Food Processing Environments Research Articles

Antimicrobial Resistance of Listeria monocytogenes from Animal Foods to First- and Second-Line Drugs in the Treatment of Listeriosis from 2008 to 2021: A Systematic Review and Meta-Analysis.

First-line drugs for the treatment of listeriosis are the same around the world, but particular conditions might reduce their efficacy, including antimicrobial resistance. Therefore, this study aimed to verify, based on a systematic review and meta-analysis, whether the prevalence of antimicrobial resistance in Listeria monocytogenes from animal foods is higher for first- or second-line antimicrobials. From the total of 302 identified studies, 16 met all the eligibility criteria from 2008 to 2021 and were included in this meta-analysis. They comprised a dataset of 1152 L. monocytogenes isolates, obtained from different animal food products, food processing environment, and live animals. The included studies were developed in South America (n = 5), Europe (n = 4), Asia (n = 3), Africa (n = 2), and North America (n = 2), testing a total of 35 different antimicrobials, 11 of them classified as first-line drugs. Complete lack of antimicrobial resistance across the studies (all L. monocytogenes isolates tested as susceptible) was only observed for linezolid, while widespread antimicrobial resistance (all L. monocytogenes isolates tested resistant) was described for amoxicillin, benzylpenicillin, cefoxitin, fusidic acid, imipenem, sulfamethoxazole, and vancomycin. Overall, the meta-analysis results indicated no evidence that antimicrobial resistance in L. monocytogenes isolated from animal-based food is higher for first-line antimicrobials compared to second-line compounds (p=0.37). A greater volume of publication, together with better characterization of the isolates, is still needed for a more precise estimate of the real prevalence of antimicrobial resistance in L. monocytogenes.

Lactic Acid Resistance and Population Structure of Escherichia coli from Meat Processing Environment.

ABSTRACTTo explore the effect of beef processing on Escherichia coli populations in relation to lactic acid resistance, this study investigated the links among acid response, phylogenetic structure, genome diversity, and genotypes associated with acid resistance of meat plant E. coli. Generic E. coli isolates (n = 700) were from carcasses, fabrication equipment, and beef products. Acid treatment was carried out in Luria-Bertani broth containing 5.5% lactic acid (pH 2.9). Log reductions of E. coli ranged from <0.5 to >5 log CFU/mL (median: 1.37 log). No difference in lactic acid resistance was observed between E. coli populations recovered before and after a processing step or antimicrobial interventions. E. coli from the preintervention carcasses were slightly more resistant than E. coli isolated from equipment, differing by <0.5 log unit. Acid-resistant E. coli (log reduction <1, n = 45) had a higher prevalence of genes related to energy metabolism (ydj, xap, ato) and oxidative stress (fec, ymjC) than the less resistant E. coli (log reduction >1, n = 133). The ydj and ato operons were abundant in E. coli from preintervention carcasses. In contrast, fec genes were abundant in E. coli from equipment surfaces. The preintervention E. coli contained phylogroups A and B1 in relatively equal proportions. Phylogroup B1 predominated (95%) in the population from equipment. Of note, E. coli collected after sanitation shared either the antigens of O8 or H21. Additionally, genome diversity decreased after chilling and equipment sanitation. Overall, beef processing did not select for E. coli resistant to lactic acid but shaped the population structure.IMPORTANCE Antimicrobial interventions have significantly reduced the microbial loads on carcasses/meat products; however, the wide use of chemical and physical biocides has raised concerns over their potential for selecting resistant populations in the beef processing environment. Phenotyping of acid resistance and whole-genome analysis described in this study demonstrated beef processing practices led to differences in acid resistance, genotype, and population structure between carcass- and equipment-associated E. coli but did not select for the acid-resistant population. Results indicate that genes coding for the metabolism of long-chain sugar acids (ydj) and short-chain fatty acids (ato) were more prevalent in carcass-associated than equipment-associated E. coli. These results suggest E. coli from carcasses and equipment surfaces have been exposed to different selective pressures. The findings improve our understanding of the microbial ecology of E. coli in food processing environments and in general.

Bacteriophages: A weapon against mixed-species biofilms in the food processing environment.

Mixed-species biofilms represent the most frequent actual lifestyles of microorganisms in food processing environments, and they are usually more resistant to control methods than single-species biofilms. The persistence of biofilms formed by foodborne pathogens is believed to cause serious human diseases. These challenges have encouraged researchers to search for novel, natural methods that are more effective towards mixed-species biofilms. Recently, the use of bacteriophages to control mixed-species biofilms have grown significantly in the food industry as an alternative to conventional methods. This review highlights a comprehensive introduction of mixed-species biofilms formed by foodborne pathogens and their enhanced resistance to anti-biofilm removal strategies. Additionally, several methods for controlling mixed-species biofilms briefly focused on applying bacteriophages in the food industry have also been discussed. This article concludes by suggesting that using bacteriophage, combined with other 'green' methods, could effectively control mixed-species biofilms in the food industry.

Antimicrobial Resistance and Virulence Characterization of Listeria monocytogenes Strains Isolated from Food and Food Processing Environments.

Listeria monocytogenes is a particularly foodborne pathogen associated with listeriosis, which can be disseminated in food and food processing environments. This study aimed to determine the serotypes and characteristics of virulence factors and antibiotic resistance among 40 L. monocytogenes strains isolated from food (n = 27) purchased in Olsztyn (Warmia and Mazury region, Poland) and food processing environments in Poland (n = 13). Isolates were assigned to serotypes 1/2a, 1/2c, 3a, and 3c using polymerase chain reaction (PCR). The results showed that serotype 1/2a (66.7%) was the most prevalent among strains from food, and serotype 1/2c (53.8%) among strains from the food processing environments. Five different virulence factors (hlyA, prfA, inlB, luxS, sigB) were detected in all isolates from the food processing environments using PCR. The hlyA (100.0%), prfA (100.0%), and inlB (96.3%) were the most prevalent in food strains. Seven (25.9%) of the strains of food and ten (76.9%) strains from the food processing environments showed the ability to form biofilm. The tested isolates were subjected to antibiotic susceptibility testing against 12 antibiotics used in the mitigation of listeriosis, using the disk diffusion method. The most frequent were intermediate resistance and resistance to clindamycin. Twelve (92.3%) strains from the food processing environments, and twenty-three (85.2%) from food were non-susceptible to clindamycin. Generally, antibacterial resistance determinants (Lde, aadB, aac(3)-IIa(aacC2)a, penA, mefA, lnuA, lnuB, sulI, sulII) were detected in sixteen (59.0%) strains from food and four (30.8%) from the food processing environments, by PCR. The most frequent were the mefA-lnuA (n = 7; 20.0%) and lnuA (n = 6; 17.1%) genotypes. From this research, we can conclude that virulent and antimicrobial-resistant strains of L. monocytogenes are present in food and the food processing environment in Poland, which may pose a potential health risk to consumers. Monitoring for the control of virulent and antimicrobial-resistant L. monocytogenes strains in the food system can contribute to effective planning and prevention of their spread.

Rapid detection of Listeria monocytogenes in dairy products by a novel chemilumonogenic approach

Routine monitoring of foodborne pathogens such as Listeria monocytogenes in food processing environments are time-consuming necessities to ensure food safety. Alternative rapid diagnostic methods for pathogen detection are increasingly used, but often demand specialized equipment, making them unsuitable for on-site testing. This short communication describes the successful demonstration of combining the sample preparation method Matrix-Lysis with a chemiluminescent based detection platform (AquaSpark™) for detection of L. monocytogenes in milk and yogurt. The proposed method was evaluated against qPCR resulting in 100% relative specificity for both foodstuffs and a relative sensitivity of 100% for milk as well as 96% for yogurt for bacterial levels >1 CFU/ml. Only at very low initial bacterial concentrations (<1 CFU/ml) diverging results were found highlighting the advantages and limitations of both methods. While being less susceptible to contamination and false positive results from non-growing or dead cells, qPCR had a slightly lower overall detection limit. However, it has to be pointed out that qPCR has an increased analytical cost and also requires an additional 24 h analysis time. This study demonstrates the first successful application of a chemilumonogenic detection approach for L. monocytogenes in food that has a high potential for on-site testing.

Response of sensitive and resistant Listeria monocytogenes strains against bacteriocins produced by different Enterococcus spp. strains

Listeria monocytogenes is a relevant foodborne pathogen causing invasive listeriosis in humans, a disease with high mortality rates. Its ubiquity and growth characteristics enable this pathogen to survive harsh food processing environments. The addition of bacteriocins, antimicrobial peptides ribosomally synthesized by certain bacteria, appears as a natural alternative to control this pathogen in food. However, the emergence of L. monocytogenes strains resistant to the inhibitory action of bacteriocins has been detected. In order to analyse the development of this resistance, different properties of L. monocytogenes strains susceptible to bacteriocins (strains 01/155, 99/287 and 99/267) and their respective resistant isolates (strains 01/155B6R, 99/287B6R, 99/286C1R, 99/287 Mo1R, 99/287 M1bR, 99/287 M2dR, 99/267B6R), were compared in this work. Differences were analysed in: a) growth of the pathogen strains in direct contact with bacteriocin solution, in co-cultures with the producing strain, or with different sugars; b) response to antibiotics typically used against listeriosis; c) changes in cell morphology, observed by transmission or scanning electron microscopy; d) expression of mobility and haemolysin activity, two of L. monocytogenes main virulence factors; and e) biofilm formation ability. For all the isolates, the acquired resistance was permanent and crossed between the different bacteriocins under study. An inhibitory effect was observed for resistant strains only when they were grown in mixed culture with any of the bacteriocin-producing strains, with an acidified medium as additional growth stress. In all cases, the decrease in viability was lower for resistant strains and followed a particular profile for each strain. The variation of sugar substrate influenced resistant variants growth ability, with a more pronounced difference in the medium supplemented with glucose. Susceptibility to antibiotics was similar or higher for resistant variants, while neither the mobility nor the haemolytic activity presented differences among resistant or susceptible strains. Finally, the resistant variants showed a greater capacity to form biofilms, although this effect was reversed when grown in the presence of bacteriocins. Each resistant isolate had a particular behaviour pattern, and the acquisition of resistance appeared to be strain and bacteriocin dependent. These results contribute to the knowledge of L. monocytogenes bacteriocin-resistance development, which is essential to favour the use of these peptides as biopreservatives.

Characterization of Listeria monocytogenes biofilm formation kinetics and biofilm transfer to cantaloupe surfaces

Listeria monocytogenes biofilm is a consistent source of cross-contamination, both in housing storage and food processing environments. This study monitored the dynamic process of L. monocytogenes ST9 and ST87 biofilms, as well as their cross-contamination behaviors at various stages of formation. Scanning Electron Microscopy (SEM) captured the honeycomb-like structures and extracellular polymeric substances (EPS) during the biofilm formation. Confocal Laser Scanning Microscopy (CLSM) images illustrated that the ST87 strain formed a closed knitted chains network later than the ST9 strain. Moreover, structural parameters including bio-volume, mean thickness, porosity, and roughness could quantified the spatio-temporal differences of the biofilms. The transfer rates of biofilm cells to cantaloupes with the single contact had no significant difference at the initial adhesion, maturation, and dispersion stage (p > 0.05). Notably, the biofilm cells remained on the glass coupons when transferred to ten cantaloupe slices, indicating that biofilm cells transferred through cohesive failure. Meanwhile, the Logistic model could describe the transfer law of biofilm cells at different formation stages, as evaluated by Root Mean Square Error (RMSE) and R2adj. Additionally, the transfer rates were positively correlated with the hydrophobicity of L. monocytogenes ST87 biofilm cells measured by xylene. However, when the hydrophobicity of n-hexadecane was measured, the correlation was negative. This study illustrated the spatial and temporal differences during L. monocytogenes biofilms formation, and the transfer and residual of biofilm cells after a single and ten successive contacts at the specific stages. Our findings help in the quantitative microbiological risk assessment of fresh produce.

Pervasive Listeria monocytogenes Is Common in the Norwegian Food System and Is Associated with Increased Prevalence of Stress Survival and Resistance Determinants

ABSTRACTTo investigate the diversity, distribution, persistence, and prevalence of stress survival and resistance genes of Listeria monocytogenes clones dominating in food processing environments in Norway, genome sequences from 769 L. monocytogenes isolates from food industry environments, foods, and raw materials (512 of which were sequenced in the present study) were subjected to whole-genome multilocus sequence typing (wgMLST), single-nucleotide polymorphism (SNP), and comparative genomic analyses. The data set comprised isolates from nine meat and six salmon processing facilities in Norway collected over a period of three decades. The most prevalent clonal complex (CC) was CC121, found in 10 factories, followed by CC7, CC8, and CC9, found in 7 factories each. Overall, 72% of the isolates were classified as persistent, showing 20 or fewer wgMLST allelic differences toward an isolate found in the same factory in a different calendar year. Moreover, over half of the isolates (56%) showed this level of genetic similarity toward an isolate collected from a different food processing facility. These were designated as pervasive strains, defined as clusters with the same level of genetic similarity as persistent strains but isolated from different factories. The prevalence of genetic determinants associated with increased survival in food processing environments, including heavy metal and biocide resistance determinants, stress response genes, and inlA truncation mutations, showed a highly significant increase among pervasive isolates but not among persistent isolates. Furthermore, these genes were significantly more prevalent among the isolates from food processing environments compared to in isolates from natural and rural environments (n = 218) and clinical isolates (n = 111) from Norway.IMPORTANCE Listeria monocytogenes can persist in food processing environments for months to decades and spread through the food system by, e.g., contaminated raw materials. Knowledge of the distribution and diversity of L. monocytogenes is important in outbreak investigations and is essential to effectively track and control this pathogen in the food system. The present study presents a comprehensive overview of the prevalence of persistent clones and of the diversity of L. monocytogenes in Norwegian food processing facilities. The results demonstrate extensive spread of highly similar strains throughout the Norwegian food system, in that 56% of the 769 collected isolates from food processing factories belonged to clusters of L. monocytogenes identified in more than one facility. These strains were associated with an overall increase in the prevalence of plasmids and determinants of heavy metal and biocide resistance, as well as other genetic elements associated with stress survival mechanisms and persistence.

Persistence of Listeria monocytogenes ST5 in Ready-to-Eat Food Processing Environment.

Most human listeriosis is foodborne, and ready-to-eat (RET) foods contaminated by Listeria monocytogenes during processing are found to be common vehicles. In this study, a total of four L. monocytogens STs (ST5, ST121, ST120, and ST2) have been identified in two RTE food plants from 2019 to 2020 in Shanghai, China. The L. monocytogenes ST5 was predominant in one RTE food processing plant, and it persists in the RTE meat processing plant with continued clone transmission. The genetic features of the four STs isolates were different. ST5 and ST121 had the three genes clpL, mdrL, and lde; however, ST120 and ST2 had two genes except for clpL. SSI-1was present in ST5, ST121, and ST120. Additionally, SSI-2 was present only in the ST121 isolates. ST120 had all six biofilm-forming associated genes (actA, prfA, lmo0673, recO, lmo2504 and luxS). The ST2 isolate had only three biofilm-forming associated genes, which were prfA, lmo0673, and recO. The four ST isolates had different biofilm formation abilities at different stages. The biofilm formation ability of ST120 was significantly higher when grown for one day. However, the biofilm formation ability of ST120 reduced significantly after growing for four days. In contrast, the biofilm formation ability of ST5 and ST121 increased significantly. These results suggested that ST5 and ST121 had stronger ability to adapt to stressful environments. Biofilms formed by all four STs grown over four days can be sanitized entirely by a disinfectant concentration of 500 mg/L. Additionally, only ST5 and ST121 biofilm cells survived in sub-lethal concentrations of chlorine-containing disinfectant. These results suggested that ST5 and ST121 were more resistant to chlorine-containing disinfectants. These results indicated that the biofilm formation ability of L. monocytogenes isolates changed at different stages. Additionally, the persistence in food processing environments might be verified by the biofilm formation, stress resistance, etc. Alternatively, these results underlined that disinfectants should be used at lethal concentrations. More attention should be paid to ST5 and ST121, and stronger surveillance should be taken to prevent and control the clonal spread of L. monocytogenes isolates in food processing plants in Shanghai.

Immunoproteome profiling of Listeria monocytogenes under mild acid and salt stress conditions.

Listeria monocytogenes is one of the main foodborne pathogens worldwide. Although its response to stress conditions has been extensively studied, it is still present in the food processing environments and is a concern for consumers. To investigate how this microorganism adapts its proteome in mild stress conditions, a combined proteomics and bioinformatics approach was used to characterize the immunogenic protein profile of a sequence type 7 (ST7) strain that caused severe listeriosis outbreaks in central Italy. Extracted proteins were analyzed by immunoblotting using positive sera against L. monocytogenes and nLC-ESI-MS/MS, and all data were examined by five software to predict subcellular localization (SCL). A total of 226 proteins were extracted from the bands of interest, 58 of which were classified as potential immunogenic antigens. Compared to control cells grown under optimal conditions, six proteins, some of which under-described, were expressed under mild acid and salt stress conditions and/or at 12°C. In particular, adaptation and shaping of the proteome mainly involved cell motility at 12°C without acid and salt stress, whereas the combination of the same temperature with mild acid and salt stress induced a response concerning carbohydrate metabolism, oxidative stress, and DNA repair. Raw data are available via ProteomeXchange with identifier PXD033519.

Isolation of Klebsiella pneumoniae and Acinetobacter radioresistens from Dish Wash Scrubbers

Aims: To identify predominant microorganisms in dish washing scrubbers collected from ten different sources.&#x0D; Study Design: Collection of dish wash scrubbers from different sources, pour plating of appropriately prepared dish wash scrub suspensions and subsequent isolation and identification of predominant isolates. Assessment of antibiotic susceptibility of the selected isolates by disc diffusion assay.&#x0D; Place and Duration of Study: April, 2018 - June 2018.&#x0D; Methodology: A total of 10 dish wash scrubbers (synthetic green scrubber pads free from any anti-bacterial preservatives belonging to the same brand) were collected from various sources. Appropriately prepared dish wash scrub suspensions in peptone water were pour plated on Plate Count Agar (PCA) and MacConkey agar. Predominant colonies selected from the plates based on the colony morphology were subjected to Grams staining, catalase, oxidase, indole, citrate, urease tests and genotypic identification by 16S ribosomal RNA sequencing. The identified isolates were tested for their susceptibility to eight antibiotics by disc diffusion method.&#x0D; Results: Irrespective of the sample source, most of the dish wash scrubbers sampled harbored similar types of colonies. From the colonies obtained two of them were identified by 16S rRNA sequencing and subsequent blasting as Klebsiella pneumoniae and Acinetobacter radioresistens. The isolates were deposited in the NCBI database with accession numbers MK032217 (Klebsiella pneumoniae RSV02) and MK032134 (Acinetobacter radioresistens RSV 01). These isolates were tested for their susceptibility to different antibiotics and Acinetobacter radioresistens RSV 01 was found to be more antibiotic susceptible than Klebsiella pneumoniae RSV02.&#x0D; Conclusion: Observations of this study confirm the potential role of dish wash scrubbers as vehicle for potential pathogens and their ability to act as cross contaminating agents in food processing environments.

Temperature, Time, and Type, Oh My! Key Environmental Factors Impacting the Recovery of Salmonella Typhimurium, Listeria monocytogenes, and Tulane Virus from Surfaces

Environmental monitoring (EM) programs are designed to detect the presence of pathogens in food manufacturing environments, with the goal of preventing microbial contamination of food. Nevertheless, limited knowledge exists regarding the influence of environmental conditions on microbial recovery during EM. This study uses a commercially available polyurethane foam EM tool to determine the influence of environmental factors on the recovery of foodborne pathogens. The specific objectives of this study were to determine if environmental conditions and surface composition impact the recovery of sought-after microorganisms found in food processing environments. These data are compared across (i) microorganism type, (ii) surface type, (iii) environmental temperature and relative humidity (RH), and (iv) exposure time. Two bacteria (Listeria monocytogenes and Salmonella Typhimurium) and one human norovirus surrogate (Tulane virus) were inoculated onto three nonporous surfaces (polypropylene, stainless steel, and neoprene). Surfaces were held in an environmental chamber for 24 or 72 h at 30°C with 30% RH, 6°C with 85% RH, and 30°C with 85% RH. Data indicate that microbial recovery from environmental surfaces significantly (P ≤ 0.05) varies by microorganism type, environmental conditions, and exposure time. For instance, all microorganisms were significantly different from each other, with the greatest mean log reduction being Tulane virus and the lesser reduction being L. monocytogenes at 4.94 ± 1.75 log PFU per surface and 2.54 ± 0.91 log CFU per surface, respectively. Overall, these data can be used to improve the effectiveness of EM programs and underscores the need to better comprehend how EM test results are impacted by food manufacturing environmental conditions.

The microbicidal potential of visible blue light in clinical medicine and public health.

Visible blue light of wavelengths in the 400–470 nm range has been observed to have microbicidal properties. A widely accepted hypothesis for the mechanism of microbial inactivation by visible blue light is that the light causes photoexcitation of either endogenous (present within the microbe) or, exogenous (present in the biological medium surrounding the microbe) photosensitizers such as porphyrins and flavins, which leads to the release of reactive oxygen species that subsequently manifests microbicidal activity. Some of the factors that have been observed to be associated with enhanced microbicidal action include increased duration of exposure, and either pre- or co-treatment with quinine hydrochloride. In case of bacteria, repetitive exposure to the blue light shows no significant evidence of resistance development. Additionally, visible blue light has exhibited the ability to inactivate fungal and viral pathogens and, multidrug-resistant bacteria as well as bacterial biofilms. Visible blue light has demonstrated efficacy in eliminating foodborne pathogens found on food surfaces and exposed surfaces in the food processing environment as well as in the decontamination of surfaces in the clinical environment to minimize the spread of nosocomial infections. We conclude from reviewing existing literature on the application of the blue light in clinical medicine and public health settings that this microbicidal light is emerging as a safer alternative to conventional ultraviolet light-based technologies in multiple settings. However, further comprehensive studies and thorough understanding of the mechanism of microbicidal action of this light in different scenarios is warranted to determine its place in human health and disease.

Ability of Two Strains of Lactic Acid Bacteria To Inhibit Listeria monocytogenes by Spot Inoculation and in an Environmental Microbiome Context.

ABSTRACTWe evaluated the ability of two strains of lactic acid bacteria (LAB) to inhibit L. monocytogenes using spot inoculation and environmental microbiome attached-biomass assays. LAB strains (PS01155 and PS01156) were tested for antilisterial activity toward 22 phylogenetically distinct L. monocytogenes strains isolated from three fruit packing environments (F1, F2, and F3). LAB strains were tested by spot inoculation onto L. monocytogenes lawns (108 and 107 CFU/mL) and incubated at 15, 20, 25, or 30°C for 3 days. The same LAB strains were also cocultured at 15°C for 3, 5, and 15 days in polypropylene conical tubes with L. monocytogenes and environmental microbiome suspensions collected from F1, F2, and F3. In the spot inoculation assay, PS01156 was significantly more inhibitory toward less concentrated L. monocytogenes lawns than more concentrated lawns at all the tested temperatures, while PS01155 was significantly more inhibitory toward less concentrated lawns only at 15 and 25°C. Furthermore, inhibition of L. monocytogenes by PS01156 was significantly greater at 15°C than higher temperatures, whereas the temperature did not have an effect on the inhibitory activity of PS01155. In the assay using attached environmental microbiome biomass, L. monocytogenes concentration was significantly reduced by PS01156, but not PS01155, when cocultured with microbiomes from F1 and F3 and incubated for 3 days at 15°C. Attached biomass microbiota composition was significantly affected by incubation time but not by LAB strain. This study demonstrates that LAB strains that may exhibit inhibitory properties toward L. monocytogenes in a spot inoculation assay may not maintain antilisterial activity within a complex microbiome.IMPORTANCE Listeria monocytogenes has previously been associated with outbreaks of foodborne illness linked to consumption of fresh produce. In addition to conventional cleaning and sanitizing, lactic acid bacteria (LAB) have been studied for biocontrol of L. monocytogenes in food processing environments that are challenging to clean and sanitize. We evaluated whether two specific LAB strains, PS01155 and PS01156, can inhibit the growth of L. monocytogenes strains in a spot inoculation and in an attached-biomass assay, in which they were cocultured with environmental microbiomes collected from tree fruit packing facilities. LAB strains PS01155 and PS01156 inhibited L. monocytogenes in a spot inoculation assay, but the antilisterial activity was lower or not detected when they were grown with environmental microbiota. These results highlight the importance of conducting biocontrol challenge tests in the context of the complex environmental microbiomes present in food processing facilities to assess their potential for application in the food industry.

Superheated steam effectively inactivates diverse microbial targets despite mediating effects from food matrices in bench-scale assessments

Sanitation in dry food processing environments is challenging due to the exclusion of water. Superheated steam (SHS) is a novel sanitation technique that utilizes high temperature steam to inactivate microorganisms. The high sensible heat of SHS prevents condensation on surfaces. Here we evaluated SHS thermal inactivation of various vegetative and spore forming bacteria and fungi and determined the effect of food matrix composition on SHS efficacy. Capillary tubes with vegetative cells (Salmonella, E. coli O157:H7, Listeria monocytogenes, or Enterococcus faecium), Aspergillus fischeri ascospores, or B. cereus spores (100 μL) were SHS treated at 135 ± 1 °C for 1 or 2 s. After 1 s, SHS achieved a reduction of 10.91 ± 0.63 log10 CFU/mL for vegetative cells, 2.09 ± 0.58 log10 ascospores/mL for A. fischeri, and 0.21 ± 0.10 log10 spores/mL for B. cereus. SHS treatment achieved significant reductions in vegetative cells and fungal ascospores (p < 0.05), however B. cereus spores were not significantly reduced after 2 s and were determined to be the most resistant of the cell types evaluated. Consequently, peanut butter compositions (peanut powder, oil, and water) and milk powder (whole and nonfat) inoculated with B. cereus spores on aluminum foil coupons (2 × 3 × 0.5 cm) were tested. The D161°C values for B. cereus spores ranged from 46.53 ± 4.48 s (6 % fat, 55 % moisture, aw: 0.927) to 79.21 ± 14.87 s (43 % fat, 10 % moisture, aw: 0.771) for various peanut butter compositions. Whole milk powder had higher D161°C (34.38 ± 20.90 s) than nonfat milk powder (24.73 ± 6.78 s). SHS (135 ± 1 °C) rapidly (1 s) inactivated most common vegetative bacterial cells; however B. cereus spores were more heat resistant. B. cereus spore inactivation was significantly affected by product composition (p < 0.05). Compared to the log-linear model (R2 0.81–0.97), the Weibull model had better fit (R2 0.94–0.99). Finally, the ease of peanut butter removal from surfaces increased while the ease of non-fat dry milk removal decreased with the increasing SHS treatment duration. However, allergen residues were detectable on surfaces regardless of SHS treatment. The findings from this study can inform the development of pilot-scale research on SHS.

Genomic analysis of Listeria monocytogenes from US food processing environments reveals a high prevalence of QAC efflux genes but limited evidence of their contribution to environmental persistence

BackgroundQuaternary ammonium compound (QAC) efflux genes increase the minimum inhibitory concentration of Listeria monocytogenes (Lm) to benzalkonium chloride sanitizer, but the contribution of these genes to persistence in food processing environments is unclear. The goal of this study was to leverage genomic data and associated metadata for 4969 Lm isolates collected between 1999 and 2019 to: (1) evaluate the prevalence of QAC efflux genes among Lm isolates from diverse US food processors, (2) use comparative genomic analyses to assess confounding factors, such as clonal complex identity and stress tolerance genotypes, and (3) identify patterns in QAC efflux gene gain and loss among persistent clones within specific facilities over time.ResultsThe QAC efflux gene cassette bcrABC was present in nearly half (46%) of all isolates. QAC efflux gene prevalence among isolates was associated with clonal complex (\U0001d6d82 < 0.001) and clonal complex was associated with the facility type (\U0001d6d82 < 0.001). Consequently, changes in the prevalence of QAC efflux genes within individual facilities were generally attributable to changes in the prevalence of specific clonal complexes. Additionally, a GWAS and targeted BLAST search revealed that clonal complexes with a high prevalence of QAC efflux genes commonly possessed other stress tolerance genes. For example, a high prevalence of bcrABC in a clonal complex was significantly associated with the presence of the SSI-1 gene cluster (p < 0.05). QAC efflux gene gain and loss were both observed among persistent populations of Lm in individual facilities, suggesting a limited direct role for QAC efflux genes as predictors of persistence.ConclusionThis study suggests that although there is evidence that QAC efflux genes are part of a suite of adaptations common among Lm isolated from some food production environments, these genes may be neither sufficient nor necessary to enhance persistence. This is a crucial distinction for decision making in the food industry. For example, changes to sanitizer regimen targeting QAC tolerance would not address other contributing genetic or non-genetic factors, such as equipment hygienic design which physically mediates sanitizer exposure.

Preliminary Investigation of the Effect of Chemical Sanitizers and UV-C Light on Listeria monocytogenes Biofilm Survivability

The ability of Listeria monocytogenes to adapt under different environments and form biofilms is a challenge for food safety. Mature biofilms are difficult to disrupt. Chemical sanitizers combined with nonthermal technologies might be an effective way to control L. monocytogenes biofilms. This study was conducted to investigate L.monocytogenes biofilm survival after treatments withchemical sanitizers and UV-C light alone or in combination. A Centers for Diseases Control and Preventionbiofilm reactor was used to grow 4-day-old multistrainL.monocytogenes biofilms on stainless steel. Biofilmsurvival was evaluated after 10 min of exposure tolactic acid (4%), peroxy acid (100 ppm), and quaternaryammonium (400 ppm) alone or in combination with 15 or30 min of exposure to UV-C light (254 nm). The sequential treatment effect was also evaluated. Reductions of2.6 to 3.6 log CFU/cm2 were observed with chemicalsanitizers, whereas a maximum of 1.8 log CFU/cm2reduction was recorded after UV-C treatment. Combinedtreatments had an enhanced effect, and the sequence ofantimicrobial treatments was significant for lactic acid and peroxy acid (P &lt; 0.05). The results obtained in this research offer an initial understanding of the response of L.monocytogenes biofilm to chemical sanitizers and contribute to development of effective strategies to controlthis pathogen in the food processing environment.

Modelling the Adhesion and Biofilm Formation Boundary of Listeria monocytogenes ST9.

Listeria monocytogenes is a major foodborne pathogen that can adhere to or form a biofilm on food contact surfaces, depending on the environmental conditions. The purpose of this work is to determine the adhesion and biofilm formation boundaries for L. monocytogenes ST9 under the combination environments of temperature (5, 15, and 25 °C), NaCl concentration (0%, 3%, 6%, and 9% (w/v)) and pH (5.0, 6.0, 7.0, and 8.0). The probability models of adhesion and biofilm formation were built using the logistic regression. For adhesion, only the terms of linear T and NaCl are significant for L. monocytogenes ST9 (p < 0.05), whereas the terms of linear T, NaCl, and pH, and the interaction between T and pH were significant for biofilm formation (p < 0.05). By analyzing contour maps and their surface plots for two different states, we discovered that high temperature promoted adhesion and biofilm formation, whereas excessive NaCl concentration inhibited both of them. With a stringent threshold of 0.1667, the accuracy rate for identifying both adhesion/no-adhesion and biofilm formation/no-biofilm formation events were 0.929, indicating that the probability models are reasonably accurate in predicting the adhesion and biofilm formation boundary of L. monocytogenes ST9. The boundary model may provide a useful way for determining and further controlling L. monocytogenes adhesion and biofilm formation in various food processing environments.

Effect of gaseous ozone treatment on biofilm of dairy-isolated Pseudomonas spp. strains.

Microbial biofilms existing in food industries have been implicated as important contamination sources of spoilage and pathogenic microorganisms in the finished products. Among the innovative strategies proposed to contrast biofilms in food environments, ozone is recognised as an environmentally friendly technology but there are few studies about its effect against bacterial biofilms. The objective of this study was to evaluate the effect of gaseous ozone (50 ppm for 6 h) in inhibition and eradication of biofilm formed by twenty-one dairyisolated Pseudomonas spp. strains. Before ozone treatments, all isolates were screened for biofilm formation according to a previously described method. Strains were then divided in four groups: weak, weak/moderate, moderate/strong, and strong biofilm producers based on the biofilm biomass value of each isolate determined using the optical density (OD - 595 nm). Inhibition treatment was effective on the strain (C1) belonging to the weak producers’ group, on all strains classified as weak/moderate producers, on two strains (C8 and C12) belonging to the group of moderate/strong producers and on one strain (C13) classified as strong producer. Conversely, eradication treatments were ineffective on all strains tested, except for the strain C4 which reduced its biofilm-forming abilities after exposure to ozone gas. In conclusion, gaseous ozone may be used to enhance existing sanitation protocols in food processing environments, but its application alone not seems sufficient to contrast Pseudomonas spp. established biofilms.

Impact of temperature, soil type and compost amendment on the survival, growth and persistence of Listeria monocytogenes of non-environmental (food-source associated) origin in soil

Listeria monocytogenes of varied sources including food-related sources may reach the soil. Associated food safety and environmental health risks of such contamination depend significantly on the capacity of L. monocytogenes to survive in the soil. This study assessed the survival of 13 L. monocytogenes strains isolated from food and food processing environments and a cocktail of three of the strains in two types of soils (loam and sandy) under controlled temperature conditions: 5, 10, 20, 25, 30℃ and ‘uncontrolled’ ambient temperature conditions in a tropical region. The impact of compost amendment on the survival of L. monocytogenes in the two different types of soils was also assessed. Soil type, temperature and compost amendment significantly (P <0.001) impacted the survival of L. monocytogenes in soil. Temperature variations affected the survival of L. monocytogenes in soil, where some strains such as strain 732, a L. monocytogenes 1/2a strain survived better at lower temperature (5°C), for which counts of up to 10.47 ± 0.005 log CFU/g were recovered in compost-amended sandy soil, 60 days post-inoculation. Some other strains such as strain 441, a L. monocytogenes 1/2a survived best at intermediate temperature (25 and 30 °C), while others such as 2739 (L. monocytogenes 1/2b) thrived at higher temperature (between 30 °C - 37 °C). There were significant correlations between the influence of temperature and soil type, where lower temperature conditions (5°C - 20°C) were generally more suitable for survival in sandy soil compared to higher temperature conditions. For some of the strains that thrived better in sandy soil at lower temperature, Pearson correlation analysis found significant correlations between temperature and soil type. Steady, controlled temperature generally favored the survival of the strains compared to uncontrolled ambient temperature conditions, except for the cocktail. The cocktail persisted until the last day of post-inoculation storage (60th day) in all test soils and under all incubation temperature conditions. Loam soil was more favorable for the survival of L. monocytogenes and compost amendment improved the survival of the strains, especially in compost-amended sandy soil. Listeria monocytogenes may exhibit variable survival capacity in soil, depending on conditions such as soil type, compost amendment and temperature.