Chemical mutagenesis of Listeria monocytogenes for increased tolerance to benzalkonium chloride shows independent genetic underpinnings and off-target antibiotic resistance.

Listeria monocytogenes, a potentially fatal foodborne pathogen commonly found in food processing facilities, creates a significant economic burden that totals more than $2 billion annually in the United States due to outbreaks. Quaternary ammonium compounds (QACs), including benzalkonium chloride (BAC), are among the most widely used sanitizers to inhibit the growth and spread of L. monocytogenes from food processing facilities. However, resistance to QACs has been increasing in L. monocytogenes and different genetic mechanisms conferring resistance have been discovered. Here, we used ethyl methanesulfonate (EMS) to chemically mutagenize the BAC-susceptible strain, L. monocytogenes FSL-N1-304. We isolated two mutants with increased tolerance to BAC compared to the parental strain. Next, we assessed the off-target effect of increased tolerance to BAC by measuring the minimum inhibitory concentrations (MICs) of a diverse set of antibiotics, revealing that mut-1 and mut-2 displayed significantly increased resistance to fluoroquinolone antibiotics compared to the parental strain. A hemolysis assay was then used to investigate a potential correlation between BAC tolerance and virulence. Interestingly, mut-1 and mut-2 both exhibited significantly higher hemolysis percentage than the parental strain. We then sequenced the genomes of the parental strain and both mutants to identify mutations that may be involved in the increased resistance to BAC. We identified 3 and 29 mutations in mut-1 and mut-2, respectively. mut-1 contained nonsynonymous mutations in dagK (a diacylglycerol kinase), lmo2768 (a permease-encoding gene), and lmo0186 (resuscitation promoting factor). mut-2 contained a nonsense mutation in the nucleotide excision repair enzyme UvrABC system protein B encoding gene, uvrB, which likely accounts for the higher number of mutations observed. Transcriptome analysis in the presence of BAC revealed that genes related to the phosphotransferase system and internalins were up-regulated in both mutants, suggesting their significance in the BAC stress response. These two mutants provide insights into alternative mechanisms for increased BAC tolerance and could further our understanding of how L. monocytogenes persists in the food processing environment.

Transcriptomic analysis using RNA sequencing and phenotypic analysis of Salmonella enterica after acid exposure for different time durations using adaptive laboratory evolution.

Introduction: This study is the final part of a two-part series that delves into the molecular mechanisms driving adaptive laboratory evolution (ALE) of Salmonella enterica in acid stress. The phenotypic and transcriptomic alterations in the acid-evolved lineages (EL) of Salmonella enterica serovar Enteritidis after 70 days of acid stress exposure were analyzed. Materials and methods: The stability of phenotypic changes observed after 70 days in acetic acid was explored after stress removal using a newly developed evolutionary lineage EL5. Additionally, the impact of short-term acid stress on the previously adapted lineage EL4 was also examined. Results: The results indicate that the elevated antibiotic minimum inhibitory concentration (MIC) observed after exposure to acetic acid for 70 days was lost when acid stress was removed. This phenomenon was observed against human antibiotics such as meropenem, ciprofloxacin, gentamicin, and streptomycin. The MIC of meropenem in EL4 on day 70 was 0.094 mM, which dropped to 0.032 mM when removed from acetic acid stress after day 70. However, after stress reintroduction, the MIC swiftly elevated, and within 4 days, it returned to 0.094 mM. After 20 more days of adaptation in acetic acid, the meropenem MIC increased to 0.125 mM. The other human antibiotics that were tested exhibited a similar trend. The MIC of acetic acid in EL4 on day 70 was observed to be 35 mM, which remained constant even after the removal of acetic acid stress. Readaptation of EL4 in acetic acid for 20 more days caused the acetic acid MIC to increase to 37 mM. Bacterial whole genome sequencing of EL5 revealed base substitutions in several genes involved in pathogenesis, such as the phoQ and wzc genes. Transcriptomic analysis of EL5 revealed upregulation of virulence, drug resistance, toxin-antitoxin, and iron metabolism genes. Unstable Salmonella small colony variants (SSCV) of S. Enteritidis were also observed in EL5 as compared to the wild-type unevolved S. Enteritidis. Discussion: This study presents a comprehensive understanding of the evolution of the phenotypic, genomic, and transcriptomic changes in S. Enteritidis due to prolonged acid exposure through ALE.

Adaptive laboratory evolution of Salmonella enterica in acid stress.

Introduction: Adaptive laboratory evolution (ALE) studies play a crucial role in understanding the adaptation and evolution of different bacterial species. In this study, we have investigated the adaptation and evolution of Salmonella enterica serovar Enteritidis to acetic acid using ALE. Materials and methods: Acetic acid concentrations below the minimum inhibitory concentration (sub-MIC) were used. Four evolutionary lineages (EL), namely, EL1, EL2, EL3, and EL4, of S. Enteritidis were developed, each demonstrating varying levels of resistance to acetic acid. Results: The acetic acid MIC of EL1 remained constant at 27 mM throughout 70 days, while the MIC of EL2, EL3, and EL4 increased throughout the 70 days. EL4 was adapted to the highest concentration of acetic acid (30 mM) and demonstrated the highest increase in its MIC against acetic acid throughout the study, reaching an MIC of 35 mM on day 70. The growth rates of the evolved lineages increased over time and were dependent on the concentration of acetic acid used during the evolutionary process. EL4 had the greatest increase in growth rate, reaching 0.33 (h-1) after 70 days in the presence of 30 mM acetic acid as compared to EL1, which had a growth rate of 0.2 (h-1) after 70 days with no exposure to acetic acid. Long-term exposure to acetic acid led to an increased MIC of human antibiotics such as ciprofloxacin and meropenem against the S. enterica evolutionary lineages. The MIC of ciprofloxacin for EL1 stayed constant at 0.016 throughout the 70 days while that of EL4 increased to 0.047. Bacterial whole genome sequencing revealed single-nucleotide polymorphisms in the ELs in various genes known to be involved in S. enterica virulence, pathogenesis, and stress response including phoP, phoQ, and fhuA. We also observed genome deletions in some of the ELs as compared to the wild-type S. Enteritidis which may have contributed to the bacterial acid adaptation. Discussion: This study highlights the potential for bacterial adaptation and evolution under environmental stress and underscores the importance of understanding the development of cross resistance to antibiotics in S. enterica populations. This study serves to enhance our understanding of the pathogenicity and survival strategies of S. enterica under acetic acid stress.

Efficacy of acidified water-in-oil emulsions against desiccated Salmonella as a function of acid carbon chain-length and membrane viscosity.

Sanitizing low-moisture food (LMF) processing equipment is challenging due to the increased heat resistance of Salmonella spp. in low-water activity (aw) environments. Food-grade oils mixed with acetic acid have been shown effective against desiccated Salmonella. In this study, different hydrocarbon chain-length (Cn) organic acids were tested against desiccated Salmonella by using 1% v/v water-in-oil (W/O) emulsion as the delivery system for 200 mM acid. Fluorescence lifetime imaging microscopy (FLIM) was utilized with a BODIPY-based molecular rotor to evaluate membrane viscosity under environmental conditions such as desiccation and temperature elevation. Drying hydrated Salmonella cells to 75% equilibrium relative humidity (ERH) increased the membrane viscosity from 1,199 to 1,309 mPa·s (cP) at 22°C. Heating to 45°C decreased the membrane viscosity of hydrated cells from 1,199 to 1,082 mPa·s, and decreased that of the desiccated cells from 1,309 to 1,245 mPa·s. At both 22°C and 45°C, desiccated Salmonella was highly susceptible (>6.5 microbial log reduction (MLR) per stainless-steel coupon) to a 30-min treatment with the W/O emulsions formulated with short carbon chain acids (C1-3). By comparison, the emulsion formulations with longer carbon chain acids (C4-12) showed little to no MLR at 22°C, but achieved >6.5 MLR at 45°C. Based upon the decreased Salmonella membrane viscosity and the increased antimicrobial efficacy of C4-12 W/O emulsions with increasing temperature, we propose that heating can make the membrane more fluid which may allow the longer carbon chain acids (C4-12) to permeate or disrupt membrane structures.

Oil-Based Sanitization in Low-Moisture Environments: Delivery of Acetic Acid with Water-in-Oil Emulsions.

Contamination with Salmonella spp. and Listeria monocytogenes is concerning across low-moisture food (LMF)-processing environments due to the pronounced survival of these organisms under dry conditions. This study treated desiccated bacteria with acetic acid delivered by oil with and without water-in-oil (W/O) emulsion. The influences of cellular desiccation, emulsion water concentration, water activity (aw), and treatment temperature were investigated. Acetic acid dissolved in oil (i.e., acidified oil) showed low levels of antimicrobial efficacy. After treatment with acidified oil (200 mM acetic acid at 22°C for 30 min), Salmonella enterica serovar Enteritidis phage type 30 cells desiccated to 75% equilibrium relative humidity (ERH) and 33% ERH were reduced by 0.69 and 0.05 log CFU/coupon, respectively. The dispersion of a low level of water (≥0.3%, vol/vol) within the acidified oil with the surfactant (i.e., acidified W/O emulsion) significantly enhanced the antimicrobial efficacy. After treatment with the acidified W/O emulsion (200 mM acetic acid at 22°C for 20 min), desiccated Salmonella (4-strain cocktail) and L. monocytogenes (3-strain cocktail) cells were reduced by >6.52 log most probable number (MPN)/coupon, regardless of the desiccation levels. Increased efficacy was observed with temperature elevation. Reduced efficacy was observed when glycerol was added to the aqueous phase of the emulsion to decrease the solution aw, indicating that the enhanced efficacy of the acidified W/O emulsion was associated with differential osmotic pressure. The antimicrobial mechanism may be due to the membrane disruption induced by acetic acid, in combination with the hypoosmotic stress provided by W/O emulsion, creating cellular lysis, as illustrated by electron micrographs. IMPORTANCE Aqueous-based cleaning and sanitation are undesirable in processing facilities that manufacture low-moisture foods such as peanut butter and chocolate. Alcohol-based sanitization is advantageous because it leaves no residue on the contact surface but requires the processing facility to close temporarily due to flammability. At >6.52 log kill of desiccated Salmonella and Listeria monocytogenes cells, the developed oil-based formulation has the potential to be an effective dry sanitation method.

Efficacy of Acidified Oils against Salmonella in Low-Moisture Environments.

When processing low-moisture, high-fat foods such as peanut butter and nuts, water-based sanitization is unsuitable due to the immiscible nature of water and fats. Dry sanitization mainly uses flammable compounds such as isopropanol, requiring equipment cooling before application. The use of oils to deliver antimicrobials against foodborne pathogens enables the use of elevated temperatures, thus eliminating processing downtimes associated with dry sanitization. This study delivered organic acids and medium-chain fatty acids (100, 250, and 500 mM) in peanut oil against Salmonella enterica serovar Enteritidis desiccated at 75% relative humidity (RH). Acetic acid in peanut oil (AO) at 45°C was the most effective food-grade acid, causing a 4.4-log reduction in S. Enteritidis at 500 mM. AO caused cellular injury and was effective against a variety of S. Enteritidis strains. Confocal microscopy demonstrated that cells treated with 50 mM and 250 mM AO had significant membrane damage and reduced cellular respiration compared to untreated controls. Treatment efficacy increased with the increase in acid concentration, treatment duration, and treatment temperature from 20 to 45°C. Transmission electron microscopy after treatment with 100 and 250 mM AO revealed membrane ruffling and leakage in cell membranes, especially at 45°C. Reduction of the RH to 33% during desiccation of S. Enteritidis caused a decrease in AO efficacy compared to that at 75% RH, while at a higher RH of 90%, there was an increase in the efficacy of AO. Acidified oils can serve as robust, cost-effective replacements for dry-sanitation methods and improve safety of low moisture foods. IMPORTANCE Currently, dry sanitization products used during food processing often contain flammable compounds which require processing to stop and equipment to cool before application. This leads to processing downtimes and consequently, economic losses. This challenge is compounded by exposure to dryness which frequently renders Salmonella resistant to heat and different antimicrobials. Thus, the development of heat-tolerant oil-based antimicrobial compounds is a novel approach for sanitizing in low-moisture (dry) environments such as those found in peanut butter, tree nuts, and chocolate manufacturing. This study shows that acidified oils, especially acetic acid in peanut oil at elevated temperatures (45°C), was highly effective against desiccated Salmonella. Acidified oils have the potential to replace dry sanitizers, increasing the frequency of sanitization, leading to an improvement in food safety.

An Experimental Adult Zebrafish Model for Shigella Pathogenesis, Transmission, and Vaccine Efficacy Studies.

Shigellosis has been a menace to society for ages. The absence of an effective vaccine against Shigella, improper sanitation, and unhygienic use of food and water allow the disease to flourish. Shigella can also be transmitted via natural water bodies. In the absence of a good animal model, the actual nature of pathogenesis and transmission remains unclear. Zebrafish larvae have previously been described as a model for Shigella pathogenesis. However, larval fish lack a mature intestinal microbiota and immune system. Here, the adult zebrafish was assessed as a potential model for Shigella pathogenesis. Their well-developed innate and adaptive immune responses mimic the mammalian immune system. Shigella showed a clear dose-, time-, and temperature-dependent colonization of the adult zebrafish gut. Efficacy of a three-dose immunization regime was tested using bath immunization with heat-killed trivalent Shigella immunogen. The present study demonstrates the efficacy of an adult zebrafish model for pathogenesis, transmission, and vaccine efficacy studies. IMPORTANCE Shigellosis is a diarrheal disease that is prevalent in developing countries and especially dangerous in young children. Currently, animal models for shigellosis are unable to model some aspects of the infectious cycle. Here, we describe a new shigellosis model in adult zebrafish, an increasingly common model organism for studying bacterial pathogens. The zebrafish model can be used to study Shigella colonization, transmission, and immune responses, as well as test vaccine efficacy.

Evaluation of the efficacy of antimicrobials against pathogens on food contact surfaces using a rapid microbial log reduction detection method.

Microbial contamination of food contact surfaces in food processing industries is a significant health hazard. Evaluating the efficacy of sanitizing agents used during food processing is essential to ensure public health and safety. This study describes an optical screening method using an oCelloScope to quantify the number of surviving bacterial cells, expressed as microbial log reduction (MLR), after antimicrobial treatment. We tested the efficacy of two sanitizing agents, sodium hypochlorite and benzalkonium chloride, against desiccated cells of three pathogens, S. Enteritidis, E. coli O157: H7, and L. monocytogenes that are of concern on food processing surfaces. Stainless steel slides were used to mimic commercial food processing surfaces. Bacterial cells were desiccated at 75% relative humidity (RH) before antimicrobial treatment on stainless steel surfaces, and survivor levels were analyzed via plate counts to calculate MLR. These were compared to MLR values generated using the oCelloScope. For analysis of MLR using the oCelloScope, cells were desiccated at 75% RH on polystyrene microtiter plates, treated with antimicrobials, and surviving cell numbers were analyzed. Our results show that MLR values of treated desiccated cells calculated using the BCA algorithm of the oCelloScope were comparable to the values generated using the traditional plate count assay for the same concentration and treatment duration of the antimicrobials against all the tested pathogens. MLR could not be calculated for a non-lytic antimicrobial (curcumin and UV-A irradiation) against E. coli O157:H7, however, modified growth curves demonstrated an antimicrobial effect of curcumin and irradiation treatment. The results indicate that this method can be used for rapid screening of MLR of lytic antimicrobial compounds. Quantification of MLR using the oCelloScope is an effective tool to rapidly identify appropriate antimicrobial treatments and can be used to study novel antimicrobial compounds in the future.

Effect of Abamectin on Fungal Growth and Its Efficacy as a Miticide in the Laboratory.

Abamectin was tested for use with solid agar media in the laboratory to eliminate or kill the common mold mite Tyrophagus spp. in fungal cultures of Phaeomoniella chlamydospora and Phaeoacremonium minimum, two important grape pathogens involved in grapevine trunk disease. Abamectin concentrations tested were at or below the recommended dosage for abamectin in greenhouse spray applications (≤625 µg/ml) to control mites and determine the following: (i) if fungal growth would be inhibited and (ii) if mites would be killed or their activity suppressed. Abamectin was either added to the media before autoclaving or filter-sterilized and added after autoclaving to test the effects of autoclaving on abamectin efficacy. Streptomycin (100 µg/ml) was also added to a set of treatments to determine whether this commonly used antibiotic would affect abamectin efficacy against mites or have an effect on fungal growth when combined with abamectin. Filter-sterilized abamectin in the range of 62.5 to 312 µg/ml, delivered to the media after autoclaving, provided the most effective control of mites while also showing limited inhibition of fungal growth on solid agar media in the absence of streptomycin. The addition of filter-sterilized streptomycin had no significant effect on fungal growth for Phaeomoniella chlamydospora, whereas for Phaeoacremonium minimum a small but significant reduction in growth with streptomycin occurred at abamectin concentrations >62.5 µg/ml.