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 Comparison of Listeria monocytogenes Exoproteomes from Biofilm and Planktonic State: Lmo2504, a Protein Associated with Biofilms

2013 ◽  
Vol 79 (19) ◽  
pp. 6075-6082 ◽  
Author(s):  
António Lourenço ◽  
Aitor de Las Heras ◽  
Mariela Scortti ◽  
Jose Vazquez-Boland ◽  
Joseph F. Frank ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Food Processing ◽  
Biofilm Formation ◽  
Ruthenium Red ◽  
Matrix Remodeling ◽  
Wild Type ◽  
Content Type ◽  
Cell Wall Binding ◽  
Physiological Processes ◽  
Planktonic State

ABSTRACTThe food-borne pathogenListeria monocytogenesis the causative agent of the severe human and animal disease listeriosis. The persistence of this bacterium in food processing environments is mainly attributed to its ability to form biofilms. The search for proteins associated with biofilm formation is an issue of great interest, with most studies targeting the whole bacterial proteome. Nevertheless, exoproteins constitute an important class of molecules participating in various physiological processes, such as cell signaling, pathogenesis, and matrix remodeling. The aim of this work was to quantify differences in protein abundance between exoproteomes from a biofilm and from the planktonic state. For this, two field strains previously evaluated to be good biofilm producers (3119 and J311) were used, and a procedure for the recovery of biofilm exoproteins was optimized. Proteins were resolved by two-dimensional difference gel electrophoresis and identified by electrospray ionization-tandem mass spectrometry. One of the proteins identified in higher abundance in the biofilm exoproteomes of both strains was the putative cell wall binding protein Lmo2504. A mutant strain with deletion of the gene for Lmo2504 was produced (3119Δlmo2504), and its biofilm-forming ability was compared to that of the wild type using the crystal violet and the ruthenium red assays as well as scanning electron microscopy. The results confirmed the involvement of Lmo2504 in biofilm formation, as strain 3119Δlmo2504showed a significantly (P< 0.05) lower biofilm-forming ability than the wild type. The identification of additional exoproteins associated with biofilm formation may lead to new strategies for controlling this pathogen in food processing facilities.

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 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 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.

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.

scholarly journals The CpAL Quorum Sensing System Regulates Production of Hemolysins CPA and PFO To Build Clostridium perfringens Biofilms

2015 ◽  
Vol 83 (6) ◽  
pp. 2430-2442 ◽  
Author(s):  
Jorge E. Vidal ◽  
Joshua R. Shak ◽  
Adrian Canizalez-Roman
Keyword(s):  
Quorum Sensing ◽  
Clostridium Perfringens ◽  
Biofilm Formation ◽  
Extracellular Dna ◽  
Wild Type ◽  
Content Type ◽  
Type C ◽  
Enteritis Necroticans ◽  
Biofilm Structure

Clostridium perfringensstrains produce severe diseases, including myonecrosis and enteritis necroticans, in humans and animals. Diseases are mediated by the production of potent toxins that often damage the site of infection, e.g., skin epithelium during myonecrosis. In planktonic cultures, the regulation of important toxins, such as CPA, CPB, and PFO, is controlled by theC. perfringensAgr-like (CpAL) quorum sensing (QS) system. Strains also encode a functional LuxS/AI-2 system. AlthoughC. perfringensstrains form biofilm-like structures, the regulation of biofilm formation is poorly understood. Therefore, our studies investigated the role of CpAL and LuxS/AI-2 QS systems and of QS-regulated factors in controlling the formation of biofilms. We first demonstrate that biofilm production by reference strains differs depending on the culture medium. Increased biomass correlated with the presence of extracellular DNA in the supernatant, which was released by lysis of a fraction of the biofilm population and planktonic cells. Whereas ΔagrBmutant strains were not able to produce biofilms, a ΔluxSmutant produced wild-type levels. The transcript levels of CpAL-regulatedcpaandpfoAgenes, but notcpb, were upregulated in biofilms compared to planktonic cultures. Accordingly, Δcpaand ΔpfoAmutants, in type A (S13) or type C (CN3685) backgrounds, were unable to produce biofilms, whereas CN3685Δcpbmade wild-type levels. Biofilm formation was restored in complemented Δcpa/cpaand ΔpfoA/pfoAstrains. Confocal microscopy studies further detected CPA partially colocalizing with eDNA on the biofilm structure. Thus, CpAL regulates biofilm formation inC. perfringensby increasing levels of certain toxins required to build biofilms.

scholarly journals Listeria monocytogenes Associated with New Zealand Seafood Production and Clinical Cases: Unique Sequence Types, Truncated InlA, and Attenuated Invasiveness

2013 ◽  
Vol 80 (4) ◽  
pp. 1489-1497 ◽  
Author(s):  
Cristina D. Cruz ◽  
Andrew R. Pitman ◽  
Sally A. Harrow ◽  
Graham C. Fletcher
Keyword(s):  
New Zealand ◽  
Listeria Monocytogenes ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Potential Health ◽  
Content Type ◽  
Processing Plants ◽  
Seafood Processing ◽  
Sequence Types ◽  
Clinical Cases

ABSTRACTListeriosis is caused by the food-borne pathogenListeria monocytogenes, which can be found in seafood and processing plants. To evaluate the risk to human health associated with seafood production in New Zealand, multi-virulence-locus sequence typing (MVLST) was used to define the sequence types (STs) of 31L. monocytogenesisolates collected from seafood-processing plants, 15 from processed foods, and 6 from human listeriosis cases. The STs of these isolates were then compared with those from a collection of seafood isolates and epidemic strains from overseas. A total of 17 STs from New Zealand clustered into two lineages: seafood-related isolates in lineages I and II and all human isolates in lineage II. None of the New Zealand STs matched previously described STs from other countries. Isolates (belonging to ST01-N and ST03-N) from mussels and their processing environments, however, were identical to those of sporadic listeriosis cases in New Zealand. ST03-N isolates (16 from mussel-processing environments, 2 from humans, and 1 from a mussel) contained aninlApremature stop codon (PMSC) mutation. Therefore, the levels of invasiveness of 22 isolates from ST03-N and the three other common STs were compared using human intestinal epithelial Caco-2 cell lines. STs carryinginlAPMSCs, including ST03-N isolates associated with clinical cases, had a low invasion phenotype. The close relatedness of some clinical and environmental strains, as revealed by identical MVLST profiles, suggests that local and persistent environmental strains in seafood-processing environments pose a potential health risk. Furthermore, a PMSC ininlAdoes not appear to giveL. monocytogenesa noninvasive profile.

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.

scholarly journals Identification of Listeria monocytogenes Contamination in a Ready-to-Eat Meat Processing Plant in China

2021 ◽  
Vol 12 ◽  
Author(s):  
Hongzhi Zhang ◽  
Fengxia Que ◽  
Biyao Xu ◽  
Linjun Sun ◽  
Yanqi Zhu ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Biofilm Formation ◽  
Raw Materials ◽  
Case Fatality ◽  
Etiologic Agent ◽  
Health Concern ◽  
Plate Count ◽  
Processing Plant ◽  
Meat Processing ◽  
Processing Plants

Listeria monocytogenes is the etiologic agent of listeriosis, which remains a significant public health concern in many countries due to its high case-fatality rate. The constant risk of L. monocytogenes transmission to consumers remains a central challenge in the food production industry. At present, there is very little known about L. monocytogenes contamination in ready-to-eat (RTE) processing plants in China. In this study, L. monocytogenes in an RTE meat processing plant in Shanghai municipality was characterized using pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS). Furthermore, the biofilm formation ability of the pathogen was also tested. Results revealed that L. monocytogenes isolates were present in 12 samples out of the 48 samples investigated. Most of them (66.7%, 8/12) were identified from the processing facilities irrespective of observed hygiene levels of aerobic plate count (APC) and coliforms. Coliforms were present in only one processing area. ST5 (1/2b) isolates were predominant (83.3%, 10/12) and were identified in two dominant pulsotypes (PTs) (three in PT3 and seven in PT4, respectively). Results of the core-genome multi-locus sequence typing (cgMLST) showed that ST5 in three PTs (PT1, PT3, and PT4) had 0–8 alleles, which confirmed that clonal transmission occurred in the RTE meat processing facilities. In addition, the biofilm formation test confirmed that the isolates from the processing facilities could form biofilms, which helped them colonize and facilitate persistence in the environment. These results indicated that common sanitation procedures regularly applied in the processing environment were efficient but not sufficient to remove L. monocytogenes isolates, especially biofilm of L. monocytogenes. Furthermore, the ST5 isolates in this study exhibited 12 alleles with one ST5 clinical isolate, which contributes to the understanding of the potential pathogenic risk that L. monocytogenes in RTE meat processing equipment posed to consumers. Therefore, strong hygienic measures, especially sanitation procedures for biofilms eradication, should be implemented to ensure the safety of raw materials. Meanwhile, continuous surveillance might be vital for the prevention and control of listeriosis caused by L. monocytogenes.

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