Development and Application of Four Foodborne Pathogens by TaqMan Multiplex Real-Time PCR.

A TaqMan multiplex real-time PCR (mRT-PCR) was developed to detect simultaneously Salmonella spp., Escherichia coli O157, Staphylococcus aureus, and Listeria monocytogenes in food samples. The method involves four sets of primers and probes tailored to the unique DNA sequences found in the invA, nuc, rfbE, and hly genes of each pathogen. The generated standard curves, correlating gene copy numbers with Ct values, demonstrated high accuracy (R2 > 0.99) and efficiency (92%-104%). Meanwhile, the limit of detection was 100 CFU/mL for the four target bacteria in artificially contaminated food samples after 6-8 h of enrichment. The assay's effectiveness was further verified by testing 80 naturally contaminated food samples, showing results largely in agreement with traditional culture methods. Overall, this newly developed TaqMan mRT-PCR, inclusive of a pre-enrichment step, proves to be a dependable and effective tool for detecting single or multiple pathogens in diverse food items, offering significant potential for in vitro diagnostics.

Population dynamics and bidirectional transfer of Listeria monocytogenes and Shiga toxin-producing Escherichia coli during cheese production in wooden vats.

Wooden vats are used in the production of some traditional cheeses as the biofilms on wooden vat surfaces are known to transfer large quantities of microbes to cheese. However, the safety of using wooden vats for cheese production remains controversial as the porous structure of wood provides an irregular surface that may protect any attached pathogen cells from cleaning and sanitation processes. On the other hand, the absence of pathogens in wooden vats has been reported in multiple studies and wooden materials have not been associated with foodborne illness outbreaks. The present study determined the survival of Listeria monocytogenes and Shiga toxin-producing Escherichia coli (STEC) during the production of an uncooked pressed cheese in wooden vats as well as their ability to transfer to the wood and then to milk used in subsequent batches of cheese production in the absence of formal cleaning. Results from the study indicate that pathogens inoculated in milk grew during production of the uncooked cheese, but showed limited ability to colonize the wooden vats and contaminate subsequent batches. These results suggest that the risks of using wooden vats to produce cheese is low if the milk is of high microbiological quality.

Airborne signals of Pseudomonas fluorescens modulate swimming motility and biofilm formation of Listeria monocytogenes in a contactless coculture system.

Bacterial volatile compounds (BVCs) facilitate interspecies communication in socio-microbiology across physical barriers, thereby influencing interactions between diverse species. The impact of BVCs emitted from Pseudomonas on the biofilm formation characteristics of Listeria monocytogenes within the same ecological niche has been scarcely investigated under practical conditions of food processing. The objective of this study was to explore the motility and biofilm formation characteristics of L. monocytogenes under the impact of Pseudomonas BVCs. It was revealed that BVCs of P. fluorescens, P. lundensis, and P. fragi significantly promoted swimming motility of L. monocytogenes (P < 0.05). As evidenced by crystal violet staining, the L. monocytogenes biofilms reached a maximum OD570 value of approximately 3.78 at 4 d, which was 0.65 units markedly higher than that of the control group (P < 0.05). Despite a decrease in adherent cells of L. monocytogenes biofilms among the BVCs groups, there was a remarkable increase in the abundance of extracellular polysaccharides and proteins with 3.58 and 4.90 µg/cm2, respectively (P < 0.05), contributing to more compact matrix architectures, which suggested that the BVCs of P. fluorescens enhanced L. monocytogenes biofilm formation through promoting the secretion of extracellular polymers. Moreover, the prominent up-regulated expression of virulence genes further revealed the positive regulation of L. monocytogenes under the influence of BVCs. Additionally, the presence of BVCs significantly elevated the pH and TVB-N levels in both the swimming medium and biofilm broth, thereby exhibiting a strong positive correlation with increased motility and biofilm formation of L. monocytogenes. It highlighted the crucial signaling regulatory role of BVCs in bacterial interactions, while also emphasizing the potential food safety risk associated with the hitchhiking behavior of L. monocytogenes, thereby shedding light on advancements in control strategies for food processing.

The ARIMA model approach for the biofilm-forming capacity prediction of Listeria monocytogenes recovered from carcasses.

The present study aimed to predict the biofilm-formation ability of L. monocytogenes isolates obtained from cattle carcasses via the ARIMA model at different temperature parameters. The identification of L. monocytogenes obtained from carcass samples collected from slaughterhouses was determined by PCR. The biofilm-forming abilities of isolates were phenotypically determined by calculating the OD value and categorizing the ability via the microplate test. The presence of some virulence genes related to biofilm was revealed by QPCR to support the biofilm profile genotypically. Biofilm-formation of the isolates was evaluated at different temperature parameters (37 °C, 22 °C, 4 °C and - 20 °C). Estimated OD values were obtained with the ARIMA model by dividing them into eight different estimation groups. The prediction performance was determined by performance measurement metrics (ME, MAE, MSE, RMSE, MPE and MAPE). One week of incubation showed all isolates strongly formed biofilm at all controlled temperatures except - 20 °C. In terms of the metrics examined, the 3 days to 7 days forecast group has a reasonable prediction accuracy based on OD values occurring at 37 °C, 22 °C, and 4 °C. It was concluded that measurements at 22 °C had lower prediction accuracy compared to predictions from other temperatures. Overall, the best OD prediction accuracy belonged to the data obtained from biofilm formation at -20 °C. For all temperatures studied, especially after the 3 days to 7 days forecast group, there was a significant decrease in the error metrics and the forecast accuracy increased. When evaluating the best prediction group, the lowest RMSE at 37 °C (0.055), 22 °C (0.027) and 4 °C (0.024) belonged to the 15 days to 21 days group. For the OD predictions obtained at -20 °C, the 15 days to 21 days prediction group had also good performance (0.011) and the lowest RMSE belongs to the 7 days to 15 days group (0.007). In conclusion, this study will guide in using indicator parameters to evaluate biofilm forming ability to predict optimum temperature-time. The ARIMA models integrated with this study can be useful tools for industrial application and risk assessment studies using different parameters such as pH, NaCl concentration, and especially temperature applied during food processing and storage on the biofilm-formation ability of L. monocytogenes.

Recent advances on the formation, detection, resistance mechanism, and control technology of Listeria monocytogenes biofilm in food industry.

Listeria monocytogenes is an important foodborne pathogen that causes listeriosis, a severe and fatal condition. Biofilms are communities of microorganisms nested within a self-secreted extracellular polymeric substance, and they protect L. monocytogenes from environmental stresses. Biofilms, once formed, can lead to the persistence of L. monocytogenes in processing equipment and are therefore considered to be a major concern for the food industry. This paper briefly introduces the recent advancements on biofilm formation characteristics and detection methods, and focuses on analysis of the mechanism of L. monocytogenes biofilm resistance; Moreover, this paper also summarizes and discusses the existing different techniques of L. monocytogenes biofilm control according to the physical, chemical, biological, and combined strategies, to provide a theoretical reference to aid the choice of effective control technology in the food industry.

Distribution and Antimicrobial Resistance Characterization of Listeria monocytogenes in Poultry Meat in Jiading District, Shanghai.

To investigate the distribution, contamination status, and antibiotic resistance of Listeria monocytogenes in four types of retail poultry meat, including chicken, duck, goose, and pigeon, sold in Jiading District, Shanghai, a total of 236 retail poultry meat samples were collected, and L. monocytogenes isolates were obtained for identification and antibiotic susceptibility testing against 14 common antibiotics. Forty-one L. monocytogenes isolates were detected from the 236 retail poultry meat samples, with detection rates of 24.47%, 19.44%, 14.75%, and 4.44% in chicken, goose, duck, and pigeon meat, respectively. Among refrigerated, frozen, and room temperature samples, refrigerated poultry had the highest detection rate at 25.40%, while frozen poultry had the lowest at 13.33%. The detection rate of L. monocytogenes in chicken meat differed significantly between the storage temperatures, while no significant differences were found for other poultry types. No significant differences in detection rates were observed between different retail locations or packaging methods. Isolates exhibited complete resistance to cefoxitin (FOX) and increasing resistance over time to tetracycline (TET) and clindamycin (CLI), while low levels of resistance were found for penicillin (PEN), oxacillin (OXA), and erythromycin (ERY). Resistance to ERY and TET suggests the potential for multidrug resistance. Significant differences in antibiotic resistance profiles were observed among L. monocytogenes from the various poultry types. In summary, contamination status and antibiotic resistance profiles differed among retail chicken, duck, goose, and pigeon meat sold and the resistance rate of strains continues to increase in Jiading District, Shanghai. Targeted control measures should be implemented to reduce the emergence of resistant strains, as retail conditions had minimal impact on L. monocytogenes prevalence in poultry meat.

Genomic characterization of Listeria monocytogenes and Listeria innocua isolated from milk and dairy samples in Ethiopia.

Listeriosis caused by Listeria monocytogenes often poses a significant threat to vulnerable populations. Dairy products have been implicated in outbreaks of listeriosis worldwide. In Ethiopia, studies have identified Listeria spp. and L. monocytogenes in various dairy products, but the genetic diversity and phylogenetic relationships of these bacteria remain largely unknown in the low- and middle-income countries. Therefore, we conducted whole-genome sequencing on 15 L. monocytogenes and 55 L. innocua isolates obtained from different levels of the dairy supply chains across three regions in Ethiopia. Genomes were assembled and used for MLST genotyping and single nucleotide polymorphism (SNP) analysis to infer phylogenetic relationships. We identified a total of 3 L. monocytogenes (i.e., 2, 145, and 18) and 12 L. innocua (i.e., 1489, 1619, 603, 537, 1010, 3186, 492, 3007, 1087, 474, 1008, and 637) MLST sequence types among the studied isolates. Some of these sequence types showed region-specific occurrence, while others were broadly distributed across regions. Through high-quality SNP analysis, we found that among 13 L. monocytogenes identified as ST 2, 11 of them were highly similar with low genetic variation, differing by only 1 to 10 SNPs, suggesting potential selection in the dairy food supply chain. The L. innocua isolates also exhibited low intra-ST genetic variation with only 0-10 SNP differences, except for the ST 1619, which displayed a greater diversity.

Unlocking the Hidden Threat: Impacts of Surface Defects on the Efficacy of Sanitizers Against Listeria monocytogenes Biofilms on Food-contact Surfaces in Tree Fruit Packing Facilities.

Food-contact surfaces showing signs of wear pose a substantial risk of Listeria monocytogenes contamination and may serve as persistent sources of cross-contamination in fresh produce packinghouses. This study offers a comprehensive exploration into the influence of surface defects on the efficacies of commonly used sanitizers against L. monocytogenes biofilms on major food-contact surfaces. The 7-day-old L. monocytogenes biofilms were cultivated on food-contact surfaces, including stainless steel, polyvinyl chloride, polyester, low-density polyethylene, and rubber, with and without defects and organic matter. Biofilms on those surfaces were subjected to treatments of 200 ppm chlorine, 400 ppm quaternary ammonium compound (QAC), or 160 ppm peroxyacetic acid (PAA). Results showed that surface defects significantly (P < 0.05) increased the population of L. monocytogenes in biofilms on non-stainless steel surfaces and compromised the efficacies of sanitizers against L. monocytogenes biofilms across various surface types. A 5-min treatment of 200 ppm chlorine caused 1.84-3.39 log10 CFU/coupon reductions of L. monocytogenes on worn surfaces, compared to 2.79-3.93 log10 CFU/coupon reduction observed on new surfaces. Similarly, a 5-min treatment with 400 ppm QAC caused 2.05-2.88 log10 CFU/coupon reductions on worn surfaces, compared to 2.51-3.66 log10 CFU/coupon reductions on new surfaces. Interestingly, PAA sanitization (160 ppm, 1 min) exhibited less susceptibility to surface defects, leading to 3.41-4.35 log10 CFU/coupon reductions on worn surfaces, in contrast to 3.68-4.64 log10 CFU/coupon reductions on new surfaces. Furthermore, apple juice soiling diminished the efficacy of sanitizers against L. monocytogenes biofilms on worn surfaces (P < 0.05). These findings underscore the critical importance of diligent equipment maintenance and thorough cleaning processes to effectively eliminate L. monocytogenes contamination on food-contact surfaces.

Biocontrol of L. monocytogenes with Selected Autochthonous Lactic Acid Bacteria in Raw Milk Soft-Ripened Cheese under Different Water Activity Conditions.

The effect of selected autochthonous Lactic Acid Bacteria (LAB) against Listeria monocytogenes was evaluated in two elaborations of soft-ripened cheese performed under high and low relative humidity (RH) elaborations, to achieve aw ranging from 0.97 to 0.94 in ripened cheeses. Two selected autochthonous strains of Lacticaseibacillus casei 31 and 116 were used. In each elaboration, 8 batches were physicochemically and microbiologically evaluated throughout the ripening process. The aw and pH decreased during ripening to final values ranging from 0.944 to 0.972 aw and 5.0 to 5.3 pH, respectively. LAB was the only microbial group that increased throughout the ripening in high and low RH elaborations. In batches that were uninoculated with LAB strains, L. monocytogenes was either maintained at the initial inoculation level or showed a slight reduction by the end of the ripening process. However, in LAB-inoculated batches in the two elaborations, steady decreases of L. monocytogenes were observed throughout maturation. L. casei 31 alone or in combination with strain 116 provoked reductions of 2 to 4 log CFU/g in L. monocytogenes over 60 days of ripening, which could be enough as a strategy for biocontrol to deal with the usual contamination by L. monocytogenes during cheese processing.

An Alternative Rapid Confirmation Method for Identifying Listeria monocytogenes from a Variety of 125 g Food Samples Within Two Days of a PCR Presumptive Positive.

Cultural confirmation following detection of a Listeria monocytogenespresumptive positive can take 3-7 days to finalize; this uncertainty is a point of frustration for food producers needing to make time-sensitive disposition decisions. To address the demand for shortened time-to-results, an alternative L. monocytogenes confirmation method consisting of two components, (i) a secondary screen using a different rapid method, and (ii) concurrent cultural isolation followed by next-day colony identification was evaluated. For the study, four food matrices (hot dogs, peanut butter, frozen vegetables, and multicomponent frozen meals) were inoculated with low levels (0.36-1.39 MPN/125 g) of L. monocytogenes per the AOAC guidelines for a matrix study. Analyses were performed on 125 g test portions and started with a PCR primary screen (Bio-Rad iQ-Check Listeria monocytogenes II). Next, all enriched food samples underwent a secondary screen by bioMérieux's GENE-UP LMO2 Real-Time PCR and VIDAS LMX ELFA along with streaking onto RAPID'L.mono Agar. Presumptive positive L. monocytogenes colonies were identified utilizing a high throughput rapid identification method (Hygiena's BAX System L. monocytogenes Real-Time PCR assay, Neogen's ANSR isothermal nucleic acid amplification assay, and Bruker's MALDI Biotyper). Importantly, this study evaluated multiple commercially available options for the secondary screen (n = 2) and rapid identification (n = 3) to allow for easy adoption by testing laboratories. Overall, there was no statistically significant difference (p ≤ 0.05) between the number of L. monocytogenes-positive 125 g samples obtained by the cultural reference method and the alternative confirmation methods (regardless of which method combinations were evaluated). Additionally, this study supports that, when both the primary and secondary screen methods yield a positive result, the sample could be considered a confirmed positive for L. monocytogenes.

Lignan-containing maple products inhibit Listeria monocytogenes biofilms on fresh produce.

Major listeriosis outbreaks have been associated with fresh produce contaminated with Listeria monocytogenes. Strains that synthesize the Pss exopolysaccharide (EPS) have an estimated 102 to 104-fold advantage over nonsynthesizing strains in causing listeriosis. They more readily attach to the surfaces of fruit and vegetables forming EPS-biofilms that better withstand stresses associated with produce storage and consumption. Here, we show that the threat to fresh produce safety posed by the listerial EPS-biofilms may be countered by broadly available maple products. We serendipitously discovered that aqueous extracts of wood from several Acer (maple) and Carya (pecan, hickory) species inhibit the formation of listerial EPS-biofilms without affecting bacterial viability. One active ingredient in maple wood was identified as nortrachelogenin-8'-O-ß-D-glucopyranoside (NTG). At 120 µM, this lignan decreased colonization of the EPS-synthesizing L. monocytogenes on cantaloupe pieces by approximately 150-fold, and on cut celery and lettuce by 10 to 11-fold. Another lignan, lariciresinol, which is abundant in a common food sweetener, maple syrup, had antibiofilm activity comparable to that of NTG. Diluted in the range of 1:200 to 1:800 maple syrup from two random manufacturers prevented formation of listeiral EPS-biofilms. Importantly, not only did maple products drastically decrease colonization of fresh produce by the EPS-synthesizing strains, they also decreased, by 6 to 30-fold, colonization by the L. monocytogenes strains that do not synthesize measurable EPS, including strains from the infamous 2011 cantaloupe listeriosis outbreak. Inhibition of surface colonization by various listerial strains, broad availability of maple sap and syrup as well as maple lumber processing waste position maple products as potential antibiofilm agents for protecting fresh produce from L. monocytogenes.

DRG2 in macrophages is crucial for initial inflammatory response and protection against Listeria monocytogenes infection.

Innate immune response is critical for the control of Listeria monocytogenes infection. Here, we identified developmentally regulated GTP-binding protein 2 (DRG2) in macrophages as a major regulator of the innate immune response against L. monocytogenes infection. Both whole-body DRG2 knockout (KO) mice and macrophage-specific DRG2 KO mice had low levels of IL-6 during early infection and increased susceptibility to L. monocytogenes infection. Following an initial impaired inflammatory response of macrophages upon i.p. L. monocytogenes infection, DRG2-/- mice showed delayed recruitment of neutrophils and monocytes into the peritoneal cavity, which led to elevated bacterial burden, inflammatory cytokine production at a late infection time point, and liver micro-abscesses. DRG2 deficiency decreased the transcriptional activity of NF-κB and impaired the inflammatory response of both bone marrow-derived and peritoneal macrophages upon L. monocytogenes stimulation. Our findings reveal that DRG2 in macrophages is critical for the initial inflammatory response and protection against L. monocytogenes infection.

Investigating the effect of the inhibitory peptide on L.monocytogenes cell invasion: an in silico and in vitro study.

AIMS: L.monocytogenes monocytogenes is an omnipresent bacterium that causes a fatal food-borne illness, listeriosis. The connection of this bacterium to E-cadherin through internalin A plays a significant role in the internalization of the bacteria. In this study, this interaction has been investigated for the design of an inhibitory peptide. METHODS: The interaction of the proteins involved in the entry of bacteria was evaluated by molecular docking. According to their interactions, an inhibitory peptide was designed to bind to internalin A by server peptiderive. Its effects on L.monocytogenes invasion on the Caco-2 cell line and biofilm formation were also assessed. FINDINGS: Docking results showed that the peptide has a high affinity for binding to Internalin A. The synthesized peptide at a concentration of 64 µg/ml inhibited 80% of the invasion of L.monocytogenes into the Caco-2 cell line. Furthermore, the studied peptide at the highest concentration had a slight inhibitory effect on biofilm formation. CONCLUSION: These results reveal that short polypeptides can impede the invasion of target cells by L. monocytogenes in vitro and could be advantageous as restoring agents in vivo.

Comprehensive metagenomic analysis of stress-resistant and -sensitive Listeria monocytogenes.

Listeria monocytogenes is a pathogenic bacterium which can live in adverse environments (low pH, high salinity, and low temperature). Even though there are various whole genome sequencing (WGS) data on L. monocytogenes, investigations on genetic differences between stress-resistant and -sensitive L. monocytogenes grown under stress environments have been not fully examined. This study aims to investigate and compare genetic characteristics between stress-resistant and -sensitive L. monocytogenes using whole genome sequencing (WGS). A total of 47 L. monocytogenes strains (43 stress-resistant and 4 stress-sensitive) were selected based on the stress-resistance tests under pH 3, 5% salt concentration, and 1 °C. The sequencing library for WGS was prepared and sequenced using an Illumina MiSeq. Genetic characteristics of two different L. monocytogenes groups were examined to analyze the pangenome, functionality, virulence, antibiotic resistance, core, and unique genes. The functionality of unique genes in the stress-resistant L. monocytogenes was distinct compared to the stress-sensitive L. monocytogenes, such as carbohydrate and nucleotide transport and metabolism. The lisR virulence gene was detected more in the stress-resistant L. monocytogenes than in the stress-sensitive group. Five stress-resistant L. monocytogenes strains possessed tet(M) antibiotic resistance gene. This is the first study suggesting that deep genomic characteristics of L. monocytogenes may have different resistance level under stress conditions. This new insight will aid in understanding the genetic relationship between stress-resistant and -sensitive L. monocytogenes strains isolated from diverse resources. KEY POINTS: • Whole genomes of L. monocytogenes isolated from three different sources were analyzed. • Differences in two L. monocytogenes groups were identified in functionality, virulence, and antibiotic resistance genes. • This study first examines the association between resistances and whole genomes of stress-resistant and -sensitive L. monocytogenes.

Growth models for Salmonella, E. coli O157:H7 and L. monocytogenes give different predictions for pathogen growth in cut leafy greens transportation, but are consistent in identifying higher risk conditions.

Leafy greens are frequently implicated in foodborne disease outbreaks and cut-leafy greens are a food that requires time and temperature control for safety. Predictive microbiology uses mathematical models to predict the growth of bacteria based on environmental conditions. The objective of our study was to compare published square root growth models for Salmonella (n = 6), pathogenic E. coli (n = 6) and Listeria monocytogenes (n = 4) using real world transport temperature data. Data from trucks transporting fresh-cut leafy greens during cross-country shipments were used as temperature inputs to the models. Bacterial growth was computed using the temperatures from each probe in every truck over the duration of transit, which resulted in 12-18 growth predictions per truck for each model. Each model generally gave significantly different predictions than other models for the same organism. The exception was for the two Salmonella models predicting the least growth and the two Salmonella models predicting the most growth which gave predictions that were not significantly different. Although different models tended to give different predictions, their ability to rank risk by truck was generally consistent across models. While absolute risk might be dependent upon choice of model, relative risk is independent of model choice.

Rapid separation and detection of Listeria monocytogenes with the combination of phage tail fiber protein and vancomycin-magnetic nanozyme.

In this work, a lateral flow assay for Listeria monocytogenes was developed based on phage tail fiber protein (TFP) and triple-functional nanozyme probes with capture-separation-catalytic activity. Inspired by interaction between phage and bacteria, TFP of L. monocytogenes phage was immobilized on test line as capture molecule, which replaced traditional antibody and aptamer. After Gram-positive bacteria was captured and separated from samples by nanozyme probes modified with vancomycin (Van), TFP specifically recognized L. monocytogenes and overcame non-specific binding of Van. Special color reaction between Coomassie Brilliant Blue and bovine serum albumin which was an amplification carrier on probe was simply utilized as control zone to replace traditional control line. Relying on enzyme-like catalytic activity of nanozyme, this biosensor realized improved sensitivity and colorimetric quantitative detection with a detection limit of 10 CFU mL-1. Analytic performance results suggested this TFP-based biosensor provided a portable, sensitive and specific strategy to detect pathogen.

Risk factors for listeriosis due to sausage consumption in Mexico: consumer practices, bacterial survival, and quantitative microbial risk assessment.

Listeriosis is a foodborne disease caused by Listeria monocytogenes (Lm), which represents a public health problem. Lm has been identified as an important contaminating bacterium of ready-to-eat meat products (RTEM) in Mexico. The objective was to explore the risk factors for acquiring listeriosis due to sausage consumption by defining the consumer profile, evaluating the survival of Lm in sausage (5, 10, and 25 °C for 32 days) and performing a quantitative microbiological risk assessment. The survey of 100 participants revealed that the factors compromising the safety of the RTEM by the consumer are the extension of the shelf life. Acquiring packaged RTEM was observed as a safe habit. All respondents stated that they were unaware of listeriosis, but 18% reported infections linked to RTEM, mainly sausage. The sausage supports the growth of Lm, whose population increases in congruence with temperature (25 °C > 10 °C >5 °C) and storage time (P ≤ 0.05). The increase in temperature decreases the adaptation time (Lag25 °C = 1.0 h, Lag10 °C= 92.5 h, Lag5 °C = 226.1 h) and increases the growth rate (µ25 °C = 4.43 CFU/h, µ10 °C = 0.075 CFU/h, µ5 °C = 0.0026 CFU/h) of Lm on the sausage. The risk of listeriosis due to sausage consumption increased according to the increase in temperature: 5.53 × 10-8-1.42 × 10-5 (5 °C), 0.00616-0.111 (10 °C), and 0.109-1.00 (25 °C). Consumer education in the hygienic management of RTEM and information on associated pathogens will minimize the risk of disease.

Efficacy of Power Ultrasound-Based Hurdle Technology on the Reduction of Bacterial Pathogens on Fresh Produce.

Minimally processed produce is frequently contaminated with foodborne bacterial pathogens. Power ultrasound is a non-thermal and cost-effective technology that can be combined with other chemical sanitization methods. This study investigated the reduction of Listeria monocytogenes and Salmonella Newport on grape tomato, romaine lettuce, and spinach washed with water, chlorine, or peroxyacetic acid alone or in combination with 25 or 40 kHz power ultrasound for 1, 2, or 5 min. Produce items were inoculated with either pathogen at 10 log CFU/g, dried for 2 h, and treated. Combined treatment of ultrasound and sanitizers resulted in 1.44-3.99 log CFU/g reduction of L. monocytogenes and 1.35-3.62 log CFU/g reduction of S. Newport, with significantly higher reductions observed on grape tomato. Synergistic effects were achieved with the hurdle treatment of power ultrasound coupled with the chemical sanitizers when compared to the single treatments; an additional 0.48-1.40 log CFU/g reduction of S. Newport was obtained with the addition of power ultrasound on grape tomato. In general, no significant differences were observed in pathogen reductions between the ultrasound frequencies, the sanitizers, or the treatment lengths. Results from this study suggest that incorporation of power ultrasound into the current washing procedure may be beneficial for the reduction, but not elimination, of bacterial pathogens on certain produce items, including tomatoes.

[Epidemiological study and serotyping by multiple PCR of Listeria monocytogenes isolated from food matrices in Argentina]. / Estudio epidemiológico y serotipificación por PCR múltiple de Listeria monocytogenes aislada de matrices alimentarias en Argentina.

Listeria monocytogenes is an opportunistic foodborne pathogen. It can resist stress conditions by adapting through the production of biofilms, which represents a serious problem for the food industry. It is classified into 14 serotypes, although only four (1/2a, 1/2b, 1/2c, and 4b) account for 89.0-98.0% of listeriosis cases worldwide. The objective of this study was to detect and serotype L.monocytogenes isolated from different food matrices from processing plants in Argentina. In the period 2016-2021, 1832 samples (meat, ready-to-eat foods, ice cream, dairy foods, and frozen vegetables) were analyzed, of which 226 (12.34%) isolates compatible with L.monocytogenes were detected. At the same time, environmental and surface samplings were performed in processing plants for ready-to-eat foods, sausages and dairy products, where environmental contamination with L.monocytogenes was detected in numerous critical points of the process, yielding a positivity rate of 22.7%. The molecular analysis of serogroups was performed, where it was observed that serogroup IIb was the most frequent with 66.5% (n=107), and in descending order IIc with 22.3% (n=36), and IIa (n=9) and IVb (n=9) with 5.6%. The serogroup mostly isolated in environmental monitoring was IIb. This work highlights the importance of the detection and serotyping of L.monocytogenes for taking actionable measures and identifying outbreaks, and is the first study in Argentina to describe an extensive study in food matrices.

Evaluation of the effect of outer primer structure, and inner primer linker sequences, in the performance of Loop-mediated isothermal amplification.

Loop-mediated isothermal amplification, or LAMP, is nowadays the most popular isothermal nucleic acid amplification technique. This technique implements a minimum of four primers, named outer (F3/B3) and inner primers (FIP/BIP). The inner primers hybridize in two distinct regions, and some studies have reported that the usage of a linker, typically composed of four thymines, in the middle of these primers can improve assay performance. In addition to this, dual-priming oligonucleotides, DPO, have been reported to provide highly specific reducing non-specific amplifications. Considering the large number of primers implemented in LAMP assays, in the current study the suitability of DPO primers replacing regular outer primers; and their combination with different linker sequences in the inner primers were explored. The results demonstrated that replacing standard F3/B3 by DPO primers does not significantly affect that overall performance of the assay, and provides additional stability to temperature changes. This observations were consistent regardless the type of linker implemented in the inner primers, out of which in the current study a linker composed of thymines significantly outperformed the other options tested, most likely due to a combination of sequence and physical structure.