Cíl práce: Cílem studie bylo určit genetickou rozmanitost humánních izolátů získaných v letech 2016–2020 z klinických laboratoří z různých lokalit České republiky (ČR) s ohledem na možné epidemické souvislosti a virulenci Listeria monocytogenes za využití dat celogenomového sekvenování. Metody: Ve studii byl použit soubor 102 humánních izolátů L. monocytogenes, u kterých byl určen sérotyp sklíčkovou aglutinací v kombinaci s multiplex PCR sérotypizací. Retrospektivně bylo provedeno celogenomové sekvenování a na základě získaných dat byla posouzena klonální příbuznost testovaných kmenů a přítomnost genů virulence pomocí softwaru Ridom SeqSphere+. Výsledky: V období let 2016–2020 bylo celkem charakterizováno 102 humánních izolátů L. monocytogenes, což činí 65 % ze všech hlášených případů listerióz registrovaných ve sledovaném období v ČR v systémech ISIN/EPIDAT. Dominantní zastoupení měl sérotyp 1/2a (57 %), následoval sérotyp 4b (30 %) a jen ojediněle byly detekovány kmeny sérotypu 1/2b (12 %) a 1/2c (1 %). Na základě analýzy celogenomových dat byly kmeny zařazeny k 26 klonálním komplexům a 27 sekvenačním typům. Porovnáním cgMLST (core genome Multi-Locus Sequence Typing) byly detekovány čtyři klastry o více jak třech kmenech vykazující vysokou příbuznost (rozdíly do 10 alel) s možnou epidemickou souvislostí. U všech kmenů byla potvrzena přítomnost klíčových genů virulence. Pouze u tří kmenů (sérotypu 1/2a, 1/2b a 1/2c) byla detekována bodová mutace v genu inlA spojovaná s expresí zkráceného proteinu internalinu A, který je zapojený do mechanismu přestupu L. monocytogenes intestinální bariérou. Závěr: Molekulární epidemiologie založená na celogenomovém sekvenování představuje účinný nástroj ke studiu populační struktury kmenů L. monocytogenes. V této studii byla zjištěna vysoká heterogenita humánních kmenů L. monocytogenes, zejména u v České republice dominantního sérotypu 1/2a. Bylo identifikováno několik klastrů s možnou epidemickou souvislostí, jejichž výskyt bude dále sledován.
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.
Biofilms are comprised of microorganisms embedded in a self-produced matrix that normally adhere to a surface. In the food processing environment they are suggested to be a source of contamination leading to food spoilage or the transmission of food-borne pathogens. To date, research has mainly focused on the presence of (biofilm-forming) bacteria within food processing environments, without measuring the associated biofilm matrix components. Here, we assessed the presence of biofilms within a meat processing environment, processing pork, poultry and beef, by the detection of microorganisms and at least two biofilm matrix components. Sampling included 47 food contact surfaces and 61 non-food contact surfaces from eleven rooms within an Austrian meat processing plant, either during operation or after cleaning and disinfection. The 108 samples were analysed for the presence of microorganisms by cultivation and targeted quantitative real-time PCR based on 16S rRNA. Furthermore, the presence of the major matrix components carbohydrates, extracellular DNA and proteins was evaluated. Overall, we identified ten biofilm hotspots, among them seven of which were sampled during operation and three after cleaning and disinfection. Five biofilms were detected on food contact surfaces (cutters and associated equipment and a screw conveyor) and five on non-food contact surfaces (drains and water hoses) resulting in 9.3 % of the sites being classified as biofilm positive. From these biofilm positive samples, we cultivated bacteria of 29 different genera. The most prevalent bacteria belonged to the genera Brochothrix (present in 80 % of biofilms), Pseudomonas and Psychrobacter (isolated from 70 % biofilms). From each biofilm we isolated bacteria from four to twelve different genera, indicating the presence of multi-species biofilms. This work ultimately determined the presence of multi-species biofilms within the meat processing environment, thereby identifying various sources of potential contamination. Especially the identification of biofilms in water hoses and associated parts highlights the need of a frequent monitoring at these sites. The knowledge gained about the presence and composition of biofilms (i.e. chemical and microbiological) will help to prevent and reduce biofilm formation within food processing environments.
- MeSH
- biofilmy klasifikace růst a vývoj MeSH
- Brochothrix izolace a purifikace MeSH
- dezinfekce metody MeSH
- drůbež mikrobiologie MeSH
- manipulace s potravinami * MeSH
- maso mikrobiologie MeSH
- nemoci přenášené potravou mikrobiologie MeSH
- potravinářská mikrobiologie MeSH
- Pseudomonas izolace a purifikace MeSH
- Psychrobacter izolace a purifikace MeSH
- RNA ribozomální 16S analýza MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Rakousko MeSH
Environmental adaptation of Listeria monocytogenes is a complex process involving various mechanisms that can contribute to their survival in the environment, further spreading throughout the food chain and the development of listeriosis. The aim of this study was to analyze whole-genome sequencing data in a set of 270 strains of L. monocytogenes derived from human listeriosis cases and food and environmental sources in order to compare the prevalence and type of genetic determinants encoding cadmium, arsenic, and benzalkonium chloride resistance. Most of the detected genes of cadmium (27.8%), arsenic (15.6%), and benzalkonium chloride (7.0%) resistance were located on mobile genetic elements, even in phylogenetically distant lineages I and II, which indicates the possibility of their horizontal spread. Although no differences were found in the prevalence of these genes between human and food strains, they have been detected sporadically in strains from the environment. Regarding cadmium resistance genes, cadA1C1_Tn5422 predominated, especially in clonal complexes (CCs) 121, 8, and 3 strains. At the same time, qacH_Tn6188-encoding benzalkonium chloride resistance was most frequently detected in the genome of CC121 strains. Genes encoding arsenic resistance were detected mainly in strains CC2 (located on the chromosomal island LGI2) and CC9 (carried on Tn554). The results indicated a relationship between the spread of genes encoding resistance to cadmium, arsenic, and benzalkonium chloride in certain serotypes and CCs and showed the need for a more extensive study of L. monocytogenes strains to better understand their ability to adapt to the food production environment.
- Publikační typ
- časopisecké články MeSH
This study was focused on characterization of the genetic diversity of Listeria monocytogenes isolated from packed fresh rabbit meat obtained from one producer via retail outlets. The partial aim was to compare the characteristics of a suspect persistent strain with strains from human cases. The occurrence of L. monocytogenes in vacuum-packed rabbit meat was monitored during 2013 to 2016. All strains were characterized by serotyping, pulsed-field gel electrophoresis, and multilocus sequence typing (MLST). Selected strains, which represented each year, were analyzed using the whole genome sequencing method. L. monocytogenes was detected in 21 (38%) of 56 originally packed rabbit meat samples from one food producer during the whole monitored period. All strains showed the identical serotype (1/2a), AscI/ApaI pulsotype (735/2), and sequence type (ST451). The clonal similarity of strains from rabbit meat was also confirmed on the basis of core genome MLST (on 1,701 loci). This fact suggests the occurrence of a suspect persistent strain in the meat processing plant. Results of core genome MLST enabled us to unambiguously exclude rabbit meat as a source of listeriosis in humans caused by the indistinguishable AscI/ApaI pulsotype and sequence type, although all strains carried all genes important for the virulence of L. monocytogenes. No specific genes that may be associated with its persistence in the food processing environment were detected among the tested strains of ST451.
- MeSH
- genetická variace MeSH
- králíci mikrobiologie MeSH
- lidé MeSH
- Listeria monocytogenes * genetika MeSH
- listeriové infekce * mikrobiologie přenos MeSH
- maso * mikrobiologie MeSH
- multilokusová sekvenční typizace MeSH
- potravinářská mikrobiologie * MeSH
- pulzní gelová elektroforéza MeSH
- sérotypizace MeSH
- zvířata MeSH
- Check Tag
- králíci mikrobiologie MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
