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.

Comparative genomics of the gut commensal Bifidobacterium bifidum reveals adaptation to carbohydrate utilization.

Bifidobacterium bifidum is one of the most abundant members of the gut microbiota at the early stage of life. The established association of the bacterium with the human gut confers health benefits. Such successful persistence of B. bifidum necessitates metabolic adaptation to the host-derived carbohydrates, a process which is poorly understood. The current study focuses on revealing the genomic-based phylogeny (phylogenomics) of B. bifidum and utilizing comparative genomics to decipher the glycolytic abilities of bifidobacterial strains isolated from different human body niches (feces, human gut, vagina, and breast milk). When the phylogenomic analysis was performed on 95 B. bifidum strains, currently available on the RefSeq database, the bacterium was clearly distinguished from other members of the Bifidobacterium genus. Furthermore, a pairwise genomic comparison indicated that a large proportion of orthologous gene families were shared among the B. bifidum strains. These findings highlight the notion that the B. bifidum species is genetically similar and may perform similar functions in their host. When 15 B. bifidum genomes representing strains from different human body niches were annotated, the resulting functional profile showed the presence of enriched proteins involved in carbohydrate utilization. Moreover, mining the 15 B. bifidum genomes for the presence of Carbohydrate-Active Enzyme (CAZY) systems, the analysis found the existence of diverse protein families which include glycosyl hydrolases, glycosyl transferases, carbohydrate-binding modules, and carbohydrate esterases. Collectively, these CAZY systems enables B. bifidum to utilize host-derived glycans (e.g., mucin) and diet-derived carbohydrates (e.g., starch). In contrast, a correlation analysis revealed that B. bifidum strains isolated from the different body niches were indistinguishable in the context of presence-absence of CAZY systems. These findings emphasize the valuable use of comparative genomics in deciphering the glycolytic abilities of B. bifidum and consequently its adaptation to carbohydrate utilization in the human gut environment.

Development and greenness assessment of a stability-indicating HPLC-DAD method for simultaneous determination of allopurinol and benzbromarone.

The growing interest in Green Analytical Chemistry (GAC) principles through the replacement of polluting analytical procedures with greener ones, has encouraged us to develop an eco-friendly stability-indicating HPLC with diode array detection method (HPLC-DAD) for simultaneous determination of allopurinol (ALP) and benzbromarone (BNZ). Effective separation was accomplished using Durashell C18 column (4.6 × 250 mm, 5 µm particle size) with gradient elution of the mobile phase composed of 0.02 M ammonium acetate (pH 5.0) and methanol. Quantification of ALP and BNZ was based on measuring their peak areas at 251 nm. ALP and BNZ peaks eluted at retention times 4.85 and 10.30 min respectively. The proposed HPLC procedure was carefully validated in terms of system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection, and quantification limits. The linearity range for both ALP and BNZ was 5-100 µg/mL with correlation coefficients >0.9999. Forced degradation conditions of neutral, acidic, and alkaline hydrolysis, oxidation, and thermal degradation were applied on both drugs. Good resolution of the drugs from their forced degradation products proved that the proposed method is stability-indicating. In addition, the resolution of both drugs from about 10 pharmacologically or chemically related pharmaceutical compounds of different medicinal categories showed the high specificity of the proposed method. The validated HPLC method was successfully applied to the simultaneous determination of both drugs in their tablet dosage forms. Furthermore, greenness assessment and comparison with previously published methods were carried out using two different GAC metrics, namely, the national environmental method index (NEMI) and the analytical Eco-Scale.

Analytical investigation of ternary mixture of phenylephrine hydrochloride, dimetindene maleate and benzalkonium chloride using validated stability indicating HPLC-DAD method.

A stability-indicating high performance liquid chromatography method with diode array detection (HPLC-DAD) was developed and validated for simultaneous determination of phenylephrine hydrochloride (PHR), dimetindene maleate (DMD) and benzalkonium chloride (BZM) in nasal drops and gel dosage forms. Effective liquid chromatographic separation was accomplished by employing Venusil XBP Cyano column (4.6 × 250 mm, 5 µm particle size) with gradient elution of the mobile phase consisting of buffer solution of potassium dihydrogen phosphate (0.025 M) and sodium 1-butane sulfonate (SBS) (0.025 M) (adjusted to pH 6.0) and acetonitrile. Peak areas of PHR, DMD and BZM at 271, 256 and 206 nm, respectively were measured and correlated to their concentrations. Peaks of PHR and DMD eluted at retention times 3.76 and 9.06 min, respectively, while BZM eluted as a couple of peaks at 11.88 and 12.51 min. The proposed HPLC procedure was carefully validated in terms of system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection and quantification limits. The linearity range for both PHR and BZM was 10-400 µg/mL and DMD was 5-300 µg/mL with correlation coefficients >0.9999. The studied compounds were subjected to stress conditions of neutral, acidic and alkaline hydrolysis, oxidation and thermal degradation. Good resolution of the three compounds from their forced degradation products proves specificity and stability-indicating merits of the proposed method. In addition, resolution of the three drugs under investigation from some pharmaceutical compounds of different medicinal categories showed the high specificity of the described method.

Antibacterial effects of antibiotics and cell-free preparations of probiotics against Staphylococcus aureus and Staphylococcus epidermidis associated with conjunctivitis.

Conjunctivitis, caused by bacterial infections, represents health concern and diagnosis of the disease is pivotal for the proper selection of the treatment. The main causes of bacterial conjunctivitis vary in different countries. The current study investigated the common bacterial causes of bacterial conjunctivitis from eye clinics' attendants and evaluated the effectiveness of different therapeutic approaches. Eye swabs from patients, diagnosed with conjunctivitis, were assessed microbiologically and the isolated bacteria were identified using the standard biochemical identification and sequencing of the 16S rRNA gene. Antibiotics' susceptibility of the conjunctivitis-associated bacterial pathogens was evaluated against nineteen broad-spectrum antibiotics. In the meanwhile, cell-free preparations from probiotic Lactobacillus and Bifidobacterium strains were used to evaluate their antagonistic activities. Findings from this study showed that out of 52 specimen, 17 eye swabs from patients with conjunctivitis were bacterial culture-positive. The identity of the bacterial species, using the biochemical identification system, was Staphylococcus aureus (4 isolates) and S. epidermidis (13 isolates). Staphylococcus spp. showed susceptibility to linezolid, vancomycin, novobiocin, and fluoroquinolones (norfloxacin, ofloxacin, ciprofloxacin and levofloxacin). However, isolates from the two Staphylococcus spp. expressed resistance to penicillin G, oxacillin, and cephalexin. As alternatives to antibiotics, the growth of Staphylococcus spp., including isolates with antibiotic resistance, was inhibited by cell-free preparations of the 4 probiotic Lactobacillus and the 2 Bifidobacterium strains. These findings provide evidence that topical antibiotics such as fluoroquinolones are still effective antimicrobial agents against staphylococci associated with conjunctivitis whereas probiotic preparations could be promising for further research to pave the way for their therapeutic applications against ophthalmic diseases.

The Microbial Lipopeptide Paenibacterin Disrupts Desiccation Resistance in Salmonella enterica Serovars Tennessee and Eimsbuettel.

Salmonella enterica is increasingly linked to disease outbreaks associated with consumption of low-water-activity (low-aw) foods. Persistence of the pathogen in these foods was attributed to its ability to implement desiccation resistance mechanisms. Published knowledge about methods that disrupt desiccation resistance in S. enterica is lacking. We hypothesize that strong membrane-active compounds disrupt the desiccation resistance that S. enterica may acquire in low-aw foods or environments. The newly discovered antimicrobial lipopeptide paenibacterin was the membrane-active agent investigated in this study. Strains of S. enterica serovars Tennessee and Eimsbuettel, with a history of association with low-moisture foods, were investigated. The viability of these strains did not decrease significantly during dehydration and subsequent storage in the dehydrated state. Considering that the paenibacterin MIC against S. enterica strains was 8 µg/ml, concentrations of 4 to 16 µg/ml paenibacterin were tested. Within this range, desiccation-adapted S. Eimsbuettel was much more tolerant to the antimicrobial agent than the desiccation-adapted S. Tennessee. Pretreatment with 8 µg/ml paenibacterin increased inactivation of S. enterica during desiccation. The use of paenibacterin at 16 µg/ml or higher concentrations resulted in leakage of intracellular potassium ions from desiccation-adapted cells. Paenibacterin significantly decreased the biosynthesis of the intracellular osmoprotectant solute, trehalose, in a concentration-dependent manner. Treatment with 64 µg/ml paenibacterin increased the permeability of the cytoplasmic membranes of desiccation-adapted cells. Transcription of the desiccation-related genes proV, STM1494, kdpA, and otsB in response to paenibacterin treatment was investigated using reverse transcription-quantitative PCR. Transcription of some of these genes was downregulated in a concentration- and strain-dependent manner.IMPORTANCESalmonella enterica adapts effectively and persists for a long time in low-aw foods or environments through resistance mechanisms to desiccation stress. Desiccation-resistant cells compromise food safety and constitute a serious health hazard. Strategies to combat desiccation resistance in S. enterica are needed to sensitize the pathogen to lethal processes used in food preservation. The study proved that the membrane-active lipopeptide paenibacterin disrupts the resistance in desiccation-adapted S. enterica, as measured by phenotypic, biochemical, and genetic analyses. This study highlighted the role of the lipopeptide paenibacterin in disrupting mechanisms employed by S. enterica to resist desiccation. This knowledge may lead to the design of novel control measures to improve the safety of low-aw foods.

Cell free preparations of probiotics exerted antibacterial and antibiofilm activities against multidrug resistant E. coli.

The sharp increase in antibiotic resistance imposes a global threat to human health and the discovery of effective antimicrobial alternatives is needed. The use of probiotics to combat bacterial pathogens has gained a rising interest. Pathogenic Escherichia coli is causative of multiple clinical syndromes such as diarrheal diseases, meningitis and urinary tract infections. In this work, we evaluated the efficacy of probiotics to control multidrug-resistant E. coli and reduce their ability to form biofilms. Six E. coli resistant to at least five antibiotics (Ceftazidime, Ampicillin, Clarithromycin, Amoxicillin + Clavulanic Acid and Ceftriaxone) were isolated in this work. Preparations of cell-free spent media (CFSM) of six probiotics belonging to the genus Bifidobacterium and Lactobacillus which were grown in Man-Rogosa-Sharpe (MRS) broth exhibited strong antibacterial activity (inhibition zones of 11.77-23.10 mm) against all E. coli isolates. Two E. coli isolates, namely E. coli WW1 and IC2, which were most resistant to all antibiotics were subjected to antibiofilm experiments. Interestingly, the CFSM of MRS fermented by all probiotics resulted in inhibition of biofilm formation while B. longum caused highest inhibition (57.94%) in case of E. coli IC2 biofilms and L. plantarum was responsible for 64.57% reduction of E. coli WW1 biofilms. On the other hand, CFSM of skim milk fermented by L. helveticus and L. rhamnosus exhibited a slight inhibitory activity against IC2 isolate (inhibition percentage of 31.52 and 17. 68, respectively) while WW1 isolate biofilms was reduced by CFSM of milk fermented by B. longum and L. helveticus (70.81 and 69.49 reduction percentage, respectively). These results support the effective use of probiotics as antimicrobial alternatives and to eradicate biofilms formed by multidrug-resistant E. coli.

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.

Genome sequences of six lactic acid bacteria from artisanal cheese reveal biosynthetic gene clusters encoding antimicrobial compounds.

Lactic acid bacteria are valuable in the production of fermented foods and as sources of antimicrobial peptides (e.g., bacteriocins). The genomes of six lactic acid bacteria, isolated from artisanal cheeses, having biosynthetic gene clusters encoding antimicrobial compounds are reported. The six strains belong to the genera Lacticaseibacillus, Lactococcus, Leuconostoc, and Enterococcus.

Multidrug-resistance and extended-spectrum beta-lactamase-producing lactose-fermenting enterobacteriaceae in the human-dairy interface in northwest Ethiopia.

BACKGROUND: Antimicrobial resistance (AMR) is among the top public health concerns in the globe. Estimating the prevalence of multidrug resistance (MDR), MDR index (MDR-I) and extended-spectrum beta-lactamase (ESBL)-producing lactose fermenting Enterobacteriaceae (LFE) is important in designing strategies to combat AMR. Thus, this study was designed to determine the status of MDR, MDR-I and ESBL-producing LFE isolated from the human-dairy interface in the northwestern part of Ethiopia, where such information is lacking. METHODOLOGY: A cross-sectional study was conducted from June 2022 to August 2023 by analyzing 362 samples consisting of raw pooled milk (58), milk container swabs (58), milker's hand swabs (58), farm sewage (57), milker's stool (47), and cow's feces (84). The samples were analyzed using standard bacteriological methods. The antimicrobial susceptibility patterns and ESBL production ability of the LFE isolates were screened using the Kirby-Bauer disk diffusion method, and candidate isolates passing the screening criteria were phenotypically confirmed by using cefotaxime (30 µg) and cefotaxime /clavulanic acid (30 µg/10 µg) combined-disk diffusion test. The isolates were further characterized genotypically using multiplex polymerase chain reaction targeting the three ESBL-encoding- genes namely blaTEM, blaSHV, and blaCTX-M. RESULTS: A total of 375 bacterial isolates were identified and the proportion of MDR and ESBL-producing bacterial isolates were 70.7 and 21.3%, respectively. The MDR-I varied from 0.0 to 0.81 with an average of 0.30. The ESBL production was detected in all sample types. Genotypically, the majority of the isolates (97.5%), which were positive on the phenotypic test, were carrying one or more of the three genes. CONCLUSION: A high proportion of the bacterial isolates were MDR; had high MDR-I and were positive for ESBL production. The findings provide evidence that the human-dairy interface is one of the important reservoirs of AMR traits. Therefore, the implementation of AMR mitigation strategies is highly needed in the area.

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.

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.

Forced Degradation and Stability-Indicating Study for the Binary Mixture of Allopurinol and Thioctic Acid Using Validated HPLC-DAD Method.

BACKGROUND: Careful review of the scientific databases revealed that no stability-indicating analytical method is available for the binary mixture of allopurinol (ALO) and thioctic acid (THA). OBJECTIVE: A comprehensive stability-indicating HPLC-DAD procedure has been executed for concurrent analysis of ALO and THA. METHOD: Successful chromatographic separation of the cited drugs was reached using a Durashell C18 column (4.6 × 250 mm, 5 µm particle size). The mobile phase consisted of a mixture of acidified water (pH 4.0) using phosphoric acid and acetonitrile pumped in gradient elution mode. For quantification of ALO and THA, their respective peak areas were recorded at 249 and 210 nm. A systematic validation of analytical performance was investigated in terms of system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection, and quantification limits. RESULTS: ALO and THA peaks emerged at retention times 4.26 and 8.15 min, respectively. Linear ranges for ALO and THA were 5-100 and 10-400 µg/mL, respectively, with correlation coefficient values exceeding 0.9999. Both drugs were exposed to conditions of neutral, acidic, and alkaline hydrolysis, oxidation, and thermal decomposition. Stability-indicating features have been demonstrated by resolution of the drugs from their forced degradation peaks. For verification of peak identity and purity, the diode-array detector (DAD) was used. In addition, degradation pathways for the cited drugs were postulated. Furthermore, separation of both analytes from about 13 medicinal compounds of different therapeutic classes disclosed optimum specificity of the proposed method. CONCLUSIONS: Advantageous application of the validated HPLC method for the concurrent analysis of ALO/THA in their tablet dosage form was accomplished. HIGHLIGHTS: So far, the described HPLC-DAD method is considered the first detailed stability-indicating analytical study for this pharmaceutical mixture.

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.

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.

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.

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.