Untargeted metabolomics unveiled the role of butanoate metabolism in the development of Pseudomonas aeruginosa hypoxic biofilm.

Pseudomonas aeruginosa is a versatile opportunistic pathogen which causes a variety of acute and chronic human infections, some of which are associated with the biofilm phenotype of the pathogen. We hypothesize that defining the intracellular metabolome of biofilm cells, compared to that of planktonic cells, will elucidate the metabolic pathways and biomarkers indicative of biofilm inception. Disc-shaped stainless-steel coupons (12.7 mm diameter) were employed as a surface for static biofilm establishment. Each disc was immersed in a well, of a 24-well microtiter plate, containing a 1-mL Lysogeny broth (LB) suspension of P. aeruginosa ATCC 9027, a strain known for its biofilm prolificacy. This setup underwent oxygen-depleted incubation at 37°C for 24 hours to yield hypoxic biofilms and the co-existing static planktonic cells. In parallel, another planktonic phenotype of ATCC 9027 was produced in LB under shaking (200 rpm) incubation at 37°C for 24 hours. Planktonic and biofilm cells were harvested, and the intracellular metabolites were subjected to global untargeted metabolomic analysis using LC-MS technology, where small metabolites (below 1.5 kDa) were selected. Data analysis showed the presence of 324 metabolites that differed (p < 0.05) in abundance between planktonic and biofilm cells, whereas 70 metabolites did not vary between these phenotypes (p > 0.05). Correlation, principal components, and partial least square discriminant analyses proved that the biofilm metabolome is distinctly clustered away from that of the two planktonic phenotypes. Based on the functional enrichment analysis, arginine and proline metabolism were enriched in planktonic cells, but butanoate metabolism was enriched in biofilm cells. Key differential metabolites within the butanoate pathway included acetoacetate, 2,3-butandiol, diacetyl, and acetoin, which were highly upregulated in the biofilm compared to the planktonic cells. Exogenous supplementation of acetoin (2 mM), a critical metabolite in butanoate metabolism, augmented biofilm mass, increased the structural integrity and thickness of the biofilm, and maintained the intracellular redox potential by balancing NADH/NAD+ ratio. In conclusion, P. aeruginosa hypoxic biofilm has a specialized metabolic landscape, and butanoate pathway is a metabolic preference and possibly required for promoting planktonic cells to the biofilm state. The butanoate pathway metabolites, particularly acetoin, could serve as markers for biofilm development.

Thermal Inactivation of Escherichia Phage OSYSP and Host Strain Escherichia coli O157:H7 EDL933: A Comparative Kinetic Analysis.

Lytic bacteriophages are promising biocontrol agents against pathogenic bacteria for food and therapeutic applications. Investigating the feasibility of combining phage and physical lethal agents, such as heat, as an effective hurdle combination could lead to beneficial applications. The current research was initiated to compare the thermal inactivation kinetics of a lytic phage (Escherichia phage OSYSP) and its host (Shiga toxin-producing Escherichia coli O157:H7 EDL933), considering they have different critical thermal targets in their structures. To provide a basis for comparison, thermal inactivation kinetics were determined on suspensions of these agents in buffered peptone water using a thermally controlled circulating water bath. Results showed that the bacteriophage virions have a remarkable heat resistance (p < 0.05) compared to their host cells. The D-values of the populations of phage (PFU/mL) and EDL933 strain (CFU/mL) were 166.7 and 7.3 min at 55°C, compared to 44.4 and 0.3 min at 60°C, respectively. Additionally, D-values were significantly (p < 0.05) more influenced by temperature changes in the case of E. coli O157:H7 EDL933 (z-value 3.7°C) compared to that for phage OSYSP (z-value 7.7°C). When the phage suspension was heat-treated in a thermal cycler instead of a water bath, no significant differences between the two treatment procedures (p > 0.05) in estimating virus D- and z-values were observed. Based on these findings, it may be feasible to combine phage OSYSP with mild heat during processing of food to selectively inactivate E. coli O157:H7 EDL933 and subsequently maintain product safety during storage by the surviving phage population; however, the feasibility of this application needs to be investigated. Additionally, the relatively heat-resistant phage OSYSP could qualify as a biological indicator to validate thermal treatments of minimally processed foods in which E. coli O157:H7 EDL933 is the pathogen-of-concern.

Egg-associated Salmonella enterica serovar Enteritidis: comparative genomics unveils phylogenetic links, virulence potential, and antimicrobial resistance traits.

Salmonella enterica serovar Enteritidis (SE) remains a frequent cause of foodborne illnesses associated with the consumption of contaminated hen eggs. Such a food-pathogen association has been demonstrated epidemiologically, but the molecular basis for this association has not been explored. Comparative genomic analysis was implemented to decipher the phylogenomic characteristics, antimicrobial resistance, and virulence potential of eggs-associated SE. Analyzing 1,002 genomes belonging to 841 sequence types of food-isolated SE strains suggests a high genomic similarity within the egg-related lineage, which is phylogenetically close to SE strains isolated from poultry but is different from those isolated from beef. Core genome- and single nucleotide polymorphism (SNP)-based phylogeny of 74 SE strains of egg origin showcased two distinct sublineages. Time-scaled phylogeny supported the possibility of a common ancestor of egg-related SE lineages. Additionally, genome mining revealed frequent antibiotic resistance due to the presence of aac(6')-Iaa and mdsAB encoded on the genomes of egg-associated SE strains. For virulence gene profiling, 103-113 virulence determinants were identified in the egg-associated SE, which were comparable to 112 determinants found in human-associated SE, emphasizing the capacity of egg-associated strains to infect humans and cause diseases. The findings of this study proved the genomic similarity of egg-associated SE strains, and these were closely related to poultry strains. The egg-associated strains also harbor virulence genes equivalent to those found in human-associated SE strains. The analysis provided critical insights into the genetic structure, phylogenomics, dynamics of virulence, and antibiotic resistance of Salmonella Enteritidis, circulating in eggs and emphasizing the necessity of implementing anti-Salmonella intervention strategies, starting at the production stage of the poultry supply chain.

Lytic Escherichia phage OSYSP acts additively and synergistically with gaseous ozone against Escherichia coli O157:H7 on spinach leaves.

Bacteriophage and gaseous ozone are evolving as meritorious alternatives to conventional sanitizers in food postharvest applications. Here, we investigated the efficacy of sequential treatments of a lytic bacteriophage and gaseous ozone, during vacuum cooling of fresh produce, against Escherichia coli O157:H7. Spinach leaves were spot-inoculated with 105-107 CFU g-1 E. coli O157:H7 B6-914 and treated with Escherichia phage OSYSP spray (109 PFU g-1), gaseous ozone, or their combination. Vacuum cooling, which preceded or followed phage application but ran concomitantly with ozone treatment, was performed in a custom-made vessel at the following process sequence: vacuum to 28.5 in. Hg, vessel pressurization to 10 psig with gas containing 1.5 g ozone/kg gas-mix, holding for 30 min, and vessel depressurization to ambient pressure. Bacteriophage or gaseous ozone inactivated E. coli O157:H7, applied at different initial populations on spinach leaves, by 1.7-2.0 or 1.8-3.5 log CFU g-1, respectively. At the high inoculum levels tested (7.1 log CFU g-1), sequential treatments of phage and ozone reduced E. coli O157:H7 population by 4.0 log CFU g-1, but when treatment order was reversed (i.e., ozone followed by bacteriophage), the combination synergistically decreased pathogen's population on spinach leaves by 5.2 log CFU g-1. Regardless the antibacterial application order, E. coli O157:H7 populations, applied initially at ~ 105 CFU g-1, were reduced below the enumeration method's detection level (i.e., < 101 CFU g-1). The study proved that bacteriophage-ozone combination, applied in conjunction with vacuum cooling, is a potent pathogen intervention strategy in fresh produce post-harvest applications.

Combating Bacterial Biofilms: Current and Emerging Antibiofilm Strategies for Treating Persistent Infections.

Biofilms are intricate multicellular structures created by microorganisms on living (biotic) or nonliving (abiotic) surfaces. Medically, biofilms often lead to persistent infections, increased antibiotic resistance, and recurrence of infections. In this review, we highlighted the clinical problem associated with biofilm infections and focused on current and emerging antibiofilm strategies. These strategies are often directed at disrupting quorum sensing, which is crucial for biofilm formation, preventing bacterial adhesion to surfaces, impeding bacterial aggregation in viscous mucus layers, degrading the extracellular polymeric matrix, and developing nanoparticle-based antimicrobial drug complexes which target persistent cells within the biofilm core. It is important to acknowledge, however, that the use of antibiofilm agents faces obstacles, such as limited effectiveness in vivo, potential cytotoxicity to host cells, and propensity to elicit resistance in targeted biofilm-forming microbes. Emerging next generation antibiofilm strategies, which rely on multipronged approaches, were highlighted, and these benefit from current advances in nanotechnology, synthetic biology, and antimicrobial drug discovery. The assessment of current antibiofilm mitigation approaches, as presented here, could guide future initiatives toward innovative antibiofilm therapeutic strategies. Enhancing the efficacy and specificity of some emerging antibiofilm strategies via careful investigations, under conditions that closely mimic biofilm characteristics within the human body, could bridge the gap between laboratory research and practical application.

Removal of Pseudomonas fluorescens biofilms from pilot-scale food processing equipment using ozone-assisted cleaning-in-place.

Biofilm formation in food processing environment and within equipment increases the risk of product spoilage and contamination with pathogens. Cleaning-in-place (CIP) operations are useful in removing soils and in sanitizing processing equipment, including eliminating biofilms. However, CIP is a resource-intensive process, particularly in the usage of chemical detergents, heat, and sanitizers. The current study was initiated to investigate the feasibility of integrating ozone into CIP operations to facilitate the elimination of Pseudomonas biofilm, with the long-term goal of decreasing the dependance on conventional cleaning and sanitizing reagents. To investigate integrating ozone into CIP, a robust biofilm of Pseudomonas fluorescens was developed on a pilot-scale food processing equipment after 2 days of incubation in 10% skim milk (skim milk-water mixture, 1:9 v/v) under stagnant conditions, followed by additional 5 days of circulation while feeding 10% fresh skim milk. CIP was applied using water prerinse at 22-25°C, alkaline cleaning with 0.2% potassium hydroxide at 50°C, and a final water rinse. These CIP operations reduced planktonic cell populations below the detection method's limit but did not fully remove P. fluorescens biofilm from either smooth or rough surfaces of the processing equipment. When the CIP process was followed by application of an aqueous ozone step (10 ppm for 10 min), the treatment reduced biofilm cell population, on smooth and rough surfaces, below the recovery method's detection limit (0.9 and 1.4 log CFU/ 100 cm2, respectively). These findings demonstrate the utility of ozone-assisted CIP in eliminating microbial biofilms on processing equipment, but further research is needed to optimize the use of cleaning agents and the application of ozone.

Inactivation kinetics of selected pathogenic and non-pathogenic bacteria by aqueous ozone to validate minimum usage in purified water.

Ozone is often used as an antimicrobial agent at the final step in purified water processing. When used in purified bottled water manufacturing, residual ozone should not exceed 0.4 mg/L, per US-FDA regulations. These regulations require the control of Escherichia coli and other coliform bacteria; however, non-coliform pathogens can contaminate bottled water. Hence, it is prudent to test the efficacy of ozone against such pathogens to determine if the regulated ozone level adequately ensures the safety of the product. Inactivation of selected pathogenic and non-pathogenic bacteria in purified water was investigated as a function of ozone dose, expressed in Ct units (mg O3*min/L). Bacterial species tested were Enterococcus faecium, E. coli (two serotypes), Listeria monocytogenes (three strains), Pseudomonas aeruginosa, and Salmonella enterica (three serovars). Resulting dose (Ct)-response (reduction in populations' log10 CFU/mL) relationships were mostly linear with obvious heteroscedasticity. This heteroscedastic relationship required developing a novel statistical approach to analyze these data so that the lower bound of the dose-response relationships can be determined and appropriate predictive models for such a bound can be formulated. An example of this analysis was determining the 95%-confidence lower bound equation for the pooled dose-responses of all tested species; the model can be presented as follows: Logpopulationreduction = 3.80Ct + 1.84. Based on this relationship, application ozone at a Ct of 0.832 and 21°C achieves ≥ 5-log reduction in the population of any of the tested pathogenic and non-pathogenic bacteria. This dose can be implemented by applying ozone at 0.832 mg/L for 1 min, 0.416 mg/L for 2 min, or other combinations. The study also proved the suitability of E. faecium ATCC 8459 as a surrogate strain for the pathogens tested in the current study for validating water decontamination processes by ozone. In conclusion, the study findings can be usefully implemented in processing validation of purified water and possibly other water types.

Heating Rate during Shell Egg Thermal Treatment Elicits Stress Responses and Alters Virulence of Salmonella enterica Serovar Enteritidis; Implications for Shell Egg Pasteurization.

Thermal pasteurization of shell eggs, at various time-temperature combinations, has been proposed previously and implemented industrially. This study was conducted to determine if shell egg heating rate, which varies with different pasteurization implementations, alters the Salmonella enterica serovar Enteritidis response to different stresses or expression of virulence. Shell eggs, containing Salmonella Enteritidis in yolk, were subjected to a low (2.4°C/min) or a high (3.5°C/min) heating rate during treatments that mimicked the pasteurization temperature come-up stage. The low heating rate protected Salmonella from the following processes: (i) lethal heat at the holding stage, (ii) loss of viability during 8-h cooling after heating, and (iii) sequential antimicrobial ozone treatment. Transcriptional analysis using Salmonella reporter strains revealed that the heat stress response gene grpE was transcribed at 3-fold-higher levels (P = 0.0009) at the low than at the high heating rate. Slow heating also significantly increased the transcription of the Salmonella virulence-related genes sopB (P = 0.0012) and sseA (P = 0.0006) in comparison to fast heating. Salmonella virulence was determined experimentally as 50% lethal dose (LD50) values in an in vivo model. The slow heat treatment mildly increased Salmonella Enteritidis virulence in mice (LD50 of 3.3 log CFU), compared to that in nontreated yolk (LD50 of 3.9 log CFU). However, when ozone application followed the slow heat treatment, Salmonella virulence decreased (LD50 of 4.2 log CFU) compared to that for heat-treated or nontreated yolk. In conclusion, heating shell eggs at a low rate can trigger hazardous responses that may compromise the safety of the final pasteurized products but following the thermal treatment with ozone application may help alleviate these concerns. IMPORTANCE Pasteurization of shell eggs is an important technology designed to protect consumers against Salmonella Enteritidis that contaminates this commodity. A low heating rate is preferred over a high rate during shell egg thermal pasteurization due to product quality concern. However, it is not known whether raising the temperature at different rates, during pasteurizing, would potentially affect product safety determinants. The current study demonstrated that slow heating during the pasteurization come-up stage increased the following risks: (i) resistance of Salmonella to pasteurization holding stage or to subsequent ozone treatment, (ii) recovery of Salmonella during the cooling that followed pasteurization, and (iii) Salmonella's ability to cause disease (i.e., virulence). Our findings inform food processors about potential safety risks to consumers resulting from improper use of processing parameters during shell egg pasteurization. Additionally, treating shell eggs with ozone after heat treatment could alleviate these hazards and protect consumers from natural Salmonella Enteritidis contaminants in shell eggs.

Maximizing Recovery of Paenibacillin, a Bacterially Produced Lantibiotic, Using Continuous Foam Separation from Bioreactors.

Industrial production of paenibacillin, and similar rare antimicrobial peptides, is hampered by low productivity of the producing microorganisms and lack of efficient methods to recover these peptides from fermentor or bioreactor end products. Preliminary data showed that paenibacillin was preferentially partitioned in foam accumulated during growth of the producer, Paenibacillus polymyxa, in aerated liquid media. This research was initiated to improve the production and recovery of paenibacillin in bioreactors by maximizing partitioning of this antimicrobial agent in the collected foam. This was completed through harvesting foam continuously during paenibacillin production, using modified bioreactor, and optimizing bioreaction conditions through response surface methodology (RSM). During initial screening, the following factors were tested using 400 mL inoculated media in 2 L bioreactors: medium (tryptic soy broth, TSB, with or without added yeast extract), airflow (0 or 0.8 L/min; LPM), stir speed (300 or 500 revolution/min; RPM), incubation temperature (30 or 36 °C), and incubation time (16 or 24 h). Results showed that airflow, time, and stir speed had significant effects (p < 0.05) on paenibacillin recovery in the collected collapsed foam (foamate). These factors were varied together to follow the path of steepest assent to maximize paenibacillin concentration. Once the local maximum was found, RSM was completed with a central composite design to fine-tune the bioreaction parameters. The optimization experiments proved that the significant parameters and their optimal conditions for paenibacillin concentration in the foam were: incubation at 30 °C for 23 h with airflow of 0.95 LPM, and agitation speed of 450 RPM. These conditions increased paenibacillin concentration, predicted by RSM, from 16 µg/mL in bioreaction without foam collection to 743 µg/mL collected in foamate. The optimized conditions also almost doubled the yield of paenibacillin measured in the foam collected from a bioreaction run (12,674 µg/400 mL bioreaction) when compared to that obtained from a run without foam collection (6400 µg/400 mL bioreaction). Results of this study could improve the feasibility of commercial production and downstream processing of paenibacillin and similar novel antimicrobial peptides. Availability of such peptides will eventually help in protecting perishable products against pathogenic and spoilage bacteria.

SARS-CoV-2 and Emerging Foodborne Pathogens: Intriguing Commonalities and Obvious Differences.

The coronavirus disease 2019 (COVID-19) has resulted in tremendous human and economic losses around the globe. The pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a virus that is closely related to SARS-CoV and other human and animal coronaviruses. Although foodborne diseases are rarely of pandemic proportions, some of the causative agents emerge in a manner remarkably similar to what was observed recently with SARS-CoV-2. For example, Shiga toxin-producing Escherichia coli (STEC), the most common cause of hemolytic uremic syndrome, shares evolution, pathogenesis, and immune evasion similarities with SARS-CoV-2. Both agents evolved over time in animal hosts, and during infection, they bind to specific receptors on the host cell's membrane and develop host adaptation mechanisms. Mechanisms such as point mutations and gene loss/genetic acquisition are the main driving forces for the evolution of SARS-CoV-2 and STEC. Both pathogens affect multiple body organs, and the resulting diseases are not completely cured with non-vaccine therapeutics. However, SARS-CoV-2 and STEC obviously differ in the nature of the infectious agent (i.e., virus vs. bacterium), disease epidemiological details (e.g., transmission vehicle and symptoms onset time), and disease severity. SARS-CoV-2 triggered a global pandemic while STEC led to limited, but sometimes serious, disease outbreaks. The current review compares several key aspects of these two pathogenic agents, including the underlying mechanisms of emergence, the driving forces for evolution, pathogenic mechanisms, and the host immune responses. We ask what can be learned from the emergence of both infectious agents in order to alleviate future outbreaks or pandemics.

The Role of Egg Yolk in Modulating the Virulence of Salmonella Enterica Serovar Enteritidis.

Contribution of food vehicles to pathogenicity of disease-causing microorganisms is an important but overlooked research field. The current study was initiated to reveal the relationship between virulence of Salmonella enterica serovar Enteritidis and egg yolk as a hosting medium. Mice were orally challenged with Salmonella Enteritidis cultured in egg yolk or tryptic soy broth (TSB). Additionally, mice were challenged with Salmonella Enteritidis cultured in TSB, followed by administration of sterile egg yolk, to discern the difference between pre-growth of the pathogen and its mere presence in egg yolk during infection. The pathogen's Lethal dose 50 (LD50) was the lowest when grown in yolk (2.8×102 CFU), compared to 1.1×103 CFU in TSB, and 4.6×103 CFU in TSB followed by administration of sterile yolk. Additionally, mice that orally received Salmonella Enteritidis grown in egg yolk expressed a high death rate. These findings were supported by transcriptional analysis results. Expression of promoters of virulence-related genes (sopB and sseA) in genetically modified Salmonella Enteritidis reporter strains was significantly higher (p < 0.05) when the bacterium was grown in the yolk, compared to that grown in TSB. Sequencing of RNA (RNA-seq) revealed 204 differentially transcribed genes in Salmonella Enteritidis grown in yolk vs. TSB. Yolk-grown Salmonella Enteritidis exhibited upregulated virulence pathways, including type III secretion systems, epithelial cell invasion, and infection processes; these observations were confirmed by RT-qPCR results. The transcriptomic analysis suggested that upregulation of virulence machinery of Salmonella Enteritidis grown in egg yolk was related to increased iron uptake, biotin utilization, flagellar biosynthesis, and export of virulence proteins encoded on Salmonella pathogenicity island 1, 2, 4, and 5. These biological responses may have acted in concert to increase the virulence of Salmonella infection in mice. In conclusion, growth in egg yolk enhanced Salmonella Enteritidis virulence, indicating the significance of this food vehicle to the risk assessment of salmonellosis.

Efficient Production of Broad-Spectrum Antimicrobials by Paenibacillus polymyxa OSY-EC Using Acid Whey-Based Medium and Novel Antimicrobial Concentration Approach.

Production of some antimicrobial peptides by bacterial producers is a resource-intensive process, thus, using inexpensive growth media and simplifying antimicrobial extraction and down-stream processing are highly desirable. Acid whey, a dairy industry waste, is explored as a medium for production of broad-spectrum antimicrobials from selected bacteriocinogenic bacteria. Neutralized and yeast extract-supplemented acid whey was suitable for production of antimicrobials by four tested strains, but Paenibacillus polymyxa OSY-EC was the most prolific antimicrobial producer. Concentrating synthesized antimicrobials during culture incubation using beads of polymeric adsorbent resin, followed by solvent extraction and freeze-drying, resulted in antimicrobials-rich powder (AMRP). Under these conditions, P. polymyxa OSY-EC produced paenibacillin, polymyxin E, and fusaricidin, which are active against Gram-positive and Gram-negative bacteria and fungi, respectively. When media containing 2x and 4x minimum inhibitory concentrations of AMRP were inoculated with Listeria innocua and Escherichia coli, microbial populations decreased by ≥4-log CFU ml-1 in tryptic soy broth and ≥3.5-log CFU ml-1 in milk. The antimicrobial mechanism of action of AMRP solutions was attributed to the disruption of cytoplasmic membrane of indicator strains, L. innocua and E. coli. These findings exemplify promising strategies for valorization of acid whey via microbial bioreactions to yield potent antimicrobials.

Detection and characterization of a rare two-component lantibiotic, amyloliquecidin GF610 produced by Bacillus velezensis, using a combination of culture, molecular and bioinformatic analyses.

AIM: To detect and characterize novel lantibiotics from a collection of Bacillus spp. using a multifaceted analytical approach. METHODS AND RESULTS: A previously completed microassay identified 45 Bacillus isolates with anti-Listeria activity. The isolates were PCR screened using degenerate primers targeting conserved sequences in lanM-type lantibiotics. B. velezensis GF610 produced a PCR product whose sequence, along with genome mining and bioinformatics, guided the liquid chromatographic analysis of strain's cell-free extracts and the mass spectrometry of purified fractions. Results revealed a new amyloliquecidin variant (designated GF610) produced by the strain. Amyloliquecidin GF610 is a two-component lantibiotic with α and ß peptides having monoisotopic masses of 3026 and 2451 Da, and molecular formulae C130 H191 N35 O39 S5 and C110 H158 N26 O30 S4 , respectively. Amyloliquecidin GF610 is active against Listeria monocytogenes, Clostridium sporogenes, Clostridioides difficile, Staphylococcus aureus and Alicyclobacillus acidoterrestris with minimum inhibitory concentrations (MICs) in the range of 0.5-7.0 µmol l-1 . CONCLUSIONS: The proposed multifaceted analytical approach was valuable to provide a deep and proper characterization of a novel bacteriocin, amyloliquecidin GF610, with high antimicrobial activity against Gram-positive bacteria. SIGNIFICANCE AND IMPACT: The discovered Amyloliquecidin GF610 is potentially useful in food, agricultural or medical applications. The analytical approach followed may facilitate future discoveries of two-component lantibiotics, which are challenging compounds to detect and characterize.

Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Enteritidis ODA 99-30581-13, a Heat-Resistant Strain Isolated from Shell Eggs.

Salmonella enterica serovar Enteritidis ODA 99-30581-13 is a relatively heat-resistant strain isolated from shell eggs. The strain has a 4,777,965-bp genome sequence (52.1% GC content) that was predicted to encode 4,455 proteins, including heat stress response proteins and stress response regulators; these may be involved in its heat resistance.

Natural Antimicrobials Suitable for Combating Desiccation-Resistant Salmonella enterica in Milk Powder.

Some Salmonella enterica strains survive well in low-water activity (low-aw) foods and cause frequent salmonellosis outbreaks in these products. Methods are needed to overcome such desiccation-resistant Salmonella and to improve the safety of low-aw foods. Building on a recent finding, we hypothesized that natural antimicrobial food additives, which are active against cytoplasmic membrane, could overcome this desiccation resistance phenomenon, and thus, sensitize the pathogen to drying and mild processing. Food additives were screened for the ability to cause leakage of intracellular potassium ions; retention of these ions is vital for protecting Salmonella against desiccation. Two antimicrobial food additives, carvacrol and thymol, caused considerable potassium leakage from the desiccation-resistant S. enterica serovars, Tennessee and Livingstone. Thus, carvacrol and thymol were investigated for their ability to sensitize the desiccation-adapted S. enterica to heat treatment. The combined use of food additives, at their minimum inhibitory concentrations, with heat treatment at 55 °C for 15 min caused 3.1 ± 0.21 to more than 5.5 log colony forming unit (CFU)/mL reduction in desiccation-adapted S. enterica, compared to 2.4 ± 0.53-3.2 ± 0.11 log CFU/mL reduction by sole heat treatment. Carvacrol was the additive that caused the greatest potassium leakage and sensitization of Salmonella to heat; hence, the application of this compound was investigated in a food model against Salmonella Typhimurium ASD200. Addition of carvacrol at 200 or 500 ppm into liquid milk followed by spray-drying reduced the strain's population by 0.9 ± 0.02 and 1.3 ± 0.1 log CFU/g, respectively, compared to 0.6 ± 0.02 log CFU/g reduction for non-treated spray-dried milk. Additionally, freeze-drying of milk treated with high levels of carvacrol (5000 ppm) reduced the population of Salmonella Typhimurium ASD200 by more than 4.5 log CFU/g, compared to 1.1 ± 0.4 log CFU/g reduction for the freeze-dried untreated milk. These findings suggest that carvacrol can combat desiccation-resistant S. enterica, and thus, potentially improve the safety of low-aw foods.

Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Livingstone 1236H, a Desiccation-Resistant Strain That Poses a Salmonellosis Hazard in Low-Moisture Foods.

Salmonella enterica serovar Livingstone 1236H was isolated originally from peanut butter and represents a health risk in low-moisture foods. The current work presents the strain's genome sequencing results, which show a 4,824,729-bp genome sequence and 4,435 protein coding sequences, including some that are involved in adaptation to low-moisture environments.

Carvacrol and Thymol Combat Desiccation Resistance Mechanisms in Salmonella enterica Serovar Tennessee.

Some Salmonella enterica serovars are frequently associated with disease outbreaks in low-moisture foods (LMF) due to their ability to adapt efficiently to desiccation stress. These serovars are often persistent during food processing. Disruption of these resistance responses was accomplished previously using the membrane-active lipopeptide, paenibacterin. This study was initiated to determine how desiccation resistance mechanisms are overcome when Salmonella Tennessee, a known resistant serovar, is treated with the membrane-active food additives carvacrol and thymol. Knowing that the minimum inhibitory concentrations (MICs) of carvacrol and thymol against Salmonella Tennessee are 200 and 100 µg/mL, the concentrations tested were 100-400 and 50-200 µg/mL, respectively. Results show that desiccation-adapted Salmonella Tennessee, prepared by air drying at 40% relative humidity and 22-25 °C for 24 h, was not inactivated when exposed for 4.0 h to less than 2xMIC of the two additives. Additionally, treatment of desiccation-adapted Salmonella Tennessee for 120 min with carvacrol and thymol at the MIC-level sensitized the cells (1.4-1.5 log CFU/mL reduction) to further desiccation stress. Treating desiccation-adapted Salmonella Tennessee with carvacrol and thymol induced leakage of intracellular potassium ions, reduced the biosynthesis of the osmoprotectant trehalose, reduced respiratory activity, decreased ATP production, and caused leakage of intracellular proteins and nucleic acids. Carvacrol, at 200-400 µg/mL, significantly downregulated the transcription of desiccation-related genes (proV, STM1494, and kdpA) as determined by the reverse-transcription quantitative PCR. The current study revealed some of the mechanisms by which carvacrol and thymol combat desiccation-resistant Salmonella Tennessee, raising the feasibility of using these additives to control desiccation-adapted S. enterica in LMF.

Assessment of Safety and Probiotic Traits of Enterococcus durans OSY-EGY, Isolated From Egyptian Artisanal Cheese, Using Comparative Genomics and Phenotypic Analyses.

An Enterococcus durans strain, designated OSY-EGY, was previously isolated from artisanal cheese. In this work, comparative genomic and phenotypic analyses were utilized to assess the safety characteristics and probiotic traits of the bacterium. The comparative genomic analysis revealed that the strain is distantly related to potentially pathogenic Enterococcus spp. The genome was devoid of genes encoding acquired antibiotic resistance or marker virulence factors associated with Enterococcus spp. Phenotypically, the bacterium is susceptible to vancomycin, ampicillin, tetracycline, chloramphenicol, and aminoglycosides and does not have any hemolytic or gelatinase activity, or cytotoxic effect on Caco-2 cells. Altogether, these findings confirm the lack of hazardous traits in E. durans OSY-EGY. Mining E. durans OSY-EGY genome, for probiotic-related sequences, revealed genes associated with acid and bile salts tolerance, adhesion, competitiveness, antioxidant activitiy, antimicrobial activity, essential amino acids production, and vitamins biosynthesis. Phenotypically, E. durans OSY-EGY was tolerant to acidic pH (3.0), and presence of 0.3% bile salts. The bacterium showed adhesion capability to Caco-2 cells, cholesterol-lowering effect, DPPH scavenging activity, and antimicrobial activity against several Gram-positive pathogenic bacteria. Based on the current work, we propose that E. durans OSY-EGY is a potentially safe strain with desirable probiotic and antimicrobial traits. Thus, the investigated strain could be a promising candidate for several industrial applications.

Draft Genome Sequence of Lactobacillus rhamnosus OSU-PECh-69, a Cheese Isolate with Antibacterial Activity.

The novel strain Lactobacillus rhamnosus OSU-PECh-69 was isolated from provolone cheese. It produces antimicrobial agents having a molecular mass of 5 to 10 kDa that are active against Gram-positive and Gram-negative bacteria. The strain has a genome sequence of 3,057,669 bp, a GC content of 46.6%, and up to two gene clusters encoding bacteriocins.

Genome-Guided Mass Spectrometry Expedited the Discovery of Paraplantaricin TC318, a Lantibiotic Produced by Lactobacillus paraplantarum Strain Isolated From Cheese.

The quest for potent alternatives to the currently used antimicrobials is urged by health professionals, considering the rapid rise in resistance to preservatives and antibiotics among pathogens. The current study was initiated to search for novel and effective bacteriocins from food microbes, preferably lactic acid bacteria (LAB), for potential use as preservatives. Advances in genome-guided mass spectrometry (MS) were implemented to expedite identifying and elucidating the structure of the recovered antimicrobial agent. A LAB strain, OSY-TC318, was isolated from a Turkish cheese, and the crude extract of the cultured strain inhibited the growth of various pathogenic and spoilage bacteria such as Bacillus cereus, Clostridium sporogenes, Enterococcus faecalis, Listeria monocytogenes, Salmonella enterica ser. Typhimurium, and Staphylococcus aureus. The antimicrobial producer was identified as Lactobacillus paraplantarum using MS biotyping and genomic analysis. Additionally, L. paraplantarum OSY-TC318 was distinguished from closely related strains using comparative genomic analysis. Based on in silico analysis, the genome of the new strain contained a complete lantibiotic biosynthetic gene cluster, encoding a novel lantibiotic that was designated as paraplantaricin TC318. The bioinformatic analysis of the gene cluster led to the prediction of the biosynthetic pathway, amino acid sequence, and theoretical molecular mass of paraplantaricin TC318. To verify the genomic analysis predictions, paraplantaricin TC318 was purified from the producer cellular crude extract using liquid chromatography, followed by structural elucidation using Fourier transform ion cyclotron resonance MS analysis. This genome-guided MS analysis revealed that the molecular mass of paraplantaricin TC318 is 2,263.900 Da, its chemical formula is C106H133N27O22S4, and its primary sequence is F-K-S-W-S-L-C-T-F-G-C-G-H-T-G-S-F-N-S-F-C-C. This lantibiotic, which differs from mutacin 1140 at positions 9, 12, 13, and 20, is considered a new member of the epidermin group in class I lantibiotics. In conclusion, the study revealed a new L. paraplantarum strain producing a novel lantibiotic that is potentially useful in food and medical applications.