scholarly journals Evolution of Listeria monocytogenes in a Food Processing Plant Involves Limited Single-Nucleotide Substitutions but Considerable Diversification by Gain and Loss of Prophages

2020 ◽  
Vol 86 (6) ◽  
Author(s):  
Anna Sophia Harrand ◽  
Balamurugan Jagadeesan ◽  
Leen Baert ◽  
Martin Wiedmann ◽  
Renato H. Orsi
Keyword(s):  
Listeria Monocytogenes ◽  
Mutation Rate ◽  
Food Processing ◽  
Purifying Selection ◽  
Processing Plant ◽  
Nucleotide Substitutions ◽  
Single Nucleotide ◽  
Content Type ◽  
Nucleotide Mutation ◽  
Tolerance Gene

ABSTRACT Whole-genome sequencing (WGS) is becoming the standard method for subtyping Listeria monocytogenes. Interpretation of WGS data for isolates from foods and associated environments is, however, challenging due to a lack of detailed data on Listeria evolution in processing facilities. Here, we used previously collected WGS data for 40 L. monocytogenes isolates obtained from a cold-smoked salmon processing facility between 1998 and 2015 to probe the L. monocytogenes molecular evolution in this facility, combined with phenotypic assessment of selected isolates. Isolates represented three clusters (1, 2, and 3); cluster 3 isolates (n = 32) were obtained over 18 years. The average mutation rate for cluster 3 was estimated as 1.15 × 10−7 changes per nucleotide per year (∼0.35 changes per genome per year); the most recent common ancestors (MRCAs) of subclusters 3a and 3b were estimated to have occurred around 1958 and 1974, respectively, within the age of the facility, suggesting long-term persistence in this facility. Extensive prophage diversity was observed within subclusters 3a and 3b, which have one shared and six unique prophage profiles for each subcluster (with 16 prophage profiles found among all 40 isolates). The plasmid-borne sanitizer tolerance operon bcrABC was found in all cluster 2 and 3 isolates, while the transposon-borne sanitizer tolerance gene qacH was found in one cluster 1 isolate; presence of these genes was correlated with the ability to survive increased concentrations of sanitizers. Selected isolates showed significant variation in the ability to attach to surfaces, with persistent isolates attaching better than transient isolates at 21°C. IMPORTANCE Knowledge about the genetic evolution of L. monocytogenes in food processing facilities over multiple years is generally lacking. This information is critical to interpret WGS findings involving food or food-associated isolates. This study suggests that L. monocytogenes that persists in processing facilities may evolve with a low single-nucleotide mutation rate mostly driven by negative (i.e., purifying) selection but with rapid diversification of prophages. Hence, isolation of L. monocytogenes with few single-nucleotide polymorphism (SNP) differences in different locations (e.g., supplier plants and receiving plants) is possible, highlighting the importance of epidemiological and detailed isolate metadata for interpreting WGS data in traceback investigation. Our study also shows how advanced WGS data analyses can be used to support root cause analysis efforts and may, for example, pinpoint the time when a persistence event started (which then potentially could be linked to facility changes, introduction of new equipment, etc.).

scholarly journals The Connection between Persistent, Disinfectant-Resistant Listeria monocytogenes Strains from Two Geographically Separate Iberian Pork Processing Plants: Evidence from Comparative Genome Analysis

2015 ◽  
Vol 82 (1) ◽  
pp. 308-317 ◽  
Author(s):  
Sagrario Ortiz ◽  
Victoria López-Alonso ◽  
Pablo Rodríguez ◽  
Joaquín V. Martínez-Suárez
Keyword(s):  
Listeria Monocytogenes ◽  
Food Processing ◽  
Pfge Type ◽  
Processing Plant ◽  
Content Type ◽  
Pork Products ◽  
Processing Plants ◽  
Pork Product ◽  
Ammonium Compounds ◽  
Insight Into

ABSTRACTThe aim of this study was to investigate the basis of the putative persistence ofListeria monocytogenesin a new industrial facility dedicated to the processing of ready-to-eat (RTE) Iberian pork products. Quaternary ammonium compounds, which included benzalkonium chloride (BAC), were repeatedly used as surface disinfectants in the processing plant. Clean and disinfected surfaces were sampled to evaluate if resistance to disinfectants was associated with persistence. Of the 14 isolates obtained from product contact and non-product contact surfaces, only five different pulsed-field gel electrophoresis (PFGE) types were identified during the 27-month study period. Two of these PFGE types (S1 and S10-1) were previously identified to be persistent and BAC-resistant (BACr) strains in a geographically separate slaughterhouse belonging to the same company. The remaining three PFGE types, which were first identified in this study, were also BACr. Whole-genome sequencing andin silicomultilocus sequence typing (MLST) analysis of five BACrisolates of the different PFGE types identified in this study showed that the isolate of the S1 PFGE type belonged to MLST sequence type 31 (ST31), a low-virulence type characterized by mutations in theinlAandprfAgenes. The isolates of the remaining four PFGE types were found to belong to MLST ST121, a persistent type that has been isolated in several countries. The ST121 strains contained the BAC resistance transposon Tn6188. The disinfection-resistantL. monocytogenespopulation in this RTE pork product plant comprised two distinct genotypes with different multidrug resistance phenotypes. This work offers insight into theL. monocytogenessubtypes associated with persistence in food processing environments.

scholarly journals Biofilm Formation by Listeria monocytogenes 15G01, a Persistent Isolate from a Seafood-Processing Plant, Is Influenced by Inactivation of Multiple Genes Belonging to Different Functional Groups

2021 ◽  
Vol 87 (10) ◽  
Author(s):  
Jessika Nowak ◽  
Sandra B. Visnovsky ◽  
Andrew R. Pitman ◽  
Cristina D. Cruz ◽  
Jon Palmer ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Food Processing ◽  
Biofilm Formation ◽  
Processing Plant ◽  
Wild Type ◽  
Content Type ◽  
Multiple Genes ◽  
Processing Plants ◽  
Biofilm Structure ◽  
Seafood Processing

ABSTRACT Listeria monocytogenes is a ubiquitous foodborne pathogen that results in a high rate of mortality in sensitive and immunocompromised people. Contamination of food with L. monocytogenes is thought to occur during food processing, most often as a result of the pathogen producing a biofilm that persists in the environment and acting as the source for subsequent dispersal of cells onto food. A survey of seafood-processing plants in New Zealand identified the persistent strain 15G01, which has a high capacity to form biofilms. In this study, a transposon library of L. monocytogenes 15G01 was screened for mutants with altered biofilm formation, assessed by a crystal violet assay, to identify genes involved in biofilm formation. This screen identified 36 transposants that showed a significant change in biofilm formation compared to the wild type. The insertion sites were in 27 genes, 20 of which led to decreased biofilm formation and seven to an increase. Two insertions were in intergenic regions. Annotation of the genes suggested that they are involved in diverse cellular processes, including stress response, autolysis, transporter systems, and cell wall/membrane synthesis. Analysis of the biofilms produced by the transposants using scanning electron microscopy and fluorescence microscopy showed notable differences in the structure of the biofilms compared to the wild type. In particular, inactivation of uvrB and mltD produced coccoid-shaped cells and elongated cells in long chains, respectively, and the mgtB mutant produced a unique biofilm with a sandwich structure which was reversed to the wild-type level upon magnesium addition. The mltD transposant was successfully complemented with the wild-type gene, whereas the phenotypes were not or only partially restored for the remaining mutants. IMPORTANCE The major source of contamination of food with Listeria monocytogenes is thought to be due to biofilm formation and/or persistence in food-processing plants. By establishing as a biofilm, L. monocytogenes cells become harder to eradicate due to their increased resistance to environmental threats. Understanding the genes involved in biofilm formation and their influence on biofilm structure will help identify new ways to eliminate harmful biofilms in food processing environments. To date, multiple genes have been identified as being involved in biofilm formation by L. monocytogenes; however, the exact mechanism remains unclear. This study identified four genes associated with biofilm formation by a persistent strain. Extensive microscopic analysis illustrated the effect of the disruption of mgtB, clsA, uvrB, and mltD and the influence of magnesium on the biofilm structure. The results strongly suggest an involvement in biofilm formation for the four genes and provide a basis for further studies to analyze gene regulation to assess the specific role of these biofilm-associated genes.

scholarly journals Genome Sequencing Identifies Two Nearly Unchanged Strains of Persistent Listeria monocytogenes Isolated at Two Different Fish Processing Plants Sampled 6 Years Apart

2013 ◽  
Vol 79 (9) ◽  
pp. 2944-2951 ◽  
Author(s):  
Anne Holch ◽  
Kristen Webb ◽  
Oksana Lukjancenko ◽  
David Ussery ◽  
Benjamin M. Rosenthal ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Food Processing ◽  
Random Amplified Polymorphic Dna ◽  
The United States ◽  
Genome Comparison ◽  
Physiological Factors ◽  
Content Type ◽  
Human Pathogenic Bacterium ◽  
A Genome ◽  
Genome Analyses

ABSTRACTListeria monocytogenesis a food-borne human-pathogenic bacterium that can cause infections with a high mortality rate. It has a remarkable ability to persist in food processing facilities. Here we report the genome sequences for twoL. monocytogenesstrains (N53-1 and La111) that were isolated 6 years apart from two different Danish fish processers. Both strains are of serotype 1/2a and belong to a highly persistent DNA subtype (random amplified polymorphic DNA [RAPD] type 9). We demonstrate usingin silicoanalyses that both strains belong to the multilocus sequence typing (MLST) type ST121 that has been isolated as a persistent subtype in several European countries. The purpose of this study was to use genome analyses to identify genes or proteins that could contribute to persistence. In a genome comparison, the two persistent strains were extremely similar and collectively differed from the reference lineage II strain, EGD-e. Also, they differed markedly from a lineage I strain (F2365). On the proteome level, the two strains were almost identical, with a predicted protein homology of 99.94%, differing at only 2 proteins. No single-nucleotide polymorphism (SNP) differences were seen between the two strains; in contrast, N53-1 and La111 differed from the EGD-e reference strain by 3,942 and 3,471 SNPs, respectively. We included a persistentL. monocytogenesstrain from the United States (F6854) in our comparisons. Compared to nonpersistent strains, all three persistent strains were distinguished by two genome deletions: one, of 2,472 bp, typically contains the gene forinlF, and the other, of 3,017 bp, includes three genes potentially related to bacteriocin production and transport (lmo2774,lmo2775, and the 3′-terminal part oflmo2776). Further studies of highly persistent strains are required to determine if the absence of these genes promotes persistence. While the genome comparison did not point to a clear physiological explanation of the persistent phenotype, the remarkable similarity between the two strains indicates that subtypes with specific traits are selected for in the food processing environment and that particular genetic and physiological factors are responsible for the persistent phenotype.

scholarly journals Whole-Genome Sequencing Allows for Improved Identification of Persistent Listeria monocytogenes in Food-Associated Environments

2015 ◽  
Vol 81 (17) ◽  
pp. 6024-6037 ◽  
Author(s):  
Matthew J. Stasiewicz ◽  
Haley F. Oliver ◽  
Martin Wiedmann ◽  
Henk C. den Bakker
Keyword(s):  
Listeria Monocytogenes ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Genetic Determinants ◽  
Single Nucleotide ◽  
Content Type ◽  
Food Borne ◽  
Clonal Spread

ABSTRACTWhile the food-borne pathogenListeria monocytogenescan persist in food associated environments, there are no whole-genome sequence (WGS) based methods to differentiate persistent from sporadic strains. Whole-genome sequencing of 188 isolates from a longitudinal study ofL. monocytogenesin retail delis was used to (i) apply single-nucleotide polymorphism (SNP)-based phylogenetics for subtyping ofL. monocytogenes, (ii) use SNP counts to differentiate persistent from repeatedly reintroduced strains, and (iii) identify genetic determinants ofL. monocytogenespersistence. WGS analysis revealed three prophage regions that explained differences between three pairs of phylogenetically similar populations with pulsed-field gel electrophoresis types that differed by ≤3 bands. WGS-SNP-based phylogenetics found that putatively persistentL. monocytogenesrepresent SNP patterns (i) unique to a single retail deli, supporting persistence within the deli (11 clades), (ii) unique to a single state, supporting clonal spread within a state (7 clades), or (iii) spanning multiple states (5 clades). Isolates that formed one of 11 deli-specific clades differed by a median of 10 SNPs or fewer. Isolates from 12 putative persistence events had significantly fewer SNPs (median, 2 to 22 SNPs) than between isolates of the same subtype from other delis (median up to 77 SNPs), supporting persistence of the strain. In 13 events, nearly indistinguishable isolates (0 to 1 SNP) were found across multiple delis. No individual genes were enriched among persistent isolates compared to sporadic isolates. Our data show that WGS analysis improves food-borne pathogen subtyping and identification of persistent bacterial pathogens in food associated environments.

scholarly journals Heavy Metal and Disinfectant Resistance of Listeria monocytogenes from Foods and Food Processing Plants

2012 ◽  
Vol 78 (19) ◽  
pp. 6938-6945 ◽  
Author(s):  
Shakir S. Ratani ◽  
Robin M. Siletzky ◽  
Vikrant Dutta ◽  
Suleyman Yildirim ◽  
Jason A. Osborne ◽  
...  
Keyword(s):  
Heavy Metals ◽  
Listeria Monocytogenes ◽  
Food Processing ◽  
Cadmium Resistance ◽  
Content Type ◽  
Ecological History ◽  
Resistance Determinants ◽  
Processing Plants ◽  
History Of ◽  
Serotype 4B

ABSTRACTThe persistence ofListeria monocytogenesin food processing plants and other ecosystems reflects its ability to adapt to numerous stresses. In this study, we investigated 138 isolates from foods and food processing plants for resistance to the quaternary ammonium disinfectant benzalkonium chloride (BC) and to heavy metals (cadmium and arsenic). We also determined the prevalence of distinct cadmium resistance determinants (cadA1,cadA2, andcadA3) among cadmium-resistant isolates. Most BC-resistant isolates were resistant to cadmium as well. Arsenic resistance was encountered primarily in serotype 4b and was an attribute of most isolates of the serotype 4b epidemic clonal group ECIa. Prevalence of the known cadmium resistance determinants was serotype associated:cadA1was more common in isolates of serotypes 1/2a and 1/2b than 4b, whilecadA2was more common in those of serotype 4b. A subset (15/77 [19%]) of the cadmium-resistant isolates lacked the known cadmium resistance determinants. Most of these isolates were of serotype 4b and were also resistant to arsenic, suggesting novel determinants that may confer resistance to both cadmium and arsenic in these serotype 4b strains. The findings may reflect previously unrecognized components of the ecological history of different serotypes and clonal groups ofL. monocytogenes, including exposures to heavy metals and disinfectants.

scholarly journals Singleton Sequence Type 382, an Emerging Clonal Group of Listeria monocytogenes Associated with Three Multistate Outbreaks Linked to Contaminated Stone Fruit, Caramel Apples, and Leafy Green Salad

2017 ◽  
Vol 55 (3) ◽  
pp. 931-941 ◽  
Author(s):  
Yi Chen ◽  
Yan Luo ◽  
James Pettengill ◽  
Ruth Timme ◽  
David Melka ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
The United States ◽  
Sequence Type ◽  
Stone Fruit ◽  
Nucleotide Polymorphisms ◽  
Outbreak Strain ◽  
Single Nucleotide ◽  
Content Type ◽  
Genomic Divergence ◽  
Singleton Sequence

ABSTRACT Three multistate outbreaks between 2014 and 2016, involving case patients in and outside the United States, were linked to stone fruit, caramel apples, and packaged leafy green salad contaminated with Listeria monocytogenes singleton sequence type 382 (ST382), a serotype IVb-v1 clone with limited genomic divergence. Isolates from these outbreaks and other ST382 isolates not associated with these outbreaks were analyzed by whole-genome sequencing (WGS) analysis. The primary differences among ST382 strains were single nucleotide polymorphisms (SNPs). WGS analysis differentiated ST382 from a clonal complex 1 outbreak strain co-contaminating the caramel apples. WGS clustered food, environmental, and clinical isolates within each outbreak, and also differentiated among the three outbreak strains and epidemiologically unrelated ST382 isolates, which were indistinguishable by pulsed-field gel electrophoresis. ST382 appeared to be an emerging clone that began to diverge from its ancestor approximately 32 years before 2016. We estimated that there was 1.29 nucleotide substitution per genome (2.94 Mbp) per year for this clone.

Single-nucleotide mutation rate increases close to insertions/deletions in eukaryotes

Nature ◽  
2008 ◽  
Vol 455 (7209) ◽  
pp. 105-108 ◽  
Author(s):  
Dacheng Tian ◽  
Qiang Wang ◽  
Pengfei Zhang ◽  
Hitoshi Araki ◽  
Sihai Yang ◽  
...  
Keyword(s):  
Mutation Rate ◽  
Single Nucleotide ◽  
Single Nucleotide Mutation ◽  
Nucleotide Mutation

scholarly journals Tolerance of Listeria monocytogenes to Quaternary Ammonium Sanitizers Is Mediated by a Novel Efflux Pump Encoded by emrE

2015 ◽  
Vol 82 (3) ◽  
pp. 939-953 ◽  
Author(s):  
Jovana Kovacevic ◽  
Jennifer Ziegler ◽  
Ewa Wałecka-Zacharska ◽  
Aleisha Reimer ◽  
David D. Kitts ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Quaternary Ammonium ◽  
Food Processing ◽  
Genomic Island ◽  
Efflux Pump ◽  
Cell Adherence ◽  
Outbreak Strain ◽  
Content Type ◽  
Increased Susceptibility

ABSTRACTA novel genomic island (LGI1) was discovered inListeria monocytogenesisolates responsible for the deadliest listeriosis outbreak in Canada, in 2008. To investigate the functional role of LGI1, the outbreak strain 08-5578 was exposed to food chain-relevant stresses, and the expression of 16 LGI1 genes was measured. LGI1 genes with putative efflux (L. monocytogenesemrE[emrELm]), regulatory (lmo1851), and adhesion (sel1) functions were deleted, and the mutants were exposed to acid (HCl), cold (4°C), salt (10 to 20% NaCl), and quaternary ammonium-based sanitizers (QACs). Deletion oflmo1851had no effect on theL. monocytogenesstress response, and deletion ofsel1did not influence Caco-2 and HeLa cell adherence/invasion, whereas deletion ofemrEresulted in increased susceptibility to QACs (P< 0.05) but had no effect on the MICs of gentamicin, chloramphenicol, ciprofloxacin, erythromycin, tetracycline, acriflavine, and triclosan. In the presence of the QAC benzalkonium chloride (BAC; 5 μg/ml), 14/16 LGI1 genes were induced, andlmo1861(putative repressor gene) was constitutively expressed at 4°C, 37°C, and 52°C and in the presence of UV exposure (0 to 30 min). Following 1 h of exposure to BAC (10 μg/ml), upregulation ofemrE(49.6-fold),lmo1851(2.3-fold),lmo1861(82.4-fold), andsigB(4.1-fold) occurred. Reserpine visibly suppressed the growth of the ΔemrELmstrain, indicating that QAC tolerance is due at least partially to efflux activity. These data suggest that a minimal function of LGI1 is to increase the tolerance ofL. monocytogenesto QACs viaemrELm. Since QACs are commonly used in the food industry, there is a concern thatL. monocytogenesstrains possessingemrEwill have an increased ability to survive this stress and thus to persist in food processing environments.

Growth of Listeria monocytogenes as a Biofilm on Various Food-Processing Surfaces

1996 ◽  
Vol 59 (8) ◽  
pp. 827-831 ◽  
Author(s):  
ISABEL C. BLACKMAN ◽  
JOSEPH F. FRANK
Keyword(s):  
Stainless Steel ◽  
Listeria Monocytogenes ◽  
Food Processing ◽  
Biofilm Formation ◽  
Minimal Medium ◽  
Processing Plant ◽  
Biofilm Growth ◽  
Sanitary Condition ◽  
Plant Environment ◽  
Substantial Risk

The objective of this research was to determine the ability of Listeria monocytogenes to grow as a biofilm on various food-processing surfaces including stainless steel, Teflon®, nylon, and polyester floor sealant. Each of these surfaces was able to support biofilm formation when incubation was at 21°C in Trypticase soy broth (TSB). Biofilm formation was greatest on polyester floor sealant (40% of surface area covered after 7 days of incubation) and least on nylon (3% coverage). The use of chemically defined minimal medium resulted in a lack of biofilm formation on polyester floor sealant, and reduced biofilm levels on stainless steel. Biofilm formation was reduced with incubation at 10°C, but Teflon® and stainless steel still allowed 23 to 24% coverage after incubation in TSB for 18 days. Biofilm growth of L. monocytogenes was sufficient to provide a substantial risk of this pathogen contaminating the food-processing plant environment if wet surfaces are not maintained in a sanitary condition.

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