Food Processing Environments Research Articles

Genomic insights into persistence of Listeria species in the food processing environment.

Listeria species may colonize and persist in food processing facilities for prolonged periods of time, despite hygiene interventions in place. To understand the genetic factors contributing to persistence of Listeria strains, this study undertook a comparative analysis of seven persistent and six presumed non-persistent strains, isolated from a single food processing environment, to identify genetic markers correlating to promoting persistence of Listeria strains, through whole genome sequence analysis. A diverse pool of genetic markers relevant to hygiene tolerance was identified, including disinfectant resistance markers qacH, emrC and the efflux cassette bcrABC. Both persistent and presumed non-persistent cohorts encoded a range of stress resistance markers, including heavy metal resistance, oxidative and pH stress, although trends were associated with each cohort (e.g., qacH and cadA1C resistance was more frequently found in persistent isolates). Persistent isolates were more likely to contain mutations associated with attenuated virulence, including a truncated InlA. Plasmids and transposons were widespread between cohorts. Results suggest that no single genetic marker identified was universally responsible for a strain's ability to persist. Persistent strains were more likely to harbour mutation associated with hypovirulence. This study provides additional insights into the distribution of genetic elements relevant to persistence across Listeria species, as well as strain virulence potential.

Genotyping of Listeria monocytogenes strains in Italian food-processing environments

Abstract Background In 2019, case fatality from Listeria monocytogenes (Lm) was higher than the previous years confirming Lm as a major public health problem. Food is the main source of Lm infection and cross-contamination from the food-processing environment (FPEs) is the most common route of contamination. The aim of this work was to characterize FPEs Lm strains collected from the National Reference Laboratory (NRL) for Lm in order to evaluate circulating clonal complexes (CCs) and their environmental adaptation. Methods NRL Lm database actually consists of 798 FPEs sequenced strains. In silico MLST was evaluated. Sequences related to prevalent CCs were further investigated using Pasteur's cgMLST scheme and detecting metal and detergent resistance genes and stress survival islet (SSI). Results MLST identified 25 CCs and 4 singleton. Nine isolates gave inexact match with existing alleles and were not classified. CC9 (17.4%) and CC121 (14.2%) were the prevalent clones. CC9 cgMLST analysis revealed a relatedness among strains isolated from the same establishment type (meat or dairy), even if sampled in different regions. Indeed, CC121 cgMLST clustering analysis was strictly associated to the point of sampling. Regarding stress adaptation genes, SSI1 and brcABC genes were detected in 100% and 2.9% of the CC9 strains, respectively. Meanwhile, SSI2 and transposon Tn6188 were detected, respectively, in 100% and 97.3% of CC121 strains. Conclusions Results confirmed, in Italian FPEs, prevalence of hypovirulent Lm strains, as previously observed in Italian food. Tested isolates highlighted adaptation to low pH and high salt concentration (SSI1 genes for CC9), to alkaline and oxidative stress (SSI2 genes for CC121) and benzalkonium chloride tolerance (Tn6188 in CC121) resulting in a best survival under stress conditions. Interestingly, CC9 clustering denoted allelic proximity among strains isolated in different Italian regions paying attention to clones spreading at national level. Key messages Listeria monocytogenes CC9 and CC121 prevalence in Italian establishment confirms environment is the main route of food contamination at production level. Prevalent food-processing environmental Italian Listeria monocytogenes strains denotes adaptation to environmental stress and clones spreading at national level.

Rare internalin A premature stop codon on Listeria monocytogenes on Italian PDO cheeses isolates

Abstract Background Listeria monocytogenes is a widespread foodborne pathogen that causes listeriosis, a human infection with low morbidity but high mortality. Internalin A (InlA), a L. monocytogenes virulence factor, has a key role in the invasion of human intestinal epithelium through the interaction with E-cadherin receptor. Truncated InlA caused by premature stop-codons (PMSC) have been correlated with attenuated virulence. To date, 29 mutations leading to a PMSC have been reported. In this study, we identified a rare PMSC mutation widespread in isolates from the production chain of the Gorgonzola and Taleggio PDO cheeses. Methods Within an ongoing monitoring plan for the characterization of L. monocytogenes, a total of 81 isolates collected in Lombardy (Northern Italy) from food, food processing environments, and clinical cases, between 2013-2021, were characterized using multilocus sequence tying (MLST) and then screened for the presence of PMSC by sequencing the 2400 pb inlA gene. Results A mutation leading to PMSC was identified on the position 277 (PMSC mutation type 26). To date, this mutation has been reported only on a CC7 strain from a clinical case. In our study, all isolates harbouring this mutation (n = 21) belong to ST325 (CC31, serotype 1/2a). Among these, 86% (n = 18) belong to the production chain of the Gorgonzola and Taleggio PDO cheeses. Conclusions To the best of our knowledge, this is the first report of PMSC mutation type 26 on food and environmental isolates. Although PMSC 26 is rarely found worldwide, our findings suggest that ST325 with this mutation is widespread in the production chain of the two PDO cheeses, and the presence of this PMSC may be correlated with the low incidence of ST325 clinical cases. Key messages A rare mutation leading to truncated InlA has been identified in a cluster of isolates correlated to the production chain of two PDO cheeses. Truncated inlA is suggestive of adaptation to the niche to the detriment of virulence.

Genomic persistence of Listeria monocytogenes strains in dairy and meat processing plants in Italy

Abstract Background Persistence of Listeria monocytogenes (Lm) in food processing plants (FPP) is still considered the major source of food contamination during processing. Correlations among different Lm strains and evaluation of the isolates persistence in FPP environments is an essential tool to improve food safety. The aim of this study was to investigate the genomic characteristics of persistent Lm strains (e.g. Metal and Detergent Resistance, SSIs and genomic island). Methods Totally 221 strains isolated from 10 FPP in the Abruzzo and Molise regions, Italy, were enrolled (4 of dairy products FPP - DP - and 6 of meat products FPP - MP). Sampling was performed in 6 sessions, from March 2017 to May 2018. Strains were sequenced using Illumina platform. The CCs, cgMLST analysis and Metal and Detergent Resistance, SSIs and genomic island (GI) detection was carried using the Pasteur scheme. Results Results returned 13 CCs and 2 singletons. cgMLST clustering showed two persistent clusters DP and 9 clusters in MP. In DP CC3 and CC9 were persistent. While in MP CC5, CC8, CC9, CC37, CC121 and CC155. The presence of SSI-1, was detected in 100% of the persistent strains from DP and 80% from MP, in particular in all strains of CC155, CC5, CC8 and CC9. In CC121 and CC37 it was absent. The presence of loci of Lm GI3, was in 100% of persistent strains of DP and in 52.3% of MP plant, in particular in 100% of CC121, CC8, CC5 and 16.3% of CC9. The 100% of CC121 in DP and MP plant harboured Tn6188 and SSI-2. The inlA PMSC mutations were detected in 100% of CC9 and CC121 strains. Conclusions The persistence of strains inside FPP was confirmed using NGS data, most of the strains were characterised by the presence of genes involved in resistance and adaptation. NGS is confirmed to be a powerful tool to study Lm persistence in FPP, looking also at strains' ecology. Key messages Most of persistent strains showed genes involved in resistance and adaptation in the food processing environment. NGS could help in facing Lm persistence in food producing plants giving new insights on the genomic characteristics of persistent strains.

Microbial colonization and resistome dynamics in food processing environments of a newly opened pork cutting industry during 1.5 years of activity

BackgroundThe microorganisms that inhabit food processing environments (FPE) can strongly influence the associated food quality and safety. In particular, the possibility that FPE may act as a reservoir of antibiotic-resistant microorganisms, and a hotspot for the transmission of antibiotic resistance genes (ARGs) is a concern in meat processing plants. Here, we monitor microbial succession and resistome dynamics relating to FPE through a detailed analysis of a newly opened pork cutting plant over 1.5 years of activity.ResultsWe identified a relatively restricted principal microbiota dominated by Pseudomonas during the first 2 months, while a higher taxonomic diversity, an increased representation of other taxa (e.g., Acinetobacter, Psychrobacter), and a certain degree of microbiome specialization on different surfaces was recorded later on. An increase in total abundance, alpha diversity, and β-dispersion of ARGs, which were predominantly assigned to Acinetobacter and associated with resistance to certain antimicrobials frequently used on pig farms of the region, was detected over time. Moreover, a sharp increase in the occurrence of extended-spectrum β-lactamase-producing Enterobacteriaceae and vancomycin-resistant Enterococcaceae was observed when cutting activities started. ARGs associated with resistance to β-lactams, tetracyclines, aminoglycosides, and sulphonamides frequently co-occurred, and mobile genetic elements (i.e., plasmids, integrons) and lateral gene transfer events were mainly detected at the later sampling times in drains.ConclusionsThe observations made suggest that pig carcasses were a source of resistant bacteria that then colonized FPE and that drains, together with some food-contact surfaces, such as equipment and table surfaces, represented a reservoir for the spread of ARGs in the meat processing facility.9rXSuE523Ksyv5BLWfnbxPVideo

Genomic characteristics of Listeria monocytogenes isolated during mushroom (Agaricus bisporus) production and processing

Listeria monocytogenes is a foodborne pathogen ubiquitously found in nature and which has been isolated from food and food processing environments. This study aimed to characterize L. monocytogenes strains isolated from the production and processing environments of frozen sliced mushrooms (Agaricus bisporus). An analysis was executed along the mushroom processing chain including one mushroom grower and two mushroom processing factories. A total of 153 L. monocytogenes strains were isolated, which could be grouped in three PCR serogroups, namely, serogroup 1/2a-3a (39.2%), serogroup 1/2b-3b-7 (34.0%) and serogroup 4b-4d-4e (26.8%). A selection of 44 L. monocytogenes strains isolated from the processing environment after cleaning and disinfection (C&D) and from frozen sliced mushrooms was genotyped by whole genome sequencing (WGS), because these strains pose a potential risk for product contamination after C&D and for human consumption. Multilocus sequence typing (MLST) revealed 11 clonal complexes (CCs), with strains belonging to CC1, CC4, CC37 and CC87 being detected in both processing factories. Comparative WGS analysis of the 44 strains showed the presence of Listeria pathogenicity island 1 (LIPI-1) with a disrupted version of actA in all CC1, CC4, CC5, CC59 strains, and all but one CC224 strains. Notably, both inlA and inlB were detected as full-length loci in every strain, except for inlA in a CC6 strain that harbored a three amino acid deletion. LIPI-3 was detected in all CC1, CC4, CC6 and CC224 strains, while LIPI-4 was detected in all CC4 and CC87 strains. In addition, antibiotic susceptibility tests showed susceptibility towards fourteen antibiotics tested. The bcrABC operon was found in one CC5 strain, that showed a higher tolerance towards benzalkonium chloride than any other strain tested with confluent growth till 12.5 μg/ml for the CC5 strain compared to 2.5 μg/ml for the other strains. This study highlights that the ecology of L. monocytogenes in the frozen sliced mushroom production chain is highly diverse, and shows the importance of hygienic measures to control L. monocytogenes along the frozen sliced mushroom production chain.

Harnessing Whole Genome Sequence Data for Facility-Specific Signatures for Listeria monocytogenes: A Case Study With Turkey Processing Plants in the United States

Listeria monocytogenes is a Gram-positive foodborne pathogen responsible for the severe disease listeriosis and notorious for its ability to persist in food processing plants, leading to contamination of processed, ready-to-eat foods. L. monocytogenes persistence in various food processing environments (FPEs) has been extensively investigated by various subtyping tools, with increasing use of whole genome sequencing (WGS). However, major knowledge gaps remain. There is a need for facility-specific molecular signatures not only for adequate attribution of L. monocytogenes to a specific FPE but also for improved understanding of the ecology and evolution of L. monocytogenes in the food processing ecosystem. Furthermore, multiple strains can be recovered from a single FPE sample, but their diversity can be underestimated with common molecular subtyping tools. In this study we investigated a panel of 54 L. monocytogenes strains from four turkey processing plants in the United States. A combination of WGS and phenotypic assays was employed to assess strain persistence as well as identify facility-specific molecular signatures. Comparative analysis of allelic variation across the whole genome revealed that allelic profiles have the potential to be specific to individual processing plants. Certain allelic profiles remained associated with individual plants even when closely-related strains from other sources were included in the analysis. Furthermore, for certain sequence types (STs) based on the seven-locus multilocus sequence typing scheme, presence and location of premature stop codons in inlA, inlB length, prophage sequences, and the sequence content of a genomic hotspot could serve as plant-specific signatures. Interestingly, the analysis of different isolates from the same environmental sample revealed major differences not only in serotype and ST, but even in the sequence content of strains of the same ST. This study highlights the potential for WGS data to be deployed for identification of facility-specific signatures, thus facilitating the tracking of strain movement through the food chain. Furthermore, deployment of WGS for intra-sample strain analysis allows for a more complete environmental surveillance of L. monocytogenes in food processing facilities, reducing the risk of failing to detect strains that may be clinically relevant and potentially novel.

Aqueous extracts of spices inhibit biofilm in Listeria monocytogenes by downregulating release of eDNA

Listeria monocytogenes is a Gram-positive mesophilic foodborne pathogen whose pathogenicity is regulated by various environmental stress factors. The ability to form robust biofilm makes it difficult to eradicate this bacteria from food processing environments. Also, there is an increase in the antibiotic resistant food isolates of L. monocytogenes. Therefore, there is a constant need for the development of novel food preservation techniques and therapeutics. In this study, we sought to determine the effectiveness of clove and cinnamon water extracts on the listerial biofilm initiation, dispersion, and expression of the virulence genes. It was observed that the spice water extracts not only inhibited the biofilm formation of L. monocytogenes but also accelerated the dispersion. The spice extracts at higher concentrations were found to be bactericidal to L. monocytogenes. The extracts downregulated the expression of virulence genes and the release of extracellular DNA (eDNA) in the biofilm. The decreased ability to form biofilm could be attributed to the inhibition of eDNA secretion. Thus the water extracts of cinnamon and clove can be used as food preservatives against the biofilm formation of L. monocytogenes.

Listeria monocytogenes: health risk and a challenge for food processing establishments.

Listeria monocytogenes is a pathogenic bacterium present in several environments able to survive and proliferate in food processing plants, contaminating products that when ingested can lead to listeriosis. Worldwide, the bacterium has been isolated from foods of animal and plant origin and its occurrence in ready-to-eat foods is a challenge for food processing establishments. Serotypes 1/2a, 1/2b, 1/2c, and 4b related to 95% of listeriosis cases have been detected in different foods and presented resistance to common antibiotics such as tetracycline and penicillin. The analysis and dissemination of the presence of L. monocytogenes in different foods are essential to anchor control and prevention measures. In Brazil, despite the lack of official data on contamination by L. monocytogenes, some studies have reported the presence of bacteria in various foods and food processing environments. Thus, this review focuses on presenting the main characteristics of the L. monocytogenes, resistance to antimicrobial agents, and occurrence in several foods and countries from 2010 to 2020.

Advances on the mechanisms regulating the formation of the biofilm of Listeria monocytogenes

Listeria monocytogenes is an important food-borne pathogen. The distribution and survival of L. monocytogenes are related to its ability to form biofilms. Biofilms are resistant to adverse environments, and bacteria separated from the biofilms may lead to persistent food contaminations. The formation, maturation and structure of biofilms depend on a variety of external and internal factors, among which a variety of regulatory mechanisms play important roles. This review summarizes the regulatory mechanisms (including intracellular, intercellular and interspecific interactions) involved in the biofilm formation of L. monocytogenes in order to control the biofilm formation in food processing environments, thus providing new intervention strategy for food safety.

Bacterial Biofilms and Their Implications in Pathogenesis and Food Safety.

Biofilm formation is an integral part of the microbial life cycle in nature. In food processing environments, bacterial transmissions occur primarily through raw or undercooked foods and by cross-contamination during unsanitary food preparation practices. Foodborne pathogens form biofilms as a survival strategy in various unfavorable environments, which also become a frequent source of recurrent contamination and outbreaks of foodborne illness. Instead of focusing on bacterial biofilm formation and their pathogenicity individually, this review discusses on a molecular level how these two physiological processes are connected in several common foodborne pathogens such as Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica and Escherichia coli. In addition, biofilm formation by Pseudomonas aeruginosa is discussed because it aids the persistence of many foodborne pathogens forming polymicrobial biofilms on food contact surfaces, thus significantly elevating food safety and public health concerns. Furthermore, in-depth analyses of several bacterial molecules with dual functions in biofilm formation and pathogenicity are highlighted.

Listeria monocytogenes Contamination Characteristics in Two Ready-to-Eat Meat Plants From 2019 to 2020 in Shanghai.

Listeria monocytogenes is a ubiquitous foodborne pathogen that causes listeriosis and is mostly linked to consumption of ready-to-eat (RTE) foods. Lack of hygiene in food processing environments may be a primary reason for contamination by L. monocytogenes isolates. In this study, L. monocytogenes strains isolated from two RTE meat processing plants in the Shanghai municipality, China, were characterized during 2019–2020 using pulsed-field gel electrophoresis and whole-genome sequencing. Results showed that 29 samples (12.2%) out of 239 were positive for L. monocytogenes, with 21 (18.9%) and 8 (6.25%) isolates from plants A and B, respectively. The packaging room at plant A had the most contamination (14, 48.3%; p < 0.05), with a peak occurrence of 76.5% in processing environments. Nineteen L. monocytogenes isolates belonging to the pulsotype (PT) 7 group were indistinguishable (≥ 95.7%). Furthermore, core-genome multiple loci sequencing typing identified up to nine allelic differences, and the closet pairwise differences among these ST5 isolates included 0–16 small nucleotide polymorphisms. Therefore, L. monocytogenes likely persisted at plant A during 2019–2020 with ongoing clone transmission. In contrast, no L. monocytogenes isolates were identified from processing environments at plant B. Most L. monocytogenes isolates were sampled from raw materials (62.5%). Several isolates (ST378, ST8, and ST120) were detected only once in 2020 and were considered as transient isolates. However, three ST121 isolates with the same PT (PT2) were detected in 2020 and should be noted for their stronger survival ability in harsh environments. These results suggest that continuous monitoring, stringent surveillance, and source tracking are crucial to guaranteeing food safety in RTE food plants.

Intensive Environmental Surveillance Plan for Listeria monocytogenes in Food Producing Plants and Retail Stores of Central Italy: Prevalence and Genetic Diversity.

Listeria monocytogenes (Lm) can persist in food processing environments (FPEs), surviving environmental stresses and disinfectants. We described an intensive environmental monitoring plan performed in Central Italy and involving food producing plants (FPPs) and retail grocery stores (RSs). The aim of the study was to provide a snapshot of the Lm circulation in different FPEs during a severe listeriosis outbreak, using whole genome sequencing (WGS) to investigate the genetic diversity of the Lm isolated, evaluating their virulence and stress resistance profiles. A total of 1217 samples were collected in 86 FPEs with 12.0% of positive surfaces at FPPs level and 7.5% at RSs level; 133 Lm isolates were typed by multilocus sequencing typing (MLST) and core genome MLST (cgMLST). Clonal complex (CC) 121 (25.6%), CC9 (22.6%), CC1 (11.3%), CC3 (10.5%), CC191 (4.5%), CC7 (4.5%) and CC31 (3.8%) were the most frequent MLST clones. Among the 26 cgMLST clusters obtained, 5 of them persisted after sanitization and were re-isolated during the follow-up sampling. All the CC121 harboured the Tn6188_qac gene for tolerance to benzalkonium chloride and the stress survival islet SSI-2. The CC3, CC7, CC9, CC31 and CC191 carried the SSI-1. All the CC9 and CC121 strains presented a premature stop codon in the inlA gene. In addition to the Lm Pathogenicity Island 1 (LIPI-1), CC1, CC3 and CC191 harboured the LIPI-3. The application of intensive environmental sampling plans for the detection and WGS analysis of Lm isolates could improve surveillance and early detection of outbreaks.

Adaptive Response of Listeria monocytogenes to the Stress Factors in the Food Processing Environment.

Listeria monocytogenes are Gram-positive, facultatively anaerobic, non-spore-forming bacteria that easily adapt to changing environmental conditions. The ability to grow at a wide range of temperatures, pH, and salinity determines the presence of the pathogen in water, sewage, soil, decaying vegetation, and animal feed. L. monocytogenes is an etiological factor of listeriosis, especially dangerous for the elderly, pregnant women, and newborns. The major source of L. monocytogenes for humans is food, including fresh and smoked products. Its high prevalence in food is associated with bacterial adaptation to the food processing environment (FPE). Since the number of listeriosis cases has been progressively increasing an efficient eradication of the pathogen from the FPE is crucial. Understanding the mechanisms of bacterial adaptation to environmental stress will significantly contribute to developing novel, effective methods of controlling L. monocytogenes in the food industry.

Genetic relationships and biofilm formation of Listeria monocytogenes isolated from the smoked salmon industry

Among pathogens, L. monocytogenes has the capability to persist on Food Processing Environment (FPE), first of all posing safety issues, then economic impact on productivity. The aim of this work was to determine the influence of biofilm forming-ability and molecular features on the persistence of 19 Listeria monocytogenes isolates obtained from FPE, raw and processed products of a cold-smoked salmon processing plant. To verify the phenotypic and genomic correlations among the isolates, different analyses were employed: serotyping, Clonal Complex (CC), core genome Multi-Locus Sequence Typing (cgMLST) and Single Nucleotide Polymorphisms (SNPs) clustering, and evaluation of the presence of virulence- and persistence-associated genes. From our results, the biofilm formation was significantly higher (*P < 0.05) at 37 °C, compared to 30 and 12 °C, suggesting a temperature-dependent behaviour. Moreover, the biofilm-forming ability showed a strain-specific trend, not correlated with CC or with strains persistence. Instead, the presence of internalin (inL), Stress Survival Islet (SSI) and resistance to erythromycin (ermC) genes was correlated with the ability to produce biofilms. Our data demonstrate that the genetic profile influences the adhesion capacity and persistence of L. monocytogenes in food processing plants and could be the result of environmental adaptation in response to the external selective pressure.

Low-Level Tolerance to Antibiotic Trimethoprim in QAC-Adapted Subpopulations of Listeria monocytogenes.

Between January and July 2021, there were as many as 30 recalls in the U.S. due to potential Listeria monocytogenes contamination from a variety of food products including muffins, kimchi, chicken salad, ready-to-eat chicken, smoked fish, mushrooms, queso fresco cheese, ice cream, turkey sandwiches, squash, and other foods. A contaminated food chain can serve as a potential vehicle for transmitting antibiotic resistant bacteria since there is a slow emergence of multi-drug antibiotic resistance in L. monocytogenes. Biocides are essential for safe food processing, but they may also induce unintended selective pressure at sublethal doses for the expression of antibiotic resistance in L. monocytogenes. To better understand the sources of such slow emergence of antibiotic resistance through biocide residues present in the food environments, we are working on the role of sublethal doses of commonly used biocides in defined broth and water models for understanding L. monocytogenes adaptation. We recently published the development of low-level tolerance to fluoroquinolone antibiotic ciprofloxacin in quaternary ammonium compound (QAC) adapted subpopulations of L. monocytogenes (Microorganisms 9, 1052). Of the six different antibiotics tested to determine heterologous stress adaptation in eight strains of L. monocytogenes, trimethoprim was the second one that exhibited low-level tolerance development after continuous exposure (by three approaches) to sublethal concentrations of QAC against actively growing planktonic cells of L. monocytogenes. When adapted to daily cycles of fixed or gradually increasing sublethal concentrations of QAC, we observed three main findings in eight L. monocytogenes strains against trimethoprim: (a) 3 of the 8 strains exhibited significant increase in short-range minimum inhibitory concentration (MIC) of trimethoprim by 1.7 to 2.5 fold in QAC-adapted subpopulations compared to non-adapted cells (p < 0.05); (b) 2 of the 8 strains exhibited significant increase in growth rate in trimethoprim (optical density (OD) by 600 nm at 12 h) by 1.4 to 4.8 fold in QAC-adapted subpopulations compared to non-adapted cells (p < 0.05); and (c) 5 of the 8 strains yielded significantly higher survival by 1.3-to-3.1 log CFU/mL in trimethoprim in QAC-adapted subpopulations compared to the non-adapted control (p < 0.05). However, for 3/8 strains of L. monocytogenes, there was no increase in the survival of QAC-adapted subpopulations compared to non-adapted control in trimethoprim. These findings suggest the potential formation of low-level trimethoprim tolerant subpopulations in some L. monocytogenes strains where QAC may be used widely. These experimental models are useful in developing early detection methods for tracking the slow emergence of antibiotic tolerant strains through food chain. Also, these findings are useful in understanding the predisposing conditions leading to slow emergence of antibiotic resistant strains of L. monocytogenes in various food production and food processing environments.

Removal of Mixed-Species Biofilms Developed on Food Contact Surfaces with a Mixture of Enzymes and Chemical Agents.

Sanicip Bio Control (SBC) is a novel product developed in Mexico for biofilms’ removal. The aims of this study were to evaluate (i) the removal of mixed-species biofilms by enzymatic (protease and α-amylase, 180 MWU/g) and chemical treatments (30 mL/L SBC, and 200 mg/L peracetic acid, PAA) and (ii) their effectiveness against planktonic cells. Mixed-species biofilms were developed on stainless steel (SS) and polypropylene B (PP) in whole milk (WM), tryptic soy broth (TSB) with meat extract (TSB+ME), and TSB with chicken egg yolk (TSB+EY) to simulate the food processing environment. On SS, all biofilms were removed after treatments, except the enzymatic treatment that only reduced 1–2 log10 CFU/cm2, whereas on PP, the reductions ranged between 0.59 and 5.21 log10 CFU/cm2, being the biofilms developed in TSB+EY being resistant to the cleaning and disinfecting process. Higher reductions in microbial load on PP were reached using enzymes, SBC, and PAA. The employed planktonic cells were markedly more sensitive to PAA and SBC than were the sessile cells. In conclusion, biofilm removal from SS can be achieved with SBC, enzymes, or PAA. It is important to note that the biofilm removal was strongly affected by the food contact surfaces (FCSs) and surrounding media.

Heterogeneity in biofilm formation and identification of biomarkers of strong biofilm formation among field isolates of Pseudomonas spp.

The biofilm formation ability of a collection of thirty-three Pseudomonas spp. isolates from food processing facilities was investigated in order to find biomarkers of strong biofilm production, a characteristic that can determine persistence in food processing environments. The strains were classified according to the colony pigmentation on solid media as green, brown or not pigmented. The biofilm production on stainless steel and polystyrene was assessed by spectrometric determination of the fixed crystal violet, and the biofilm formed on glass, through confocal laser scanning microscopy. Besides, pyoverdine production, catalase activity, RpoS status and cellular hydrophobicity were also monitored. A significantly higher biofilm production level on stainless steel and polystyrene was observed for green-pigmented strains as compared to brown or not pigmented strains. The influence of iron availability on biofilm formation on stainless steel was studied through the addition of the iron scavenger 2,2-bipyridine resulting in a decrease of 40 % in biofilm formation for the not pigmented strains. For most of the potential biomarkers studied (i.e., pyoverdine production, catalase activity, cellular hydrophobicity), the phenotypic heterogeneity observed among strains was mainly dependent on the Pseudomonas species and no strong associations with the biofilm formation capacity were detected. However, the green colony pigmentation on solid media showed good potential as a biomarker of strong biofilm formation on stainless steel and polystyrene both in P. aeruginosa and Pseudomonas spp.

Antimicrobial tolerance, biofilm formation, and molecular characterization of Salmonella isolates from poultry processing equipment

Foodborne pathogens, such as Salmonella are problematic in food processing environments, and understanding the means of persistence is critical in the development of effective control measures. This study determined the antimicrobial tolerance of Salmonella isolates from the processing environment and characterized their biofilm production and antibiotic resistance. Twenty-five Salmonella isolates were previously recovered from poultry processing equipment in commercial production facilities after sanitation. The minimum inhibitory concentration of 2 antimicrobials; chlorine and quaternary ammonium compounds that were frequently used for sanitation was determined for these isolates using the Clinical and Laboratory Standards Institute guidelines. Biofilm forming ability was assessed using the crystal violet assay and antibiotic susceptibility was also determined. These isolates were further characterized based on their genes that were responsible for biofilm formation and resistance to sanitizers and antibiotics. Minimum inhibitory concentration values between 500 and 1,000 parts per million for chlorine, or 3 to 25 parts per million for quaternary ammonium compounds were observed amongst these Salmonella isolates. These isolates possessed strong (24%), moderate (28%), and weak (48%) biofilm forming ability. All isolates were resistant to multiple antibiotics, and 64% exhibited resistance to aminoglycosides and β-lactams. Molecular characterization showed that the isolates possessed specific genes for biofilm formation, sanitizer tolerance, and antibiotic resistance. These results suggest that Salmonella isolates with low tolerance to sanitizers may remain on surfaces because of their strong biofilm forming ability.

Antibiofilm Effects of Nanoparticles and Visible Light Illumination Against Listeria monocytogenes.

Listeria monocytogenes bacteria pose a particular risk to the food industry as the species is known to form biofilm and to survive in a wide range of challenging environmental conditions. L. monocytogenes can cause listeriosis, a serious food-borne disease, and effective and safe antibiofilm materials and sanitary methods for food processing environments are intensively sought. A variety of nanoparticle materials have been recognized as safe to use in food environments, which allows the application of nanomaterials also for food safety purposes. Nanoparticles together with light illumination generate reactive oxygen species which inactivate bacteria by breaking down cell membranes, proteins, and DNA. The main objective of this study was to evaluate the efficacy of nanomaterials and blue light illumination for L. monocytogenes ATCC 7644 biofilm inactivation. Biofilm was allowed to form for 72 h on nanocoated stainless steel and aluminum plates, after which the plates were illuminated. Non-coated control plates were used to evaluate the antibiofilm efficacy of nanocoating. Plate count method was used to evaluate bacteria counts after illumination. Nanocoating did not affect initial biofilm formation compared to the control plates. Biofilm was significantly (p < 0.05) reduced on stainless steel, aluminum, and TiO2-coated aluminum plates after 72-h illumination by 1.9, 3.2, and 5.9 log, respectively. Nanocoating with visible light illumination could be an effective and safe method for enhancing food safety in food processing facilities to control biofilm formation. Evidence of antibiofilm properties of nanomaterials together with visible light illumination is limited; hence, future studies with variable light intensities and nanomaterials are needed.