mléce odebraném na farmách a z prodejních automatu.

BACKGROUND: Evaluation of the incidence and characteristics of L. monocytogenes in samples of raw cow's milk collected on farms (bulk tank milk samples) and from vending machines. MATERIAL AND METHODS: Detection of L. monocytogenes and enumeration were carried out according to EN/ISO 11290--1, 2. Strains were characterised by serotyping and macrorestriction analysis using pulsed-field gel electrophoresis. RESULTS: The presence of L. monocytogenes was detected in 3,2 % (11/346) of bulk tank milk samples and 1,8 % (4/219) samples of raw cow's milk from vending machines. Findings of L. monocytogenes in raw milk were sporadic. Only on one farm strains of L. monocytogenes were detected repeatedly. Thirteen strains of L. monocytogenes belonged to serotype 1/2a, two strains to serotype 1/2b and one to serotype 4b. Macrorestriction analysis revealed considerable heterogeinity of profiles, with nine different pulsotypes being detected. Pulsotype 711 was the most frequent. This pulsotype was found on three different farms. CONCLUSIONS: The incidence of L. monocytogenes in raw cow's milk is relatively low in the Czech Republic. The results confirmed that some clones of L. monocytogenes from raw milk are identical with food and human strains.

Cases of Salmonella Urbana in Finland, the Czech Republic and Latvia, January-February 2010.

A cluster of 14 cases of Salmonella Urbana cases in Finland, the Czech Republic and Latvia were identified in January-February, 2010. The majority of cases (11) were male and children under 16 years of age. The investigation is currently ongoing and comparison of pulsed-field gel electrophoresis (PFGE) profiles of the isolates suggests that the cases may have a common source of infection.

Update: Multinational listeriosis outbreak due to 'Quargel', a sour milk curd cheese, caused by two different L. monocytogenes serotype 1/2a strains, 2009-2010.

We previously reported an outbreak of listeriosis in Austria and Germany due to consumption of Quargel cheese. It comprised 14 cases (including five fatalities) infected by a serotype 1/2a Listeria monocytogenes (clone 1), with onset of illness from June 2009 to January 2010. A second strain of L. monocytogenes serotype 1/2a (clone 2) spread by this product could be linked to further 13 cases in Austria (two fatal), six in Germany (one fatal) and one case in the Czech Republic, with onset of disease from December 2009 to end of February 2010.

[Prevalence and characteristics of Salmonella in selected localities of the Czech Republic--comparison of epidemiological and laboratory data]. / Výskyt a charakteristika salmonel ve vybraných lokalitách Ceské republiky--porovnání epidemiologických a laboratorních dat.

UNLABELLED: The study objectives were to characterize in detail human isolates of Salmonella in selected localities of the Czech Republic and to consider the actual epidemiological situation in the country based on the comparison with the reported cases of salmonellosis (source EPIDAT). METHODS: The source of the national epidemiological data was EPIDAT (communicable disease notification system). Laboratory data was obtained by analysis of Salmonella isolates from five clinical laboratories in Bohemia and Moravia. The isolates were collected at regular intervals from January to August and their numbers were indicative of the local epidemiological situation. The Salmonella strains were serotyped and those of serotypes Enteritidis and Typhimurium were phage typed. RESULTS: Epidemiological data analysis confirmed that the predominant causative serotype in the study period was Enteritidis, implicated in 5 329 (89.8%) of 5 934 reported cases of salmonellosis, followed by Typhimurium (302 cases, i.e. 5.1%). Laboratory data analysis revealed a lower incidence of the serotype Enteritidis (found in 231, i.e. in 84.6%, of 273 tested strains) and the same incidence of the serotype S. Typhimurium (14 strains, i.e. 5.1%). The most common phage types among strains of serotype Enteritidis were PT8, PT4, PT13a and PT6 while phage type DT104 was most often detected among strains of serotype Typhimurium. Moreover, the study revealed differences between the numbers of reported (EPIDAT) and laboratory diagnosed cases of salmonellosis. Within the study period, 111 (40%) cases of salmonellosis remained unreported in the study localities. CONCLUSION: The study has shown that the characteristics of Salmonella isolates (serotyping, phage typing and antimicrobial resistance data) relevant to the epidemiological data collection are either inaccurate or unavailable. Therefore, a national system needs to be created for the collection of Salmonella clinical isolates that should be further typed to obtain data for the monitoring at both the national and international levels.

Biofilm formation in field strains of Salmonella enterica serovar Typhimurium: identification of a new colony morphology type and the role of SGI1 in biofilm formation.

In this study we examined the extent of biofilm formation in field strains of Salmonella enterica serovar Typhimurium (S. Typhimurium), an important foodborne pathogen. Ninety-four field strains of S. Typhimurium were tested for their ability to form biofilm and components contributing to its formation. Most S. Typhimurium strains were highly capable of biofilm formation except for strains of phage type DT2 originating from pigeons. The most efficient biofilm forming strains were those of phage type DT104 positive for Salmonella genomic island 1 (SGI1). A comparison of SGI1 positive and negative strains indicated that the increased biofilm formation of SGI1 positive strains was associated with the presence of this genomic island. Finally, in five strains we found an alternative strategy of biofilm formation independent of curli fimbriae and cellulose production but solely dependent on an overproduction of capsular polysaccharide. Due to a mucoid and brown appearance on Congo Red agar we designated these strains as belonging to the SBAM (smooth brown and mucoid) morphotype.

[Characterization and prevalence of clones of Listeria monocytogenes isolated from patients in 2001-2008 in the Czech Republic]. / Charakteristika a prevalence klonu Listeria monocytogenes izolovaných od pacientu v letech 2001-2008 v Ceské republice.

OBJECTIVES: To characterize human isolates of Listeria monocytogenes by typing methods and to test them for use in epidemiological investigations. METHODS: Typing was carried out in 78 human isolates obtained in 2001-2008. Slide agglutination and multiplex PCR were used for serotyping. Genome macrorestriction analysis of the analyzed strains was performed. RESULTS: Serotype 1/2a was predominant, serotypes 1/2b and 4b were detected sporadically. DNA macrorestriction analysis yielded 20 pulsotypes. The comparison of these results with the data obtained for food isolates suggested possible linkage between sporadic cases of listeriosis and suspected vehicles of infection. CONCLUSION: Based on the obtained results, all human isolates should be typed and analyzed at the national level. It would be helpful in preventing late detection of listeriosis outbreaks, rapidly detecting sources of infection and reducing the number of cases.

Prevalence, characterization and antibiotic resistance of Salmonella isolates in large corvid species of europe and north America between 2010 and 2013.

It is well understood that Salmonella is carried by animals and in majority of cases as asymptomatic hosts. Surveillance efforts have focused on the role of agriculture and contamination points along the food chain as the main source of human infection; however, very little attention has been paid to the contribution of wildlife in the dissemination of Salmonella and what effect anthropogenic sources have on the circulation of antibiotic resistant Salmonella serovars in wildlife species. A purposive survey was taken of large corvids roosting yearly between November and March in Europe and North America. Two thousand and seven hundred and seventy-eight corvid faecal specimens from 11 countries were submitted for Salmonella spp. culture testing. Presumptive positive isolates were further serotyped, susceptibility tested and analysed for antibiotic resistance genes. Overall, 1.40% (39/2778) (CI = 1.01, 1.90) of samples were positive for Salmonella spp. Salmonella Enteritidis was the most prevalent serovar followed by S. Infantis, S. Montevideo and S. Typhimurium. No significant difference (P > 0.05) was found in the proportion of Salmonella recovered in Europe versus North America. The most variability of serovars within a site was in Kansas, USA with five different serovars recovered. European sites were significantly more likely to yield Salmonella resistant to more than one antibiotic (OR 71.5, P < 0.001, CI = 3.77, 1358) than North American sites, where no resistance was found. Resistance to nalidixic acid, a quinolone, was recovered in nine isolates from four serovars in four different sites across Europe. Large corvids contribute to the transmission and dissemination of Salmonella and resistance genes between human and animal populations and across great distances. This information adds to the knowledge base of zoonotic pathogen prevalence and antibiotic resistance ecology in wild birds.

Application of a chromogenic medium and the PCR method for the rapid confirmation of L. monocytogenes in foodstuffs.

Detection of Listeria monocytogenes in foodstuffs by conventional cultivation methods carried out according to EN ISO guidelines is rather time-consuming. Therefore, two alternative methods were applied for rapid confirmation of L. monocytogenes in foodstuffs. Inoculum from liquid selective broth was plated on PALCAM and OXFORD agar and on chromogenic agar medium RAPID L. mono. Suspect colonies from PALCAM were confirmed according to EN ISO standards and by the multiplex PCR method. In total, 990 samples of foodstuffs were investigated and 63 strains of L. monocytogenes were isolated. The chromogenic medium RAPID L. mono provided results comparable to PCR, it is easier to handle and provides considerable financial savings.

Subdivision of Salmonella enterica serovar enteritidis phage types PT14b and PT21 by plasmid profiling.

We have shown that plasmid profiling is a sensitive method for further identification of strains of Salmonella enterica serovar enteritidis (S. enteritidis) phage type PT21 and to a lesser extent the strains of phage type PT14b. Five and three plasmid types were identified within 33 strains of phage type PT21 and 19 strains of phage type PT14b, respectively. Plasmid types in strains of phage type PT21 showed significant correlation with geographical origin of the strain. In strains of phage type PT14b a single isolate predominated suggesting that the plasmid designated as 'C' can be directly linked with S. enteritidis PT14b strains. Application of IS200 fingerprinting did not reveal any other differences and showed just one copy of IS200 in all the 52 analysed strains. All the strains were tested for antibiotic resistance and only four strains were resistant to ampicillin, cefotaxime, cefuroxime and cotrimoxazole. This indicates that low molecular weight plasmids in Salmonella enteritidis are not responsible for the spread of antibiotic resistance.