Resistance In Foodborne Pathogens Research Articles

Enterococcus spp. in fish: Analysis of the presence and resistance in samples from Tri-City, Poland.

The growing concern over antibiotic resistance in foodborne pathogens necessitates comprehensive assessments of its prevalence and associated risks in various food products. The present study aimed to assess the occurrence of Enterococcus spp. in samples of fish purchased at various points of sale in the Tricity region. The selection of products (n = 74) was based on their availability and included both fish caught in the Baltic region and products imported from, Vietnam, China, Norway, and European Union (EU) countries. For bacterial isolation, samples were inoculated into selective broth, and the growth of enterococci was assessed based on turbidity. Positive cultures were confirmed by a change in color in bromocresol purple broth and were isolated on Slanetz-Bartley agar. Bacteria were present in all tested samples regardless of the degree of raw material processing as follows: frozen (F)- 55%, fresh/raw (FS)- 70.6%, thawed (DF)- 30%, smoked (S)- 50%, and the packaging methods, modified atmosphere packaging (MAP)- 34.4%, unit packaging (UP)- 75%, and sold in bulk (SB)- 76.9%, with an overall frequency of occurrence of 58.1%. The number of bacteria ranged from not detected to 4.28-log cfu/g, with the lowest mean values for thawed fish and those packed in MAP. Tests conducted on 24 strains isolated from samples showed their varied sensitivity to tetracyclines. Single cases of multidrug resistance of the tested strains were also observed. The conducted statistical analysis did not show statistically significant differences in the count of enterococci based on the origin, degree of processing, or packaging (p < 0.05). Moreover, differences in strain sensitivity to ampicillin were observed. Detected cases of resistance, especially to tetracycline, require careful monitoring and action to limit the health risks associated with resistant bacterial strains in food products.

The prevalent dynamic and genetic characterization of mcr-1 encoding multi-drug resistant Escherichia coli strains recovered from poultry in Hebei, China

ObjectivesColistin is known as the last resort antibiotic to treat the infections caused by multi-drug resistant (MDR) foodborne pathogens. The emergence and widespread dissemination of plasmid-mediated colistin resistance gene mcr-1 in the E. coli incurs potential threat to public health. Here, we investigated the epidemiology, transmission dynamics, and genetic characterization of mcr-1 harboring E. coli isolates from poultry origin in Hebei province, China. MethodsA total of 297 fecal samples were collected from the two large poultry farms in Hebei province, China. The samples were processed for E. coli identification by MALDI-TOF-MS and 16S rD4A sequencing. Then, mcr-1 gene harboring E. coli strains were identified by PCR and subjected to antimicrobial susceptibility testing by broth microdilution assay. The genomic characterization of the isolates was done by whole genome sequencing using the various bioinformatics tools, and multi-locus sequence typing (MLST) was done by sequence analysis of the seven housekeeping genes. The conjugation experiment was done to check the transferability of mcr-1 along with the plasmid stability testing. ResultsA total of six mcr-1 E. coli isolates with MIC of 4 μg/mL were identified from 297 samples (2.02%). The mcr-1 harboring E. coli were identified as MDR and belonged to ST101 (n=4) and ST410 (n=2). The genetic environment of mcr-1 presented its position on IncHI2 plasmid in four isolates and p0111 in two isolates which is rarely reported plasmid type for mcr-1. Moreover, both type of plasmids was transferable to recipient J53, and mcr-1 was flanked by three mobile elements ISApl1, Tn3, and IS26 forming a novel backbone Tn3–IS26-mcr-1– pap2-ISApl1 on p0111 plasmid. The phylogenetic analysis shared a common lineage with mcr-1 harboring isolates from the environment, human and animals which indicate its horizontal spread among the diverse sources, species, and hosts. ConclusionThis study recommends the one health approach for future surveillance across multiple sources and bacterial species to adopt relevant measures and reduce global resistance crises.

Genetic determinants of thermal resistance in foodborne bacterial pathogens

AbstractThermal food processing stands as the conventional and prevalent intervention utilized by food industries to uphold food safety standards. Some bacteria are known to withstand thermal treatments up to definite extents. This ability primarily stems from genetically determined traits that can be influenced by environmental conditions. This review focuses on the genetic determinants of thermal resistance in foodborne pathogens including the locus of heat resistance (LHR). Genes contributing to the expression of proteins that might indirectly contribute to thermal resistance have also been covered. The emergence of thermal tolerance in these pathogens raises significant food safety concerns, given global worries about bacterial resistance. The review consolidates existing research on genetic components and loci of heat resistance and highlights potential areas of gaps that warrant further research. Furthermore, acquired cross‐protection leading to resistance across multiple interventions has also been highlighted as an emerging concern. Acquiring insights into the environmental conditions that promote the selection of the LHR will empower the adoption of more efficient strategies to obstruct the pathways by which these pathogens persist within processing chains.

Polyphenols and CRISPR as Quorum Quenching Agents in Antibiotic-Resistant Foodborne Human Pathogens (Salmonella Typhimurium, Campylobacter jejuni and Escherichia coli 0157:H7).

Antibiotic resistance in foodborne pathogens is an increasing threat to global human health. Among the most prevalent antibiotic-resistant bacteria are Salmonella enterica serovar Typhimurium, Campylobacter jejuni and E. coli 0157:H7. Control of these and other pathogens requires innovative approaches, i.e., discovering new molecules that will inactivate them, or render them less virulent without inducing resistance. Recently, several polyphenol molecules have been shown to possess such characteristics. Also, the use of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) approaches has recently been proposed for such purpose. This review summarizes the main findings regarding the application of both approaches to control the above-mentioned foodborne pathogens by relying on Quorum Sensing interference (Quorum Quenching) mechanisms and highlights the avenues needed for further research.

Antimicrobial resistance in food-borne pathogens at the human-animal interface: Results from a large surveillance study in India

BackgroundThe burden of foodborne diseases and antimicrobial resistance carried by key foodborne pathogens in India is unknown due to a lack of an integrated surveillance system at the human-animal interface. MethodsWe present data from the WHO-AGISAR (Advisory Group on Integrated Surveillance of Antimicrobial Resistance), India project. Concurrent human and animal sampling was done across a large area across north India. Community-acquired diarrhea cases (n = 1968) of all age groups were included. Cross-sectional sampling of stool/ intestinal contents (n = 487) and meat samples (n = 419) from food-producing animals was done at farms, retail shops, and slaughterhouses. Pathogens were cultured and identified, and antimicrobial susceptibility was performed. ResultsOver 80% of diarrhoeal samples were obtained from moderate to severe diarrhea patients, which yielded EAEC (5%), ETEC (4.84%), EPEC (4.32%), and Campylobacter spp. (2%). A high carriage of EPEC (32.11%) and Campylobacter spp. (24.72%) was noted in food animals, but the prevalence of ETEC (2%) and EAEC (1%) was low. Atypical EPEC (aEPEC, 84.52%, p ≤0.0001) were predominant and caused milder diarrhea. All EPEC from animal/poultry were aEPEC. Overall, a very high level of resistance was observed, and the MDR rate ranged from 29.2% in Campylobacter spp., 53.6% in EPEC, and 59.8% in ETEC. Resistance to piperacillin-tazobactam, cefepime, ceftriaxone, and co-trimoxazole was significantly higher in human strains. In contrast, resistance to ciprofloxacin, aminoglycosides, and tetracycline was higher in animal strains, reflecting the corresponding usage in human and animal sectors. ESBL production was commoner in animal isolates than in humans, indicating high use of third-generation cephalosporins in the animal sector. C. hyointestinalis is an emerging zoonotic pathogen, first time reported from India. ConclusionIn one of the most extensive studies from India, a high burden of key foodborne pathogens with MDR and ESBL phenotypes was found in livestock, poultry, and retail meat.

Tolerance variations and mechanisms of Salmonella enterica serovar Newport in response to long-term hypertonic stress

Abstract Objectives To assess the variations in hypertonic tolerance among Salmonella enterica serovar Newport (S. Newport) strains isolated from diverse sources and investigate the mechanism underlying the tolerance differences between the sensitive and tolerant strain. Materials and Methods In this work, various S. Newport strains were cultured in 5% and 10% NaCl solutions (hypertonic stress), and the most sensitive and tolerant strains were selected using a Weibull model. The regulatory mechanisms underlying the hypertonic tolerance differences of S. Newport were preliminarily investigated in the aspects of cell phenotype, intracellular solute concentration, and gene expression level. Results With prolonged hypertonic stress time, the leakage of nucleic acids and proteins of S. Newport increased gradually, and the membrane potential of S. Newport declined after increasing. Compared with the sensitive strain, the tolerant counterpart exhibited the ability to maintain the integrity of the cell membrane and sustain a high membrane potential level. The expression levels of the upstream genes proV and otsB in the tolerant strain were significantly lower than those in the sensitive strain; but the Kdp and Trk systems and downstream genes proX, proW, and otsA were highly expressed in the tolerant strain compared with the sensitive strain, leading to higher concentrations of intracellular K+ and trehalose, enabling better survival in a hypertonic environment. Conclusions The findings of this work offer valuable insights into pathogen survival mechanisms under hypertonic stress and contribute to the development of strategies for mitigating microbiological risks during long-term processing and storage in the cured food industry.

Biocontrol of Salmonella Typhimurium in milk, lettuce, raw pork meat and ready-to-eat steamed-chicken breast by using a novel bacteriophage with broad host range

Salmonella spp., one of the most frequently reported bacteria, causes foodborne illness and economic losses. Due to the threat of increasing antibiotic resistant foodborne pathogens, application of bacteriophages as novel antibacterial agents in food matrices has become an emerging strategy. In this study, a novel Salmonella phage PS3-1 with high lytic activity against Salmonella Typhimurium was identified from previously isolated phages. PS3-1 belonged to the class Caudoviricetes with a broad host range, and had relatively short latent period (15 min), large burst size (92 PFU/cell), high pH stability (pH 3.0–11.0) and thermal tolerance (4–60 °C). Genome sequencing analysis showed that PS3-1 genome consisted of 107,110 bp DNA, without antibiotic resistance and virulence related genes. The results of growth curve and time-kill assay showed that PS3-1 not only inhibited the growth of S. Typhimurium, but also effectively decreased the viable cell counts (0.30–4.72 log) after 24-h incubation at 7, 25 and 37 °C (P < 0.05). Moreover, >1.28 log of established biofilm cells were effectively removed after 24-h treatment with PS3-1. Besides, PS3-1 significantly reduced the viability of S. Typhimurium in milk, lettuce, raw pork meat and ready-to-eat steamed-chicken breast at different temperatures (P < 0.05). These results demonstrated that PS3-1 may be an excellent antibacterial agent for controlling S. Typhimurium in food industry.

Characterization of lactic acid bacteria isolated from street pickles of Dhaka, Bangladesh

Traditionally fermented pickles are a popular street food in Bangladesh famous for their unique flavors and health benefits. Pickles are often prepared by fermentation using lactic acid bacteria (LAB) that can act as probiotics. The study was aimed to isolate and characterize lactic acid bacteria from pickle samples collected from streets of Dhaka city, as well as assess the microbial quality of pickles for food safety.A total of 30 pickle samples of different kinds were collected from streets of Dhaka city. Isolation and identification were conducted using conventional cultural and biochemical tests, followed by molecular confirmation of identity. Antibiotic susceptibility of isolates was investigated against 7 antibiotics of different groups. Antimicrobial activity of LAB isolates was analyzed by well-diffusion assay and phenotypic enterocin activity assay. Physiological characterizations of LAB were performed to determine their tolerance to temperature, salt, pH, bile, carbohydrate fermentation pattern, proteolytic activity and biofilm formation.Fifty isolates were obtained from pickle samples, of which 18% was identified as LAB, including Enterococcus faecalis (6) and Enterococcus faecium (3). The rest included S. aureus (18), E. coli (11), Klebsiella spp. (5), Salmonella (3), Shigella (3) and Pseudomonas aeruginosa (1). Antibiotic resistance pattern revealed higher occurrence of resistance against azithromycin among the non-LAB isolates, but none of the LAB isolates were found to resist any of the antibiotics used. Antimicrobial activity of LAB isolates was not observed against the foodborne isolates. All LAB isolates fermented a wide range of carbohydrates and showed adequate tolerance to salt, pH, temperature and bile. Out of 9 isolates, 5 displayed proteolytic activity, and 6 were found as strong biofilm producer.These results suggest that although the LAB isolates from street pickles collected from Dhaka does not have antimicrobial activities, they still have potential to be used as probiotics. It also shows high occurrence of antibiotic resistant foodborne pathogens in pickles, indicating that consumption of such street food can be serious health hazard.

Occurrence and Multidrug Resistance in Strains of Listeria monocytogenes Recovered from the Anaerobic Co-Digestion Sludge Contained in a Single Stage Steel Biodigester: Implications for Antimicrobial Stewardship.

L. monocytogenes is a zoonotic foodborne pathogen with inherent adaptability to tolerate environmental and physiological stresses, thereby causing severe disease outbreaks. Antibiotic resistant foodborne pathogens are a challenge to the food industry. A total of 18 samples were pooled from a bio-digester co-digesting swine manure/pinewood sawdust, and evaluated for the occurrence of bacterium plus total viable counts using the spread plate method. The recovered bacterial isolates were presumptively identified by growth on selective medium and confirmed by biochemical characterisation, leading to the isolation of 43 L. monocytogenes. The isolates were characterized based on their susceptibility to antibiotics via the Kirby-Bauer disc diffusion technique against a panel of 14 antibiotics. Equally, the multiple antibiotic resistance (MAR) index was calculated, and MAR phenotypes generated. The bacterial counts were between 102 and104 cfu/mL. Complete susceptibility (100%) was demonstrated to ampicillin, gentamicin and sulfamethoxazole, which are the drugs of choice in the treatment of listeriosis. In addition, intermediate sensitivity occurred at 25.58% to cefotaxime, and the highest resistance (51.16%) was exhibited against nalidixic acid. The MAR index ranged from 0 to 0.71. Overall, 41.86% of the Listeria isolates displayed multidrug resistance, with 18 different MAR phenotypes, demonstrating CIP, E, C, TET, AUG, S, CTX, NA, AML, NI as the greatest MAR phenotype. It can be concluded that the isolates yielding MAR > 0.2 originated from the farm, where antibiotics had been in routine use. Therefore, strict monitoring of antibiotics use in the farm is crucial to mitigate further increase in antibiotic resistance amongst these bacterial isolates.

Carriage of Antibiotic Resistant Bacteria and Associated Factors Among Food Handlers in Tamale Metropolis, Ghana: Implications for Food Safety.

Bacteria pathogens constitute a significant proportion of diarrhoea-causing food contaminants. Transmission of antibiotic resistant foodborne pathogens to humans is a major threat to food safety, especially in developing countries where quality hygiene and sanitation facilities are lacking. Factors related to antibiotic use, sanitation and hand hygiene have been associated with the spread of infectious diseases as well as antibiotic resistant bacteria. Proper food handling ensures that food is not contaminated with potential pathogenic bacteria. This study assessed the carriage of antibiotic resistant bacteria and associated factors. A cross-sectional study was conducted among food handlers who sell ready to eat food in the Tamale metropolis of the Northern Region of Ghana. Food vending stations with huge customer base were randomly selected and the food handlers recruited using written informed consent. Structured questionnaires were used to collect participants sociodemographic details and information on sanitation, hand hygiene practice and antibiotic use. Sterile cotton swabs soaked in phosphate buffered saline was used to swab the palms of participating food handlers for bacteria isolation. All identified bacteria were tested for susceptibility to 12 antibiotics. In all, 406 food handlers participated in this study, the mean (SD) age was 26.5 (2.64) years. Bacteria isolated were predominantly Staphylococci 60 (14.8%) and Escherichia coli 54 (13.3%). All the isolates were resistant to at least one antibiotic tested. The isolates showed high resistance to broad-spectrum antibiotics such as ampicillin (40.0%-75.0%), tetracycline (40.0%-80.0%), amoxiclav (20.0%-80.0%) and chloramphenicol (7.7%-50.0%). Logistic regression model revealed that the carriage of antibiotic resistant bacteria by food handlers was significantly associated with age, educational level, years on the job, training in food preparation, hygiene practice, water source, type of toilet facility used and antibiotic use. Street food handlers could be potential sources of food-borne transmission of antibiotic resistant bacteria.

PHYTOCHEMICAL SCREENING AND ANTIBACTERIAL ACTIVITY OF Zingiber officinale AND Syzygium aromaticum ON BACTERIA PATHOGENS ISOLATED FROM KUNUN-AYA

The increased use of antibiotics in agricultural practices is accompanied with the development of resistant foodborne pathogens. Spices and herbs have been reported to be potent source of natural antioxidants. This study aimed to assess the antibacterial activity of Zingiber officinale (Ginger) and Syzygium aromaticum (Clove) against bacterial food pathogens. Fresh ginger andclove were collected, identified, air dried and extracted the bioactive compound using distilled water. The extract was used for phytochemical analysis of flavonoids, tannins, saponins, steroids, glycosides, alkaloids and total phenols and determination of inhibition zone. Bacteria and fungi were isolated and identified from locally prepared Pap (Kunun aya) and later used for theantimicrobial analyses. Phytochemical screening shows clove contain flavonoids, tannins, saponins, steroids, glycosides, alkaloids and total phenols whereas glycosides and tannins were absent in ginger. Ginger has more antibacterial activity with the highest zone of inhibition observed in E coli at 16.73mm at 2000µg and the lowest in Salmonella at 13.83mm at 2000µg, clove also shows antibacterial activity with the highest zone of inhibition (14.8mm) in S. aureus at 2000µg and the lowest 11.5mm in E. coli at 2000µg. Thus, from the result obtained, it can be concluded that Zingiber officinale and Syzygium aromaticum extracts possesses effective antimicrobial agents against food pathogens.

Combined transcriptomic and proteomic analysis of the antibacterial mechanisms of an antimicrobial substance produced by Lactobacillus paracasei FX-6 against colistin-resistant Escherichia coli

The emergence and spread of plasmid-mediated mobile colistin resistance in food-borne pathogens threatens food safety and human health, necessitating the development of novel antimicrobial agents. Our previous studies indicate that an antimicrobial substance (AMS) produced by Lactobacillus paracasei FX-6 could be a promising candidate due to its broad-spectrum antimicrobial activities. This study aimed to investigate the antibacterial effects and mechanisms of AMS against colistin-resistant Escherichia coli SHP45. Our results showed that the minimum inhibitory concentration (MIC) of AMS against E. coli SHP45 was 4.0 mg/mL. Flow cytometry showed that AMS disrupts the structural integrity of the cell membrane. According to transmission electron microscope (TEM) observations, the bacterial morphology was severely damaged, and hollow areas formed in the cytoplasm. Further transcriptomic and proteomic analysis revealed that AMS inhibits E. coli SHP45 by co-upregulating critical genes and proteins involved in membrane damage (FtsH, ClpX) and protein hydrolysis (Lon, DnaJ, GroEL), causing DNA damage and metabolic disorders, as well as by inhibiting nutrient transport and energy metabolism. Our results provide new insights into the antibacterial effects of AMS against E. coli SHP45 and suggest that AMS derived from Lactobacillus paracasei FX-6 may be a promising antimicrobial agent for controlling colistin-resistant pathogens in food.

Distribution and Characterization of Antimicrobial Resistant Pathogens in a Pig Farm, Slaughterhouse, Meat Processing Plant, and in Retail Stores.

The emergence of antibiotic resistance in foodborne pathogens isolated from meat pro-ducts and their producing environment has been an increasing and leading threat to public health. The aim of the study was to identify pathogens and their antimicrobial resistance isolated from pig production to pork meat distribution phases. Through this study, food spoilage and foodborne or clinical pathogenic bacteria were isolated and identified from pork (belly and neck) meat product and its related environmental samples that include pig swabs, diets, feces, liquid manure, workers' gloves, dust fan swabs, carcass swabs, floor swabs, and drain water in the affiliated farm, slaughterhouse, meat processing plant, and in retail stores. All carcasses at the slaughterhouse and meat products at the meat processing plant were tracked from pigs at a targeted farm. Nine different selective media agars were used to effectively isolate various pathogenic bacteria. A total of 283 presumptive pathogenic bacteria isolated from 126 samples were selected and identified using MALDI-ToF MS. Twenty-three important foodborne pathogens were identified, and some of them, Shiga-toxin-producing E. coli (STEC), Listeria monocytogenes, Staphylococcus aureus, and Yersinia enterocolitica, were further confirmed using PCR. The PFGE patterns of 12 STEC isolates were grouped by sample source or site. All the foodborne pathogens used in the study were not resistant to amoxicillin/clavulanate, ciprofloxacin, and gentamicin, whereas some of the STEC, L. monocytogenes, and S. aureus isolates were resistant to various antibiotics, including ampicillin, erythromycin, tetracycline, and vancomycin. The most common antimicrobial resistance pattern in the pathogenic STEC isolates was AMP-KAN-STR-SXT-TET. Consequently, this study provides valuable information for the distribution of antimicrobial-resistant pathogens along the pork meat production chain and can assist farmers and stakeholders to develop a systematic strategy for reducing the current emergence and spread of antimicrobial resistance in the different phases of pig production and distribution.

Antimicrobial activity of tofu whey and steam blanching pea water for enhancement of shelf-life of 3D printed mashed potatoes

Using naturally-occurring antimicrobials against the resistance of foodborne pathogens towards antibiotics and chemicals, as well as their insensitivity is a promising research field. Water extracts of legumes have been proven to inhibit microbial development due to the presence of phenolic compounds, protein-derived peptides, saponins and defensin. This study examined the antimicrobial effects of two types of legume water, such as tofu whey (TW) and steam blanching pea water (SBPW), by adding into three-dimensional (3D) printed mashed potato. Composition, moulding, microbial growth assessment and the inhibition zone analysis were conducted. TW and SBPW contained 4 g/100 ml of solid matter, consisting of protein, soluble carbohydrates, minerals and saponins. Results have shown decreased microbial spoilage and enhanced Lactobacillus growth of mashed potato formulated with legume water. Limited inhibition zone (1 mm) was present on TW saponin extract against Staphylococcus aureus and Lactobacillus spp. TW and SBPW addition to 3D printed mashed potatoes encouraged the development of probiotic bacteria, which can be applied to extend shelf-life of food products as well as to develop fermented foods.

Virulence Profiling, Multidrug Resistance and Molecular Mechanisms of Campylobacter Strains from Chicken Carcasses in Tunisia.

Antibiotic resistance in foodborne pathogens is an emergent global health concern. The objectives of this study were to assess antimicrobial resistance (AMR) in Campylobacter isolates from chicken carcasses and to investigate the AMR molecular mechanisms as well as the presence of virulence determinants. The study was performed on 257 samples collected from abattoirs and retail shops in northeastern Tunisia. Forty-eight Campylobacter isolates were recovered and identified as C. jejuni (n = 33) and C. coli (n = 15). Antibiotic resistance was tested against eight antibiotics and high resistance rates were observed against tetracycline (100%), erythromycin (97.9%), ciprofloxacin (73%), nalidixic acid (85.4%), ampicillin (83.3%), amoxicillin/clavulanic acid (22.9%), chloramphenicol (75%), and gentamicin (27.1%). All isolates were multidrug-resistant, and 22 resistance patterns were found. All isolates were screened for AMR genes (tet(O), tet(A), tet(B), tet(L), cmeB, ermB, blaOXA-61, and aphA-3), and for point mutations in gyrA (C257T substitution) and 23SrRNA (A2075G/A2074C) genes. All screened AMR genes, as well as the C257T and the A2075G mutations, were detected. The virulence genotypes were also determined, and all isolates carried the motility (flaA) and invasion (cadF) genes. Most of them also harbored the cdtA, cdtB, and cdtC genes, encoding the Campylobacter toxin. The screening of the cgtB and the wlaN genes, involved in Guillain-Barré Syndrome expression, revealed the presence of the cgtB in 21.2% of C. jejuni strains, whereas none of them carried the wlaN gene. Our findings highlight the emergence of Campylobacter strains simultaneously harboring several virulence and AMR determinants, which emphasizes the risk of transmission of MDR strains to humans via the food chain. Hence, controlling the dissemination of foodborne pathogens “from the farm to the fork” as well as restricting the use of antimicrobials in husbandry are mandatory to prevent the risk for consumers and to mitigate the dissemination of MDR pathogens.

Green synthesis of silver nanoparticles by Pisum sativum L. (pea) pod against multidrug resistant foodborne pathogens

The consumption of foods contaminated with various foodborne organisms such as, viruses, fungi and bacteria are recognized as the important sources of foodborne illness in animals and humans. Multidrug resistant foodborne bacteria increased morbidity, mortality rates, and severe economic loss and associated with prolong hospitalization. The development of natural and novel antibacterial agent is much needed as there is an increasing concern on multidrug resistant bacteria. This study aimed to synthesize silver nanoparticles using aqeous Pisum sativum L. (pea) pod extract and to characterize the nanoparticles. It was further used on various drug resistant foodborne bacterial pathogens and antimicrobial activity was determined. The synergistic antibacterial activity, antioxidant activity and biocompatibility were also assessed. Green synthesized nanoparticles applied in this study were synthesized using aqueous P. sativum L. (pea) pod extract with particle size 30 nm. The green synthesized silver nanoparticles showed antimicrobial activity against Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 700802, Streptococcus gordonii ATCC 49818, Staphylococcus aureus (MRSA) ATCC 4330 and Staphylococcus aureus ATCC 25923. Among the pathogenic bacterial strains, silver nanoparticles were highly active against S. aureus ATCC 25923 than other bacterial strains (22 ± 2 mm). The antibacterial activity varied between 15 ± 2 mm and 22 ± 2 mm diameter zone of inhibition. The MIC value of the nanoparticles ranged from 6.25 µg/mL to 75 µg/mL and MBC value ranged from 50 µg/mL to 150 µg/mL. The green synthesized nanoparticles showed antioxidant potential. Normal fibroblast cell lines (L-929) was used to test the cytotoxic activity and the increased IC50 value (750.5 ± 5.5 μg/mL) indicated good biological compatibility. The present findings revealed that green synthesized silver nanoparticles exhibited potent activity against various food borne pathogenic bacteria. These nanoparticles might be well developed as novel antibacterial substances for the treatment of food borne multidrug resistant bacterial infection.

Developing Qualitative Plasmid DNA Reference Materials to Detect Mechanisms of Quinolone and Fluoroquinolone Resistance in Foodborne Pathogens

The aim of this study was to develop homogeneous and stable plasmid DNA reference materials for detecting the mechanisms of resistance to quinolones and fluoroquinolones in foodborne pathogens. The DNA fragments of 11 target genes associated with quinolone and fluoroquinolone resistance were artificially synthesized, inserted into plasmid vectors, and transferred into recipient cells. PCR and sequencing of DNA were performed to assess the genetic stability of the target DNA in recombinant Escherichia coli DH5α cells during subculturing for 15 generations. The limit of detection (LOD) of the target DNA was determined using PCR and real-time qualitative PCR (qPCR). The homogeneity and storage stability of plasmid DNA reference materials were evaluated in terms of plasmid DNA quantity, PCR-measured gene expression, and qPCR threshold cycle. All 11 target DNAs were successfully synthesized and inserted into vectors to obtain recombinant plasmids. No nucleotide mutations were identified in the target DNA being stably inherited and detectable in the corresponding plasmids during subculturing of recombinant strains. When the target DNA was assessed using PCR and qPCR, the LOD was ≤1.77 × 105 and 3.26 × 104 copies/μL, respectively. Further, when the reference materials were stored at 37 °C for 13 days, 4 °C for 90 days, and −20 °C for 300 days, each target DNA was detectable by PCR, and no mutations were found. Although the threshold cycle values of qPCR varied with storage time, they were above the LOD, and no significant differences were found in the quantity of each plasmid DNA at different timepoints. Further, the homogeneity and stability of the materials were highly consistent with the requirements of standard reference materials. To summarize, considering that our plasmid DNA reference materials conformed to standard requirements, they can be used to detect the mechanisms of quinolone and fluoroquinolone resistance in foodborne pathogens.

Biofabrication of ZnO nanoparticles using Acacia arabica leaf extract and their antibiofilm and antioxidant potential against foodborne pathogens.

Emergence of multidrug resistant pathogens is increasing globally at an alarming rate with a need to discover novel and effective methods to cope infections due to these pathogens. Green nanoparticles have gained attention to be used as efficient therapeutic agents because of their safety and reliability. In the present study, we prepared zinc oxide nanoparticles (ZnO NPs) from aqueous leaf extract of Acacia arabica. The nanoparticles produced were characterized through UV-Visible spectroscopy, scanning electron microscopy, and X-ray diffraction. In vitro antibacterial susceptibility testing against foodborne pathogens was done by agar well diffusion, growth kinetics and broth microdilution assays. Effect of ZnO NPs on biofilm formation (both qualitatively and quantitatively) and exopolysaccharide (EPS) production was also determined. Antioxidant potential of green synthesized nanoparticles was detected by DPPH radical scavenging assay. The cytotoxicity studies of nanoparticles were also performed against HeLa cell lines. The results revealed that diameter of zones of inhibition against foodborne pathogens was found to be 16–30 nm, whereas the values of MIC and MBC ranged between 31.25–62.5 μg/ml. Growth kinetics revealed nanoparticles bactericidal potential after 3 hours incubation at 2 × MIC for E. coli while for S. aureus and S. enterica reached after 2 hours of incubation at 2 × MIC, 4 × MIC, and 8 × MIC. 32.5–71.0% inhibition was observed for biofilm formation. Almost 50.6–65.1% (wet weight) and 44.6–57.8% (dry weight) of EPS production was decreased after treatment with sub-inhibitory concentrations of nanoparticles. Radical scavenging potential of nanoparticles increased in a dose dependent manner and value ranged from 19.25 to 73.15%. Whereas cytotoxicity studies revealed non-toxic nature of nanoparticles at the concentrations tested. The present study suggests that green synthesized ZnO NPs can substitute chemical drugs against antibiotic resistant foodborne pathogens.

Alternative and Complementary Therapies against Foodborne Salmonella Infections.

The limitations in the therapeutic options for foodborne pathogens lead to treatments failure, especially for multidrug-resistant (MDR) Salmonella sp., worldwide. Therefore, we aimed to find alternative and complementary therapies against these resistant foodborne pathogens. Out of 100 meat products samples, the prevalence rate of salmonella was 6%, serotyped only as S. Typhimurium and S. Enteritidis. According to the antibiotic susceptibility assays, the majority of our isolates were MDR and susceptible to cefotaxime. Out of the 13 tested plant extracts, five only showed an inhibition zone in the range of 8–50 mm against both serotypes. Based on their promising activity, the oily extract of cinnamon and aqueous extract of paprika represented the highest potency. Surprisingly, a significant synergistic effect was detected between cinnamon oil and cefotaxime. Depending on Gas Chromatography/Mass Spectrometry (GC-MS), the antimicrobial activity of cinnamon oil was attributed to four components including linalool, camphor, (Z)-3-Phenylacrylaldehyde and its stereoisomer 2-Propenal-3-phenyl. The anti-virulence activities of these compounds were confirmed on the basis of computational molecular docking studies. Accordingly, we recommended the use of cinnamon oil as a food additive to fight the resistant foodborne pathogens. Additionally, we confirmed its therapeutic uses, especially when co-administrated with other antimicrobial agents.

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.