Ultra-shear technology (UST) is a hybrid thermal-nonthermal, semi-continuous high-pressure food processing method. This study aims to develop and validate a clean-in-place (CIP) procedure for pilot-scale UST equipment. The CIP procedure for UST equipment involves rinsing with water (approximate to 60 degrees C, 14 min), applying an alkaline wash (Alconox, 1% w/v, 43 degrees C, 10 min), followed by another water rinse (approximate to 60 degrees C, 14 min), an acid wash (peracetic acid, 0.25% w/v, 42 degrees C, 10 min), a disinfectant wash (hydrogen peroxide, 0.3% w/v, 39 degrees C, 10 min), and a final water rinse (approximate to 60 degrees C, 14 min). Processing pressure during CIP was 138 MPa. Listeria innocua ATCC33090 cells (6.3 +/- 0.1 log CFU/mL) and Clostridium sporogenes PA3679 spores (4.3 +/- 0.2 log CFU/mL) suspended in a pressure-stable buffer solution were processed with two different pressure-thermal UST conditions to evaluate sanitation efficiency. It was further verified using representative liquid foods (raw milk, dairy-plant protein dispersions, and ice cream mix). Three critical microbial testing points were used for sampling after UST treatments, with or without CIP. Surviving cells and spores were enumerated using suitable agar growth media. Results showed that the tested CIP procedure successfully reduced L. innocua (5.0, 3.7, and 3.1 log CFU/cm2) and C. sporogenes spores (2.5, 1.1, and 0.7 log CFU/cm(2)) populations at different testing locations below the detection limit. The application of the developed sanitation procedures after UST processing of representative food matrices also effectively sanitized the equipment. However, residual food deposits at the bottom of the bladder within the pressure vessel required manual removal, which is a limitation of the current method. These findings will be useful for food processors interested in developing sanitation procedures for industrial-scale UST equipment.

Contamination of milk is a serious public health risk, particularly in developing countries such as Ethiopia. Training is a tool for improving the quality and safety of milk. However, its effect on the microbial quality and safety of milk has not been well documented. This study assessed the impact of training interventions on the microbial quality and safety of milk, with a specific focus on smallholder dairy farms. The study was conducted from January to June 2022 in four locations in Central Ethiopia. Milk samples were collected from 120 dairy farmers one week before and four to six weeks after the training and analyzed for total coliforms, thermotolerant coliforms, Escherichia coli, Shiga toxin-producing E. coli (STEC), Salmonella enterica, and Campylobacter jejuni. Total and thermotolerant coliforms were determined by the Most Probable Number (MPN) method; categorized as high (>= 1,001 MPN/ml), medium (101 103 MPN/ml), low (21-102 MPN/ml), and very low (<= 20 MPN/ml); and analyzed using generalized linear mixed models. Prevalence of E. coli, STEC, S. enterica, and C. jejuni in raw milk samples were 67, 12, 3, and 4% pretraining and 45, 4, 3, and 2% posttraining, respectively. The prevalence of E. coli (p = 0.0389) and STEC (p = 0.0005) was significantly lower posttraining compared to pretraining. Most samples had total coliform counts exceeding 103 MPN/ml pretraining (71%); this proportion decreased to 62% posttraining. The estimated cumulative probability of being in the high category was marginally significantly higher (p = 0.0581) pretraining (76%) compared to posttraining (64%). In conclusion, reductions in the detection of some milk quality and safety parameters were observed. However, a considerable proportion of the milk samples were highly contaminated even after the training. Hence, comprehensive and continued risk mitigation strategies are needed to ensure milk safety for consumers.

Mild food processing could trigger stress response, or even elevated virulence, in the targeted pathogen. Such conditions could be encountered during improper pasteurization of shell eggs. Hence, changes in the virulence of Salmonella enterica serovar Enteritidis, driven by sublethal processing of shell eggs, were investigated. Shell eggs were inoculated with similar to 10(3) CFU Salmonella Enteritidis/egg and incubated to a pathogen's population of similar to 10(9) CFU/egg. These eggs were heated to an internal temperature of 42 degrees C, applied alone or followed by a 30-min ozone exposure (7.5 %, wt./wt. ozone in ozone-oxygen mixture), to mimic egg sub-pasteurization conditions. Levels of Salmonella Enteritidis were standardized in yolk of processed or untreated eggs (3.4-3.7 log CFU/mL) and 200 mu L of the standardized yolk were orally administered in streptomycin-treated C57BL/6 mice. On day one post-infection, mice that received the heat-ozone treated yolk exhibited faster Salmonella colonization compared to those received heated-only or untreated yolk, but on day four, pathogen's fecal populations did not differ significantly (p > 0.05) among the three groups. On day five post-infection, populations of Salmonella Enteritidis were not significantly different (7.7-7.9 log CFU/g; p > 0.05) in ceca of mice of the three treatment groups; however, histological examination revealed considerable elevation of colonic inflammation in mice receiving the processed yolk, relative to the untreated yolk. These findings suggest that sub-pasteurization treatments compromises egg safety by elevating Salmonella virulence and consequently increasing the risk of Salmonella infection. Hence, the results emphasize the need for careful implementation of egg pasteurization methods.

This work explored the use of ohmic heating (OH) for inactivating Alicyclobacillus acidoterrestris spores in apple and cranberry juices in comparison to conventional heating (CH). Experiments were conducted at different field strengths (30, 40, 50 V/cm) and end-point temperatures of 90 degrees C, 100 degrees C, 110 degrees C, with CH trials designed to match OH's heating rates. The spore inactivation kinetics were determined at 100 degrees C, and modeled using loglinear, Weibull, modified Gompertz, and empirical sigmoid models. OH, consistently achieved faster and more complete spore inactivation compared to CH, particularly at 110 degrees C, where OH reduced viable spores below the detection limit (100 CFU/ml) from an initial spore population in the range of 107-108 CFU/ml, even at the lowest field strength tested. A key finding was that regardless of whether the mode of heating is OH or CH, spore inactivation was influenced not only by temperature but also by the rate at which the target temperature is reached-highlighting the importance of heating rate. In addition, the synergy between electric field strength and temperature resulted in further acceleration of lethality. Our work showed that accelerated inactivation may be obtained without a holding period, especially when high field strengths and temperatures are used simultaneously to achieve a final set point, suggesting that A. acidoterrestris may be controlled using this approach, with potential improvements in juice quality.

Driven by the need for pilot-scale aerobiology chambers, we constructed and tested a free-standing 0.5-m(3) cuboid chamber, complete with accessories needed to control experimental variables, measure treatment parameters, and sample treated and untreated air. Parameters affecting bioaerosol longevity were tested; these included target bacterium species and their initial population counts, relative humidity, and the presence of hot spots within the chamber. When 2 mL (10(8)-10(9) CFU/mL) of cell suspensions of tested bacteria were aerosolized, airborne concentration in the chamber averaged 4.3 (+/- 0.28 SD) log(10) CFU/L-air immediately after nebulization, indicating acceptable transferability of cells from the liquid medium into the aerosolized state. A sizable portion of this population retained viability in the chamber; average population decay during 40-min aerosol holding was 0.836 (+/- 0.498 SD) log10 CFU/L-air. The population decay was likely attributed to both biological and physical losses. Aerosols of Staphylococcus aureus ATCC 6583 and Pseudomonas aeruginosa ATCC 10145 were significantly (p < 0.05) more stable than those of Escherichia coli K12 and Klebsiella pneumoniae 556. When compared to the other species, S. aureus produced the most stable aerosols during a 40-minute hold. Longevity of the aerosolized S. aureus increased with increasing initial nebulizer cell concentration and with high chamber relative humidity. Chamber air temperature did not affect bioaerosol longevity, but the introduction of hot spots (>200 degrees C) inside the chamber greatly affected the recovery of viable aerosolized cells. In conclusion, the developed midsize aerobiology chamber was successfully used to investigate bioaerosol stability, and it is potentially useful in studying bioaerosol control agents.

The study was initiated to characterize the degradation of four pesticides, which target different pest categories, by aqueous ozone and to define their degradation kinetics. Pesticide's degradation was quantified using liquid chromatographic-mass spectrometric technique and residual level served as models' dependent variable. Applied ozone dose, measured as concentration-time (Ct, min·mg/L) values, served as the independent variable. Ozone degraded the pesticides at different efficiencies, which were elucidated by data modeling. The first-order reaction kinetic model was inadequate for all tested compounds (R2, 0.1355-0.7413). Degradation kinetics of the fungicide, prochloraz, and the neonicotinoid insecticide, acetamiprid, were best described by the exponential decay model, where the rate constant, k, was 16.1 and 25.1, respectively. Notably, the offset parameter for acetamiprid kinetic model was 0.497, indicating a substantial fraction of the pesticide remaining undegraded even after prolonged exposure to ozone. Degradation of the organophosphorus insecticide, chlorpyrifos-methyl, was best fitted using an exponential-linear combination model, characterized by a dominant exponential decay with a small linear contribution. Degradation of the acaricide, fenbutatin oxide, followed Weibull decay model, indicating an initial lag before the onset of rapid degradation. Ozone Ct values for 50 % pesticides degradation (Ct50), derived from best-fit non-linear kinetic models, were 0.045, 0.601, and 2.79 min·mg/L for prochloraz, chlorpyrifos-methyl, and fenbutatin oxide, respectively. These findings suggest that (i) ozone may be effective against pesticide residue in water and food, (ii) ozone efficacy depends on the structure of the pesticide, and (iii) compound-specific kinetic models for ozone-based pesticide residue removal need to be developed.

Shiga toxin-producing Escherichia coli (STEC), a significant cause of foodborne illnesses, is often associated with the consumption of fresh produce, including alfalfa sprouts. This study was executed to determine how quickly STEC grows, adapts, and colonizes alfalfa sprouts during production and storage, and whether the pathogen's virulence and infectious doses are affected by physiological adaptation to sprouts as an environment. A reporter STEC O157:H7 EDL933 strain was developed to track the transcription of eae, a virulence gene involved in colonizing human intestinal enterocytes. When the seeds were inoculated with 2.1 x 10(3) CFU/g of the reporter strain, the pathogen's population increased to 1.5 x 10(6) CFU/g sprouts within 1.38 days and then remained stable during the remainder of the 5-day sprouting, indicating physiological adaptation to this environment. Seeds were inoculated with similar to 10(8) CFU/g and subsequently treated with 2000 ppm calcium hypochlorite solution, followed by a water-rinse (treated seeds), or just rinsed with water (untreated seeds). After 5 days of sprouting, the resulting fresh sprouts were refrigerated for three days at 4 degrees C. Sprout samples were collected and treated with 2000 ppm calcium hypochlorite solution and rinsed thoroughly with water before counting internalized STEC, or just water-washed before measuring total STEC. The transcription of eae (normalized to cell count) was the highest on the second day of sprouting, but the transcription of other virulence and stress-related genes varied, with sodA being upregulated in STEC cells. Lethal dose 50 (LD50) to Galleria mellonella, a STEC infection animal model, was lower (i.e., virulence was higher) in total STEC collected from fresh sprouts produced from treated seeds, compared to that from untreated seeds (1.9 x 10(0) and 6.0 x 10(1) CFU/larva, respectively). Compared to refrigerated sprouts, the LD50 of STEC from freshly produced sprouts was lower. Based on these findings, it can be concluded that (a) STEC quickly adapts physiologically to sprouts as an environment, (b) transcription of STEC virulence genes changed during sprouts production but generally decreased during refrigeration, and (c) STEC from fresh sprouts grown from sanitizer-treated seeds were more virulent in the animal model, but STEC from refrigerated sprouts were less virulent.

Novel starter cultures are coveted by the dairy industry for enhancing the sensory, quality, and safety attributes of fermented dairy products. In this study, artisanal cheeses were investigated as a source of novel starter cultures. Lactic acid bacteria (LAB) were isolated from the cheese and underwent an antimicrobial activity screening. Six potential antimicrobial producers were selected for whole genome sequencing and were confirmed to be new LAB strains belonging to the genera Lacticaseibacillus, Lactococcus, Leuconostoc, and Enterococcus. Genes associated with carbohydrate utilization, proteolytic enzyme production, and exopolysaccharide synthesis were identified. Additionally, in silico analysis revealed safety-related traits including absence of antibiotic resistance genes, intact prophage regions, and biogenic amine production genes. Genome-guided phenotypic confirmation of important identified traits was completed, which included utilization of multiple carbohydrates, hydrolysis of casein, coagulation of milk, and susceptibility to antibiotic. Among these LAB strains, Lactococcus lactis OSY-92 contained and expressed the necessary genes for a dairy starter culture. The strain displayed anti-Gram-positive antimicrobial activity, a strong preference for lactose and galactose, limited casein hydrolysis, and ability to coagulate milk in 7.5 h. When all these findings were considered, L. lactis OSY-92 qualified as a potential novel and safe starter culture for dairy fermentations.

Current food preservation practice involves a single intensive kill-step followed by isolation of the product within a package: an open-loop control strategy. Alternatively, a closed-loop control strategy, wherein the food is minimally processed as an initial stabilization (first kill-step) but the option for processing later as-needed is available, can improve food safety and quality levels. For this purpose, unpasteurized apple juice (330 mL), packed in sterilized electrode-embedded laminate pouches, was stored at 4 degrees C for 45 days after processed by pasteurization (water-bath; at 71.1 degrees C for 3 s) and moderate processing (ohmic heating; 25 V/cm; 65 degrees C for 30 s) once or with periodic reprocessing, by ohmic heating. Reprocessing was conducted three times (after Day 10, 24, and 42) based on microbial counts, and resulted in microbial counts maintained lower than 2.1 log CFU/mL over about 5 weeks. Results of sensory analysis showed that apple juice could be maintained at an acceptable quality in the selected sensory attributes over 6 weeks of storage by using multiple mild electrothermal processes, instead of one terminal kill-step. This approach enables proactive response to microbial growth during storage and promotes reduction of waste.

Advances in bacteriophage genome sequencing and regulatory approvals of some bacteriophages in various applications have renewed interest in these antibacterial viruses as a potential solution to persistent food safety challenges. Here, we analyzed in depth the genome of the previously studied Escherichia bacteriophage OSYSP (phage OSYSP), revealed its application-related characteristics, and optimized its enumeration techniques for facilitating industrial implementation. We previously sequenced phage OSYSP genome completely by combining results from Illumina Miseq and Ion Torrent sequencing platforms and completing the remaining sequence gaps using PCR. Based on the genomics analysis completed herein, phage OSYSP was confirmed as an obligate lytic phage of the Caudoviricetes class. The genome encodes 81 proteins of identifiable functions, including two endolysins and 45 proteins that support host-independent DNA replication, transcription, and repair. Despite its similarities to T5-like phages, unique genome arrangements confirm phage OSYSP's novelty. The genomic analysis also confirmed the absence of DNA sequences encoding virulence or antibiotic resistance factors. For optimizing phage detection and quantification in the conventional plaque assay, it was observed that decreasing the concentration of agar or agarose, when used as a medium gelling agent, increased phage recovery (p < 0.05), but using agarose resulted in smaller plaque diameters (p < 0.05). Phage OSYSP inactivated pathogenic and non-pathogenic strains of E. coli and some Salmonella enterica serovars, with more pronounced effect against E. coli O157:H7. Phage titers remained fairly unchanged throughout a 24-month storage at 4 degrees C. Incubation for 30 min at 4 degrees C-47 degrees C or pH 4-11 had no significant detrimental effect (p > 0.05) on phage infectivity. In vitro application of phage OSYSP against E. coli O157:H7 EDL933 decreased the pathogen's viable population by >5.7-log CFU/mL within 80 min, at a multiplicity of infection as low as 0.01. The favorable genome characteristics, combined with improved enumeration methodology, and the proven infectivity stability, make phage OSYSP a promising biocontrol agent against pathogenic E. coli for food or therapeutic applications.

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.

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.

The discovery and biotechnological application of new antimicrobial peptides are impeded by a lack of sensitive methods for peptide quantification. Paenibacillin is an emerging antimicrobial lantibiotic that was discovered in Paenibacillus polymyxa OSY-DF ATCC PTA-7852, isolated from the fermented vegetable Kimchee. This lantibiotic has potency against many foodborne pathogenic and spoilage bacteria. To advance the research and application of paenibacillin, a rapid, specific, and sensitive detection and quantification immunoassay was developed. After anti-paenibacillin polyclonal antibodies (pAbs) were generated and purified, a competitive enzyme-linked immunosorbent assay (cELISA) was developed and optimized for paenibacillin quantification. The dynamic range of the cELISA was determined by using a three-parameter nonlinear regression model, achieving a correlation (R2) value of 0.95. The cELISA displayed high sensitivity, with the ability to detect paenibacillin at levels as low as 15.6 ng/mL, which is significantly lower than the limit of detection of the conventional antimicrobial assay (20 µg/mL paenibacillin). The cELISA successfully differentiated paenibacillin concentrations in cell-free crude supernatants of P. polymyxa wild type and its mutant strain when grown at 30 °C and 37 °C; higher paenibacillin levels were found in the mutant (0.248–0.276 µg/mL) than in the wild type (0.122–0.212 µg/mL) culture. These findings were validated by the transcriptional analysis of 11 paenibacillin biosynthetic genes, which were significantly upregulated (≥2-fold increase) in the mutant compared with the wild strain. Additionally, the cELISA exhibited high sensitivity by recovery of paenibacillin titers spiked at 2.5 and 10 µg/mL in de Man, Rogosa, and Sharpe (MRS) broth and diluted skim milk. These results suggest that the anti-paenibacillin pAbs and the developed cELISA could be valuable in quantifying paenibacillin in complex matrices and in aiding the discovery of paenibacillin-producing natural microbiota.

Pressure-assisted thermal processing (PATP) technology is an emerging sterilization method for food processing, ensuring microbiological safety with minimal heat damage. This study investigated the potential antimicrobial efficacy of 25 compounds, including enzymes, polysaccharides, cyclodextrins, cationic surfactants, polymers, and plant and fruit extracts, for the inactivation of Clostridium sporogenes PA 3679 spores during PATP. Experiments were conducted using a laboratory-scale, high-pressure processor. Spores suspended in pressure-stable buffer containing a potential antimicrobial agent were subjected to pressure (600 MPa) at 90 degrees C or 105 degrees C for a holding time of 3- and 6-min. Spore survivors were enumerated by spread-plating on Trypticase-Peptone-Glucose-Yeast Extract (TPGY) agar and incubated anaerobically at 32 degrees C for 5 days. PATP treatment at 600 MPa, 90 degrees C(3-min holding time) and 105 degrees C (3 or 6-min holding time) inactivated the spore by 1.5-, 3.8-, and 5.8-log spores/mL, respectively. Among all the antimicrobials tested, low- and high-molecular-weight chitosan enhanced PATP (600 MPa, 105 degrees C,6-min holding time) inactivation of C. sporogenes spores by 7.9- and 6.9-log reductions, respectively. The treatment also decreased particle size and increased zeta potential of chitosan. Overall, combining PATP with chitosan is a promising synergistic strategy for spore elimination, possibly due to damage to the spore protective layers by PATP followed by chitosan electrostatic interaction.

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.

Non-typhoidal Salmonella (NTS) is a zoonotic pathogen that exerts huge public health and economic impacts in the world. The severity of illness is mainly related to the serovars involved, the presence of virulence genes, and antimicrobial resistance (AMR) patterns. However, data are scarce on serovars, virulence genes, and AMR among NTS identified from the human-dairy interface in Northwest Ethiopia. Thus, this study investigated the serovars, common virulence genes, and AMR patterns of NTS isolates in the area. The study was conducted from June 2022 to August 2023 among randomly selected 58 dairy farms. A total of 362 samples were processed to detect NTS using standard bacteriological methods. The presumptive positive colonies were confirmed by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight (MALDi-ToF). Polymerase chain reaction (PCR) was used to detect virulence genes, including invA and spvC. A slide agglutination test according to the White-Kauffmann-Le Minor scheme was employed to identify the serovars of the NTS isolates. The Kirby-Bauer disk diffusion method was used to assess the antimicrobial susceptibility patterns. Of the processed samples (362), 28 (7.7%) NTS isolates were detected. When distributed among samples, the proportions were 11.9%, 10.5%, 10.3%, 5.2%, 4.3%, and 1.7% among cows’ feces, dairy farm sewage, pooled raw milk, milk container swabs, milkers’ stool, and milkers’ hand swab samples, respectively. Six serovars were detected with the dominancy of S. Uganda (39.3%), followed by S. enterica subsp. diarizonae (25.0%) and S. Typhimurium (21.4%). Among the 28 NTS isolates, 100% and 21.4% had the virulence genes invA and spvC, respectively. The susceptibility profile showed that 89.3% of the NTS isolates were resistant to at least one antimicrobial agent and 46.4% were resistant to three or more classes of antimicrobials (multidrug-resistant). Among antimicrobials, isolates were highly resistant to ampicillin (57.1%), followed by tetracycline (42.9%) and chloramphenicol (35.7%). On the other hand, the NTS isolates were 100%, 96.4%, and 96.4% susceptible to ceftriaxone, azithromycin, and norfloxacin, respectively. In conclusion, we detected NTS from humans, dairy cows, raw milk, dairy utensils, and the environment (sewage), showing the potential of the human-dairy farm-environment nexus in the NTS circulation. These further highlight that the interface is a good point of intervention in the control and prevention of NTS infection. The susceptibility profiles of the isolate necessitate interventions including the prudent use of the antimicrobials.

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.

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.

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 10 5 –10 7 CFU g −1 E. coli O157:H7 B6-914 and treated with Escherichia phage OSYSP spray (10 9 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 ~ 10 5 CFU g −1 , were reduced below the enumeration method’s detection level (i.e., < 10 1 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.

Ultra-shear technology (UST) is a semi-continuous high-pressure method for processing liquid foods. By pressurizing liquid foods up to 400 MPa and decompressing them through a shear valve, UST is designed for pasteurizing or sterilizing foods and modifying their structure and rheological characteristics. This study evaluated the lethal effects of pressure, holding time, temperature, shear, and their interactions for inactivating endospores of Clostridium sporogenes PA3679 suspended in pressure-stable 4-(2-hydroxyethyl)-1-piper-azineethanesulfonic acid (HEPES) buffer solution (0.1 M, pH 7.0, initial concentration = 6.3 log CFU/mL). Results indicated while thermal treatment at 105 degrees C did not have appreciable spore inactivation, isostatic treatment at 400 MPa and 105 degrees C for 5 min resulted in a 3.3-log inactivation. UST treatment at 400 MPa and 85 degrees C for 5 min, followed by 125 degrees C shear discharge resulted in 3.3-log reduction. Our analysis also revealed that thermal-pressure treatment history as well as their intensities also influence magnitude of spore reduction. This study revealed the sporicidal capability of UST and evaluated the relative importance of different lethal factors on spore inactivation.Industrial relevance: Ultra-shear technology (UST) is a novel processing method that has a potential to help produce value-added liquid foods, sauces, gels, and nanoemulsions, while helping to preserve the foods by inactivating undesirable microorganisms using the combination of pressure, shear, and temperature. This research provided an insight on the effects of key UST lethal factors on spores of C. sporogenes PA3679 for commercial sterilization validation. The results of this study could help the industry select the safe-harbor UST processing parameters for sterilizing food products in the future.