Molecular characterization of Listeria monocytogenes isolates from a small-scale meat processor in Montenegro, 2011-2014.

The presence of Listeria monocytogenes was evaluated in a small-scale meat processing facility in Montenegro during 2011-2014. L. monocytogenes isolates from traditional meat products and environmental swabs were subjected to a) molecular characterization b) serotyping by both multiplex PCR and next generation sequencing (NGS) c) potential antimicrobial resistance (AMR) was assessed by extraction of specific genes from NGS data and d) screening for the presence of some disinfectant resistance markers. Overall, traditional meat products were contaminated, most likely from incoming raw materials, with 4 major specific STs of L. monocytogenes (ST515, ST8, ST21, ST121) representing 4 clonal complexes (CC1, CC8, CC21, CC121) identified during the four-year period. These strains belonged to serogroup IIa which predominated, followed by IVb (ST515, CC1). The strains from environmental swabs belonged, exclusively, to ST21 and were isolated from cutting board and floor swabs in 2011. Furthermore, we found Tn6188, a novel transposon conferring tolerance to BC, to be specific to sequence type ST121. In addition, antimicrobial resistance genes mprF and fosX were present in clonal complexes CC21 and CC121, while complexes CC8 and CC1 exclusively harbored the mprF antimicrobial resistance gene.

Wholegenome sequencing as the gold standard approach for control of Listeria monocytogenes in the food chain.

Listeria monocytogenes has been implicated in numerous outbreaks and related deaths of listeriosis. In food production, L. monocytogenes occurs in raw food material and above all, through postprocessing contamination. The use of next-generation sequencing technologies such as whole-genome sequencing (WGS) facilitates foodborne outbreak investigations, pathogen source tracking and tracing geographic distributions of different clonal complexes, routine microbiological/epidemiological surveillance of listeriosis, and quantitative microbial risk assessment. WGS can also be used to predict various genetic traits related to virulence, stress, or antimicrobial resistance, which can be of great benefit for improving food safety management as well as public health.

Effects of Selected Essential Oils on Listeria monocytogenes in Biofilms and in a Model Food System.

The composition of 18 essential oils was determined using gas chromatography-mass spectrometry, and their antilisterial activity was evaluated by the disk diffusion method, followed by the determination of the minimum inhibitory and minimum bactericidal concentrations. The most active essential oils were oregano, thyme, cinnamon, winter savory, and clove, with MIC values ranging from 0.09 to 1.78 µL/mL. We investigated the biofilm-forming potential of Listeria monocytogenes on polystyrene at 5 °C, 15 °C, and 37 °C in three different media. The formation of biofilm was found to be dependent on the temperature and the availability of nutrients. After treatment with selected essential oils, the reduction in biofilm biomass was in the range of 32.61% and 78.62%. Micromorphological changes in the L. monocytogenes treated by oregano and thyme essential oils were observed in the form of impaired cell integrity and cell lyses by using scanning electron microscope. Oregano and thyme essential oils (MIC and 2MIC) significantly (p < 0.05) reduced the population of L. monocytogenes in minced pork meat during storage at 4 °C. In conclusion, the obtained results indicated the good activity of some selected essential oils on L. monocytogenes, with bacteriostatic, bactericidal, and antibiofilm effects at very low concentrations.

Landscape of Stress Response and Virulence Genes Among Listeria monocytogenes Strains.

The pathogenic microorganism Listeria monocytogenes is ubiquitous and responsible for listeriosis, a disease with a high mortality rate in susceptible people. It can persist in different habitats, including the farm environment, the food production environments, and in foods. This pathogen can grow under challenging conditions, such as low pH, low temperatures, and high salt concentrations. However, L. monocytogenes has a high degree of strain divergence regarding virulence potential, environmental adaption, and stress response. This review seeks to provide the reader with an up-to-date overview of clonal and serotype-specific differences among L. monocytogenes strains. Emphasis on the genes and genomic islands responsible for virulence and resistance to environmental stresses is given to explain the complex adaptation among L. monocytogenes strains. Moreover, we highlight the use of advanced diagnostic technologies, such as whole-genome sequencing, to fine-tune quantitative microbiological risk assessment for better control of listeriosis.

Tracking the Origin of Austrian Human Brucellosis Cases Using Whole Genome Sequencing.

Brucellosis is a zoonotic disease caused by Brucella spp. and a major concern for livestock. Most human cases are caused by B. melitensis and clinical presentation is usually a mild febrile illness. However, treatment failure is frequent and more severe complications can occur. In Austria, every human brucellosis is investigated to determine whether it was imported from endemic areas or is the sign of an undetected autochthonous transmission. For this study, 21 B. melitensis strains isolated in Austria between 2005 and 2019 were collected, 17 strains from 15 different patients and four strains from cattle. Whole genome sequencing combined with core-genome MLST analysis was used to characterize these strains. A cluster of seven isolates from 2018 (three human and four cattle isolates) was identified, with fewer than two allelic differences. They corresponded to the only Austrian B. melitensis outbreak that happened over the past 15 years. The other 12 Austrian brucellosis cases were single cases, and geographical origins were available for 8/12. Genomic data was used to locate probable geographical origins and compared with the results of the epidemiological investigations. Austrian strains were compared with 67 published B. melitensis sequences available on NCBI. The result of genomic analysis matched for 7/8 cases with documented conclusion of the epidemiological investigation. Genome analysis also pointed to the geographical origin for three of the four cases with missing epidemiological data. Strains from six cases were grouped together (<40 allelic differences) with 4/6 cases imported from the Balkans. Additional B. melitensis isolates from Serbian animals were analyzed and grouped with this branch, suggesting frequent importation from Balkan countries to Austria. Overall, this study highlights the specificities of human brucellosis in Austria. It also underlines the value of whole genome sequencing as a tool to investigate brucellosis cases, allowing to identify and investigate outbreaks but also to support epidemiological investigation of imported cases. However, the reliability of such methods depends on the number of strains for comparison, which can be challenging in low incidence countries. Increasing the availability of published sequences with documented geographical origins would help establishing genomic-based methods for investigating brucellosis cases.

Tracking of Listeria monocytogenes in meat establishment using Whole Genome Sequencing as a food safety management tool: A proof of concept.

Repeated Listeria outbreaks particularly associated with Ready-To-Eat (RTE) delicatessen meat products have been reported annually at global level. The most frequent scenario that led to foodborne outbreaks was the post-thermal treatment cross-contamination of deli meat products during slicing and modified atmosphere packaging (MAP). The precondition for such cross contamination is the previous introduction of Listeria into meat processing facilities and subsequent colonization of the production environment, associated with formation of biofilms resilient to common sanitation procedures regularly applied in meat establishments. The use of Whole Genome Sequencing (WGS) can facilitate the understanding of contamination and colonization routes of pathogens within the food production environment and enable efficient pathogen tracking among different departments. This study aimed to: a) provide a proof of concept on practical use of WGS in a meat establishment to define the entry routes and spread pattern of L. monocytogenes, and b) to consider the regular use of WGS in meat processing establishments as a strong support of food safety management system. The results revealed that Listeria spp. was present in slaughter line, chilling chambers, deboning, slicing, MAP, as well as in corridors and dispatch (53 positive samples, out of 240). Eight L. monocytogenes isolates (out of 53) were identified from the slaughterhouse, chilling chambers, deboning, MAP and dispatch. L. monocytogenes isolates were of three different serotypes (1/2a, 1/2c, 4b) and correspondingly of three MLST sequence types. Overall, two pairs of L. monocytogenes isolates were genetically identical, i.e. two serotype 4b isolates (ST1), isolated from water drain at dispatch unit and two isolates obtained from slaughterhouse (floorwall junction at the carcass wash point) and MAP (water drain). These findings indicated that L. monocytogenes isolates identified in meat processing units (MAP, chilling chamber and dispatch) originated from the slaughter line. Further, all eight L. monocytogenes isolates were confirmed to be biofilm producers on glass and stainless steel surfaces. The identification of the main entry routes of L. monocytogenes into meat establishments and tracking the routes for spread of the pathogen are of essential importance to define appropriate risk mitigation strategies for L. monocytogenes in meat production environment. The routine use of WGS for bacterial characterization, as a strong support of food safety management system in meat establishments, will require the cost-effective approach. It may encompass in-house sequencing when sequencing equipment is used for multiple applications (e.g. WGS of pathogens, starter cultures and spoilage organisms).

WITHDRAWN: Comparison of high performance liquid chromatography and liquid chromatography/tandem mass spectrometry methods in the quantification and confirmation of ochratoxin A in pig tissue.

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.