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 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 Conservation and Distribution of the Benzalkonium Chloride Resistance CassettebcrABCin Listeria monocytogenes

2013 ◽  
Vol 79 (19) ◽  
pp. 6067-6074 ◽  
Author(s):  
Vikrant Dutta ◽  
Driss Elhanafi ◽  
Sophia Kathariou
Keyword(s):  
Listeria Monocytogenes ◽  
Food Processing ◽  
Benzalkonium Chloride ◽  
Clinical Environment ◽  
Outbreak Strain ◽  
Content Type ◽  
Chloride Resistance ◽  
Processing Plants ◽  
Hot Dog ◽  
Widespread Dissemination

ABSTRACTAnalysis of a panel of 116Listeria monocytogenesstrains of diverse serotypes and sources (clinical, environment of food processing plants, and food) revealed that all but one of the 71 benzalkonium chloride-resistant (BCr) isolates harboredbcrABC, previously identified on a large plasmid (pLM80) of the 1998-1999 hot dog outbreak strain H7858. In contrast,bcrABCwas not detected among BC-susceptible (BCs) isolates. ThebcrABCsequences were highly conserved among strains of different serotypes, but variability was noted in sequences flankingbcrABC. The majority of the BCrisolates had either the pLM80-type of organization of thebcrABCregion or appeared to harborbcrABCon the chromosome, adjacent to novel sequences. Transcription ofbcrABCwas induced by BC (10 μg/ml) in strains of different serotypes and diversebcrABCregion organization. These findings reveal widespread dissemination ofbcrABCacross BCrL. monocytogenesstrains regardless of serotype and source, while also suggesting possible mechanisms ofbcrABCdissemination acrossL. monocytogenesgenomes.

Prevalence and Genetic Diversity of Listeria monocytogenes in Vacuum-Packaged Ready-to-Eat Meat Products at Retail Markets in Latvia

2009 ◽  
Vol 72 (6) ◽  
pp. 1283-1287 ◽  
Author(s):  
AIVARS BĒRZIŅŠ ◽  
MARGARITA TERENTJEVA ◽  
HANNU KORKEALA
Keyword(s):  
Listeria Monocytogenes ◽  
Shelf Life ◽  
Meat Products ◽  
Processing Plant ◽  
Meat Processing ◽  
Pork Products ◽  
Beef Products ◽  
Processing Plants ◽  
Serotype 1 ◽  
Packaged Beef

Nine groups of different retail ready-to-eat vacuum-packaged meat products from 10 Baltic meat processing plants were analyzed for presence and numbers of Listeria monocytogenes at the end of shelf life. A total of 38 (18%) of 211 samples tested positive for L. monocytogenes serotype 1/2a (88%) or 1/2c (12%). The prevalence of L. monocytogenes in cold-smoked, sliced, vacuum-packaged beef and pork products (42%) was significantly higher than in cooked, sliced, vacuum-packaged meat products (0.8%) (P < 0.001). Enumeration of L. monocytogenes showed that 84% of the positive samples contained <100 CFU/g upon expiry of product shelf life. The numbers of L. monocytogenes exceeded 100 CFU/g only in cold-smoked, sliced, vacuum-packaged beef products. Identical pulsed-field gel electrophoresis types were recovered from different production lots of cold-smoked vacuum-packaged beef and pork products produced by the same meat processing plant, demonstrating L. monocytogenes contamination as a recurrent problem within one meat processing plant.

scholarly journals comKProphage Junction Fragments as Markers for Listeria monocytogenes Genotypes Unique to Individual Meat and Poultry Processing Plants and a Model for Rapid Niche-Specific Adaptation, Biofilm Formation, and Persistence

2011 ◽  
Vol 77 (10) ◽  
pp. 3279-3292 ◽  
Author(s):  
Bindhu Verghese ◽  
Mei Lok ◽  
Jia Wen ◽  
Valentina Alessandria ◽  
Yi Chen ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Food Processing ◽  
Biofilm Formation ◽  
Content Type ◽  
Niche Adaptation ◽  
Processing Plants ◽  
Poultry Processing ◽  
Conditioning Film ◽  
Conditioning Films ◽  
Different Strains

ABSTRACTDifferent strains ofListeria monocytogenesare well known to persist in individual food processing plants and to contaminate foods for many years; however, the specific genotypic and phenotypic mechanisms responsible for persistence of these unique strains remain largely unknown. Based on sequences incomKprophage junction fragments, different strains of epidemic clones (ECs), which included ECII, ECIII, and ECV, were identified and shown to be specific to individual meat and poultry processing plants. ThecomKprophage-containing strains showed significantly higher cell densities after incubation at 30°C for 48 h on meat and poultry food-conditioning films than did strains lacking thecomKprophage (P< 0.05). Overall, the type of strain, the type of conditioning film, and the interaction between the two were all highly significant (P< 0.001). Recombination analysis indicated that thecomKprophage junction fragments in these strains had evolved due to extensive recombination. Based on the results of the present study, we propose a novel model in which the concept of defectivecomKprophage was replaced with the rapid adaptation island (RAI). Genes within the RAI were recharacterized as “adaptons,” as these genes may allowL. monocytogenesto rapidly adapt to different food processing facilities and foods. If confirmed, the model presented would help explainListeria's rapid niche adaptation, biofilm formation, persistence, and subsequent transmission to foods. Also,comKprophage junction fragment sequences may permit accurate tracking of persistent strains back to and within individual food processing operations and thus allow the design of more effective intervention strategies to reduce contamination and enhance food safety.

scholarly journals Persistent and Nonpersistent Listeria monocytogenes Contamination in Meat and Poultry Processing Plants

2003 ◽  
Vol 66 (11) ◽  
pp. 2062-2069 ◽  
Author(s):  
JANNE M. LUNDÉN ◽  
TIINA J. AUTIO ◽  
A.-M. SJÖBERG ◽  
HANNU J. KORKEALA
Keyword(s):  
Heat Treatment ◽  
Listeria Monocytogenes ◽  
Sampling Site ◽  
Pfge Type ◽  
Processing Plant ◽  
Post Heat Treatment ◽  
Common Location ◽  
Heat Treated ◽  
Processing Plants ◽  
Poultry Processing

Contamination analysis of persistent and nonpersistent Listeria monocytogenes strains in three meat processing plants and one poultry processing plant were performed in order to identify factors predisposing to or sustaining persistent plant contamination. A total of 596 L. monocytogenes isolates were divided into 47 pulsed-field gel electrophoresis (PFGE) types by combining the restriction enzyme patterns of AscI (42 patterns) and ApaI (38 patterns). Persistent and nonpersistent strains were found in all plants. Nonpersistent PFGE types were found mostly at one sampling site, with the processing environment being the most common location, whereas the persistent strains were found at several sampling sites in most cases. The processing machines were frequently contaminated with persistent L. monocytogenes PFGE types, and it was of concern that surfaces having direct contact with the products were contaminated. The role of the processing machines in sustaining contamination and in contaminating the products appeared to be important because the final product of several processing lines was contaminated with the same L. monocytogenes PFGE type as that found in the processing machine. The proportion of persistent PFGE types in heat-treated products was eight times higher than in the raw products, showing the importance of the persistent PFGE types as contaminants of the final heat-treated products. The contamination status of the processing lines and machines appeared to be influenced by the compartmentalization of the processing line, with poor compartmentalization increasing L. monocytogenes contamination. The separation of raw and post–heat treatment areas seemed especially important in the contamination status of post–heat treatment lines.

scholarly journals Genetic Characterization of Plasmid-Associated Benzalkonium Chloride Resistance Determinants in a Listeria monocytogenes Strain from the 1998-1999 Outbreak

2010 ◽  
Vol 76 (24) ◽  
pp. 8231-8238 ◽  
Author(s):  
Driss Elhanafi ◽  
Vikrant Dutta ◽  
Sophia Kathariou
Keyword(s):  
Listeria Monocytogenes ◽  
Food Processing ◽  
Benzalkonium Chloride ◽  
Genetic Basis ◽  
Genetic Characterization ◽  
Chloride Resistance ◽  
Processing Plants ◽  
Resistant Strains ◽  
Ammonium Compounds

ABSTRACT Quaternary ammonium compounds such as benzalkonium chloride (BC) are widely used as disinfectants in both food processing and medical environments. BC-resistant strains of Listeria monocytogenes have been implicated in multistate outbreaks of listeriosis and have been frequently isolated from food processing plants. However, the genetic basis for BC resistance in L. monocytogenes remains poorly understood. In this study, we have characterized a plasmid (pLM80)-associated BC resistance cassette in L. monocytogenes H7550, a strain implicated in the 1998-1999 multistate outbreak involving contaminated hot dogs. The BC resistance cassette (bcrABC) restored resistance to BC (MIC, 40 μg/ml) in a plasmid-cured derivative of H7550. All three genes of the cassette were essential for imparting BC resistance. The transcription of H7550 BC resistance genes was increased under sublethal (10 μg/ml) BC exposure and was higher at reduced temperatures (4, 8, or 25°C) than at 37°C. The level of transcription was higher at 10 μg/ml than at 20 or 40 μg/ml. In silico analysis suggested that the BC resistance cassette was harbored by an IS1216 composite transposon along with other genes whose functions are yet to be determined. The findings from this study will further our understanding of the adaptations of this organism to disinfectants such as BC and may contribute to the elucidation of possible BC resistance dissemination in L. monocytogenes.

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 Physiological and Transcriptional Characterization of Persistent and Nonpersistent Listeria monocytogenes Isolates

2011 ◽  
Vol 77 (18) ◽  
pp. 6559-6569 ◽  
Author(s):  
Edward M. Fox ◽  
Nola Leonard ◽  
Kieran Jordan
Keyword(s):  
Listeria Monocytogenes ◽  
Antimicrobial Agents ◽  
Cetylpyridinium Chloride ◽  
Phenotype Microarray ◽  
Content Type ◽  
Benzethonium Chloride ◽  
Transcriptomic Sequencing ◽  
Ammonium Compounds ◽  
Compare Gene Expression

ABSTRACTThis study aimed to characterize physiological differences between persistent and presumed nonpersistentListeria monocytogenesstrains isolated at processing facilities and to investigate the molecular basis for this by transcriptomic sequencing. Full metabolic profiles of two strains, one persistent and one nonpersistent, were initially screened using Biolog's Phenotype MicroArray (PM) technology. Based on these results, in which major differences from selected antimicrobial agents were detected, another persistent strain and two nonpersistent strains were characterized using two antimicrobial PMs. Resistance to quaternary ammonium compounds (QACs) was shown to be higher among persistent strains. Growth of persistent and nonpersistent strains in various concentrations of the QACs benzethonium chloride (BZT) and cetylpyridinium chloride (CPC) was determined. Transcriptomic sequencing of a persistent and a presumed nonpersistent strain was performed to compare gene expression among these strains in the presence and absence of BZT. Two strains, designated “frequent persisters” because they were the most frequently isolated at the processing facility, showed overall higher resistance to QACs. Transcriptome analysis showed that BZT induced a complex peptidoglycan (PG) biosynthesis response, which may play a key role in BZT resistance. Comparison of persistent and nonpersistent strains indicated that transcription of many genes was upregulated among persistent strains. This included three gene operons:pdu,cob-cbi, andeut. These genes may play a role in the persistence ofL. monocytogenesoutside the human host.

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.

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