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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Surveillance and examination of microbial contamination in ice cream in China

This study investigated the microbial contamination of ice cream in China.A total of 2887 ice cream samples were collected from different regions of China.Contamination by the aerobic plate count(APC),coliforms,and three foodborne pathogens,Listeria monocytogenes(L.monocytogenes),Staphylococcus aureus(S.aureus),and Salmonella spp.,was detected in the samples.L.monocytogenes isolates were further analyzed for antibiotic susceptibility and multi-locus sequence typing(MLST).The results showed that APC was more than 10^(5) colony forming units(CFU)/g in 6.10%(176/2887)and coliform was more than 10^(2) CFU/g in 15.69%(453/2887)of all samples.The positive rates of S.aureus,L.monocytogenes,and Salmonella spp.were 0.66%,0.62%,and 0.10%,respectively.Among these,S.aureus contamination was more than 102 CFU/g in two samples,and L.monocytogenes in the positive sample was in the range of 0.3-240 most probable number(MPN)/g.with a median of 4.3 MPN/g.The hygienic status of the packaged samples was much better than that of the bulk samples(P<0.05).Catering samples were more frequently and heavily contaminated than samples from retail and wholesale outlets(P<0.05).No significant differences were observed in samples bought from urban and rural areas(P>0.05).For 18 L.monocytogenes strains isolated from ice cream,the resistance rate of nine antibiotics was 5.56%(1/18).By MLST,the L.monocytogenes strains were classified into nine sequence types(STs),of which ST8 was the most common(six isolates).These results indicate that a potential health risk to the public may be caused by ice cream,particularly in susceptiblepopulations.

Research progress on antibiotic resistance of Salmonella

Antibiotic abuse results in various antibiotic resistance among a number of foodborne bacteria,posing a severe threat to food safety.Antibiotic resistance genes are commonly detected in foodborne pathogens,which has sparked much interest in finding solutions to these issues.Various strategies against these drug-resistant pathogens have been studied,including new antibiotics and phages.Recently,a powerful tool has been introduced in the fight against drug-resistant pathogens,namely,clustered regularly interspaced short palindromic repeats-CRiSPR associated(CRISPR-Cas)system aggregated by a prokaryotic defense mechanism.This review summarizes the mechanism of antibiotic resistance in Salmonella and resistance to common antibiotics,analyzes the relationship between Salmonella CRiSPR-Cas and antibiotic resistance,discusses the changes in antibiotic resistance on the structure and function of CRiSPR-Cas,andfinally predicts the mechanism of CRISPR-Cas intervention in Salmonella antibiotic resistance.In the future,CRiSPR-Cas is expected to become an important tool to reduce the threat of antibiotic-resistant pathogens in food safety.

Combined antibacterial and antibiofilm activity of phenyllactic acid and bacteriocin XJS01 against Shigella flexneri

This study investigated antibacterial and antibiofilm activity of the combined use of phenyllactic acid(PLA)and bacteriocin XJS01 against Shigella flexneri_14.The minimum inhibitory concentration(MIC)of PLA and XJS01 against S.flexneri_14 was 2.45 mg/mL and 18.75μg/mL,respectively.Growth and kill kinetics assays showed that the combined use of 1/2MIC PLA plus 1/2MIC XJS01 had a better activity against planktonic S.flexneri_14 compared to treatment with PLA and XJS01 used singly(1/2MIC and 2MIC).Cellular biochemical and morphological analysis revealed the remarkable ability of the combination in disrupting cell appearance and promoting deformation of planktonic S.flexneri_14 compared to single use.Moreover,S.flexneri_14 biofilm formation was inhibited and degraded by the combination,which showed a more remarkable antibiofilm activity than PLA and XJS01 when used singly.This study demonstrates the synergistic antibacterial activity of PLA and XJS01 against S.flexneri_14 in either planktonic or biofilm states in foods.

Microbiological safety of spices and their interaction with antibiotics:implications for antimicrobial resistance and their role as potential antibiotic adjuncts

Objectives:A study was undertaken to:1.examine contaminating bacteria on a variety of spices purchased at retail market;2.investigate if spice bacterial enrichments alter the phenotype of 13 bacterial foodborne and clinical pathogens and 1 probiotic organism;and 3.investigate if spices can alter antimicrobial activity of seven clinical antibiotics against 16 bacterial foodborne/clinical pathogens.Materials and Methods:Microbiological examination was undertaken employing 27 spice varieties with four antibiotics and 15 bacterial pathogens.Results:Bacteriological contamination levels varied amongst spice varieties,ranging from Kasmin chilli powder(7.5×10^(6)cfu/g;log10^(6).88 cfu/g)to ginger(1.5×10^(4)cfu/g;log_(10)4.18 cfu/g);mean contamination was 1.38×10^(6)cfu/g(log_(10)6.14 cfu/g).Four species within the genus Bacillus were identified(Bacillus megaterium,Bacillus subtilis,Bacillus licheniformis,and Bacillus cereus).There was no phenotypic difference with the 14 bacteria,with bacterial colony growth/proliferation,pigment production,or with adhesin and mucoid production.None of the spice cultures inhibited any of the 14 bacterial species examined.In the case of doxycycline,amoxicillin,colistin,erythromycin,and piperacillin/tazobactam,the zone of inhibition increased with the inclusion of the 26 spice varieties,suggesting that the spices were interacting synergistically with the antibiotic,thus making the antibiotic more potent against the bacteria tested.Conclusions:This study demonstrates a positive interaction between spices and conventional antibiotics.Given the burden of antimicrobial resistance(AMR)worldwide,but particularly in South Asian countries(India and Pakistan),any food-related innovation that can help maximize the potency of existing antibiotics is to be encouraged and developed.The specific mechanism as to how spices increase the potency of antibiotics needs to be elucidated,as well as novel food(spice)delivery modalities including novel medicinal foodstuffs or functional foods,that can harness this beneficial effect for medicine and society.