Overuse of food-grade disinfectants threatens a global spread of antimicrobial-resistant bacteria.

Food-grade disinfectants are extensively used for microbial decontamination of food processing equipment. In recent years, food-grade disinfectants have been increasingly used. However, the overuse of disinfectants causes another major issue, which is the emergence and spread of antimicrobial-resistant bacteria on a global scale. As the ongoing pandemic takes global attention, bacterial infections with antibiotic resistance are another ongoing pandemic that often goes unnoticed and will be the next real threat to humankind. Here, the effects of food-grade disinfectant overuse on the global emergence and spread of antimicrobial-resistant bacteria were reviewed. It was found that longtime exposure to the most common food-grade disinfectants promoted resistance to clinically important antibiotics in pathogenic bacteria, namely cross-resistance. Currently, the use of disinfectants is largely unregulated. The mechanisms of cross-resistance are regulated by intrinsic molecular mechanisms including efflux pumps, DNA repair system, modification of the molecular target, and metabolic adaptation. Cross-resistance can also be acquired by mobile genetic elements. Long-term exposure to disinfectants has an impact on the dissemination of antimicrobial resistance in soil, plants, animals, water, and human gut environments.

Induction of viable but nonculturable Salmonella spp. in liquid eggs by mild heat and subsequent resuscitation.

Salmonella spp. is one of the leading causes of foodborne outbreaks worldwide. Salmonella spp. has been associated with a variety of food sources, particularly egg products. They can enter a viable but nonculturable (VBNC) state in response to harsh stress. VBNC cells still retain membrane integrity and metabolic activity, which may pose health risks. However, the formation mechanism and resuscitation ability of VBNC cells are not well understood. In this work, Salmonella spp. cocktails, including Salmonella enterica serovar Newport and Salmonella enterica serovar Enteritidis, in liquid egg products was induced into a VBNC state by mild heat treatment, a commonly used method to inhibit the growth of pathogenic in liquid egg industry. Mild heat induced VBNC cells were found to resuscitate in liquid egg yolk (LEY) and liquid whole egg (LWE), but they failed to recover in liquid egg white (LEW). In addition, a certain number of cells remained as VBNC state after in vitro digestion. The membrane vesicle (MV) protein encoding gene pagC, two-component system encoding genes phoP/Q and sigma factor encoding gene rpoS were highly expressed in VBNC cells compared with the culturable counterparts. The results of this study can contribute to a better understanding of the health risks associated with Salmonella spp. in VBNC state and provide a theoretical basis for formation mechanism of VBNC state.

Dynamic Fluctuation and Niche Differentiation of Fungal Pathogens Infecting Bell Pepper Plants.

The plant microbiome is shaped by plant development and microbial interaction. Fungal pathogens infecting bell pepper plants may fluctuate across the growing seasons. Dynamic fluctuation of the microbiome and fungal pathogens in bell pepper plants is poorly understood, and the origin of fungal pathogens causing fruit rot and leaf wilt has been barely investigated. In this study, we used amplicon sequencing (i.e., 16S rRNA and internal transcribed spacer [ITS] sequencing) to explore the compositional variations of the microbiome in bell pepper plants and studied the fluctuation of fungal pathogens across the growing seasons. Co-occurrence network analysis was applied to track the origin and dissemination route of fungal pathogens that infected bell pepper plants. ITS and 16S rRNA sequencing analyses demonstrated that fungal pathogens infecting fruits and leaves probably belonged to the Penicillium, Cladosporium, Fusarium, and unclassified_Sclerotiniaceae genera rather than one specific genus. The dominant fungal pathogens were different, along with the development of bell pepper plants. Both plant development and fungal pathogens shaped microbial communities in bell pepper plants across the growing seasons. Fungal pathogens decreased species richness and diversity of fungal communities in fungus-infected fruit and leaf tissues but not the uninfected stem tissues. Bacterial metabolic functions of xenobiotics increased in fungus-infected leaves at a mature developmental stage. Competitive interaction was present between fungal and bacterial communities in leaves. Co-occurrence network analysis revealed that the origins of fungal pathogens included the greenhouse, packing house, and storage room. Niche differentiation of microbes was discovered among these locations. IMPORTANCE Bell peppers are widely consumed worldwide. Fungal pathogen infections of bell peppers lead to enormous economic loss. To control fungal pathogens and increase economic benefit, it is essential to investigate the shifting patterns of the microbiome and fungal pathogens in bell pepper plants across the growing seasons. In this study, bell pepper plant diseases observed in fruits and leaves were caused by different fungal pathogens. Fungal pathogens originated from the greenhouse, packing house, and storage room, and niche differentiation existed among microbes. This study improves the understanding of dynamic fluctuation and source of fungal pathogens infecting bell pepper plants in the farming system. It also facilitates precise management of fungal pathogens in the greenhouse.

Viable but Nonculturable Escherichia coli O157:H7 and Salmonella enterica in Fresh Produce: Rapid Determination by Loop-Mediated Isothermal Amplification Coupled with a Propidium Monoazide Treatment.

Escherichia coli O157:H7 and Salmonella enterica are leading causes of foodborne outbreaks linked to fresh produce. Both species can enter the "viable but nonculturable" (VBNC) state that precludes detection using conventional culture-based or molecular methods. In this study, we assessed propidium monoazide-quantitative PCR (PMA-qPCR) assays and novel methods combining PMA and loop-mediated isothermal amplification (LAMP) for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. The performance of PMA-LAMP assays targeting the wzy gene of E. coli O157:H7 and the agfA gene of S. enterica and the performance of PMA-qPCR assays were compared in pure culture and spiked tomato, lettuce, and spinach. No cross-reaction was observed in the specificity tests. The values representing the limit of detection (LOD) seen with PMA-LAMP were 9.0 CFU/reaction for E. coli O157:H7 and 4.6 CFU/reaction for S. enterica in pure culture and were 5.13 × 103 or 5.13 × 104 CFU/g for VBNC E. coli O157:H7 and 1.05 × 104 or 1.05 × 105 CFU/g for VBNC S. enterica in fresh produce, representing results comparable to those obtained by PMA-qPCR. Standard curves showed correlation coefficients ranging from 0.925 to 0.996, indicating a good quantitative capacity of PMA-LAMP for determining populations of both bacterial species in the VBNC state. The PMA-LAMP assay was completed with considerable economy of time (30 min versus 1 h) and achieved sensitivity and quantitative capacity comparable to those seen with a PMA-qPCR assay. PMA-LAMP is a rapid, sensitive, and robust method for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce.IMPORTANCE VBNC pathogenic bacteria pose a potential risk to the food industry because they do not multiply on routine microbiological media and thus can evade detection in conventional plating assays. Both E. coli O157:H7 and S. enterica have been reported to enter the VBNC state under a range of environmental stress conditions and to resuscitate under favorable conditions and are a potential cause of human infections. PMA-LAMP methods developed in this study provide a rapid, sensitive, and specific way to determine levels of VBNC E. coli O157:H7 and S. enterica in fresh produce, which potentially decreases the risks related to the consumption of fresh produce contaminated by enteric pathogens in this state. PMA-LAMP can be further applied in the field study to enhance our understanding of the fate of VBNC pathogens in the preharvest and postharvest stages of fresh produce.

Active Packaging of Immobilized Zinc Oxide Nanoparticles Controls Campylobacter jejuni in Raw Chicken Meat.

Zinc oxide nanoparticles (ZnO NPs) are regarded as a safe and stable antimicrobial that can inactivate bacteria by several potential working mechanisms. We aimed to incorporate ZnO NPs into packaging material to control Campylobacter in raw chicken meat. ZnO NPs were first incorporated into three-dimensional (3D) paper tubes to identify the lethal concentration against Campylobacter jejuni, which was selected as the working concentration to develop 2D functionalized absorbing pads by an ultrasound-assisted dipping technique. The functionalized pad was placed underneath raw chicken meat to inactivate C. jejuni and the predominant chicken microbiota at 4°C within 8 days of storage. Immobilized ZnO NPs at 0.856 mg/cm2 reduced C. jejuni from ∼4 log CFU/25 g raw chicken meat to an undetectable level after 3 days of storage. Analysis by inductively coupled plasma-optical emission spectroscopy showed that the Zn level increased from 0.02 to 0.17 mg/cm2 in treated raw chicken meat. Scanning electron microscopy validated the absence of nanoparticle migration onto raw chicken meat after treatment. Inactivation of C. jejuni was associated with the increase of lactic acid produced by Lactobacillus in raw chicken meat in a pH-dependent manner. Less than 5% of Zn2+ was released from ZnO NPs at neutral pH, while up to 88% was released when the pH was <3.5 within 2 days. Whole-transcriptome sequencing (RNA-Seq) analysis demonstrated a broad effect of ZnO NPs on genes involved in various cellular developmental processes as annotated by gene ontology. Taken together, the results indicate that functionalized absorbing pads inactivated C. jejuni in raw chicken meat by immobilized ZnO NPs along with the controllable released Zn2+IMPORTANCE Prevalence of Campylobacter in raw poultry remains a major food microbiological safety challenge. Novel mitigation strategies are required to ensure the safety and quality of poultry products. Active food packaging can control pathogens without directly adding antimicrobials into the food matrix and extend the food's shelf life. The functionalized absorbing pad with ZnO NPs developed in this study was able to inactivate C. jejuni in raw chicken meat and keep the meat free from C. jejuni contamination during shelf life without any observed migration of nanoparticles. The controllable conversion of immobilized ZnO NPs to free Zn2+ makes this approach safe and eco-friendly and paves the way for developing a novel intervention strategy for other high-risk foods. Our study applied nanotechnology to exploit an effective approach for Campylobacter control in raw chicken meat products.

Environmental Stress-Induced Bacterial Lysis and Extracellular DNA Release Contribute to Campylobacter jejuni Biofilm Formation.

Campylobacter jejuni is a microaerophilic bacterium and is believed to persist in a biofilm to antagonize environmental stress. This study investigated the influence of environmental conditions on the formation of C. jejuni biofilm. We report an extracellular DNA (eDNA)-mediated mechanism of biofilm formation in response to aerobic and starvation stress. The eDNA was determined to represent a major form of constitutional material of C. jejuni biofilms and to be closely associated with bacterial lysis. Deletion mutation of the stress response genes spoT and recA enhanced the aerobic influence by stimulating lysis and increasing eDNA release. Flagella were also involved in biofilm formation but mainly contributed to attachment rather than induction of lysis. The addition of genomic DNA from either Campylobacter or Salmonella resulted in a concentration-dependent stimulation effect on biofilm formation, but the effect was not due to forming a precoating DNA layer. Enzymatic degradation of DNA by DNase I disrupted C. jejuni biofilm. In a dual-species biofilm, eDNA allocated Campylobacter and Salmonella at distinct spatial locations that protect Campylobacter from oxygen stress. Our findings demonstrated an essential role and multiple functions of eDNA in biofilm formation of C. jejuni, including facilitating initial attachment, establishing and maintaining biofilm, and allocating bacterial cells.IMPORTANCECampylobacter jejuni is a major cause of foodborne illness worldwide. In the natural environment, the growth of C. jejuni is greatly inhibited by various forms of environmental stress, such as aerobic stress and starvation stress. Biofilm formation can facilitate the distribution of C. jejuni by enabling the survival of this fragile microorganism under unfavorable conditions. However, the mechanism of C. jejuni biofilm formation in response to environmental stress has been investigated only partially. The significance of our research is in identifying extracellular DNA released by bacterial lysis as a major form of constitution material that mediates the formation of C. jejuni biofilm in response to environmental stress, which enhances our understanding of the formation mechanism of C. jejuni biofilm. This knowledge can aid the development of intervention strategies to limit the distribution of C. jejuni.

Significance of oxygen carriers and role of liquid paraffin in improving validamycin A production.

Validamycin A (Val-A) synthesized by Streptomyces hygroscopicus 5008 is widely used as a high-efficient antibiotic to protect plants from sheath blight disease. A novel fermentation strategy was introduced to stimulate Val-A production by adding oxygen carriers. About 58 % increase in Val-A production was achieved using liquid paraffin. Further, biomass, carbon source, metabolic genes, and metabolic enzymes were studied. It was also found that the supplementation of liquid paraffin increased the medium dissolved oxygen and intracellular oxidative stress level. The expression of the global regulators afsR and soxR sensitive to ROS, ugp catalyzing synthesis of Val-A precursor, and Val-A structural genes was enhanced. The change of the activities of glucose-6-phosphate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase was observed, which reflected the redirection of carbon metabolic flux. Based on these results, liquid paraffin addition as an oxygen carrier could be a useful technique in industrial production of Val-A and our study revealed a redox-based secondary metabolic regulation in S. hygroscopicus 5008, which provided a new insight into the regulation of the biosynthesis of secondary metabolites.

Modernization of digital food safety control.

Foodborne illness remains a pressing global issue due to the complexities of modern food supply chains and the vast array of potential contaminants that can arise at every stage of food processing from farm to fork. Traditional food safety control systems are increasingly challenged to identify these intricate hazards. The U.S. Food and Drug Administration's (FDA) New Era of Smarter Food Safety represents a revolutionary shift in food safety methodology by leveraging cutting-edge digital technologies. Digital food safety control systems employ modern solutions to monitor food quality by efficiently detecting in real time a wide range of contaminants across diverse food matrices within a short timeframe. These systems also utilize digital tools for data analysis, providing highly predictive assessments of food safety risks. In addition, digital food safety systems can deliver a secure and reliable food supply chain with comprehensive traceability, safeguarding public health through innovative technological approaches. By utilizing new digital food safety methods, food safety authorities and businesses can establish an efficient regulatory framework that genuinely ensures food safety. These cutting-edge approaches, when applied throughout the food chain, enable the delivery of safe, contaminant-free food products to consumers.

Biofilm formation in food industries: Challenges and control strategies for food safety.

Various pathogens have the ability to grow on food matrices and instruments. This grow may reach to form biofilms. Bacterial biofilms are community of microorganisms embedded in extracellular polymeric substances (EPSs) containing lipids, DNA, proteins, and polysaccharides. These EPSs provide a tolerance and favorable living condition for microorganisms. Biofilm formations could not only contribute a risk for food safety but also have negative impacts on healthcare sector. Once biofilms form, they reveal resistances to traditional detergents and disinfectants, leading to cross-contamination. Inhibition of biofilms formation and abolition of mature biofilms is the main target for controlling of biofilm hazards in the food industry. Some novel eco-friendly technologies such as ultrasound, ultraviolet, cold plasma, magnetic nanoparticles, different chemicals additives as vitamins, D-amino acids, enzymes, antimicrobial peptides, and many other inhibitors provide a significant value on biofilm inhibition. These anti-biofilm agents represent promising tools for food industries and researchers to interfere with different phases of biofilms including adherence, quorum sensing molecules, and cell-to-cell communication. This perspective review highlights the biofilm formation mechanisms, issues associated with biofilms, environmental factors influencing bacterial biofilm development, and recent strategies employed to control biofilm-forming bacteria in the food industry. Further studies are still needed to explore the effects of biofilm regulation in food industries and exploit more regulation strategies for improving the quality and decreasing economic losses.

Stress response of Salmonella Newport with various sequence types toward plasma-activated water: Viable but nonculturable state formation and outer membrane vesicle production.

This study aims to investigate the response of Salmonella Newport to plasma-activated water (PAW), a novel disinfectant that attracts attention due to its broad-spectrum antimicrobial efficacy and eco-friendliness. In this work, we demonstrated that S. Newport of different sequence types (STs) could be induced into the viable but nonculturable (VBNC) state by PAW treatment. Notably, a remarkable 99.96% of S. Newport ST45 strain entered the VBNC state after a 12-min PAW treatment, which was the fastest observed among the five S. Newport STs (ST31, ST45, ST46, ST166, ST2364). Secretion of outer membrane vesicles was observed in ST45, suggesting a potential strategy against PAW treatment. Genes related to oxidative stress (sodA, katE, trxA), outer membrane proteins (ompA, ompC, ompD, ompF) and virulence (pagC, sipC, sopE2) were upregulated in the PAW-treated S. Newport, especially in ST45. A reduction of 38-65% in intracellular ATP level after PAW treatment was observed, indicating a contributor to the formation of the VBNC state. In addition, a rapid method for detecting the proportion of VBNC cells in food products based on pagC was established. This study contributes to understanding the formation mechanism of the VBNC state in S. Newport under PAW stress and offers insights for controlling microbial risks in the food industry.

Whole transcriptome sequencing analysis of synergistic combinations of plant-based antimicrobials and zinc oxide nanoparticles against Campylobacter jejuni.

The emergence of antibiotic resistance among animal farms impels the development of novel antimicrobials or strategies for agri-food production. The combinational use of agents to achieve a synergistic antimicrobial effect provides many advantages such as dosage reduction, shortened treatment time, and avoidance of antimicrobial resistance. In this study, we evaluated the killing efficacy of single agent or combinational use of three antimicrobials, including cinnamon oil, encapsulated curcumin and zinc oxide nanoparticles (ZnO NPs), against a leading foodborne pathogen Campylobacter jejuni. We then investigated the antimicrobial mechanism using whole transcriptome sequencing analysis (RNA-Seq). The single-agent treatment of cinnamon oil, encapsulated curcumin, or ZnO NPs showed a significant antimicrobial effect against C. jejuni by generating more than 8-log reduction within 3 h. The transcriptional signatures of C. jejuni in response to these agents varied, indicating that these agents shared distinct mechanisms of action and were likely to generate synergistic effects. Cinnamon oil affected the integrity of cell membrane, which might lead to an increase in cell permeability. Encapsulated curcumin and ZnO NPs disrupted bacterial outer membranes and cell membranes against the same membrane protein targets. The combinational use of these agents showed synergistic antimicrobial effects and distinct mechanisms of action compared to single treatment. The combination of cinnamon oil and encapsulated curcumin provoked the expression of cellular signaling, but repressed the chemotaxis-associated genes. The antimicrobial resistance associated genes showed a low expression level in the combination of encapsulated curcumin and ZnO NPs. The tri-combination treatment systematically overexpressed genes involved in the amino acid synthesis, protein translation, and membrane protein synthesis. This study provides new insights in combating Campylobacter with minimizing the development of antimicrobial resistance in long-term usage.

Pre-Exposure of Foodborne Staphylococcus aureus Isolates to Organic Acids Induces Cross-Adaptation to Mild Heat.

Staphylococcus aureus is a typical enterotoxin-producing bacterium that causes food poisoning. In the food industry, pasteurization is the most widely used technique for food decontamination. However, pre-exposure to an acidic environment might make bacteria more resistant to heat treatment, which could compromise the bactericidal effect of heat treatment and endanger food safety. In this work, the organic acid-induced cross-adaptation of S. aureus isolates to heat and the associated mechanisms were investigated. Cross-adaptation area analysis indicated that pre-exposure to organic acids induced cross-adaptation of S. aureus to heat in a strain-dependent manner. Compared with other strains, S. aureus strain J15 showed extremely high heat resistance after being stressed by acetic acid, citric acid, and lactic acid. S. aureus strains J19, J9, and J17 were found to be unable to develop cross-adaptation to heat with pre-exposure to acetic acid, citric acid, and lactic acid, respectively. Analysis of the phenotypic characteristics of the cell membrane demonstrated that the acid-heat-cross-adapted strain J15 retained cell membrane integrity and functions through enhanced Na+K+-ATPase and FoF1-ATPase activities. Cell membrane fatty acid analysis revealed that the ratio of anteiso to iso branched-chain fatty acids in the acid-heat-cross-adapted strain J15 decreased and the content of straight-chain fatty acids exhibited a 2.9 to 4.4% increase, contributing to the reduction in membrane fluidity. At the molecular level, fabH was overexpressed with preconditioning by organic acid, and its expression was further enhanced with subsequent heat exposure. Organic acids activated the GroESL system, which participated in the heat shock response of S. aureus to the subsequent heat stress. IMPORTANCE Cross-adaptation is one of the most important phenotypes in foodborne pathogens and poses a potential risk to food safety and human health. In this work, we found that pretreatment with acetic acid, citric acid, and lactic acid could induce subsequent heat tolerance development in S. aureus. Various S. aureus strains exhibited different acid-heat cross-adaptation areas. The acid-induced cross-adaptation to heat might be attributable to membrane integrity maintenance, stabilization of the charge equilibrium to achieve a normal internal pH, and membrane fluidity reduction achieved by decreasing the ratios of anteiso to iso fatty acids. The fabH gene, which is involved in fatty acid biosynthesis, and groES/groEL, which are related to heat shock response, contributed to the development of the acid-heat cross-adaptation phenomenon in S. aureus. The investigations of the stress cross-adaptation phenomenon in foodborne pathogens could help optimize food processing to better control S. aureus.

Extended lag phase indicates the dormancy of biphenyl degrading Rhodococcus biphenylivorans TG9 under heat stress.

Microbial remediation is a green and sustainable technology, but harsh environmental conditions could lead to microbial dormancy, such as entering a viable but non-culturable (VBNC) state. However, the evidence of VBNC is controversial and limited. In this study, heat stress (60 °C), one of the leading challenges for mesophilic degrading bacteria, was mimicked to investigate the physiological response of Rhodococcus biphenylivorans TG9. After 2 h of heat stress, the culturable TG9 cell count decreased from 108 cells/mL to undetectable while the viable cell count was still 105 cells/mL. The biphenyl degradation efficiency of stressed TG9 dropped by 50% compared to that of cells at logarithmic phase. During heat stress, the respiratory activity of TG9 declined dramatically while the intracellular ATP level initially increased and then decreased. Notably, the corresponding indicators recovered when restored to 30 °C. These characteristics were in consistent with bacteria entering into VBNC state. Furthermore, fluorescence activated cell sorting together with single cell as seed culture detection verified the unculturability and viability of VBNC state of TG9 cells. Also, we found that single cells in VBNC state could resuscitate and regrowth with significantly extended lag phase (LP). Our results highlight the potential of TG9 for microbial remediation and hint LP duration as an indicator for survival state of bacteria.

Benzalkonium chloride forces selective evolution of resistance towards antibiotics in Salmonella enterica serovar Typhimurium.

BACKGROUND: Although food-grade disinfectants are extensively used worldwide, it has been reported that the long-term exposure of bacteria to these compounds may represent a selective force inducing evolution including the emergence of antibiotic resistance. However, the mechanism underlying this correlation has not been elucidated. This study aims to investigate the genomic evolution caused by long-term disinfectant exposure in terms of antibiotic resistance in Salmonella enterica Typhimurium. METHODS: S. Typhimurium isolates were exposed to increasing concentrations of benzalkonium chloride (BAC) and variations of their antibiotic susceptibilities were monitored. Strains that survived BAC exposure were analyzed at whole genome perspective using comparative genomics, and Sanger sequencing-confirmed mutations in ramR gene were identified. Next, the efflux activity in ramR-mutated strains shown as bisbenzimide accumulation and expression of genes involved in AcrAB-TolC efflux pump using quantitative reverse transcriptase PCR were determined. RESULTS: Mutation rates of evolved strains varied from 5.82 × 10-9 to 5.56 × 10-8, with fold increase from 18.55 to 1.20 when compared with strains evolved without BAC. Mutations in ramR gene were found in evolved strains. Upregulated expression and increased activity of AcrAB-TolC was observed in evolved strains, which may contribute to their increased resistance to clinically relevant antibiotics. In addition, several indels and point mutations in ramR were identified, including L158P, A37V, G42E, F45L, and R46H which have not yet been linked to antimicrobial resistance. Resistance and mutations were stable after seven consecutive cultivations without BAC exposure. These results suggest that strains with sequence type (ST) ST34 were the most prone to mutations in ramR among the three STs tested (ST34, ST19, ST36). CONCLUSIONS: This work demonstrated that disinfectants, specifically BAC forces S. Typhimurium to enter a specific evolutionary trajectory towards antibiotic resistance illustrating the side effects of long-term exposure to BAC and probably also to other disinfectants. Most significantly, this study provides new insights in understanding the emergence of antibiotic resistance in modern society.

Long-term exposure to food-grade disinfectants causes cross-resistance to antibiotics in Salmonella enterica serovar Typhimurium strains with different antibiograms and sequence types.

BACKGROUND: Disinfectants are important in the food industry to prevent the transmission of pathogens. Excessive use of disinfectants may increase the probability of bacteria experiencing long-term exposure and consequently resistance and cross-resistance to antibiotics. This study aims to investigate the cross-resistance of multidrug-resistant, drug-resistant, and drug-susceptible isolates of Salmonella enterica serovar Typhimurium (S. Typhimurium) with different sequence types (STs) to a group of antibiotics after exposure to different food-grade disinfectants. METHODS: A panel of 27 S. Typhimurium strains with different antibiograms and STs were exposed to increasing concentrations of five food-grade disinfectants, including hydrogen peroxide (H2O2), benzalkonium chloride (BAC), chlorine dioxide (ClO2), sodium hypochlorite (NaClO), and ethanol. Recovered evolved strains were analyzed using genomic tools and phenotypic tests. Genetic mutations were screened using breseq pipeline and changes in resistance to antibiotics and to the same disinfectant were determined. The relative fitness of evolved strains was also determined. RESULTS: Following exposure to disinfectants, 22 out of 135 evolved strains increased their resistance to antibiotics from a group of 14 clinically important antibiotics. The results also showed that 9 out of 135 evolved strains had decreased resistance to some antibiotics. Genetic mutations were found in evolved strains. A total of 77.78% of ST34, 58.33% of ST19, and 66.67% of the other STs strains exhibited changes in antibiotic resistance. BAC was the disinfectant that induced the highest number of strains to cross-resistance to antibiotics. Besides, H2O2 induced the highest number of strains with decreased resistance to antibiotics. CONCLUSIONS: These findings provide a basis for understanding the effect of disinfectants on the antibiotic resistance of S. Typhimurium. This work highlights the link between long-term exposure to disinfectants and the evolution of resistance to antibiotics and provides evidence to promote the regulated use of disinfectants.

Campylobacter biofilms.

Campylobacter infection is one of the most widespread foodborne gastroenteritis worldwide. As a commensal microbe in the intestinal tracts of food-producing animals, Campylobacter easily enters the food chain and eventually transmits to human hosts through the consumption of contaminated food products. The survival of Campylobacter in food chain remains as a paradox considering its fastidious growth requirement in laboratory settings and ubiquitous presence in unfavorable environment. Biofilm is suggested as a key persistence mechanism used by Campylobacter during its transmission from animals to humans. This review summarizes Campylobacter biofilm characteristics, identifies biological and non-biological factors that influence biofilm formation, and discusses the control strategies. Overall, biofilm formation ability shows strain-to-strain variation in Campylobacter and is affected by the presence of other co-cultivated bacteria. Carbohydrates and eDNA are recognized as significant portions of extracellular polymeric substances (EPS) in Campylobacter biofilm. Advanced techniques such as super resolution microscopy and chemical imaging platforms can demonstrate the structure and EPS biochemical compositions. Biofilm formation relies on various biological factors (e.g., flagella, cell surface hydrophobicity, and quorum sensing) and environmental factors (e.g., substrate properties, hydrodynamic condition, and nutrients). Different biological and inorganic compounds can inactivate Campylobacter biofilm along with the feasibility of discovering specific chemicals to quench quorum sensing of this microbe so as to indirectly reduce its biofilm. Taken together, Campylobacter biofilm research is still in its infancy and future research can focus on investigating dormancy state and antimicrobial resistance evolution in biofilms as well as in vivo characterization.

Cyclodextrin metal-organic framework by ultrasound-assisted rapid synthesis for caffeic acid loading and antibacterial application.

Cyclodextrin metal-organic framework by ultrasound-assisted rapid synthesis for caffeic acid (CA) loading and antibacterial application (U-CD-MOF) was successfully studied and this method shortened the preparation time to a few minutes. It was found that the ultrasonic power, reaction time and temperature would affect the morphology and size of the obtained crystal. Under the optimal conditions, U-CD-MOF had a cubic structure with uniform size of 8.60 ± 1.95 µm. U-CD-MOF was used to load the antibacterial natural product CA to form the composite (CA@U-CD-MOF) and the loading rate of CA@U-CD-MOF to CA could reach 19.63 ± 2.53%, which was more than twice that of γ-CD. Various techniques were applied to characterize the synthesized crystal, including Powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and N2 adsorption. In addition, antibacterial tests were performed on the obtained crystal. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of CA@U-CD-MOF for Escherichia coli O157: H7 (E. coli O157: H7) were both 25 mg·mL-1, and the MIC for Staphylococcus aureus (S. aureus). was 25 mg·mL-1. The sustained release behavior of CA@U-CD-MOF to CA in ethanol fitted well to Higuchi model and the loading of CA was supported by molecular docking results. In general, U-CD-MOF was successfully achieved by ultrasound-assisted rapid synthesis and the obtained crystal was further evaluated for potential antibacterial application.

Detection and Quantification of Viable but Non-culturable Campylobacter jejuni.

Campylobacter can enter a viable but non-culturable (VBNC) state to evade various stresses, and this state is undetectable using traditional microbiological culturing techniques. These VBNC bacterial cells retain metabolism and demonstrate pathogenic potential due to their ability to resuscitate under favorable conditions. Rapid and accurate determination of VBNC Campylobacter is critical to further understand the induction and resuscitation of the dormancy state of this microbe in the agri-food system. Here, we integrated propidium monoazide (PMA) with real-time polymerase chain reaction (qPCR) targeting the rpoB gene to detect and quantify Campylobacter jejuni in the VBNC state. First, we optimized the concentration of PMA (20 µM) that could significantly inhibit the amplification of dead cells by qPCR with no significant interference on the amplification of viable cell DNA. PMA-qPCR was highly specific to C. jejuni with a limit of detection (LOD) of 2.43 log CFU/ml in pure bacterial culture. A standard curve for C. jejuni cell concentrations was established with the correlation coefficient of 0.9999 at the linear range of 3.43 to 8.43 log CFU/ml. Induction of C. jejuni into the VBNC state by osmotic stress (i.e., 7% NaCl) was rapid (<48 h) and effective (>10% population). The LOD of PMA-qPCR for VBNC C. jejuni exogenously applied to chicken breasts was 3.12 log CFU/g. In conclusion, PMA-qPCR is a rapid, specific, and sensitive method for the detection and quantification of VBNC C. jejuni in poultry products. This technique can give insight into the prevalence of VBNC Campylobacter in the environment and agri-food production system.

Whole Transcriptome Sequencing Analysis of the Synergistic Antimicrobial Effect of Metal Oxide Nanoparticles and Ajoene on Campylobacter jejuni.

Two metal oxide (i.e., Al2O3 and TiO2) nanoparticles and ajoene, a garlic-derived organosulfur compound, were identified to be effective antimicrobials against Campylobacter jejuni, a leading cause of human gastrointestinal diseases worldwide. A significant synergistic antimicrobial effect was observed using ajoene and Al2O3/TiO2 nanoparticles in a combined manner to cause at least 8 log10 CFU/mL reduction of C. jejuni cells. Whole transcriptome sequencing (RNA-seq) and confocal micro-Raman spectroscopic analyses revealed the antimicrobial mechanism and identified the roles of ajoene and metal oxide nanoparticles in the synergistic treatment. Ajoene and metal oxide nanoparticles mediated a two-phase antimicrobial mechanism. Ajoene served as the inducing factor at the first phase that caused injury of cell membranes and increased the susceptibility of C. jejuni to stress. Metal oxide nanoparticles served as the active factor at the second phase that targeted sensitive cells and physically disrupted cell structure. This synergistic antimicrobial treatment demonstrates a potential to reduce the prevalence of C. jejuni and other pathogens on food contact surfaces and in the food chain.

Effects of meat juice on biofilm formation of Campylobacter and Salmonella.

Campylobacter and Salmonella are leading causes of foodborne illnesses worldwide, vastly harboured by raw meat as their common food reservoir. Both microbes are prevalent in meat processing environments in the form of biofilms that contribute to cross-contamination and foodborne infection. This study applied raw meat juice (chicken juice and pork juice) as a minimally processed food model to study its effects on bacterial biofilm formation. Meat juice was collected during the freeze-thaw process of raw meat and sterilized by filtration. In 96-well polystyrene plates and glass chambers, supplementation of over 25% meat juice (v/v) in laboratory media led to an increase in biofilm formation of Campylobacter and Salmonella. During the initial attachment stage of biofilm development, more bacterial cells were present on surfaces treated with meat juice residues compared to control surfaces. Meat juice particulates on abiotic surfaces facilitated biofilm formation of Campylobacter and Salmonella under both static and flow conditions, with the latter being assessed using a microfluidic platform. Further, the deficiency in biofilm formation of selected Campylobacter and Salmonella mutant strains was restored in the presence of meat juice particulates. These results suggested that meat juice residues on the abiotic surfaces might act as a surface conditioner to support initial attachment and biofilm formation of Campylobacter and Salmonella. This study sheds light on a possible survival mechanism of Campylobacter and Salmonella in meat processing environments, and indicates that thorough cleaning of meat residues during meat production and handling is critical to reduce the bacterial load of Campylobacter and Salmonella.