Major Foodborne Pathogens Research Articles

Isolation and antimicrobial resistance of motileSalmonella entericafrom the poultry hatchery environment.

Salmonella is a globally distributed major food-borne pathogen and poultry is one of the predominant sources of salmonellosis in humans. To investigate the presence of motile Salmonella in the poultry hatchery environment, we collected 97 fluff samples from four selected broiler breeder chicken hatcheries from Chattogram, Bangladesh during July-December 2015. To isolate motile Salmonella enterica, we used conventional bacteriological techniques followed by serological verification using anti-Salmonella Poly A-E serum and species confirmation by conventional PCR assay. Antimicrobial susceptibility testing by Kirby-Bauer disc diffusion method for 10 commonly used antibiotics was performed on all isolates. Isolates displaying phenotypic resistance to ampicillin were tested by PCR for blaTEM gene, whereas those resistant to tetracycline were tested for the presence of tetA, tetB and tetC genes. A total of 24 samples (24.7%; 95% CI: 16.5-34.5, N = 97) from 3 hatcheries were positive for motile Salmonella. Of them, 21 (87.5%) and 12 (50.0%) were resistant to ampicillin and tetracycline, respectively, 9 (37.5%) to nalidixic acid and sulphamethoxazole/trimethoprim. No resistance was detected to ceftriaxone, cefoxitin, gentamicin, neomycin, ciprofloxacin and colistin. Ten (42%) of 24 isolates from 2 hatcheries were multi-drug resistant (i.e. resistant to ≥ 3 antimicrobial classes). Six of 21 ampicillin resistant isolates contained blaTEM gene and 10 of 12 tetracycline resistant isolates contained tetA gene. This study highlights the circulation of multi-drug resistant motile Salmonella in the hatchery environment for the first time in Bangladesh. Further epidemiological and molecular studies are therefore needed to identify the serotypes and source of the bacteria in hatcheries.

Shiga Toxin-Producing Escherichia coli in Wheat Grains: Detection and Isolation by Polymerase Chain Reaction and Culture Methods.

Shiga toxin-producing Escherichia coli (STEC) are major foodborne pathogens and seven serogroups, O26, O45, O103, O111, O121, O145, and O157, often called top-7 STEC, account for the majority of the STEC-associated human illnesses in the United States. Two Shiga toxins, Shiga toxins 1 and 2, encoded by stx1 and stx2 genes, are major virulence factors that are involved in STEC infections. Foodborne STEC infections have been linked to a variety of foods of both animal and plant origin, including products derived from cereal grains. In recent years, a few STEC outbreaks have been linked to contaminated wheat flour. The microbiological quality of the wheat grains is a major contributor to the safety of wheat flour. The objective of the study was to utilize polymerase chain reaction (PCR)- and culture-based methods to detect and isolate STEC in wheat grains. Wheat grain samples (n = 625), collected from different regions of the United States, were enriched in modified buffered peptone water with pyruvate (mBPWp) or E. coli (EC) broth, and they were then subjected to PCR- and culture-based methods to detect and isolate STEC. Wheat grains enriched in EC broth yielded more samples positive for stx genes (1.6% vs. 0.32%) and STEC serogroups (5.8% vs. 2.4%) than mBPWp. The four serogroups of top-7 detected and isolated were O26, O45, O103, and O157 and none of the isolates was positive for the Shiga toxin genes. A total of five isolates that carried the stx2 gene were isolated and identified as serogroups O8 (0.6%) and O130 (0.2%). The EC broth was a better medium to enrich wheat grains than mBPWp for the detection and isolation of STEC. The overall prevalence of virulence genes and STEC serogroups in wheat grains was low. The stx2-positive serogroups isolated, O8 and O130, are not major STEC pathogens and have only been implicated in sporadic infections in animals and humans.

The combined effect of stressful factors (temperature and pH) on the expression of biofilm, stress, and virulence genes in Salmonella enterica ser. Enteritidis and Typhimurium.

Salmonella enterica is a major food borne pathogen that creates biofilm. Salmonella biofilm formation under different environmental conditions is a public health problem. The present study was aimed to evaluate the combined effects of stressful factors (temperature and pH) on the expression of biofilm, stress, and virulence genes in Salmonella Enteritidis and Salmonella Typhimurium. In this study, the effect of temperature (2, 8, 22.5, 37, 43°C) and pH (2.4, 3, 4.5, 6, 6.6) on the expression of biofilm production genes (adr A, bap A), virulence genes (hil A, inv A) and the stress gene (RpoS) of S. Enteritidis and S. Typhimurium was evaluated. The response surface methodology (RSM) approach was used to evaluate the combined effect of the above factors. The highest expression of adr A, bap A, hil A, and RpoS gene for S. Typhimurium was at 22°C-pH 4.5 (6.39-fold increase), 37°C-pH 6 (3.92-fold increase), 37°C-pH 6 (183-fold increase), and 37°C-pH 3 (43.8-fold increase), respectively. The inv A gene of S. Typhimurium was decreased in all conditions. The adr A, bap A, hil A, inv A, and RpoS gene of S. Enteritidis had the highest expression level at 8°C-pH 3 (4.09-fold increase), 22°C-pH 6 (2.71-fold increase), 8°C pH 3 (190-fold increase), 22°C-pH 4.5 (9.21-fold increase), and 8°C-pH 3 (16.6-fold), respectively. Response surface methodology (RSM) indicated that the temperature and pH had no significant effect on the expression level of adr A, bap A, hil A, Inv A, and RpoS gene in S. Enteritidis and S. Typhimurium. The expression of biofilm production genes (adr A, bap A), virulence genes (hil A, inv A) and the stress gene (RpoS) of S. Enteritidis and S. Typhimurium is not directly and exclusively associated with temperature and pH conditions.

Prevalence, Antibiogram and Genetic Characterization of Listeria monocytogenes from Food Products in Egypt.

World Health Organization classified Listeria monocytogenes as a major notable foodborne pathogen associated with high mortality and hospitalization. The study reports the prevalence, antibiogram, virulence determination and genetic characterization of L. monocytogenes from different food products. A total of 250 food samples, fifty samples each from raw milk, ice cream, minced meat, fish fillet and sausage were collected from the Menoufiya governorate in Egypt. L. monocytogenes was detected in 17 (6.8%) of the tested food samples including minced meat (14%), fish fillet (8%), sausage (6%) and raw milk (6%). The antimicrobial susceptibility assay of 17 L. monocytogenes isolates against seventeen antibiotics belonging to eight antibiotics classes revealed a high susceptibility to norfloxacin (82.3%), amoxicillin-clavulanic acid (76.4%), cefotaxime (70.5%), erythromycin (64.6%), amoxicillin (64.6%), gentamicin (58.7%) and vancomycin (58.7%). While, high resistance was observed against oxytetracycline (76.4%), trimethoprim-sulfamethoxazole (76.4%), chloramphenicol (70.5%), doxycycline (64.6%), levofloxacin (41.2%) and azithromycin (41.2%). Of note, all L. monocytogenes isolates were multidrug-resistant. The multiplex PCR successfully amplified L. monocytogenes in all tested isolates. Screening of the five virulence-related genes revealed the hlyA and iap as the most prevalent genes followed by actA gene, however, the inlA and prfA genes were not detected in any of the studied isolates. The partial 16S rRNA gene sequencing of three L. monocytogenes isolates showed a high nucleotide similarity (99.1–99.8%) between the study isolates and various global clones, and phylogenetic analysis clustered these L. monocytogenes strains with other Listeria species including L. welshimeri, L. seeligeri and L. innocua. This study demonstrates the impact of L. monocytogenes as a major contaminant of various food products and suggests more attention to the awareness and hygienic measures in the food industry.

Novel Sequence Types of Listeria monocytogenes of Different Origin Obtained in the Republic of Serbia.

Listeria monocytogenes, the causative agent of listeriosis, is amongst the major food-borne pathogens in the world that affect mammal species, including humans. This microorganism has been associated with both sporadic episodes and large outbreaks of human listeriosis worldwide, with high mortality rates. In this study, the main sequence types (STs) and clonal complexes (CCs) were investigated in all of the 13 L. monocytogenes strains originating from different sources in the Republic of Serbia in 2004–2019 and that were available in the BIGSdb-Lm database. We found at least 13 STs belonging to the phylogenetic lineages I and II. These strains were represented by ST1/ST3/ST9 of CC1/CC3/CC9, which were common in the majority of the European countries and worldwide, as well as by eight novel STs (ST1232/ST1233/ST1234/ST1235/ST1238/ST1236/ST1237/ST1242) of CC19/CC155/CC5/CC21/CC3/CC315/CC37, and the rare ST32 (clonal complex ST32) and ST734 (CC1), reported in the Republic of Serbia, the EU, for the first time. Our study confirmed the association of CC1 with cases of neuroinfection and abortions among small ruminants, and of CC3 and CC9 with food products of animal origin. The strains isolated in 2019 carried alleles of the internalin genes (inlA/inlB/inlC/inlE) characteristic of the most virulent strains from the hypervirulent CC1. These findings demonstrated the genetic relatedness between L. monocytogenes strains isolated in the Republic of Serbia and worldwide. Our study adds further information about the diversity of the L. monocytogenes genotypes of small ruminants and food products, as the strain distribution in these sources in Serbia had not previously been evaluated.

Inhibitory effects of Flavourzyme on biofilm formation, quorum sensing, and virulence genes of foodborne pathogens Salmonella Typhimurium and Escherichia coli

Salmonella enterica and Shiga toxin-producing (or verotoxin-producing) Escherichia coli are major foodborne pathogens, posing substantial food safety risks. Due to the negative effects of chemical treatment against foodborne pathogens, the application of enzyme-based techniques is currently receiving great attention. Here, we evaluated the inhibitory properties of Flavourzyme, a commercial peptidase, against these two foodborne pathogens. We noticed 4.0 and 5.5 log inhibition of biofilm formation by S. Typhimurium and E. coli, respectively, while treated with sub-minimum inhibitory concentrations of Flavourzyme for 24 h. For both bacteria, the enzyme exhibited quorum-quenching activity, preventing autoinducer-2 production completely by E. coli. In addition, Flavourzyme significantly suppressed the relative expression levels of biofilm-forming, quorum sensing, and virulence regulatory genes as measured by qRT-PCR. Based on our results, we suggest the use of Flavourzyme as a preventive agent against foodborne pathogens that possibly acts by inhibiting bacterial self-defense mechanisms following disruption of cellular proteins. This finding may shed light on how enzymes can be applied as a novel weapon to control foodborne illnesses to ensure food safety and public health.

Isolation and Characterization of Potential Salmonella Phages Targeting Multidrug-Resistant and Major Serovars of Salmonella Derived From Broiler Production Chain in Thailand.

Salmonella is a major foodborne pathogen that causes foodborne disease in humans through consumption of contaminated foods, especially those of animal origin. Multiple Salmonella strains are antibiotic-resistant due to the common use of antibiotics in farm animals, including broiler farms. In this study, an alternative strategy using phage-based treatment was evaluated against Salmonella isolated from the broiler production. The prevalence of Salmonella spp. showed up to 46.2 and 44.4% in bedding samples from the broiler farms located in eastern and southern Thailand, respectively. Overall, 21 samples (36.2%) were positive for Salmonella and eight serovars were recovered from cloacal swabs, bedding materials (rice husk), and boot swabs collected from five farms. Up to 20 Salmonella phages were isolated from seven water samples from wastewater treatment ponds, a river, and a natural reservoir in Songkhla province. Isolated phages were investigated, as well as their lysis ability on eight target Salmonella serovars derived from broiler farms, five foodborne outbreak-related serovars, and 10 multidrug-resistant (MDR) serovars. All phages showed a strong lytic ability against five serovars of Salmonella derived from broiler farms including Kentucky, Saintpaul, Schwarzengrund, Corvalis, and Typhimurium; three foodborne outbreak serovars including Enteritidis, Typhimurium, and Virchow; and eight MDR serovars including Agona, Albany, Give, Kentucky, Typhimurium, Schwarzengrund, Singapore, and Weltevreden. Three phages with the highest lysis potential including vB_SenS_WP109, vB_SenS_WP110, and vB_SenP_WP128 were selected for a phage cocktail preparation. Overall, a phage cocktail could reduce Salmonella counts by 2.2–2.8 log units at 6 h of treatment. Moreover, Salmonella did not develop a resistant pattern after being treated with a phage cocktail. Findings here suggest that a phage cocktail is an effective biocontrol to combat Salmonella derived from broiler production chain, other serovars linked to foodborne outbreaks, and MDR serovars.

First Isolation and Molecular Characterization of bla CTX-M-121 -Producing Escherichia coli O157:H7 From Cattle in Xinjiang, China.

The bovine Escherichia coli O157:H7 is a major foodborne pathogen causing severe bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans. Cattle are recognized major reservoir and source of E. coli O157:H7. We investigated the antibiotic resistance, molecular profiles, and intrinsic relationship between 21 isolates of E. coli O157:H7 from cattle farms and slaughtering houses in Xinjiang. Using pulsed-field gel electrophoresis (PFGE) molecular typing, two types of PFGE were revealed through cluster analysis, including clusters I and II, with 66 and 100% similarity of PFGE spectra between 21 isolates. We also detected that 18 isolates (86%) carried at least one virulence gene, 16 isolates (76%) carried the eae gene, and 7 (33%) carried the stx1 + stx2 + eae + hly + tccp genes. Eighteen isolates were susceptible to antibiotics. Three isolates were resistant to antibiotics, and two were multidrug resistant. One of the two multidrug-resistant isolates detectably carried the blaCTX−M−121 gene. This is the first finding of the blaCTX−M−121 gene detected in E. coli O157:H7 isolated from cattle in Xinjiang. The blaCTX−M−121 gene is transferable between the bacterial strains via plasmid transmission. The results indicated that E. coli O157:H7 may have undergone clonal propagation in cattle population and cross-regional transmission in Xinjiang, China.

Screening the Presence of Non-Typhoidal Salmonella in Different Animal Systems and the Assessment of Antimicrobial Resistance.

Simple SummaryIn this study, for the first time in Chile, we compared resistance profiles of Salmonella strains isolated from 4047 samples from domestic and wild animals. A total of 106 Salmonella strains (2.61%) were isolated, and their serogroups were characterized and tested for susceptibility to 16 different antimicrobials. This study reports 47 antimicrobial-resistant (AMR) Salmonella strains (44.3% of total strains). Of the 47, 28 corresponded to single-drug resistance (26.4%) and 19 to multidrug resistance (17.9%). The association between AMR and a subset of independent variables was evaluated using multivariate logistic models. Interestingly, S. Enteritidis was highly persistent in animal production systems; however, we report that serogroup D strains were 18 times less likely to be resistant to at least one antimicrobial agent than the most common serogroup (serogroup B). The antimicrobials presenting the greatest contributions to AMR were ampicillin, streptomycin and tetracycline.Salmonella is a major bacterial foodborne pathogen that causes the majority of worldwide food-related outbreaks and hospitalizations. Salmonellosis outbreaks can be caused by multidrug-resistant (MDR) strains, emphasizing the importance of maintaining public health and safer food production. Nevertheless, the drivers of MDR Salmonella serovars have remained poorly understood. In this study, we compare the resistance profiles of Salmonella strains isolated from 4047 samples from domestic and wild animals in Chile. A total of 106 Salmonella strains (2.61%) are isolated, and their serogroups are characterized and tested for susceptibility to 16 different antimicrobials. The association between antimicrobial resistance (AMR) and a subset of independent variables is evaluated using multivariate logistic models. Our results show that 47 antimicrobial-resistant strains were found (44.3% of the total strains). Of the 47, 28 correspond to single-drug resistance (SDR = 26.4%) and 19 are MDR (17.9%). S. Enteritidis is highly persistent in animal production systems; however, we report that serogroup D strains are 18 times less likely to be resistant to at least one antimicrobial agent than the most common serogroup (serogroup B). The antimicrobials presenting the greatest contributions to AMR are ampicillin, streptomycin and tetracycline. Additionally, equines and industrial swine are more likely to acquire Salmonella strains with AMR. This study reports antimicrobial-susceptible and resistant Salmonella in Chile by expanding the extant literature on the potential variables affecting antimicrobial-resistant Salmonella.

Investigation of the antimicrobial mode of action of sodium acid sulfate and potassium acid sulfate

This study aims to identify the antimicrobial mode of action of sodium acid sulfate (SAS) and potassium acid sulfate (KAS) against three major foodborne pathogens, Escherichia coli O157:H7, Salmonella Typhimurium DT 104, and Listeria monocytogenes. The overnight grown cultures of the target pathogens were subjected to SAS and KAS treatments to identify the minimum inhibition concentrations of each antimicrobial. The impact of SAS and KAS treatments on the target pathogens at bio-molecular levels was also studied. To help determine if the mode of action is pH-driven, SAS and KAS's antimicrobial efficacies were also compared against hydrochloric acid (HCL). The MIC values of SAS and KAS for all three pathogens were 7.5 mg/mL while for HCL, the MICs were greater than 30 mg/mL. Similarly, SAS and KAS treatments resulted in more damage to all target pathogens and increased leakage of nucleic acid, protein, and ions than HCL treatments. Transmission electron microscope images also revealed that 7.5 mg/mL SAS and KAS treatments resulted in significantly more visible cell damage in S. Typhimurium and E. coli O157:H7 than HCL treatments. The study's findings indicate that the SAS and KAS could be the function of pH, oxidation of cell components, and osmolarity.

Draft Genome Sequence of a Campylobacter coli WL22 Isolate Possessing erm(B) with gyrA Mutations, Isolated from Poultry in China.

Campylobacter coli is a major foodborne pathogen worldwide that causes campylobacteriosis cases in humans and is an emerging threat in developing countries. The rapid dissemination of the macrolide resistance gene erm(B) poses a significant threat to the clinical therapy of campylobacteriosis. Here, we report the draft genome sequences of one Campylobacter coli strain possessing erm(B), isolated from the cecal contents of poultry in Jinhua, China.

Comparative genomics reveals an SNP potentially leading to phenotypic diversity of Salmonella enterica serovar Enteritidis.

An SNP is a spontaneous genetic change having a potential to modify the functions of the original genes and to lead to phenotypic diversity of bacteria in nature. In this study, a phylogenetic analysis of Salmonella enterica serovar Enteritidis, a major food-borne pathogen, showed that eight strains of S. Enteritidis isolated in South Korea, including FORC_075 and FORC_078, have almost identical genome sequences. Interestingly, however, the abilities of FORC_075 to form biofilms and red, dry and rough (RDAR) colonies were significantly impaired, resulting in phenotypic differences among the eight strains. Comparative genomic analyses revealed that one of the non-synonymous SNPs unique to FORC_075 has occurred in envZ, which encodes a sensor kinase of the EnvZ/OmpR two-component system. The SNP in envZ leads to an amino acid change from Pro248 (CCG) in other strains including FORC_078 to Leu248 (CTG) in FORC_075. Allelic exchange of envZ between FORC_075 and FORC_078 identified that the SNP in envZ is responsible for the impaired biofilm- and RDAR colony-forming abilities of S. Enteritidis. Biochemical analyses demonstrated that the SNP in envZ significantly increases the phosphorylated status of OmpR in S. Enteritidis and alters the expression of the OmpR regulon. Phenotypic analyses further identified that the SNP in envZ decreases motility of S. Enteritidis but increases its adhesion and invasion to both human epithelial cells and murine macrophage cells. In addition to an enhancement of infectivity to the host cells, survival under acid stress was also elevated by the SNP in envZ. Together, these results suggest that the natural occurrence of the SNP in envZ could contribute to phenotypic diversity of S. Enteritidis, possibly improving its fitness and pathogenesis.

Dissemination and characteristics of Klebsiella spp. at the processed cheese plant

The genus Klebsiella is not generally considered as a major foodborne pathogen and with regard to food production it represents also a hygiene indicator. This study was focused on the monitoring of Klebsiella spp. dissemination at the processed cheese plant and determination of virulence determinants and antibiotic resistance of obtained strains. Klebsiella spp. were detected in 43% (37/87) of samples: swabs from the cheese processing environment (34/69), personnel (2/3) and raw material (milk powder) from an opened packaging (1/7). All tested samples of final processed cheeses were negative for the presence of Klebsiella spp. Obtained strains were identified as K. oxytoca (n = 32) and K. pneumoniae (n = 23). Typing results enabled to reveal the presence of two predominant PFGE clusters of K. pneumoniae and K. oxytoca suggesting the occurrence of suspect persistent strains, and spread of Klebsiella spp. between the production environment and the personnel. The melting process of processed cheese production was able to eliminate Klebsiella spp. in the final products, although some K. oxytoca and K. pneumoniae strains carried genes of the locus of heat resistance 1 (LHR1), which may lead to their increased heat resistance. K. pneumoniae and K. oxytoca isolated from the processed cheese plant did not represent any hypervirulent or multidrug-resistant strains which could be a potential threat to public health.

Whole-genome sequencing analysis of uncommon Shiga toxin-producing Escherichia coli from cattle: Virulence gene profiles, antimicrobial resistance predictions, and identification of novel O-serogroups

Shiga toxin-producing E. coli (STEC) are major foodborne pathogens. While many studies have focused on the “top-7 STEC”, little is known for minor serogroups. A total of 284 non-top-7 STEC strains isolated from cattle feces were subjected to whole-genome sequencing (WGS) to determine the serotypes, the presence of virulence genes and antimicrobial resistance (AMR) determinants. Nineteen typeable and three non-typeable serotypes with novel O-antigen loci were identified. Twenty-one AMR genes and point mutations in another six genes that conferred resistance to 10 antimicrobial classes were detected, as well as 46 virulence genes. The distribution of 33 virulence genes and 15 AMR determinants exhibited significant differences among serotypes (p < 0.05). Among all strains, 81.7% (n = 232) and 14.1% (n = 40) carried stx2 and stx1 only, respectively; only 4.2% (n = 12) carried both. Subtypes stx1a, stx1c, stx2a, stx2c, stx2d, and stx2g were identified. Forty-six strains carried eae and stx2a and therefore had the potential cause severe diseases; 47 strains were genetically related to human clinical strains inferred from a pan-genome phylogenetic tree. We were able to demonstrate the utility of WGS as a surveillance tool to characterize the novel serotypes, as well as AMR and virulence profiles of uncommon STEC that could potentially cause human illness.

The emergence of extended-spectrum β-lactamase (ESBL)-producing Salmonella London isolates from human patients, retail meats and chickens in southern China and the evaluation of the potential risk factors of Salmonella London

Salmonella is a major foodborne pathogen that causes a broad range of infection in humans and animals. This research was undertaken to investigate the antimicrobial resistance, virulence genes and genotyping of the extended-spectrum β-lactamase (ESBL)-producing Salmonella London (S. London) isolates originating from human patients, retail meats and chickens in southern China. The high antimicrobial resistance rates to trimethoprim/sulfamethoxazole (100%), methoxypyrimidine (100%), sulfaisoxazole (100%), ampicillin (73.68%) and amoxicillin (63.16%) were observed among a total of 19 S. London isolates, and the particular concern is that all isolates exhibited multidrug resistance (MDR). The ESBL-positive phenotype, which is resistant to antibiotics such as ampicillin, ceftazidime and cefotaxime, was observed in the isolates (15/19). These strains harbor the emerging ESBL-coding genes blaSHV, blaTEM-1, blaCTX-M and blaOXA, and co-harbor the virulence genes stn, invA, sseL and virK. In addition, the predominant genotypic virulence profile was characterized by the presence of stn, fimA, virK, invA, sseL, mgtC, siiE and sopB genes in the strains. The profiles of enterobacterial repetitive intergenic consensus (ERIC)-PCR revealed that S. London isolates with genetic diversity were widely disseminated. This study highlights the potential threat to public health posed by the foodborne transmission of the multidrug-resistant S. London isolates from diverse sources.

Development of a Selective Agar for Improving Campylobacter jejuni Detection in Food.

Campylobacter jejuni is a major gastroenteritis-causing foodborne pathogen. However, it is difficult to isolate when competing bacteria or cold-damaged cells are present. Herein, a medium (Campylobacter selective agar, CSA) was developed and supplemented with catalase, L-serine, L-cysteine, and quercetin for the selective detection of C. jejuni in food. The C. jejuni-detection efficiency in media broth and chicken tenders was evaluated. The pathogen was enumerated on modified charcoal-cefoperazone-deoxycholate agar (mCCDA), CSA supplemented with 4 µM catalase (CSA-C4), 8 µM catalase (CSA-C8), 20 mM L-serine (CSA-S20) or 50 mM L-serine (CSA-S50), and mCCDA supplemented with 0.5 mM L-cysteine (mCCDA-LC0.5), 1 mM L-cysteine (mCCDA-LC1), 40 µM quercetin (mCCDA-Q40) or 320 µM quercetin (mCCDA-Q320). The detection efficiency was then evaluated by counting colonies on the selective agar media. Quantitative assessment was also performed using chicken and duck carcasses. The C. jejuni detection efficiencies were higher (P < 0.05) in the groups CSA-C4 or CSA-C8, and CSA-S20 or CSA-S50, than mCCDA, and the detection efficiencies were maintained even in the presence of Acinetobacter baumannii, a competing bacterium. In the quantitative test, CSA-C8 and CSA-S50 demonstrated higher C. jejuni-detection efficiencies than mCCDA (control). Therefore, CSA-C8 and CSA-S50 improved the detection efficiency of C. jejuni in poultry products by promoting the recovery of cold-damaged cells. When using CSA-C8 or CSA-S50 developed in this study for detection of C. jejuni in food, detection efficiency was higher than mCCDA.

Detection of group A rotavirus in oyster tissues by in situ capture RT-qPCR

Group A rotavirus (RVA) is the major foodborne pathogen that could be transferred via oysters. There are limitations of current methods for detecting rotavirus in oysters, such as the long duration of the traditional cell culture-based plaque assay, the false positive due to the free RNA using RT-PCR-based molecular methods, and the complex process of RNA extraction from the food matrix. Therefore, we developed a PGM-mediated in situ capture reverse transcription quantitative polymerase chain reaction (ISC-RT-qPCR) assay to investigate the encapsulated RVA in oysters. In parallel comparing tests for the cell-cultured viral samples, the ISC-RT-qPCR exhibited 10-fold greater sensitivity and better accuracy of distinguishing the encapsulated RVA than RT-qPCR. Furthermore, the ISC-RT-qPCR demonstrated a higher detection rate than RT-qPCR when applied to detect RVA in the artificially contaminated oyster. In practical application, the detection rates of RVA in oysters collected from four different farms were respectively higher by ISC-RT-qPCR (37.9%, 28.6%, 27.3%, and 0.0%) than by RT-qPCR (0.0%, 13.8%, 4.5%, and 0.0%), making the ISC-RT-qPCR a better assay for disease monitoring and control. Overall, all results indicate that ISC-RT-qPCR is a promising method for detecting encapsulated RVA in oysters and simplifies the steps of virus concentration, virus extraction with higher sensitivity and accuracy than the conventional RT-qPCR assay.

Designing of a chimeric protein contains StxB, intimin and EscC against toxicity and adherence of enterohemorrhagic Escherichia coli O157:H7 and evaluation of serum antibody titers against it

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain is known as one of the major human foodborne pathogens. Lack of effective clinical treatment for human diarrheal diseases confirms the need for vaccine production against enteric bacteria such as E.coli O157:H7. Shiga-like toxin (Stx), EscC, and Intimin are the main important virulent factors of this enteric pathogen. In the present study, a comparative Omics analysis was conducted to identify most invasion EHEC antigenic factors as a potential immunogen. SEI (Stx-EscC-Intimin) trivalent chimeric protein was designed from the exposed and epitope rich part of these virulence factors. Sequence optimization, physicochemical properties, mRNA folding, three-dimensional structure and immunoinformatics data were investigated. The chimeric gene was synthesized with codon bias of E. coli. Recombinant protein was expressed and confirmed by western blot analysis. To evaluate the immunogenicity of the designed protein, the protein was administered to BALB/c mice and the serum IgG was determined by ELISA. Based on the Ramachandran plot, the validation data showed that 90.1 % of residues lie in the favored region. The high antigenicity of the multimeric protein was predicted by the immunoinformatic analysis. Epitope prediction had shown the proper distribution of linear and conformational B-cell epitopes and the competition of T-cell epitopes to bind MHC molecules too. Recombinant ESI Protein with 74.5 kDa was expressed in E. coli. Western blot analysis by anti-Stx antibody, confirmed a single band of chimeric protein. Consequently, the chimeric gene was designed and constructed after assessments. From in silico approach, the protein deduced from this cassette can be an immunogen candidate, and act against toxicity and adherence of EHEC.

Hurdle Effects of Ethanolic Plant Extracts with Antimicrobials Commonly Used in Food against Foodborne Pathogenic Escherichia coli

Escherichia coli (E. coli) O157:H7 is a major foodborne pathogen that causes severe human infections. Plant extracts, glycine, and sodium acetate (NaOAc) exert antimicrobial effects that can be used to control pathogenic E. coli. However, their combinations have not been investigated. Thus, this study investigates the combination of ethanolic plant extracts with glycine and NaOAc against E. coli at various pH and temperature levels. Clove and rosemary extracts exhibited significant (p ≤ 0.05) antimicrobial activity against E. coli. At neutral pH, the combination of plant extracts with 1.0% glycine or 0.1% NaOAc reduced the minimum inhibitory concentration of clove from 0.4% to 0.2%; at pH 5.5, clove (0.1%) and rosemary (0.2%) extracts supplemented with NaOAc (0.1%) showed an additive effect. The population of E. coli O157:H7 in phosphate-buffered saline with 0.2% clove extract, 2% glycine, and 2% NaOAc showed a more than 5 log reduction after incubation at 15 °C for 96 h, while the combination of 0.1% clove extract with 2% NaOAc at pH 5.5 completely inhibited E. coli within 24 h at 35 °C. Thus, the combination of plant extracts with glycine and NaOAc could serve as a promising hurdle technology in controlling the growth of E. coli.

Prevalence and Molecular Characterization of Salmonella Isolated from Broiler Farms at the Tolima Region-Colombia.

Simple SummarySalmonella spp. is a major foodborne pathogen with a worldwide distribution that is responsible for salmonellosis in animals and humans. Salmonella contamination of poultry and derivative products occurs at different levels. The aim of this study was to estimate the prevalence of Salmonella in broilers farms and to conduct a phenotypic and molecular characterization of Salmonella isolates. The prevalence at the broiler farm level was 26.67%, and all isolates were found to belong to the serovar Salmonella Paratyphi B. These results suggest a common source of Salmonella contamination between broiler farms, presumably via feed, parent flocks or hatchery machines. Salmonella Paratyphi B is present in different segments of the poultry chain in the Tolima region. Additional studies are needed to identify the main source of Salmonella in broilers, chicken carcasses, and eggs commercialized in the Tolima region.Salmonella is an important animal and human pathogen responsible for Salmonellosis, and it is frequently associated with the consumption of contaminated poultry products. The aim of this study was to estimate the prevalence of Salmonella in the poultry farms and to determine the genetic relationship. A total of 135 samples collected from fifteen broiler farms, including cloacal, feed, water, environmental and farm operator faeces samples were subjected to microbiological isolation. Molecular confirmation of Salmonella isolates was carried out by amplification of the invA gene, discrimination of d-tartrate-fermenting Salmonella isolates using multiplex PCR, and subsequently analysed by pulsed-field gel electrophoresis (PFGE). A survey questionnaire was conducted to identify potential risk factors for Salmonella presence in broiler farms. The prevalence of Salmonella at the farm level was 26.67%, and Salmonella isolates were serotyped as S. Paratyphi B and all isolates were d-tartrate-fermenting (dT+). PFGE showed three highly similar clusters and one significantly different Salmonella isolate. S. Paratyphi B continued to be present in different links of the poultry chain in the Tolima region, and identification of its main source is necessary to control its dissemination.