Dynamic responses of Salmonella Typhimurium to re-exposure to sublethal ciprofloxacin.

This study was designed to evaluate the history-dependent behaviors of Salmonella Typhimurium re-exposed to sublethal levels of ciprofloxacin. The S. Typhimurium cells were pre-exposed to 0 (CON), 1/16 (LOW), 1/8 (MED), and 1/4 (HIGH) minimum inhibitory concentrations (MICs) of ciprofloxacin, followed by re-exposure to the same concentrations. The bacterial growth, postantibiotic effect (PAE), relative fitness, and swimming motility of treatments were evaluated in the absence of ciprofloxacin. The lag phase duration (LPD) was estimate to assess bacterial recovery under ciprofloxacin exposure. A disk diffusion assay was used to determine the cross-resistance and collateral sensitivity of CON, LOW, MED, and HIGH treatments to ciprofloxacin (CIP), ceftriaxone (CEF), erythromycin (ERY), gentamicin (GEN), and polymyxin B (POL). The S. Typhimurium cells pre-exposed to ciprofloxacin were susceptible in antibiotic-free media, showing delayed growth. The highest PAE (>1 h) and bacterial fluctuation (CV = 5%) were observed at the High treatment compared to the CON. The HIGH treatment had the lowest relative fitness levels (0.87) and swimming motility (55 mm). The LPD was significantly decreased at the LOW treatment (1.8 h) when re-exposed to 1/16 × MIC of ciprofloxacin. The LOW, MED, and HIGH treatments showed the cross-resistance to POL and the collateral sensitivity to CEF, ERY, and GEN. The pre-exposure to ciprofloxacin could induce phenotypic diversity, corresponding to the history-dependent behaviors. These results provide important insights for the dynamic nature of bacterial populations when re-exposed to sublethal concentrations of antibiotics.

Growth fitness, virulence, and heat tolerance of Salmonella Typhimurium variants resistant to food preservation methods.

To study potential ramifications of antimicrobial resistance, we carried out adaptive laboratory evolution assays (ALE) to isolate three resistant variants (RVs) of Salmonella enterica Typhimurium, employing three different types of food preservation methods: 1) an emergent technology, plasma-activated water (PAW), leading to variant RV-PAW; a traditional method, heat, leading to variant RV-HT, and a natural antimicrobial compound, carvacrol, leading to variant RV-CAR. The variant resistant to plasma-activated water, RV-PAW, had mutations in rpoA and rpoD; it showed increased tolerance to heat in orange juice but ultimately did not pose a significant threat, as it exhibited a fitness cost at refrigeration temperature (8 °C), whereas its virulence against Caenorhabditis elegans decreased. The variant resistant to heat, RV-HT, had mutations in flhC, dnaJ: it exhibited a fitness cost at high growth temperatures (43 °C) and induced morphofunctional alterations in C. elegans. The variant resistant to carvacrol, RV-CAR, had mutations in sseG, flhA, wbaV, lon; this variant not only exhibited significantly higher thermotolerance in both laboratory media and food models but also effectively increased its growth fitness at refrigeration temperatures while retaining its virulence, evidenced by the highest percentage of Smurf phenotype in C. elegans. To address these challenges, we applied a process combining thermal treatment with citral, with the aim of leveraging the sublethal damage caused in RVs by heat treatments in orange juice. This approach achieves enhanced microbial inactivation without having to escalate the intensity of the thermal treatment. The result was particularly encouraging in the case of RV-CAR, the most challenging strain, for which we improved lethality by up to 3 log10 inactivation cycles.

Klebsiella oxytoca inhibits Salmonella infection through multiple microbiota-context-dependent mechanisms.

The Klebsiella oxytoca species complex is part of the human microbiome, especially during infancy and childhood. K. oxytoca species complex strains can produce enterotoxins, namely, tilimycin and tilivalline, while also contributing to colonization resistance (CR). The relationship between these seemingly contradictory roles is not well understood. Here, by coupling ex vivo assays with CRISPR-mutagenesis and various mouse models, we show that K. oxytoca provides CR against Salmonella Typhimurium. In vitro, the antimicrobial activity against various Salmonella strains depended on tilimycin production and was induced by various simple carbohydrates. In vivo, CR against Salmonella depended on toxin production in germ-free mice, while it was largely toxin-independent in mice with residual microbiota. This was linked to the relative levels of toxin-inducing carbohydrates in vivo. Finally, dulcitol utilization was essential for toxin-independent CR in gnotobiotic mice. Together, this demonstrates that nutrient availability is key to both toxin-dependent and substrate-driven competition between K. oxytoca and Salmonella.

The synergistic bactericidal effect of simultaneous 222 nm krypton-chlorine excilamp and 307 nm UVB light treatment on sliced cheese and its mechanisms.

In this study, we investigated the combined effect of 222 nm krypton-chlorine excilamp (EX) and 307 nm ultraviolet-B (UVB) light on the inactivation of Salmonella Typhimurium and Listeria monocytogenes on sliced cheese. The data confirmed that simultaneous exposure to EX and UVB irradiation for 80 s reduced S. Typhimurium and L. monocytogenes population by 3.50 and 3.20 log CFU/g, respectively, on sliced cheese. The synergistic cell count reductions in S. Typhimurium and L. monocytogenes in the combined treatment group were 0.88 and 0.59 log units, respectively. The inactivation mechanism underlying the EX and UVB combination treatment was evaluated using fluorescent staining. The combination of EX and UVB light induced the inactivation of reactive oxygen species (ROS) defense enzymes (superoxide dismutase) and synergistic ROS generation, resulting in synergistic lipid peroxidation and destruction of the cell membrane. There were no significant (P > 0.05) differences in the color, texture, or sensory attributes of sliced cheese between the combination treatment and control groups. These results demonstrate that combined treatment with EX and UVB light is a potential alternative strategy for inactivating foodborne pathogens in dairy products without affecting their quality.

Sanitization of hydroponic farming facilities in Singapore: what, why, and how.

This study performed microbial analysis of nutrient film technique (NFT) hydroponic systems on three indoor farms in Singapore (the "what"). To justify the necessity of sanitizing hydroponic systems, strong biofilm-forming bacteria were isolated from the facility and investigated for their influence on Salmonella colonization on polyvinyl chloride (PVC) coupons in hydroponic nutrient solutions (the "why"). Finally, sanitization solutions were evaluated with both laboratory-scale and field-scale tests (the "how"). As a result, the microbiome composition in NFT systems was found to be highly farm specific. The strong biofilm formers Corynebacterium tuberculostearicum C2 and Pseudoxanthomonas mexicana C3 were found to facilitate the attachment and colonization of Salmonella on PVC coupons. When forming dual-species biofilms, the presence of C2 and C3 also significantly promoted the growth of Salmonella (P < 0.05). Compared with hydrogen peroxide (H2O2) and sodium percarbonate (SPC), sodium hypochlorite (NaOCl) exhibited superior efficacy in biofilm removal. At 50 ppm, NaOCl reduced the Salmonella Typhimurium, C2, and C3 counts to <1 log CFU/cm2 within 12 h, whereas neither 3% H2O2 nor 1% SPC achieved this effect. In operational hydroponic systems, the concentration of NaOCl needed to achieve biofilm elimination increased to 500 ppm, likely due to the presence of organic matter accumulated during crop cultivation and the greater persistence of naturally formed multispecies biofilms. Sanitization using 500 ppm NaOCl for 12 h did not impede subsequent plant growth, but chlorination byproduct chlorate was detected at high levels in the hydroponic solution and in plants in the sanitized systems without rinsing. IMPORTANCE: This study's significance lies first in its elucidation of the necessity of sanitizing hydroponic farming systems. The microbiome in hydroponic systems, although mostly nonpathogenic, might serve as a hotbed for pathogen colonization and thus pose a risk for food safety. We thus explored sanitization solutions with both laboratory-scale and field-scale tests. Of the three tested sanitizers, NaOCl was the most effective and economical option, whereas one must note the vital importance of rinsing the hydroponic systems after sanitization with NaOCl.

Antimicrobial activity of chitosan- edible films containing a combination of carvacrol and rosemary nano-emulsion against Salmonella enterica serovar Typhimurium and Listeria monocytogenes for ground meat.

This research aimed to assess the potential of active food packaging as an innovative approach to enhance the quality of fresh food products. Specifically, our focus was on developing chitosan edible films combined with rosemary nanoemulsion (Ch-RNE) and carvacrol nano-emulsion (Ch-CNE) as effective antibacterial food packaging solutions. The efficacy of these films against artificially inoculated L. monocytogenes (NCTC 13372\ ATCC® 7644) as a Gram-positive bacterium, and S. enterica serovar Typhimurium (ATCC 14028) as a Gram-negative bacterium, in ground meat was investigated. The size of the prepared nano-emulsions was characterized using zeta sizer, FTIR and HRTEM. The MIC of both nano-emulsions against both pathogens was found to be 0.78 % and 1.56 %. Filmogenic mixtures were casted using these concentrations, which were then dried and evaluated for their physical and mechanical properties.

Dust sprinkling as an effective method for infecting layer chickens with wild-type Salmonella Typhimurium and changes in host gut microbiota.

Role of dust in Salmonella transmission on chicken farms is not well characterised. Salmonella Typhimurium (ST) infection of commercial layer chickens was investigated using a novel sprinkling method of chicken dust spiked with ST and the uptake compared to a conventional oral infection. While both inoculation methods resulted in colonisation of the intestines, the Salmonella load in liver samples was significantly higher at 7 dpi after exposing chicks to sprinkled dust compared to the oral infection group. Infection of chickens using the sprinkling method at a range of doses showed a threshold for colonisation of the gut and organs as low as 1000 CFU/g of dust. Caecal content microbiota analysis post-challenge showed that the profiles of chickens infected by the sprinkling and oral routes were not significantly different; however, both challenges induced differences when compared to the uninfected negative controls. Overall, the study showed that dust sprinkling was an effective way to experimentally colonise chickens with Salmonella and alter the gut microbiota than oral gavage at levels as low as 1000 CFU/g dust. This infection model mimics the field scenario of Salmonella infection in poultry sheds. The model can be used for future challenge studies for effective Salmonella control.

Characterization of Antimicrobial Activities of Bifidobacterium lactis BB-12 and Their Inhibitory Effect Against Some Foodborne Pathogens.

Bifidobacterium animalis subsp. lactis BB-12, a probiotic, has shown potential to promote health benefits and control pathogens. This study aimed to investigate the effectiveness of BB-12 and its cell-free supernatant (CFS) in inhibiting the growth of Listeria monocytogenes and Salmonella enterica serovar Typhimurium. To assess the antimicrobial activity of BB-12, agar well diffusion, disk diffusion, and minimum inhibitory concentration (MIC) tests were conducted. The bicinchoninic acid (BCA) assay was performed to measure the protein concentration in CFS. The study's results indicated that the BB-12 strain inhibited the pathogens' growth. The disk diffusion test using BB-12 showed inhibitory results ranging from 11 to 14 mm for both bacteria. The agar well diffusion test reported the zone of inhibition ranging from 11.6 to 16 mm for both bacteria. The MIC test was conducted as a confirmatory test, which demonstrated the highest inhibitory zone using 2 McFarland (6 × 108 CFU/mL) concentrations of probiotics on L. monocytogenes (44.98%) and S. Typhimurium (66.41%). The disk diffusion test revealed that the probiotic CFS had a significant inhibitory impact on S. Typhimurium with a 16.6 mm zone of inhibition. The BCA test findings indicated that the 24- and 48-h CFSs exhibited inhibitory properties against infections. Notably, the 24-h CFS, including a protein level of 78.47 µg/mL, demonstrated a more pronounced inhibitory impact on both pathogens. The findings highlight that utilizing the BB-12 strain and its CFS can serve as a viable approach to battle infections, enhancing food safety and public health.

Microbiome diversity protects against pathogens by nutrient blocking.

The human gut microbiome plays an important role in resisting colonization of the host by pathogens, but we lack the ability to predict which communities will be protective. We studied how human gut bacteria influence colonization of two major bacterial pathogens, both in vitro and in gnotobiotic mice. Whereas single species alone had negligible effects, colonization resistance greatly increased with community diversity. Moreover, this community-level resistance rested critically upon certain species being present. We explained these ecological patterns through the collective ability of resistant communities to consume nutrients that overlap with those used by the pathogen. Furthermore, we applied our findings to successfully predict communities that resist a novel target strain. Our work provides a reason why microbiome diversity is beneficial and suggests a route for the rational design of pathogen-resistant communities.

Bacterial retrons encode phage-defending tripartite toxin-antitoxin systems.

Retrons are prokaryotic genetic retroelements encoding a reverse transcriptase that produces multi-copy single-stranded DNA1 (msDNA). Despite decades of research on the biosynthesis of msDNA2, the function and physiological roles of retrons have remained unknown. Here we show that Retron-Sen2 of Salmonella enterica serovar Typhimurium encodes an accessory toxin protein, STM14_4640, which we renamed as RcaT. RcaT is neutralized by the reverse transcriptase-msDNA antitoxin complex, and becomes active upon perturbation of msDNA biosynthesis. The reverse transcriptase is required for binding to RcaT, and the msDNA is required for the antitoxin activity. The highly prevalent RcaT-containing retron family constitutes a new type of tripartite DNA-containing toxin-antitoxin system. To understand the physiological roles of such toxin-antitoxin systems, we developed toxin activation-inhibition conjugation (TAC-TIC), a high-throughput reverse genetics approach that identifies the molecular triggers and blockers of toxin-antitoxin systems. By applying TAC-TIC to Retron-Sen2, we identified multiple trigger and blocker proteins of phage origin. We demonstrate that phage-related triggers directly modify the msDNA, thereby activating RcaT and inhibiting bacterial growth. By contrast, prophage proteins circumvent retrons by directly blocking RcaT. Consistently, retron toxin-antitoxin systems act as abortive infection anti-phage defence systems, in line with recent reports3,4. Thus, RcaT retrons are tripartite DNA-regulated toxin-antitoxin systems, which use the reverse transcriptase-msDNA complex both as an antitoxin and as a sensor of phage protein activities.

Salmonella enterica serovar Typhimurium uses anaerobic respiration to overcome propionate-mediated colonization resistance.

The gut microbiota benefits the host by limiting enteric pathogen expansion (colonization resistance), partially via the production of inhibitory metabolites. Propionate, a short-chain fatty acid produced by microbiota members, is proposed to mediate colonization resistance against Salmonella enterica serovar Typhimurium (S. Tm). Here, we show that S. Tm overcomes the inhibitory effects of propionate by using it as a carbon source for anaerobic respiration. We determine that propionate metabolism provides an inflammation-dependent colonization advantage to S. Tm during infection. Such benefit is abolished in the intestinal lumen of Salmonella-infected germ-free mice. Interestingly, S. Tm propionate-mediated intestinal expansion is restored when germ-free mice are monocolonized with Bacteroides thetaiotaomicron (B. theta), a prominent propionate producer in the gut, but not when mice are monocolonized with a propionate-production-deficient B. theta strain. Taken together, our results reveal a strategy used by S. Tm to mitigate colonization resistance by metabolizing microbiota-derived propionate.

The effect of Thymus vulgaris and Thymbra spicata essential oils and/or extracts in pectin edible coating on the preservation of sliced bolognas.

The aim of this research was to determine the effect of pectin coating made with essential oils and/or extracts of Thymus vulgaris (thyme) and Thymbra spicata (thymbra) on the preservation of aerobically packaged sliced bolognas during cold storage. The treatment made with essential oils resulted in a reduction of 1.73 log CFU/g of Salmonella typhimurium ATCC 14028. Also, pectin coating made with essential oil-treated sliced bolognas had the lowest total mesophilic bacteria (6.27 log CFU/g), and total lactic acid bacteria (1.72 CFU/g), in comparison to non-treated bolognas, with 7.65 log CFU/g for total mesophilic bacteria and 4.99 log CFU/g for lactic acid bacteria. Application of an emulsion significantly (P < 0.05) affected L*(lightness), a*(redness), and b*(yellowness) values. The essential oil treatment had the highest TBARS values at the end of the storage period. The pH was not affected by the treatment (P > 0.05), but storage had a significant (P < 0.05) effect on the pH values.

Antibacterial Properties of Melanoidins Produced from Various Combinations of Maillard Reaction against Pathogenic Bacteria.

Novel melanoidins are produced by the Maillard reaction. Here, melanoidins with high antibacterial activity were tested by examining various combinations of reducing sugars and amino acids as reaction substrates. Twenty-two types of melanoidins were examined by combining two reducing sugars (glucose and xylose) and eleven l-isomers of amino acids (alanine, arginine, glutamine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and valine) to confirm the effects of these melanoidins on the growth of Listeria monocytogenes at 25°C. The melanoidins produced from the combination of d-xylose with either l-phenylalanine (Xyl-Phe) or l-proline (Xyl-Pro), for which absorbance at 420 nm was 3.5 ± 0.2, completely inhibited the growth of L. monocytogenes at 25°C for 48 h. Both of the melanoidins exhibited growth inhibition of L. monocytogenes which was equivalent to the effect of nisin (350 IU/mL). The antimicrobial spectrum of both melanoidins was also investigated for 10 different species of bacteria, including both Gram-positive and Gram-negative species. While Xyl-Phe-based melanoidin successfully inhibited the growth of Bacillus cereus and Brevibacillus brevis, Xyl-Pro-based melanoidin inhibited the growth of Salmonella enterica Typhimurium. However, no clear trend in the antimicrobial spectrum of the melanoidins against different bacterial species was observed. The findings in the present study suggest that melanoidins generated from xylose with phenylalanine and/or proline could be used as potential novel alternative food preservatives derived from food ingredients to control pathogenic bacteria. IMPORTANCE Although the antimicrobial effect of melanoidins has been reported in some foods, there have been few comprehensive investigations on the antimicrobial activity of combinations of reaction substrates of the Maillard reaction. The present study comprehensively investigated the potential of various combinations of reducing sugars and amino acids. Because the melanoidins examined in this study were produced simply by heating in an autoclave at 121°C for 60 min, the targeted melanoidins can be easily produced. The melanoidins produced from combinations of xylose with either phenylalanine or proline exhibited a wide spectrum of antibiotic effects against various pathogens, including Listeria monocytogenes, Bacillus cereus, and Salmonella enterica Typhimurium. Since the antibacterial effect of the melanoidins on L. monocytogenes was equivalent to that of a nisin solution (350 IU/mL), we might expect a practical application of melanoidins as novel food preservatives.

ZnO, TiO2 and Ag nanoparticles impact against some species of pathogenic bacteria and yeast.

The economic approaches for manufacturing the nanoparticles with physical and chemical effects and limited resistance to antibiotics have been progressed recently due to the rise of microbial resistance to antibiotics. This research aimed to study the antimicrobial efficacy of silver nanoparticles Ag, ZnO, and Tio2 nanoparticles against Salmonella typhimurium and Brucella abortus and Candida albicans. Two isolates of Salmonella and two isolates of Brucella abortus were isolated from food spastically meat and blood specimens, respectively. Candida albicans were isolated from the patient's mouth with oral candidiasis (oral thrush) and confirmed diagnosis by API 20C test. The antimicrobial susceptibility of Salmonella typhimurium and B. abortus isolates were performed against nine different antibiotics. Silver nanoparticles consisting of AgNPs size (90) nm, ZnO NPs size (20, 50) nm as well as TiO2 NPs size (10, 50) nm, were used. UV-Visible spectrophotometer was used to characterize silver nanoparticles. The highest resistance of Candida albicans was seen for fluconazole, Clotrimazole and Itraconazole. The results of the Minimum Inhibitory Concentration (MIC) of nanoparticles against Salmonella typhimurium showed the average MIC of Tio2-10nm and Tio2-50nm were 5000 and 2500 µg\ml for S1 and S2 isolates, respectively. The isolated Brucella abortus (B1 and B2) showed sensitivity to NPs with different MIC. The average MIC for Ag-90nm was 5000 and 2500 µg/ml for B1 and B2 isolates, respectively. The findings suggest NP solution has fungicidal and bactericidal impacts on the tested microorganisms so they can be suitable for multiple applications of the biomedical field such as developing new antimicrobial agents.

Efflux Impacts Intracellular Accumulation Only in Actively Growing Bacterial Cells.

For antibiotics with intracellular targets, effective treatment of bacterial infections requires the drug to accumulate to a high concentration inside cells. Bacteria produce a complex cell envelope and possess drug export efflux pumps to limit drug accumulation inside cells. Decreasing cell envelope permeability and increasing efflux pump activity can reduce intracellular accumulation of antibiotics and are commonly seen in antibiotic-resistant strains. Here, we show that the balance between influx and efflux differs depending on bacterial growth phase in Gram-negative bacteria. Accumulation of the fluorescent compound ethidium bromide (EtBr) was measured in Salmonella enterica serovar Typhimurium SL1344 (wild type) and efflux deficient (ΔacrB) strains during growth. In SL1344, EtBr accumulation remained low, regardless of growth phase, and did not correlate with acrAB transcription. EtBr accumulation in the ΔacrB strains was high in exponential phase but dropped sharply later in growth, with no significant difference from that in SL1344 in stationary phase. Low EtBr accumulation in stationary phase was not due to the upregulation of other efflux pumps but instead was due to decreased permeability of the envelope in stationary phase. Transcriptome sequencing (RNA-seq) identified changes in expression of several pathways that remodel the envelope in stationary phase, leading to lower permeability. IMPORTANCE This study shows that efflux is important for maintaining low intracellular accumulation only in actively growing cells and that envelope permeability is the predominant factor in stationary-phase cells. This conclusion means that (i) antibiotics with intracellular targets may be less effective in complex infections with nongrowing or slow-growing bacteria, where intracellular accumulation may be low; (ii) efflux inhibitors may be successful in potentiating the activity of existing antibiotics, but potentially only for bacterial infections where cells are actively growing; and (iii) the remodeling of the cell envelope prior to stationary phase could provide novel drug targets.

Structural optimization of natural product fusaric acid to discover novel T3SS inhibitors of Salmonella.

Type III secretion system (T3SS) plays a critical role in host cell invasion and pathogenesis of Salmonella. We recently identified the mycotoxin fusaric acid (FA) as a T3SS inhibitor of Salmonella. Herein, twenty-two diphenylsulfane derivatives were designed and synthesized using FA as a lead compound through scaffold hopping. Among them, SL-8 and SL-19 possessing strong anti-T3SS and anti-invasion activity were identified as T3SS inhibitors with improvement in potency as compared to FA. The inhibitory mechanisms on SPI-1 did not depend on the HilD-HilC-RtsA-HilA or PhoP-PhoQ pathway or the assembly of T3SS needle complex. Accordingly, we proposed that the inhibitory effects of SL-8 and SL-19 on SPI-1 probably influence the formation of SicA/InvF-effector complex or other related proteins.

Systematic reconstruction of an effector-gene network reveals determinants of Salmonella cellular and tissue tropism.

The minimal genetic requirements for microbes to survive within multiorganism communities, including host-pathogen interactions, remain poorly understood. Here, we combined targeted gene mutagenesis with phenotype-guided genetic reassembly to identify a cooperative network of SPI-2 T3SS effector genes that are sufficient for Salmonella Typhimurium (STm) to cause disease in a natural host organism. Five SPI-2 effector genes support pathogen survival within the host cell cytoplasm by coordinating bacterial replication with Salmonella-containing vacuole (SCV) division. Unexpectedly, this minimal genetic repertoire does not support STm systemic infection of mice. In vivo screening revealed a second effector-gene network, encoded by the spv operon, that expands the life cycle of STm from growth in cells to deep-tissue colonization in a murine model of typhoid fever. Comparison between Salmonella infection models suggests how cooperation between effector genes drives tissue tropism in a pathogen group.

Evaluation of the Stress Tolerance of Salmonella with Different Antibiotic Resistance Profiles.

Disease caused by antibiotic-resistant Salmonella is a serious clinical problem that poses a great threat to public health. The present study is aimed at assessing differences in bacterial kinetics with different antibiotic resistance profiles under environmental stress and at developing microbial tolerance models in lettuce during storage from 4 to 36°C. The drug-resistance phenotypes of 10 Salmonella Typhimurium (S. Typhimurium) isolates were examined using the broth microdilution method. The results of 10 S. Typhimurium isolates in the suspensions showed that a slow trend towards reduction of drug-sensitive (DS) isolates in relation to the others though without statistical difference. Compared to DS S. Typhimurium SA62, greater bacterial reduction was observed in multidrug-resistant (MDR) S. Typhimurium HZC3 during lettuce storage at 4°C (P < 0.05). It was likely that a cross-response between antibiotic resistance and food-associated stress tolerance. The greater growth in lettuce at 12°C was observed for DS S. Typhimurium SA62 compared to MDR S. Typhimurium HZC3 and was even statistically different (P < 0.05), while no significant difference was observed for bacterial growth between MDR S. Typhimurium HZC3 and DS S. Typhimurium SA62 strains in lettuce storage from 16 to 36°C (P > 0.05). The goodness-of-fit indices indicated the Log-linear primary model provided a satisfactory fit to describe the MDR S. Typhimurium HZC3 and DS S. Typhimurium SA62 survival at 4°C. A square root secondary model could be used to describe the effect of temperature (12, 16, 28, and 36°C) on the growth rates of S. Typhimurium HZC3 (adj - R 2 = 0.91, RMSE = 0.06) and S. Typhimurium SA62 (adj - R 2 = 0.99, RMSE = 0.01) derived from the Huang primary model. It was necessary to pay attention to the tolerance of antibiotic resistant bacteria under environmental stress, and the generated models could provide parts of the input data for microbial risk assessment of Salmonella with different antibiotic resistance profile in lettuce.

Refrigeration of eggs influences the virulence of Salmonella Typhimurium.

Salmonella Typhimurium is a human pathogen associated with eggs and egg-derived products. In Australia, it is recommended that eggs should be refrigerated to prevent condensation that can enhance bacterial penetration across the eggshell. Except for the United States, the guidelines on egg refrigeration are not prescriptive. In the current study, in-vitro and in-vivo experiments were conducted to understand the role of egg storage temperatures (refrigerated vs ambient) on bacterial load and the virulence genes expression of Salmonella Typhimurium. The in-vitro egg study showed that the load of Salmonella Typhimurium significantly increased in yolk and albumen stored at 25 °C. The gene expression study showed that ompR, misL, pefA, spvA, shdA, bapA, and csgB were significantly up-regulated in the egg yolk stored at 5 °C and 25 °C for 96 h; however, an in-vivo study revealed that mice infected with egg yolk stored at 25 °C, developed salmonellosis from day 3 post-infection (p.i.). Mice fed with inoculated egg yolk, albumen, or eggshell wash stored at refrigerated temperature did not show signs of salmonellosis during the period of the experiment. Data obtained in this study highlighted the importance of egg refrigeration in terms of improving product safety.

Itaconic acid exerts anti-inflammatory and antibacterial effects via promoting pentose phosphate pathway to produce ROS.

Itaconic acid is produced by immune responsive gene 1 (IRG1)-coded enzyme in activated macrophages and known to play an important role in metabolism and immunity. In this study, mechanism of itaconic acid functioning as an anti-inflammatory metabolite was investigated with molecular biology and immunology techniques, by employing IRG1-null (prepared with CRISPR) and wild-type macrophages. Experimental results showed that itaconic acid significantly promoted the pentose phosphate pathway (PPP), which subsequently led to significantly higher NADPH oxidase activity and more reactive oxygen species (ROS) production. ROS production increased the expression of anti-inflammatory gene A20, which in turn decreased the production of inflammatory cytokines IL-6, IL-1ß and TNF-α. NF-κB, which can up-regulate A20, was also vital in controlling IRG1 and itaconic acid involved immune-modulatory responses in LPS-stimulated macrophage in this study. In addition, itaconic acid inhibited the growth of Salmonella typhimurium in cell through increasing ROS production from NADPH oxidase and the hatching of Schistosoma japonicum eggs in vitro. In short, this study revealed an alternative mechanism by which itaconic acid acts as an anti-inflammatory metabolite and confirmed the inhibition of bacterial pathogens with itaconic acid via ROS in cell. These findings provide the basic knowledge for future biological applications of itaconic acid in anti-inflammation and related pathogens control.