Common Foodborne Pathogens Research Articles

Detection of Antimicrobial Resistance Genes from Shiga Toxin Producing Escherichia coli by Multiplex PCR

Background: Shiga toxin-producing Escherichia coli (STEC) strains are considered to be most common food-borne enteric zoonotic pathogen, causing various disease conditions in both animals and humans and are highly pathogenic to human in low infectious doses. Resistance against antibiotics by STEC is also a big concern now a days. Hence in view of the public health significance of STEC, present work was planned to know the combination of phenotypic and genotypic resistance patterns against certain most commonly used antibiotics by using disk diffusion method and multiplex PCR respectively. Methods: A total of 426 PCR confirmed STEC isolates isolated from pooled samples (animal faecal(179), farm water(122) and human faecal samples (125) of different livestock farms in and around Proddatur andhra Pradesh, were subjected to antibiotic sensitivity test by using disc diffusion method. The isolates that showed resistance for tetracycline, streptomycin, sulphonamides and ampicillin were selected and subjected to multiplex PCR for the detection of resistance genes. Result: Disk diffusion assay revealed highest phenotypic resistance for STEC isolates against Cepahlothin (100%), followed by Ampicillin (99.06%), Tetracycline (97.2%), Streptomycin (94.3%), Sulphonamides (90.8%) and Trimethoprim (84.5%). Pooled samples also revealed the presence of antimicrobial resistance genes like tetA(59.2%), tetB(43.5%), tetC (9.2%), strA (39.3%), strB(54.1%), sul1 (40.8%), sul2 (58.7%), sul3 (3.8%) and blaTEM(83.4%). These findings indicate the highest prevalence of antimicrobial resistance among the STEC isolates, which alarms indiscriminate use of antibiotics both for therapeutic purpose and as growth promoters. Strict hygienic, sanitation and HACCP programmes should be applied to counter STEC prevalence.

Antioxidant and antibacterial activity of Apis laboriosa honey against Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a common food-borne pathogen that commonly causes gastroenteritis in humans and animals. Apis laboriosa honey (ALH) harvested in China has significant antibacterial activity against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis. We hypothesize that ALH has antibacterial activity against S. Typhimurium. The physicochemical parameters, minimum inhibitory and bactericidal concentrations (MIC and MBC) and the possible mechanism were determined. The results showed that there were significantly different physicochemical parameters, including 73 phenolic compounds, among ALH samples harvested at different times and from different regions. Their antioxidant activity was affected by their components, especially total phenol and flavonoid contents (TPC, TFC), which had a high correlation with antioxidant activities except for the O2- assay. The MIC and MBC of ALH against S. Typhimurium were 20-30% and 25-40%, respectively, which were close to those of UMF5+ manuka honey. The proteomic experiment revealed the possible antibacterial mechanism of ALH1 at IC50 (2.97%, w/v), whose antioxidant activity reduced the bacterial reduction reaction and energy supply, mainly by inhibiting the citrate cycle (TCA cycle), amino acid metabolism pathways and enhancing the glycolysis pathway. The results provide a theoretical basis for the development of bacteriostatic agents and application of ALH.

A New Duplex Recombinase Polymerase Amplification (D-RPA) Method for the Simultaneous and Rapid Detection of Shigella and Bacillus cereus in Food.

Shigella and Bacillus cereus are two common foodborne pathogens that cause intestinal diseases and seriously affect human life and health. Traditional microbiological culture methods are time-consuming and laborious, and polymerase chain reaction (PCR)-based methods rely on expensive thermal cyclers and lengthy reaction times. In this study, on the basis of the specific gene ipaH7 of Shigella and the virulence gene nheABC of B. cereus, a duplex detection system was established for the first time by using the recombinase polymerase amplification technique (D-RPA). After optimization, D-RPA could be effectively amplified at 42 °C for 25 min with excellent specificity, and the detection limits of D-RPA for Shigella and B. cereus in artificially contaminated samples were 2.7 × 101 and 5.2 × 102 CFU/mL, respectively. This study provides a certain research basis for multiple detection with RPA, an isothermal amplification technology. Furthermore, it lays a good foundation for high-throughput rapid detection of foodborne pathogens.

Impact of chlorine or peracetic acid on inactivation of Salmonella, Escherichia coli, and Listeria monocytogenes in agricultural water

Preharvest agricultural water has been recognized as one of the routes of contamination for foodborne pathogens during fruit and vegetable production. Several strategies have been proposed to reduce the risk of pathogens, including preharvest water chemigation, but literature is lacking with regards to microbiological inactivation of common bacterial foodborne pathogens associated with fresh produce contamination, Salmonella enterica, Shiga-toxigenic Escherichia coli (STEC), and Listeria monocytogenes, in surface irrigation water after exposure to chlorine and peracetic acid (PAA). Surface water supplied by a local irrigation district was collected over the summer of 2019. Water was autoclaved, divided into 100 mL samples, and inoculated with a cocktail of five Salmonella, STEC, or Listeria monocytogenes strains or a single strain non-pathogenic E. coli. Samples were then treated with 3, 5, or 7 ppm of free chlorine or PAA, and surviving populations were evaluated using a time-kill assay. A first-order kinetic model was used to fit the inactivation data and obtain the D-values. A secondary model was used to explain the changes due to the type of water, treatment, and microorganism. At a concentration of 3 ppm, the observed and predicted D-values of free chlorine treatments were higher than that of PAA treatments for ground and surface water. Results indicated that PAA was more effective inactivating bacteria than sodium hypochlorite at concentrations of 3 and 5 ppm for both water sources (surface and ground). However, at 7 ppm, the effectiveness of PAA and sodium hypochlorite showed no statistically significant difference for both surface and groundwater. Findings will provide information regarding efficacy of chemical sanitizers like chlorine and PAA for inactivation of Salmonella, Listeria, and STEC in surface water from which treatments can be derived. Ultimately benefitting growers in the selection of an appropriate method for in-field treatment of irrigation water if deemed necessary.

Antimicrobial activities of untreated and grape vinegar treated selected vegetables against common food borne pathogens

In the post-Covid-19 era, plant-based diets and products have become more popular. Fruit vinegar is considered a healthy drink, rich in bioactive compounds like organic acids, polyphenols, flavonoids and alkaloids that provide several beneficial properties. Fruit vinegar also contains several beneficial nutrients like amino acids, sugars, vitamins and minerals. They provide energy, regulate cell metabolism, immunity, antioxidation, anticoagulation and help in the improvement of brain development. Vinegar is also known for its strong antimicrobial properties against foodborne pathogens. The aim of the present investigation was to find out the antimicrobial activities of grape vinegar against common food-borne pathogens and to see whether common vegetable lose their antimicrobial activities when soaked with grape vinegar or gain. The vegetables used in the present investigation were randomly collected from the local market. We have evaluated the antimicrobial properties of untreated and grape vinegar (GV) treated selected vegetables against Escherichia coli (ATCC8739), Bacillus subtilis, Staphylococcus aureus (ATCC 6539), Shigella flexneri (ATCC 12022), Salmonella typhi (ATCC 14028), Cronobacter sakazakii (ATCC 29544), Vibrio parahaemolyticus (ATCC 17802), Vibrio cholera (ATCC 3906) and Pseudomonas aeruginosa. Based on the randomized survey, commonly used vegetable ginger (Zingiber officinale), garlic (Allium sativum), onion (Allium cepa), raw papaya (Carica papaya), white radish (Raphanus sativus) and green chilli (Capsicum annuum) were selected for the assessment of their antimicrobial activities. Different phytochemicals were found in water extract and grape vinegar-treated vegetables. Our results suggested that in general, grape vinegar-treated vegetables showed higher antimicrobial activities against all tested food-borne pathogens, but onion, garlic, green chillies and white radish revealed drastically higher activities. Hence, it is suggested that onion, garlic, green chillies and white radish should preferably be used after soaking with grape vinegar. All vegetables except garlic and white radish showed very little antimicrobial activity against B. subtilis. V. cholera was highly sensitive to grape vinegar-treated vegetables except for garlic. Similarly, V. parahaemolyticus was highly sensitive to grape vinegar-treated green chillies, white radish, raw papaya, and onion. Shigella flexneri also showed greater sensitivity to GV-treated green chillies and white radishes. Pseudomonas aeruginosa was sensitive to ginger and green chillies only. It may safely be concluded that grape vinegar-treated vegetables are beneficial to fight against food-borne infections. The vegetable when soaked with grape vinegar did not lose their antimicrobial activities rather they gained the antimicrobial components from grape vinegar. Our studies revealed that white radish, ginger, green chilies, and onion should be used after soaking in grape vinegar while garlic and raw papaya should be eaten without treatment with grape vinegar.

Isolation of a foodborne Bacillus cereus strain and its effect on intestinal mucosal immunity-associated factors and gut microbial community in mice

Bacillus cereus is a common foodborne pathogen. Accidently eating food contaminated by B. cereus will cause vomiting or diarrhea, and even death in severe cases. In the present study, a B. cereus strain was isolated from spoiled rice by streak culture. The pathogenicity and drug resistance of the isolated strain were analyzed by drug sensitivity test and PCR amplification of virulence-associated gene respectively. Cultures of the purified strain were injected intraperitoneally into mice to examine their effects on intestinal immunity-associated factors and gut microbial communities, to provide references for the pathogenic mechanism and medication guidance of these spoilage microorganisms. The results showed that the isolated B. cereus strain was sensitive to norfloxacin, nitrofurantoin, tetracycline, minocycline, ciprofloxacin, spectinomycin, clindamycin, erythrocin, clarithromycin, chloramphenicol, levofloxacin, and vancomycin, but resistant to bactrim, oxacillin and penicillin G. The strain carries seven virulence-associated genes including hblA, hblC, hblD, nheA, nheB, nheC and entFM, which are involved in diarrhea-causing toxins production. After infecting mice, the isolated B. cereus strain was found to cause diarrhea in mice, and the expression levels of immunoglobulins and inflammatory factors in the intestinal mucosae of the challenged mice were significantly up-regulated. Gut microbiome analysis showed that the composition of gut microbial community in mice changed after infection with B. cereus. The abundance of the uncultured_bacterium_f_Muribaculaceae in Bacteroidetes, which is a marker of body health, was significantly decreased. On the other hand, the abundance of uncultured_bacterium_f_Enterobacteriaceae, which is an opportunistic pathogen in Proteobacteria and a marker of dysbacteriosis, was significantly increased and was significantly positively correlated with the concentrations of IgM and IgG. These results showed that the pathogenic B. cereus carrying diarrhea type virulence-associated gene can activate the immune system by altering the composition of gut microbiota upon infection.

Characteristics of a Temperature-Sensitive Mutant Strain of Salmonella Enteritidis and Its Potential as a Live Vaccine Candidate.

Salmonella Enteritidis is a common foodborne pathogen transmitted through poultry products, which are its main carriers. Poultry are vaccinated against Salmonella Enteritidis in many countries, despite the absence of clinical symptoms, using commercially available live-attenuated vaccines. We previously constructed a highly attenuated temperature-sensitive (ts) Salmonella Enteritidis mutant, 2S-G10. In the present study, we describe the construction and attenuation-associated characteristics of 2S-G10. We infected 1-day-old chicks with 2S-G10 and the parental strains to evaluate the attenuation. One week after infection, 2S-G10 was not detected in the liver, cecum, or cecal tonsil tissues of the orally inoculated chicks, contrary to the parental strain. This indicates that 2S-G10 was highly attenuated when compared to the parental stain. In vitro experiments revealed the inability of 2S-G10 to grow at the normal body temperature of chickens and invade chicken liver epithelial cells. Moreover, single nucleotide polymorphism (SNP) analysis between the complete genome sequence of 2S-G10 and its parental strain revealed SNPs in bcsE, recG, rfaF, and pepD_1 genes, which are involved in epithelial cell invasion and persistence in host systems, growth, lipopolysaccharide core biosynthesis, and cellular survival under heat stress, respectively. These potential characteristics are consistent with the findings of in vitro experiments. Conclusively, chemical treatment-induced random genetic mutations highly attenuated 2S-G10, implying its potential to be developed as a novel live-attenuated vaccine against Salmonella Enteritidis.

Human Salmonellosis: A Continuous Global Threat in the Farm-to-Fork Food Safety Continuum.

Salmonella is one of the most common zoonotic foodborne pathogens and a worldwide public health threat. Salmonella enterica is the most pathogenic among Salmonella species, comprising over 2500 serovars. It causes typhoid fever and gastroenteritis, and the serovars responsible for the later disease are known as non-typhoidal Salmonella (NTS). Salmonella transmission to humans happens along the farm-to-fork continuum via contaminated animal- and plant-derived foods, including poultry, eggs, fish, pork, beef, vegetables, fruits, nuts, and flour. Several virulence factors have been recognized to play a vital role in attaching, invading, and evading the host defense system. These factors include capsule, adhesion proteins, flagella, plasmids, and type III secretion systems that are encoded on the Salmonella pathogenicity islands. The increased global prevalence of NTS serovars in recent years indicates that the control approaches centered on alleviating the food animals' contamination along the food chain have been unsuccessful. Moreover, the emergence of antibiotic-resistant Salmonella variants suggests a potential food safety crisis. This review summarizes the current state of the knowledge on the nomenclature, microbiological features, virulence factors, and the mechanism of antimicrobial resistance of Salmonella. Furthermore, it provides insights into the pathogenesis and epidemiology of Salmonella infections. The recent outbreaks of salmonellosis reported in different clinical settings and geographical regions, including Africa, the Middle East and North Africa, Latin America, Europe, and the USA in the farm-to-fork continuum, are also highlighted.

Detection and characterization of the most common foodborne pathogens by using multiplex PCR procedure

Food Microbial contamination is one of the most serious problems. A large percentage of food-borne illnesses are caused by food-borne pathogens, and diarrheal agents comprise more than half of the overall prevalence of food-borne illnesses in the globe, and more commonly in developing countries. This study aimed to identify the most-common foodborne organisms from foods in Khartoum state by PCR.A total of 207 food samples (raw milk, fresh cheese, yogurt, fish, sausage, mortadella, and eggs) were collected. DNA was extracted from food samples by guanidine chloride protocol, and then species-specific primers were used to identify Escherichia coli O157: H7, Listeria monocytogenes, Salmonella spp., Vibrio cholerae, V. parahaemolyticus, and Staphylococcus aureus. Out of 207 samples, five (2.41%) were positive for L. monocytogenes, one (0.48%) was positive for S. aureus, and one (0.48%) was positive for both Vibrio cholerae and Vibrio parahaemolyticus. From 91 fresh cheese samples, 2 (2.19%) were positive for L. monocytogenes, and one (1.1%) sample was positive for two different foodborne pathogens (V. cholerae and V. parahaemolyticus). Out of 43 Cow's milk samples, three (7%) samples were positive for L. monocytogenes, and out of 4 sausage samples, one (25 %) was positive for S. aureus. Our study revealed the presence of L. monocytogenes and V. cholera in raw milk and fresh cheese samples. Their presence is considered a potential problem and needs intensive hygiene efforts and standard safety measures before, during, and after food processing operations.

Antibiofilm effects of punicalagin against Staphylococcus aureus in vitro

Staphylococcus aureus is a common foodborne pathogen which can form biofilms to help them resist to antimicrobials. It brings great harm to human health. Punicalagin has good antimicrobial activities against S. aureus, but its effect on biofilm formation has not been clearly illustrated. The aim of this study was to explore the antibiofilm effects of punicalagin against S. aureus. Results showed that punicalagin did not significantly interfere with the growth of S. aureus at the concentrations of 1/64 MIC to 1/16 MIC. The biomass and metabolic activity of biofilms were significantly reduced when exposed to sub-inhibitory concentrations of punicalagin. The number of viable cells in the biofilms was also decreased after punicalagin treatment. Scanning electron microscopy and confocal laser scanning microscopy images confirmed that punicalagin damaged the structure of biofilms. The antibiofilm mechanism was partly due to the modification of the cell surface which led to the reduction of cell surface hydrophobicity. These findings suggest that punicalagin has the potential to be developed as an alternative to control S. aureus biofilms.

Characterization of a broad-spectrum endolysin rLysJNwz and its utility against Salmonella in foods.

Salmonella is a common foodborne pathogen worldwide. The use of bacteriophage-encoded endolysins as antimicrobial agents is a promising approach for controlling pathogenic contamination. In this context, a recombinant endolysin named rLysJNwz, consisting of a single domain falling with the L-alanogyl-D-glutamate peptidase-like family, was cloned, expressed, and characterized. The yield of rLysJNwz was about 25mg/L. Synergy between 7.5μg/mL rLysJNwz and 0.5mmol/L EDTA could decrease the viable counts of Salmonella NCTC 8271 by 93.28%. A synergistic effect between rLysJNwz and polymyxin B was demonstrated, exhibiting the MIC of polymyxin B decreased by twofold. Specifically, rlysJNwz had strong thermostability at temperatures (4-95°C) and maintained high activity at pHs from 5.0 to 11.0. rlysJNwz was a metal ion-dependent peptidase, which activated by divalent metal ions such as Zn2+, Mn2+, or Ca2+. Moreover, it was also found that the synergism of rlysJNwz and EDTA had bactericidal activities against a broad range of Gram-negative bacteria, including several multidrug-resistant bacteria. The application of rLysJNwz combined with EDTA was evaluated on contaminated eggs and lettuce for 60min, displaying more than 86.7% and 86.5% reduction of viable Salmonella, respectively. Hence, these results suggest that rLysJNwz is a potential antibacterial agent to control Salmonella, especially antibiotic-resistant pathogen contamination in the field of food safety. KEY POINTS: • rLysJNwz shows lytic activities against a broad range of Gram-negative bacteria. • Endolysin rLysJNwz is a stable metalloenzyme and has high thermostability. • rLysJNwz and 0.5mmol/L EDTA synergistically inactivate Salmonella on eggs and lettuce.

First insights into antimicrobial resistance, toxigenic profiles, and genetic diversity in Bacillus cereus isolated from Chinese sausages

Bacillus cereus is a common food-borne bacterial pathogen. This study was to designed the prevalence of B. cereus in Chinese sausages and evaluate its genetic diversity, toxigenic profiles, and antimicrobial resistance. Using bacteriological and biochemical detection tests, B. cereus was isolated from 12.63% (N = 49) of the total 388 samples. However, only 37 of the 49 isolates were simultaneously classified as the strictly B. cereus based on the classification of their genomes. Multi-locus sequence typing showed that the clonal complex was highly concentrated in CC142. Enterotoxin-encoding genes (hblABCD, nheABC, entFM, and cytK) were detected in all the strains. A total of 25 genes, associated with biofilm synthesis, were confirmed according to a computerized approach. In total, 89.19% of the isolates possessed only four resistance genes (BcⅠ, BcⅡ, fosB, and vanZF). Nevertheless, all isolates displayed resistance to three or more kinds of antibiotics. Moreover, three isolates were discovered to have tet(45) gene variants and were resistant to tigecycline. This study revealed the powerful potential of B. cereus to cause food poisoning in humans. The presence of multidrug-resistant B. cereus, especially with resistance to tigecycline, mediated by the tet(45) variant, poses a major threat to food safety and public health.

Plant-mediated Synthesis of Silver Nanoparticles using Mangosteen Pericarp Extract and their Antimicrobial Potential

Introduction: Silver nanoparticles (AgNPs) were synthesized using mangosteen pericarp ethanolic extract (MPEE) as a source of bioreductants and their antimicrobial activity against common foodborne pathogens was evaluated. Methods: Characterization of MPEE was conducted using phytochemical screening, total phenolic content analysis, and DPPH (antioxidant) assay. Synthesis AgNPs and optimization studies were monitored using UV-Vis spectrophotometry. Transmission electron microscopy was used to characterize the AgNPs, and resazurin microtiter assay was used for antimicrobial testing. Results: Alkaloids, flavonoids, saponins, quinones, anthraquinones, and tannins were confirmed present in the extract. TPC and IC50 of MPEE were 0.192 mg GAE/mg extract and 0.277 mg/mL, respectively. A surface plasmon resonance (SPR) peak within 450-403 nm confirmed the formation of AgNPs. At pH 7, the optimum reaction conditions were 45°C and 3 h. Meanwhile, at pH 9, the optimum reaction conditions were 27°C and 0.5 h. The sizes of nanoparticles synthesized at pH 7 and pH 9 were 13-35 nm and 7- 38 nm, respectively. The minimum inhibitory concentration (MIC90) of AgNPs produced at pH 7 were 1.45, 2.81, and 2.93 ug/mL for S. aureus, E.coli, and B. cereus, respectively. For AgNPs synthesized at pH 9, the MIC90 were 2.93, 3.02, and 5.24 ug/mL, for the same microorganisms, respectively. Conclusion: MPEE was able to successfully synthesize AgNPs. Compared to chloramphenicol, AgNPs exhibited better antimicrobial activity, which can address the growing concern of drug resistance in certain pathogenic microorganisms. Furthermore, the use of MPEE provides a green and sustainable alternative to synthesizing AgNPs.

Antimicrobial resistance profiles of Salmonella isolates from human diarrhea cases in China: an eight-year surveilance study

Salmonella is widely known as one of the most common foodborne pathogens, and antibiotics remain effective in clinical therapies against its infections. To guide better clinical antibiotic treatment, we analyzed the antimicrobial resistance (AMR) profiles of a nation-wide collection of 36,822 Salmonella isolates derived from sporadic diarrhea cases in China from 2014 to 2021. A panel of 15 antibiotics, including 10 critically important and 5 highly important antimicrobial agents for human medicine based on the WHO CIA List, was selected for AMR surveillance. Salmonella enterica serovar Typhimurium, Enteritidis, I 4,[5],12:i:-, London, and Stanley were turns to be the top five serotypes from human diarrhea cases in China. Antimicrobial susceptibility testing revealed that the majority of the isolates (87.2%) were resistant to at least one antimicrobial agent, and 66.5% were multidrug resistant (MDR). Salmonella isolates were found to be highly resistant to ampicillin (73.4%) while sensitive to imipenem (98.73%). Over the eight years, the isolates were demonstrated generally an increase in resistance to ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, and azithromycin, but displayed varied resistance profiles in terms of serotypes. Stanley (0.66–27.18%) and Agona (0.78–45.30%) had lower resistance rates compared to Typhimurium (1.11–85.6%), Enteritidis (1.55–91.29%), and I 4,[5],12:i:- (1.02–94.28%). In conclusion, our results provide systematic data on the resistance characteristics of Salmonella isolates from human diarrhea cases in China. Furthermore, this data identifies priorities for the clinical treatments of antibiotics.

Dual recognition strategy for the rapid and precise detection of Bacillus cereus using post-modified nano-MOF and aptamer

Bacillus cereus is one of the most common foodborne pathogens and is widely distributed in foods, soil, water, plants and fodder. As an aerobic Gram-positive bacteria, B. cereus also plays an important role in liquor production, generating flavor enhancers and antibacterial substances. Therefore, a rapid and precise analysis strategy for this microbe is highly desired in food safety and sustainable food production. In this study, we propose an effective approach for B. cereus quantification using a dual recognition biosensor. B. cereus was specifically enriched with aptamer-functionalized magnetic beads, and then detected using the broad-spectrum fluorescent material, fragmented crystalline mannose-conjugated lectin-modified nano-MIL-53(Al)-NH2 (FcMBL@NM5AN). The dual recognition method was applied for specific quantification of B. cereus in the range of 20–2 × 108 CFU/mL with a limit of detection of 4 CFU/mL within 60 min. Recoveries ranged from 93.8%−100.8% in milk, rice, chicken meat, eggs, and distilled yeast as real samples were also obtained.

Screening and Identification of Goat-Milk-Derived Lactic Acid Bacteria with Bacteriocin-like Activity and Probiotic Potentials.

In the current study, we screened 46 isolates of lactic acid bacteria (LAB) derived from goat milk for bacteriocin producers that can inhibit common foodborne pathogens (Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus). The following three strains that showed antimicrobial activity against all indicators were identified: Enterococcus faecalis DH9003 and DH9012, and Lactococcus lactis DH9011. Their antimicrobial products exhibited typical bacteriocin characteristics, such as heat stability and proteinase nature. The bacteriostatic activity of concentrated bacteriocins produced by these LAB was observed at low concentrations (half-minimum inhibitory concentration [MIC50] and 4MIC50), whereas complete inhibition activity against Listeria monocytogenes was detected at high concentrations (16MIC50) of the two Enterococcus faecalis strains (DH9003 and DH9012). Furthermore, the probiotic potentials of the three strains were investigated and described. The results revealed that none of the strains had hemolytic activity, whereas all: were sensitive to ampicillin (50 mg/mL) and streptomycin sulfate (100 mg/mL); were resistant to bile, artificial simulated intestinal tract, and gastric juice at different pH levels (2.5, 3.0, 3.5); and had β-galactosidase activity. Furthermore, all strains exhibited an auto-aggregating phenotype, with self-aggregation ranging from 30% to 55%. DH9003 and DH9012 co-aggregated well with Listeria monocytogenes and Escherichia coli (52.6% and 63.2%, 68.5% and 57.6%, respectively), whereas DH9011 co-aggregated poorly with Listeria monocytogenes (15.6%) and did not co-aggregate with Escherichia coli. Furthermore, our results showed that all three isolates exhibited strong antibacterial activity, tolerance to bile and simulated gastrointestinal environments, adhesion capability, and safety. Finally, DH9003 was selected and used for gavage in rats. By observing the pathological characteristics of rat intestinal and liver tissue sections, DH9003 showed no harmful effects on the intestine and liver of rats, but rather resulted in a denser and longer intestinal mucosa, as well as improving the intestinal mucosa of rats. Considering their substantial prospective applications, we concluded that these three isolates are potential probiotic candidates.

Biofilm Formation and Control of Foodborne Pathogenic Bacteria.

Biofilms are microbial aggregation membranes that are formed when microorganisms attach to the surfaces of living or nonliving things. Importantly, biofilm properties provide microorganisms with protection against environmental pressures and enhance their resistance to antimicrobial agents, contributing to microbial persistence and toxicity. Thus, bacterial biofilm formation is part of the bacterial survival mechanism. However, if foodborne pathogens form biofilms, the risk of foodborne disease infections can be greatly exacerbated, which can cause major public health risks and lead to adverse economic consequences. Therefore, research on biofilms and their removal strategies are very important in the food industry. Food waste due to spoilage within the food industry remains a global challenge to environmental sustainability and the security of food supplies. This review describes bacterial biofilm formation, elaborates on the problem associated with biofilms in the food industry, enumerates several kinds of common foodborne pathogens in biofilms, summarizes the current strategies used to eliminate or control harmful bacterial biofilm formation, introduces the current and emerging control strategies, and emphasizes future development prospects with respect to bacterial biofilms.

A caprylate esterase-activated fluorescent probe for sensitive and selective detection of Salmonella enteritidis.

Salmonella enteritidis is one of the most common foodborne pathogens. Many methods have been developed to detect Salmonella, but most of them are expensive, time-consuming, and complex in experimental procedures. Developing a rapid, specific, cost-effective, and sensitive detection method is still demanded. In this work, a practical detection method is presented using salicylaldazine caprylate as the fluorescent probe, which could be hydrolyzed by caprylate esterase liberated from Salmonella lysed by phage, to form strong fluorescent salicylaldazine. The Salmonella could be detected accurately with a low limit of detection of 6CFU/mL and a broad concentration range of 10-106CFU/mL. Moreover, this method was successfully used for the rapid detection of Salmonella in milk within 2h through pre-enrichment by ampicillin-conjugated magnetic beads. The novel combination of fluorescent turn-on probe salicylaldazine caprylate and phage ensures this method has excellent sensitivity and selectivity.

Antibacterial Pattern of Rosa roxburghii Tratt Pomace Crude Extract Against Staphylococcus aureus and Its Application in Preservation of Cooked Beef

Staphylococcus aureus is a common foodborne pathogen and spoilage bacterium in meat products. To develop a natural preservative for meat products, this study revealed the antibacterial activity and mechanism of Rosa roxburghii Tratt pomace crude extract (RRPCE) against S. aureus, and applied RRPCE to the preservation of cooked beef. The diameter of inhibition zone, minimum inhibitory concentration (MIC), and minimum bactericide concentration of RRPCE against S. aureus were 15.85 ± 0.35 to 16.21 ± 0.29 mm, 1.5 mg/mL, and 3 mg/mL, respectively. The growth curve of S. aureus was completely stalled by treatment with RRPCE at 2 MIC. RRPCE results in the decrease of intracellular adenosine 5'-triphosphate (ATP) content, depolarization of cell membrane, leakage of cell fluid including nucleic acid and protein, and destruction of cell membrane integrity and cell morphology. During storage, RRPCE significantly reduced S. aureus viable counts, pH, and total volatile basic nitrogen of cooked beef compared with untreated samples (p < 0.05). In addition, RRPCE could significantly increase the redness (a*) value, decrease lightness (L*) and yellowness (b*) values, and slow down the color change of cooked beef (p < 0.05). These findings suggest that RRPCE can effectively inhibit S. aureus, and has the potential as a natural preservative for the preservation of cooked beef.

Development of a 3-plex droplet digital PCR for identification and absolute quantification of Salmonella and its two important serovars in various food samples

Salmonella is one of the most common foodborne pathogens that cause diarrhea in human, in which Enteritidis and Typhimurium are the top serovars frequently isolated from foodstuffs. A novel 3-plex droplet digital PCR (ddPCR) was successfully developed in this study for the simultaneous identification and absolute quantification of Salmonella and its two important serovars (Enteritidis and Typhimurium). The specificity of all the primers and probes was validated by testing 24 strains of Salmonella and 10 strains of other bacteria. Meanwhile, the anti-interference ability of this method was also evaluated using high proportion of non-target strains or various bacterial combinations. The detection limit of this 3-plex ddPCR was as low as 5 fg/μL for gDNA and 101 CFU/mL for pure cultures, respectively. Moreover, compared with quantitative PCR (qPCR), the detection limit of this ddPCR was much lower when it was applied to the detection of artificially contaminated food samples, which was 101 CFU/mL for lettuce, and 102 CFU/mL for milk and chicken juice samples, respectively. Additionally, a total of 100 retailed food samples were tested by this validated 3-plex ddPCR, which was also compared with traditional cultivation method. Notably, 19 of 20 samples showed consistent positive results by both methods, while the rest with the lowest concentration of Salmonella cells was detectable only by ddPCR. These results demonstrated that the 3-plex ddPCR established in this study presented highly specific, sensitive, and absolute quantitative capability, which had the potential to identify Salmonella, and to differentiate the two important serovars Enteritidis and Typhimurium in a wide variety of food samples.