The cascade regulation of small RNA and quorum sensing system: Focusing on biofilm formation of foodborne pathogens in food industry

Biofilms containing foodborne pathogens can adhere to food ingredients or food processing equipment.Due to their enhanced resilience to cleaning and chance of cross-contamination,there is an increased risk of food poisoning to consumers.The life course of biofilms is generally thought to be regulated by a quorum sensing(QS)system by secreted autoinducing peptides(AIPs).Recent studies have revealed that small non-coding RNA(sRNA)also plays an important role in regulating biofilm formation at the post-transcriptional level.Moreover,sRNA can act as an intermediate regulating hub between the quorum sensing system and biofilm.This review summarizes the latest findings on sRNA in biofilms of common foodborne pathogens and their potential impact.We also highlight the different mechanism of cascade regulation of sRNA and QS system in biofilm formation in several typical foodborne pathogens.In this process,sRNA responds to dynamic external environmental signals finely and accurately,and can feedback information on the bacterial cell environment to modify the QS system.These findings will deepen our understanding of biofilm formation and provide new perspectives for eliminating biofilm of foodborne pathogens based on the cascade regulation of small RNA and QS for developing biofilm-free food-processing systems.

Estimates and burden of foodborne pathogens in RTE beverages in relation to vending practices

Objectives:Growing trend of street-vended food in underdeveloped countries offers low-cost food to many sections of population.Although it provides job opportunities to many urban dwellers,several health hazards are associated with this business.The present study investigates the burden of foodborne pathogens in Ready-To-Eat(RTE)beverages in relation to vending practices among street vendors of Rawalpindi City,Pakistan according to standardized methods and protocols.Materials and Methods:Six densely populated locations of Rawalpindi city were selected.Commonly consumed sugar cane juice(SCJ)and tamarind prune(dried plums)drink(TPD)(locally called as Imli Alu Bukhara sherbet)from five vendors from each location were chosen in summer season where the temperature reaches above 40℃.Mean and the standard deviation were obtained by univariate and bivariate analyses.Association between the study variables was assessed through cross-tabulations,chi-square,and correlation tests.Results:All the samples were found unsatisfactory in comparison to guidelines of aerobic plate count.Total coliform was observed in 86.7 per cent of SCJ and 70.0 per cent of TPD samples.Fourteen samples of SCJ exceeded the limit of>1100 MPN/ml value,whereas samples of TPD exceeded this limit for Escherichia coli.All of SCJ and 93.3 per cent of TPD samples depicted the presence of Salmonella aureus.Salmonella spp.were found significantly high in 73.3 per cent samples of SCJ and 23.3 per cent samples of TPD.Conclusions:The incidence of high bioloads attributes towards a potential reservoir of foodborne pathogens due to unhygienic vending practices.

Comparing invasive effects of five foodborne bacterial pathogens in human embryonic intestine 407 cells and human ileocecum HCT-8 cells

Objective: To refine the infectious doses of enteric bacterial pathogens in animal assays and vaccine clinical trials by studying the invasion kinetics of five bacterial pathogens with human intestinal cells.Methods: Utilizing in vitro cultured cell invasion assays with gentamicin-killing step,the invasive effects were analyzed in foodborne pathogens including Salmonella,Shigella, Yersinia, Escherichia coli(E. coli) O157 and opportunistic pathogens Citrobacter in human embryonic intestine 407 cells and ileocecum HCT-8 cells at multiplicities of infection(MOIs) of 0.04–4 000.00 E. coli HS served as a noninvasive control.Results: The study results showed that the bacterial invasive efficiency and the average number of internalized bacteria per host cell changed with different starting MOIs. Higher starting MOIs did not always produce more bacterial internalization. The bacterial invasion effects varied with different bacterial strains and host cell lines. E. coli O157:H7 did invade human ileocecum HCT-8 cells.Conclusions: This study shows that these bacteria possess different invasive patterns at various starting MOIs and also in different cell lines. The results could help to figure out the appropriate infectious doses of the bacteria in animal assays and in vaccine clinical trials. The bacterial invasion kinetics is also valuable in evaluating the safety and efficacy of live attenuated bacterial vaccines.

Recent progress on lateral flow immunoassays in foodborne pathogen detection

Foodborne pathogens cause diseases in humans.The traditional methods of detecting foodborne pathogens are time-consuming.The lateral flow immunoassay(LFIA)has become a widely used detection platform for onsite testing of various foodborne pathogens due to its time-efficiency,cost-effectiveness,portability,and ease of use.With the development of novel nanomaterials,the sensitivity of the LFIA has improved tremendously compared with traditional colorimetric LFIA sensors.This review first summarizes the principles and corresponding formats of the LFIA.Then,a detailed classification of nanomaterial label(e.g.,metallic,carbon and selenium,fluorescent,and magnetic nanoparticles)synthesis,signal amplification strategy,and detection principles are discussed as related to food safety.Subsequently,the LFIA used in the detection of pathogenic bacteria,including Escherichia coli,Vibrio parahaemolyticus,Staphylococcus aureus,Listeria monocytogenes,and Salmonella,are classified and summarized.Multiple signal modes have been explored that improve the sensitivity of foodborne pathogen detection.Further improvement should focus on the design and preparation of high signal-to-noise ratio nanomaterials to achieve highly sensitive detection,and multitarget and multimode sensing.

Rapid label-free SERS detection of foodborne pathogenic bacteria based on hafnium ditelluride-Au nanocomposites

Two-dimensional(2D)nanomaterials have captured an increasing attention in biophotonics owing to their excellent optical features.Herein,2D hafnium ditelluride(HfTe_(2)),a new member of transition metal tellurides,is exploited to support gold nanoparticles fabricating HfTe_(2)-Au nanocomposites.The nanohybrids can serve as novel 2D surface-enhanced Raman scattering(SERS)substrate for the label-free detection of analyte with high sensitivity and reproducibility.Chemical mechanism originated from HfTe_(2) nanosheets and the electromagnetic enhancement induced by the hot spots on the nano-hybrids may largely contribute to the superior SERS effect of HfTe_(2)-Au nanocomposites.Finally,HfTe_(2)-Au nanocomposites are utilized for the label-free SERS analysis of foodborne pathogenic bac-teria,which realize the rapid and ultrasensitive Raman test of Escherichia coli,Listeria mono-cytogenes,Staphylococcus aureus and Salmonella with the limit of detection of 10 CFU/mL and the maximum Raman enhancement factor up to 1.7×10^(8).Combined with principal component analysis,HfTe_(2)-Au-based SERS analysis also completes the bacterial classification without extra treatment.

PCR-based methodologies for detection and characterization of Listeria monocytogenes and Listeria ivanovii in foods and environmental sources

Listeria monocytogenes is an important foodborne pathogen responsible for listeriosis,a fatal disease.It is widely distributed in various foods and environmental sources.In this review,we focused on addressing PCR-based technologies,including conventional PCR,qPCR and droplet digital PCR(ddPCR).Specifically,we described(a)conventional PCR and mono-,duplex-and multiplex-qPCR methodologies;(b)development and applications of gene HlyA-,Iap-,PrfA–and SsrA-based conventional and qPCR assays as well as PCR assays targeting newly identified gene targets for specific detection of L.monocytogenes;differentiation of viable from dead L.monocytogenes by qPCR in conjugation with propidium monoazide pretreatment;PCR-based serotype identification of L.monocytogenes isolates;PCR-based detection of L.ivanovii,infecting ruminants,differentiation of L.monocytogenes from other Listeria species;and sigB-gene based PCR identification of Listeria spp;(c)applications of ddPCR in detection of L.monocytogenes;and(d)application of qPCR assays in detection and subtyping of L.monocytogenes in milk and dairy products;meats,meat products and meat-processing environment;and seafood,seafood products and processing environment.Our goal was to provide a relatively comprehensive overview of PCR-based methodologies available in detection,characterization and subtyping of various strains of L.monocytogenes in foods and environmental sources.

Prevalence and methodologies for detection,characterization and subtyping of Listeria monocytogenes and L.ivanovii in foods and environmental sources

Listeria monocytogenes,one of the most important foodborne pathogens,can cause listeriosis,a lethal disease for humans.L.ivanovii,which is closely related to L.monocytogenes,is also widely distributed in nature and infects mainly warm-blooded ruminants,causing economic loss.Thus,there are high priority needs for methodologies for rapid,specific,cost-effective and accurate detection,characterization and subtyping of L.monocytogenes and L.ivanovii in foods and environmental sources.In this review,we(A)described L.monocytogenes and L.ivanovii,world-wide incidence of listeriosis,and prevalence of various L.monocytogenes strains in food and environmental sources;(B)comprehensively reviewed different types of traditional and newly developed methodologies,including culture-based,antigen/antibody-based,LOOP-mediated isothermal amplification,matrix-assisted laser desorption ionization-time of flight-mass spectrometry,DNA microarray,and genomic sequencing for detection and characterization of L.monocytogenes in foods and environmental sources;(C)comprehensively summarized different subtyping methodologies,including pulsed-field gel electrophoresis,multi-locus sequence typing,ribotyping,and phage-typing,and whole genomic sequencing etc.for subtyping of L.monocytogenes strains from food and environmental sources;and(D)described the applications of these methodologies in detection and subtyping of L.monocytogenes in foods and food processing facilities.

Microbial profile of some ready-to-cook frozen food items sold in Dhaka city,Bangladesh

This study aimed to assess the microbiological quality of 159 animal-and plant-based ready-to-cook(RTC)frozen food items currently popular in Dhaka.Microbiological analyses were carried out to enumerate total aerobic counts of mesophilic bacteria,yeast and mold and to detect some common foodborne pathogens according to standard microbiological criteria.Higher microbial loads in all animal-based food types,other than chicken strips were found.In contrast,plant-based items were relatively safe except paratha,dal-puri,rooti chapati,and aloo-puri.Several common pathogenic bacterial species were screened by analyzing their morphological,cultural,and biochemical characteristics.Escherichia coli was prevalent in most samples,while Salmonella species were least reported.A considerable number of samples exhibited enough microbial loads at infection or intoxication levels.

Subinhibitory Levels of Fluoroquinolones Result in Enrichment of the Membrane Proteome of Staphylococcus aureus

Staphylococcus aureus is a common marine foodborne pathogen.In this study,antibiotics ciprofloxacin and enrofloxacin were used to induce drug-resistance in S.aureus.The differentially expressed proteins(DEPs)were analyzed and compared with those in the bacteria cultured without antibiotics.The primary proteomic alterations were in the levels of cell membrane components and proteins related to lysine and folic acid biosynthesis,which were all significantly up-regulated.The minimal inhibitory concentrations(MIC)for both test drugs were elevated to 10μg m L^(-1)following serial passaging.These results indicated that,for both ciprofloxacin and enrofloxacin,drug-resistance were developed even in the subinhibitory levels and the primary response was a major alteration in the cell membrane proteome.These changes were similar to those observed in S.aureus cultured with super-MIC levels of these antibiotics.The current study provides a theoretical basis for in-depth study of the related changes of marine foodborne pathogens in subinhibitory concentrations that are commonly found in situ.

Antibacterial properties of citral against Staphylococcus aureus:From membrane damage to metabolic inhibition

Citral,a natural antimicrobial agent,has been found to induce severe membrane damage resulting in bacterial death.Despite its effectiveness,there is a lack of in-depth research on its interaction with bacterial cells,espe-cially at low concentrations.This work aimed to investigate the impact of citral concentration on its antibacterial effect against Staphylococcus aureus(S.aureus)and to analyze the relevance between membrane damage and cell death.In this study,the minimal inhibitory concentration of citral against S.aureus was measured at 0.25 mg/mL.At a concentration of 0.5 mg/mL,citral significantly disturbed the cell membrane fluidity and permeability,leading to the leakage of intracellular macromolecules and rapid cell death.However,when the concentration was decreased to 0.25 mg/mL,the membrane damage was greatly reduced,and a complex biological process between citral and membrane components was found.Under continuous drug exposure,the cell membrane underwent gradual rigidification,concomitant with the loss of membrane function,ultimately causing cell death.Despite the fact that no notable disturbance to membrane functions was discovered,the presence of 0.125 mg/mL citral could still weaken the metabolic vitality and growth of S.aureus.Therefore,it was suggested that there could be multiple mechanisms of citral action on S.aureus cells,with drug concentration being a critical factor affecting the mode of action.Overall,the findings of this study provide valuable insights into the antibacterial mechanisms of natural compounds and broaden the potential applications of citral as a natural antimicrobial.

Analysis and Evaluation of Microbial Contamination of Raw Beef in Supermarkets in Phnom Penh

The study focused on analysis and evaluation of microbial contamination of raw beef was designed to （1） determine the presence of foodborne pathogens in raw beef; （2） compare the prevalence of microbial contamination among the three supermarkets, and （3） evaluate the sanitary quality of raw beef products. Also, six kind of microorganisms including Total Plate Count, Total Coliform and Fecal Coliform, which represented sanitary quality and E. coli, S. Aureus and B. cereus, which determined the presence of foodborne pathogens, were analyzed. The procedures used to analyse experimental samples taken from three supermarket （in every one week for three weeks） in Phnom Penh were based on Merck＇s study in 2005. The results had shown that there were no significant differences in Total Plate Counts, Total Coliform and Fecal Coliform in all samples, and they were found in unacceptable numbers in the raw beef products. However, the prevalence of foodborne pathogens including E. coli, S. aureus and B. cereus were found in acceptable numbers. Furthermore, the samples taken from the second supermarket had the highest level of microbial contamination among the three supermarkets, while the samples taken from the first supermarket had the lowest level of microbial contamination. This experimental finding demonstrated the need for sanitary improvement in the beef retails markets and strict sanitary guideline and implementation of these practices could guarantee consumers＇ health by consuming raw beefs with the lowest risk of foodborne pathogens.

Animals as sources of food-borne pathogens: A review

Food-producing animals are the major reservoirs for many foodborne pathogens such as Campylobacter species, non-Typhi serotypes of Salmonella enterica, Shiga toxin-producing strains of Escherichia coli, and Listeria monocytogenes. The zoonotic potential of foodborne pathogens and their ability to produce toxins causing diseases or even death are sufficient to recognize the seriousness of the situation. This manuscript reviews the evidence that links animals as vehicles of the foodborne pathogens Salmonella,Campylobacter, Shiga toxigenic E. coli, and L. monocytogenes, their impact, and their current status. We conclude that these pathogenic bacteria will continue causing outbreaks and deaths throughout the world, because no effective interventions have eliminated them from animals and food.

Cross-Cutting Approach for the Characterization of Microbial Emerging Hazards in Agriculture Settings from Circular Economy-Driven Wastewater Streams

The recycling of biowaste from municipal wastewater treatment plants(WWTPs)in agriculture represents a circular economy-driven source of water and nutrients to support food system sustainability.However,biowaste may represent the source of emerging hazards of anthropogenic and animal origin that can transfer from agricultural soils to related food production,posing a risk to consumers’health,as in the case of outbreaks due to the consumption of ready-to-eat leafy vegetables contaminated with pathogenic E.coli.From this perspective,we propose a combined strategy based on both classical methods and culture-independent metagenomics approaches to identify microbial hazards relevant to foodborne diseases in WWTP-related biowastes.The virulence genes targeted by real-time PCR,performed before and after the enrichment of the raw samples,may represent a proxy for the viability of pathogens,the presence of which is then confirmed via classical microbiological methods.Bioinformatics analysis of shotgun metagenomic sequences could assess the presence of genes associated with resistance to specific antimicrobials followed by phenotypic confirmation via cultivation of the raw samples in the presence of the predicted molecules.Bacterial 16S rDNA analysis supports biowaste traceability based on their taxonomic composition.This strategy would support a“One Health”Action based on a cross-cutting assessment of emerging food-borne risks along the food chain.

Synthesis and antibacterial activity of 8-ketoberberine derivatives

Natural berberine(BBR)exhibits good antimicrobial efficacy:however,its limited bioavailability greatly hinders its clinical application.The aim of this study is to synthesize a series of novel lipophilic C-8 substituted BBR prodrugs,conduct in vitro antibacterial activity tests on four common clinical pathogens,and further investigate their inhibitory mechanisms through bacterial membrane formation ability,cell permeability,hydrophobicity,and self-cohesion.Compound 14 showed the best activity with minimum inhibitory concentration(MiC)of 15.62μg/mL and minimum bactericidal concentration(MBC)of 31.25μg/mL against Staphylococcus aureus(ATCC 29213)and Listeria monocytogenes(4b G).In the mechanism study,compound 14 significantly inhibited bacterial biofilm formation by over 60% at a concentration of 0.5 MiC and enhanced cell membrane permeability by 40%.Meanwhile,compound 14 can interact with the cell membrane,exposing its hydrophobic regions and reducing the intercellular self-coagulation index to approximately 20%.The study indicates that the novel compound 14 enhanced the antibacterial efficacy by significantly reducing bacterial surface hydrophobicity and self-cohesion,as well as disrupting bacterial biofilm.In summary,we have created a series of 8-ketoberberine derivatives,which opens new prospects for BBR derivatives in the treatment of bacterial infections.

Antibiofilm activity and synergistic effects of DNase I and lysostaphinaggainst Staphylococcus aureus biofilms

Background:In recent years,food safety has become a global public health concern.Microbial contamination is one of the most common food safety issues.Staphylococcus aureus is a common foodborne pathogen that can form biofilms on the surface of food processing equipment,leading to greater resistance to antimicrobial agents than occurs with planktonic bacteria.Materials and methods:In this work,recombinant Escherichia coli BL21(DE3)cells expressing optimized lysostaphin(Lst)were constructed,recombinant Lst was produced and purified,and Lst enzymatic assays were performed,followed by antimicrobial testing of Lst.Finally,a mixtureof Lst and DNase I was tested for antibiofilmactivity.Results:The protein content of purified Lst was 0.6 mg/mL and the enzyme activity was 240 U/mL.The minimum inhibitory concentration(MiC)of Lst against S.aureus was O.1μg/mL.At 1 MiC,Lst exerted an effect on the growth,cell wall integrity and cell membrane permeability of S.aureus.Conclusions:Although Lst alone also showed good inhibition and disruption of S.aureus biofilms,the inhibition and disruption of S.aureus biofilms were significantly greater when Lst was mixed with DNase l.This is probably because DNase I removes extracellular DNA,affecting biofilm formation and dispersing mature biofilms,and thereby facilitating the penetration of Lst.

Research on pH-responsive antibacterial materials using citral-modified zinc oxide nanoparticles

Objectives:With the increasing damage caused by foodborne pathogens to human health and the increasing attention given to healthy diets,novel food antimicrobial agents have been widely studied.Materials and Methods:In this study,three different morphologies of citral-modified ZnO nanoparticle antimicrobial materials were prepared,and the citral-modified porous ZnO nanorod antimicrobial materials with the highest loading(60.35%)and the strongest inhibitory effect(MIC=0.2-0.1 mg/mL)were screened through a series of characterization and bacterial inhibition experiments.This novel antimicrobial material has excellent and long-lasting antimicrobial properties.It inhibited Escherichia coliby 100% when stowed at 25℃ and protected from light for 10 d and inhibited the growth of E.coli by 58.17% after being stowed under the same conditions for 60 d.Furthermore,we tested the pH change during 24 h of E.coli growth and the pH responsiveness of the materials.Results:The results demonstrated that under the acid-producing condition of E.coli growth,the pH-sensitive imine bond(-CH=N-)formed by the condensation of the amino of functionalized ZnO nanoparticles and citral was hydrolyzed to release the citral,which indicated that the release mechanism of citral in the antibacterial material was pH-sensitive.Conclusions:The antibacterial materials in this study have broad application prospects in the field of food production and packaging in the future.Moreover,this study provides a theoretical basis for guaranteeing food quality and safety.

Systematic review of Listeria monocytogenes from food and clinical samples in Chinese mainland from 2010 to 2019

Listeria monocytogenes(L.monocytogenes),a foodborne pathogen,can cause human listeriosis.Listeriosis is a potentially fatal gastrointestinal illness,which is closely related to the spread of food to humans.We review the literature published during 2010-2019 to better understand the prevalence of L.monocytogenes in food products,incidence of human listeriosis,and their characteristics in Chinese mainland.We found the main sequence types(STs)strains from foods are similar globally,and the prevalence of L.monocytogenes from raw meat was the highest among all food products.The most common STs in food products and clinical cases were ST9(serogroup I.2)strains and ST87(serogroup Il.2)strains,respectively.The ST87 strains being the most common STs of clinical cases might be related to the existence of Listeria pathogenicity island 4 gene and Chinese eating habits for ready-to-eat foods,among which the prevalence of ST87 strain was the highest in ready-to-eat foods.Therefore,more research should be conducted to explore the reasons for the L.monocytogenes isolates differences in food and clinical sources.

A Novel Aptamer Lateral Flow Strip for the Rapid Detection of Gram-positive and Gram-negative Bacteria

Diseases caused by foodborne pathogens are one of the main burdens of public health and seriously hinder global social and economic development.In this research,a novel lateral flow strip was successfully constructed for the simultaneous detection of Gram-positive and Gram-negative bacteria based on the fluidity and color aggregation effect of Au nanoparticles(AuNPs).The lateral flow strip first combined ampicillin(AMP)antigens with AuNPs to form AuNPs-AMP antigens as the first recognition molecule.Then vancomycin specifically recognizing Gram-positive bacteria,and aptamers specifi-cally recognizing Gram-negative bacteria were used as the second recognition molecules.Finally,Gram-positive bacteria and Gram-negative bacteria were detected rapidly using this sandwich mode.The strip can rapidly test the samples within 5 min.Using a lateral flow strip detector,the limit of detection was 4 CFU/mL,and the recovery rates in honey samples were 78.2-88.6%.The lateral flow strip constructed can detect foodborne pathogens quickly,accurately and e fficiently,which is of great significance for food safety detection.

Milk phospholipids-based nanostructures functionalized with rhamnolipids and bacteriocin:Intrinsic and synergistic antimicrobial activity for cheese preservation

Milk fat globule membrane (MFGM) phospholipids-based nanostructures were developed and their functionalization with rhamnolipids (RLs) was carried out to enhance the preservation of cheese against resistant foodborne pathogens i.e.,Listeria monocytogenes and Escherichia coli .Void (without nisin) and nisin-loaded RLs functionalized MFGM nanostructures (RLs-MFGM-NS) were fabricated by ultrasonication-assisted self-assembly method.Cubic morphology of void and loaded RLs-MFGM-NS and pristine MFGM nanostructures (MFGM-NS) was observed under scanning electron microscopy (SEM),which indicated uniform size ranging from 43 nm (void RLs-MFGM-NS) to 194 nm (loaded RLs-MFGM-NS).FTIR analyses confirmed the electrostatic interaction,predominantly H-bonding and linkage of carboxyl ester group of MFGM with C–H group in RLs after functionalization of NS.Furthermore,quantitative antimicrobial assay on cheese slices confirmed the broad-spectrum potential of intrinsically active nanostructures (due to RLs) having synergistic activity with nisin against L.monocytogenes and E.coli .Hence,nisin-loaded RLs-MFGM-NS can be applied as promising bioactive additives for sustained preservation of cheese.

A high-efficiency pretreatment method for elution of pathogenic bacteria in lettuce

Many current studies on rapid detection of pathogenic bacteria in foods have focused on the construction of detection methods,neglecting pretreatment.It is also a key step to efficiently elute pathogenic bacteria from food samples for rapid detection and can even determine the success or failure of an assay.In this study,we used Escherichia coli(E.coli),Salmonella enteritidis(S.enteritidis),and Listeria monocytogenes(L.monocytogenes)as model bacteria to compare the elution efficiency of different eluants;explore the effect of surfactant,ionic strength,protein(or amino acid and peptide),and enzyme on the recovery rate of bacteria in lettuce;and compare the compound effect caused by multiple factors.Finally,we developed an efficient bacterial recovery method and confirmed the superiority of this method to analyze the bacterial diversity of eluants from lettuce.The results showed that the recovery efficiency of E.coli,S.enteritidis,and L.monocytogenes,which were artificially contaminated with approximately 10^(5)CFU/g in lettuces,could reach 94.4%,90.6%,and 93.7%by using 10 mmol/L Tris·HCl(pH 9.5)with 0.1%peptone and 300 U/100 mL of cellulase,and furthermore,the elution efficiency could reach 99.6%,98.6%,and 100%with the aid of a 2-min stomaching.For the lettuce samples with only native bacteria,the recovery rate reached 92.1%for viable aerobic bacteria by this method,which was approximately 10%higher than that of the modified previous method.The bacterial diversity of the eluted solution was analyzed,and the result was significantly improved.Considering these advantages,it is important to improve the elution efficiency to achieve rapid and accurate detection of pathogenic bacteria in lettuces.