Bacterial Cocktail Research Articles

Spiking immunotherapy with a bacterial cocktail brings T cells back to the fight

A recent study by Montalban-Arques et al.1 in Cell Host & Microbe shows that augmenting the function of the gut microbiota reduces tumor burden. Four Clostridiales species blocked tumor growth as efficient as chemotherapy or immunotherapy in colorectal cancer and melanoma.

Wild Halophytic Phragmites karka Biomass Saccharification by Bacterial Enzyme Cocktail.

Biofuel derived from halophytic biomass is getting attention owing to the concerns of energy versus food crisis. The disadvantages associated with edible bioenergy resources necessitate the need to explore new feedstocks for sustainable biofuel production. In this study, biomass from locally available abundant halophytes (Panicum antidotale, Phragmites karka, Halopyrum mucronatum, and Desmostachya bipinnata) was screened for saccharification by an enzyme cocktail composed of cellulase, xylanase, and pectinase from Brevibacillus borstelensis UE10 and UE27, Bacillus aestuarii UE25, Aneurinibacillus thermoaerophilus UE1, and Bacillus vallismortis MH 1. Two types of pretreatment, i.e., with dilute acid and freeze-thaw, were independently applied to the halophytic biomass. Saccharification of acid-pretreated P. karka biomass yielded maximum reducing sugars (9 mg g–1) as compared to other plants. Thus, the factors (temperature, pH, substrate concentration, and enzyme units) affecting its saccharification were optimized using central composite design. This statistical model predicted 49.8 mg g–1 of reducing sugars that was comparable to the experimental value (40 mg g–1). Scanning electron microscopy and Fourier-transform infrared spectroscopy showed significant structural changes after pretreatment and saccharification. Therefore, halophytes growing in saline, arid, and semi-arid regions can be promising alternative sources for bioenergy production.

A Broad-Spectrum Antimicrobial and Antiviral Membrane Inactivates SARS-CoV-2 in Minutes.

SARS‐CoV‐2, the virus that caused the COVID‐19 pandemic, can remain viable and infectious on surfaces for days, posing a potential risk for fomite transmission. Liquid‐based disinfectants, such as chlorine‐based ones, have played an indispensable role in decontaminating surfaces but they do not provide prolonged protection from recontamination. Here a safe, inexpensive, and scalable membrane with covalently immobilized chlorine, large surface area, and fast wetting that exhibits long‐lasting, exceptional killing efficacy against a broad spectrum of bacteria and viruses is reported. The membrane achieves a more than 6 log reduction within several minutes against all five bacterial strains tested, including gram‐positive, gram‐negative, and drug‐resistant ones as well as a clinical bacterial cocktail. The membrane also efficiently deactivated nonenveloped and enveloped viruses in minutes. In particular, a 5.17 log reduction is achieved against SARS‐CoV‐2 after only 10 min of contact with the membrane. This membrane may be used on high‐touch surfaces in healthcare and other public facilities or in air filters and personal protective equipment to provide continuous protection and minimize transmission risks.

The effects of environmental factors on the prevalence and diversity of bacteriophages lytic against the top six non‐O157 Shiga toxin‐producing Escherichia coli on an organic farm

AbstractBacteriophages (or phages) specific to Shiga toxin‐producing Escherichia coli (STEC) strains are frequently isolated from animal‐associated environments primarily because ruminant animals are the natural reservoir of STEC. However, little is known about these phages in produce‐growing environments. The objective of this study was to determine the effects of environmental factors on the prevalence of the phages lytic against O157 and the top six non‐O157 STEC on an organic farm. A total of 370 samples were collected from an organic farm, containing animal‐active and produce‐growing areas, for 1 year. A bacterial cocktail, including nonpathogenic and pathogenic E. coli strains, was used for phage isolation. Meanwhile, culture methods and PCR were used to isolate STEC strains. Weather information was also collected for each sampling trip. Twenty‐eight samples contained phages lytic against STEC (or STEC‐infecting phages), of which 26 were collected from the animal‐active area. Moreover, the winter season had a higher phage prevalence than other seasons, likely due to high rain precipitation. The phages belonging to the Myoviridae family and those lytic against STEC O103 were the most prevalent. One E. coli O103:H2 was isolated from a water sample where no STEC O103‐infecting phages were found. Additionally, no STEC O103 was present in the samples containing STEC O103‐infecting phages. The findings indicate that animal is the primary factor contributing to the prevalence of the STEC‐infecting phages in the surrounding environment of the organic farm, and the presence of these phages contributes to a negative correlation with their STEC hosts.

BIOREMEDIATION OF DIESEL CONTAMINATED SOIL USING BACTERIAL COCKTAIL AND ORGANIC NUTRIENTS

Bioremediation is a process of contaminant degradation in the environment using microorganisms. Bioremediation of diesel contaminated soil was studied using bacterial cocktail and organic nutrients from cow dung and poultry droppings at interval of 21 days for a total period of 84 days. Two hundred grams (200g) of soil were weighed into clay pots polluted with 10% (w/w) diesel oil and left undisturbed for 48 hours in an open field. After 48 hours, the clay pots were inoculated with bacterial cocktail (two bacteria isolates from diesel contaminated soil), 10% (w/w) of cow dung (CD), 10% (w/w) of poultry droppings (PD) and 5% (w/w) of sodium azide (NaN3). The two bacterial isolates were identified as Micrococcus luteus trpE16 and Bacillus subtilis DNK UT 02 after screening for hydrocarbon utilization using standard methods. The counts of hydrocarbon utilizing bacteria (THB) in the amended soil ranged from 20.2×107 and 63.5×107 cfu/g while unamended soil had the least count of THB ranging between 8.4×107 and 19.0× 107 cfu/g. Soil bioremediated with bacterial cocktail (BC) + 10% (CD+PD) recorded highest total petroleum hydrocarbon (TPH) degradation of 48.76%, 56.32%, 72.89% and 96.80% at the end of days 21, 42, 63 and 84 respectively while autoclaved soil with 5% NaN3 recorded the least (10.03%, 13.38%, 14.02% and 18.42%) respectively. First order kinetic model showed that soil bioremediated with BC with 10% (CD+PD) recorded highest biodegradation rate constant of 0.2096 day-1 and half-life of 3.31 days. Statistical analysis indicated that the results obtained were significantly (P < 0.05) different during the 84 days of this study. Amendment of diesel contaminated soil with bacterial cocktail and organic wastes caused changes in the soil physiochemical properties and accelerated the rate of biodegradation in the soil. However, poultry droppings and cow dung can serve as a potential and viable biostimulant for enhanced biodegradation of diesel in soil.

Synergistic efficacy of alkyltrimethylammonium bromide, chlorhexidine digluconate on diverse bacterial strains causing red-heat and purple-stain deteriorations of leather.

This investigation assessed the synergistic effectiveness of alkyltrimethylammonium bromide (ATMB) and chlorhexidine digluconate (CDG) on selected microbes causing red-heat and purple stain degradations on salt-cured hides and leather products. This biological phenomenon ultimately deteriorates the finished leather quality and grounds considerable economic losses for leather industry. In tryptic soy broth, combination of ATMB and/CDG at the concentration of 900/90ppm for predefined time interval of 1, 3, and 6min caused in 0.73, 2.45, and 2.95 log CFU/mL reductions of bacterial cocktail population cell survival, respectively. Whereas in saturated brine solution (SBS), the bacterial cocktail treated with 600/60 and 900/90ppm of ATMB/CDG for 18h resulted ~ 74% and 98% reduction, respectively, in comparison to their respective controls. Furthermore, lessening of individual bacterial cultivations by combined ATMB and CDG in SBS was also elucidated.

An oral bacterial cocktail for kidney protection.

The oral delivery of a microencapsulated bacterial cocktail into animal models of kidney disease promotes the degradation of nitrogenous waste in the gut, thereby supporting renal function.

An encapsulated bacterial cocktail for the removal of nitrogenous metabolic waste.

An encapsulated bacterial cocktail for the removal of nitrogenous metabolic waste.

An orally delivered microbial cocktail for the removal of nitrogenous metabolic waste in animal models of kidney failure.

Patients with kidney failure commonly require dialysis to remove nitrogenous wastes and to reduce burden to the kidney. Here, we show that a bacterial cocktail orally delivered in animals with kidney injury can metabolize blood nitrogenous waste products before they diffuse through the intestinal mucosal barrier. The microbial cocktail consists of three strains of bacteria isolated from faecal microbiota that metabolize urea and creatinine into amino acids, and is encapsulated in calcium alginate microspheres coated with a polydopamine layer that is selectively permeable to small-molecule nitrogenous wastes. In murine models of acute kidney injury and chronic kidney failure, and in porcine kidney failure models, the encapsulated microbial cocktail significantly reduced urea and creatinine concentrations in blood, and did not lead to any adverse effects.

Skin microbiome profiling and cultivation of healthy adult Indonesian skin

Studies on the impact of skin microbiota on human health have been gaining more attention. The skin microbiome is considered to provide several probiotics for skin therapeutic. Beneficial bacteria mixtures (bacterial cocktail) isolated from targeted organs have shown promising modulatory activities for use in skin therapeutics. The objectives of this study were to determine and identify the microbial communities on the skin that can be potentially used as probiotics-postbiotics. Determination and identification of skin microbiota were carried out simultaneously by employing next-generation sequencing (NGS) of direct sampling, as well as by bacterial cultivation; twenty bacterial isolates with different characteristics were selected and identified by both culture-based methods and 16sRNA sequencing. We found that Actinobacteria and Firmicutes are the most abundant phylum present on the skin as presented by NGS data, which constitute to 67% and 28.59% of the whole bacterial population, consecutively. Three strains, i.e., Staphylococcus hominis (AN MK968325.1), Staphylococcus warneri (AN MK968315.1), and Micrococcus luteus (MK968318.1), were obtained from cultivable samples. They were potential to be developed further as probiotics in skin microbiome therapeutic as well as for postbiotic formulation. However, the promising formula of bacterial cocktail for skin microbiome therapeutic must be elucidated thoroughly to avoid unwanted effects by performing visual observation on agar plate and by molecular approach, q-PCR.

Peptide-Based Formulation from Lactic Acid Bacteria Impairs the Pathogen Growth in Ananas Comosus (Pineapple)

Worldwide, street vending commerce has grown exponentially, representing in some countries, including Ecuador, a significant proportion of food consumed by the urban population. Pineapple is one of the common fruits sold as ready-to-eat slices by ambulant vendors in the street or on public transport at risk of contamination by various microorganisms. Previously, we selected Lactobacillus plantarum UTNCys5-4 and Lactococcus lactis subsp. lactis Gt28 strains producing peptides with high capacity to inhibit pathogen growth in vitro. In this study, the effect of different edited formulations containing a mixture of Cys5-4/Gt28 peptides was evaluated in vitro and ex vitro against a pathogenic cocktail containing E. coli (2), Salmonella (2) and Shigella (1). The growth of bacterial cocktail co-inoculated with cell-free supernatant containing peptides (formulation T1) and precipitated peptides (formulation T6), in a ratio of Cys5-4/Gt28:1:1 (v/v), results in a decrease of total cell viability with 1.85 and 1.2 log CFU/mL orders of magnitude at 6 h of incubation. About the same decrease (1.9 log CFU/g) was observed when pineapple slices artificially inoculated with the pathogenic cocktail were coated with T1 formulation, indicating the capacity to diminish simultaneous pathogens in situ, thus demonstrating its great biological control and protection. However, the E. coli cell counts reduced by 2.08 log CFU/g while Salmonella and Shigella cell counts reduced by 1.43 and 1.91 log CFU/g, respectively, at 5 days of refrigeration. In the untreated pineapple slices, the total cell density was maintained during storage, suggesting the adaptation of the pathogens to the fruit matrix. The peptide-based formulation exerted a bacteriolytic mode of action inducing pathogenic cell death. The results indicate that coating pineapple slices with peptide-based formulation is a promising approach to protect them from further contamination by microbial spoilage as well as an alternative to increase the food safety.

Simultaneous profiling and cultivation of the skin microbiome of healthy young adult skin for the development of therapeutic agents

BackgroundStudies on the impact of the skin microbiota on human health have been gaining more attention. Bacteria are associated with various diseases, although certain strains of bacteria, which are known as probiotics, are considered beneficial. Mixtures of several bacteria (bacterial cocktail) isolated from targeted organs have shown promising modulatory activities for use in skin therapeutics. The objectives of this study were to determine and identify the microbial communities on the skin that can potentially be used as probiotics, as determined by bacterial isolation and cultivation, followed by next-generation sequencing (NGS). ResultsSamples were collected by swabbing on forehead and cheek skin. Genomic DNA from bacterial swab samples were directly extracted to be further processed into NGS. Cultivation of skin bacteria was carried out in subsequent medium. Thus, around twenty bacterial isolates with different characteristics were selected and identified by both culture-based method and 16sRNA sequencing. We found that Actinobacteria and Firmicutes are the most abundant phylum present on the skin as presented by NGS data, which constitute to 67% and 28.59% of the whole bacterial population, consecutively. However, Staphylococcus hominis, Staphylococcus warneri, and Micrococcus luteus (AN MK968325.1; AN MK968315.1; and MK968318.1 respectively) were able to be obtained in the samples of cultivable, and could be potentially developed as probiotics in skin microbiome therapeutic as well as for postbiotic formulation. ConclusionSkin microbiome is considered to provide several probiotics for skin therapeutic. However, some opportunistic pathogens were discovered in this study population. Thus, the promising formula of bacterial cocktail for skin microbiome therapeutic must be thoroughly elucidated to avoid unwanted species. Our study is the first human skin microbiome profile of Indonesia resulted from a Next Generation Sequencing as an effort to show a representative of tropical country profile.

P074 HUMAN-DERIVED CLOSTRIDIUM VE202 STRAINS REDUCE ENTEROBACTERIACEAE AND FUSOBACTERIA AND REVERSE EXPERIMENTAL COLITIS INDUCED BY HUMAN GUT MICROBIOTA

Abstract Background 17 human-derived Clostridium strains (VE202), belonging to a bacterial cluster under-represented in active IBD, induce colonic IL-10-producing FOXP3+ regulatory T cells and prevent colitis in murine IBD models (Nature 2013). The VE202 consortium is a promising live biotherapeutic product for clinical application. However, its 1) therapeutic benefit on established colitis and 2) effect on intestinal microbiota composition remain unclear. Methods Models: We established colitis in ex-germ-free (exGF) Rag2−/− mice with naïve T cell transfer (Hu-nT) and Il10−/− (Hu-Il10−/−) mice colonized with healthy human fecal microbiota, using stool with low abundance of Clostridiales. In Hu-Il10−/−, we tested two other healthy donor stools (one with low Clostridiales and another with Fusobacteria, which is a potential pathobiont in IBD). For a selected bacteria-colonized model, we selected three aggressive Enterobacteriaceae strains cultured from the human donor stool used for the previous studies, based on colonic Th1 response and colitis in monoassociated exGF-Il10−/−. We then established an aggressive Klebsiella (K), Enterobacter (E) and E. coli (E) strain -colonized exGF Il10−/− colitis model (KEE-colitis). In addition, we established a human IBD-derived E.coli and Fusobacterium varium consortium-colonized Il10−/− (EF-colitis) model. In vitro, we anaerobically co-cultured human stools with VE202 and analyzed bacterial composition. Therapy: We treated mice by oral administration of VE202 or vehicle twice weekly for 2–4 weeks. Evaluation: Colitis severity was assessed by blinded histological score, fecal lipocalin-2, stool consistency score and colonic IFNγ +CD4+ T cells. Bacteria profiles in cecal contents were analyzed by metagenomics or qPCR. Results Hu-nT and Hu-Il10−/− (Fig.1A, B): VE202 significantly reversed histological colitis and other inflammatory endpoints compared to vehicle. Consistently VE202 attenuated colitis induced by three different donor stools. Bacteria profiling revealed that VE202 attenuated the colitis-associated bloom of Enterobacteriaceae and Fusobacteria. In KEE-colitis (Fig.1C) VE202 reversed colitis with reduction of all Enterobacteriaceae strains. In EF-colitis (Fig.1D) VE202 reversed colitis with reduction of E. coli and F. varium. Co-culture (Fig.1E): VE202 reduced abundance of Enterobacteriaceae in a dose dependent manner. Conclusion Our findings suggest a novel IL-10-independent protective mechanism for human Clostridium VE202 strains, i.e. correction of dysbiosis with reduction of levels of Enterobacteriaceae and Fusobacteria. In addition, VE202 treatment is consistently effective for different human donor microbiota in vivo and in vitro. These results provide a rationale and target for therapeutic use of rationally selected resident protective bacterial cocktails in IBD patients.

Use of Customized Bacteriophages in the Treatment of Chronic Nonhealing Wounds: A Prospective Study.

Nonhealing ulcers are a great challenge to surgeons as they may occasionally culminate in amputation of the affected part. Mostly nonhealing of wounds results due to infection by antibiotic-resistant bacteria and subsequent biofilm formation. However, customized bacteriophage therapy may take care of both of the above-mentioned hurdles. A total of 48 study subjects of age group 12 to 70 years, having minimum one eligible full-thickness wound and failed to heal in 6-week duration with conventional therapy, were included in this exploratory prospective study. Patients with systemic diseases, that is, burn, malignancy, dermatological disorders, and ulcers with leprosy or tuberculosis, were excluded. However, subjects having diabetes and hypertension were included in the study. The customized monophage for single bacterial infection and cocktail of phages specific to 2 or more infecting bacteria were applied on an alternate day over the wound surface. A total of 5 to 7 applications were made till the wound became free of infecting bacteria. The study period extended from August 2018 to May 2019. The study subjects were followed for 3 months since the start of therapy. A cure rate of 81.2% could be obtained, of which 90.5% (19/21) patients were nondiabetic and 74.1% (20/27) diabetic. The wounds infected with Klebsiella pneumoniae were observed with relatively delayed healing. Post phage therapy, the mean hemoglobin level and percentage of lymphocytes increased significantly. The customized local phage therapy is very promising in nonhealing ulcers.

Experimental Inoculation of Coral Recruits With Marine Bacteria Indicates Scope for Microbiome Manipulation in Acropora tenuis and Platygyra daedalea.

Coral-associated microorganisms are essential for maintaining the health of the coral holobiont by participating in nutrient cycling and protecting the coral host from pathogens. Under stressful conditions, disruption of the coral prokaryotic microbiome is linked to increased susceptibility to diseases and mortality. Inoculation of corals with beneficial microbes could confer enhanced stress tolerance to the host and may be a powerful tool to help corals thrive under challenging environmental conditions. Here, we explored the feasibility of coral early life stage microbiome manipulation by repeatedly inoculating coral recruits with a bacterial cocktail generated in the laboratory. Co-culturing the two species Acropora tenuis and Platygyra daedalea allowed us to simultaneously investigate the effect of host factors on the coral microbiome. Inoculation cocktails were regularly prepared from freshly grown pure bacterial cultures, which were hence assumed viable, and characterized via the optical density measurement of each individual strain put in suspension. Coral early recruits were inoculated seven times over 3 weeks and sampled once 36 h following the last inoculation event. At this time point, the cumulative inoculations with the bacterial cocktails had a strong effect on the bacterial community composition in recruits of both coral species. While the location of bacterial cells within the coral hosts was not assessed, metabarcoding using the 16S rRNA gene revealed that two and six of the seven bacterial strains administered through the cocktails were significantly enriched in inoculated recruits of A. tenuis and P. daedalea, respectively, compared to control recruits. Despite being reared in the same environment, A. tenuis and P. daedalea established significantly different bacterial communities, both in terms of taxonomic composition and diversity measurements. These findings indicate that coral host factors as well as the environmental bacterial pool play a role in shaping coral-associated bacterial community composition. Host factors may include microbe transmission mode (horizontal versus maternal) and host specificity. While the long-term stability of taxa included in the bacterial inocula as members of the host-associated microbiome remains to be evaluated, our results provide support for the feasibility of coral microbiome manipulation, at least in a laboratory setting.

Effects of pulsed light and sanitizer wash combination on inactivation of Escherichia coli O157:H7, microbial loads and apparent quality of spinach leaves

The purpose of this study was to investigate the efficacy of pulsed light (PL), a new formula of sanitizer (HEN) consisting of hydrogen peroxide, EDTA and Nisin, as well as synergy of PL and HEN sanitizer (PL-HEN) wash in inactivating E. coli O157:H7 on spinach. The treatment effect on microbial loads and apparent quality during 13 days storage at 4 °C was also determined. A bacterial cocktail containing three strains of E. coli O157:H7 was used as inoculum based on their association with produce-related outbreaks. Spinach leaves were spot inoculated on surface before treating with PL (1–63J/cm2), HEN sanitizer wash (2 min) or their combinations. PL inactivation was influenced significantly at low doses. Treatment dose of 15.75 J/cm2, equivalent to 15 s intense PL treatment, was found optimal above which adverse quality effect was evident. The optimal PL dose resulted 2.7 log CFU/g reduction of E. coli O157:H7 while a rapid 2 min wash in sanitizer formulation HEN, provided comparatively low, 1.8 log CFU/g, reduction of the pathogen. Two different sequences of PL and HEN treatment combinations were tested. In PL-HEN treatment, inoculated leaves were first treated at optimal PL dose (15.75 J/cm2) followed by 2 min immersion in HEN whereas in HEN-PL treatment, leaves were first washed in HEN before PL exposure. HEN-PL treatment indicated a compound inactivation activity (4.6 logs reduction) while PL-HEN treatment indicated a strong synergistic inactivation as E. coli cells were not detectable after treatment indicating >5 log reduction. The PL-HEN treatment not only significantly reduced spoilage microbial populations on spinach but also slowed their growth during storage. Furthermore, the visual and firmness quality of spinach were not significantly affected by the PL-HEN treatment. Overall, our results demonstrate that integrated PL-HEN technology can be used to enhance microbial safety of spinach.

Nutritional and Microbiological Quality of Tiger Nut Tubers (Cyperus esculentus), Derived Plant-Based and Lactic Fermented Beverages

Tiger nut (Cyperus esculentus) is a tuber that can be consumed raw or processed into beverages. Its nutritional composition shows a high content of lipid and dietary fiber, close to those of nuts, and a high content of starch, like in other tubers. Tiger nuts also contain high levels of phosphorus, calcium, and phenolic compounds, which contribute to their antioxidant activity. From those characteristics, tiger nuts and derived beverages are particularly relevant to limit food insecurity in regions where the plant can grow. In Europe and United States, the tiger nut derived beverages are of high interest as alternatives to milk and for gluten-free diets. Fermentation or addition of probiotic cultures to tiger nut beverages has proven the ability of lactic acid bacteria to acidify the beverages. Preliminary sensory assays concluded that acceptable products are obtained. In the absence of pasteurization, the safety of tiger nut-based beverages is not warranted. In spite of fermentation, some foodborne pathogens or mycotoxigenic fungi have been observed in fermented beverages. Further studies are required to select a tailored bacterial cocktail which would effectively dominate endogenous flora, preserve bioactive compounds and result in a well-accepted beverage.

Treatment With Probiotic Bacteria Does Not Diminish the Impact of a Cystoisospora suis Challenge in Suckling Piglets.

Colonization of newborn piglets with beneficial and ubiquitous microorganisms in combination with colostral passive immunity is the prerequisite for development of immunity and gut maturation. In this study living strains of Clostridium perfringens type A (CpA) and non-pathogenic Escherichia (E.) coli strains harvested from healthy piglets were administered to piglets prior to first colostrum intake in order to prevent disease caused by pathogenic variants of the same bacterial species by competitive exclusion. In addition, it was investigated whether these potential beneficial colonizers were able to prevent harmful effects of infection with Cystoisospora (C.) suis as a primary invasive pathogen. In a first trial, half of the piglets from four litters were treated with a bacterial cocktail consisting of two E. coli and four CpA strains immediately after birth on two consecutive days, while the other half of the litters served as control group. In a second trial, piglets were treated following the protocol of the first trial, and additionally all piglets were infected 4 h after the end of littering with ~1,000 sporulated oocysts of a C. suis laboratory strain. General health, body weight development, fecal consistency and, in the second trial, oocyst excretion were monitored from birth until weaning. No adverse effects of the cocktail on the health status were observed. Treated piglets of the first trial showed a higher average daily weight gain until weaning. In the second trial, no significant differences were found with respect to average daily weight gain, fecal consistency, amount, and duration of oocyst excretion assessed in daily samples. In treatment group 51.1% and in the control group 38.5% of the fecal samples were positive for oocysts in autofluorescence. The average duration of oocyst excretion was longer in treatment group (7.7 days) than in control group (5.6 days). Application of bacterial cocktail could not effectively minimize disease symptoms caused by C. suis. There was a trend toward an increase in severity of disease symptoms in treated pigs, suggesting that the synergism between CpA and C. suis was independent of the bacterial strains, but is exclusively dominated by the pathogenic effect of C. suis.

Application of bacteriophages in simultaneously controlling Escherichia coli O157:H7 and extended-spectrum beta-lactamase producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 and extended-spectrum beta-lactamase (ESBL) producing E. coli (ESBLEC) are important bacteria of public health concern and frequently isolated from raw beef products. Bacteriophage-based methods have been increasingly exploited to control bacterial contamination in meats. Here, we describe the isolation, characterization, and application of a lytic phage PE37 for the simultaneous bio-control of STEC O157:H7 and ESBLEC. Phage PE37, isolated from the bovine intestine, was morphologically characterized as a member of the Myoviridae family, with a broad host range and great stability under various stress conditions. Sequencing analysis revealed that the genomic DNA of phage PE37 contains genes that contribute to virion structure, replication, assembly, and host lysis. PE37 significantly reduced the viable counts of STEC O157:H7 by 4.9 and 2.6logCFU/mL in broth after 6h of incubation at 25 and 8°C, respectively. Application of phage PE37 to raw beef artificially contaminated with STEC O157:H7 resulted in significant reductions in the viable counts by 2.3 and 0.9logCFU/piece after 24h of storage at 25 and 8°C, respectively. Treatment of raw beef contaminated with a bacterial cocktail of STEC O157:H7 and ESBLEC with PE37 also significantly decreased the viable counts of the bacterial mixture by 1.4 and 1.0logCFU/piece after 24h of incubation at 25 and 8°C, respectively. These findings suggest that bacteriophage PE37 may be a potential bio-agent for controlling STEC O157:H7 and ESBLEC contamination in raw beef.

Antibacterial activity of acidified sodium benzoate against Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes in tryptic soy broth and on cherry tomatoes

Concerns about undesirable by-products from chlorine sanitation of fresh produce and the limited efficacy with the presence of organic matter, have led to studies on alternative washing solutions. The aim of this study was to evaluate the antibacterial activities of acidified sodium benzoate (NaB) solutions against Escherichia coli O157:H7, Salmonella enterica and Listeria monocytogenes in growth medium and on cherry tomatoes. Experimentally, the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs, >3 Log reduction) of NaB against E. coli O157:H7 ATCC 43895, S. Enteritidis, and L. monocytogenes Scott A were determined at pH 7.0–4.0 using micro-broth dilution method and agar plating method, respectively. The reduction of the three bacteria in tryptic soy broth (TSB) by 500 and 1000 ppm NaB at pH 2.0, 2.5 and 3.0 for 30 min at 21 °C was compared. Residual bacterial cocktails inoculated on cherry tomatoes were determined after soaking in 3000 ppm NaB solution adjusted to pH 2.0 for 3 min at 21 °C. Results showed that the MBC of NaB reduced from >10,000 ppm at pH 7.0 to 1000 ppm at pH 4.0 and was identical for the three bacteria. The log reduction of bacteria in TSB indicated that 1000 ppm NaB at pH 2.0 was the most effective in killing the three pathogens. The respective reduction of E. coli O157:H7 and S. enterica cocktails inoculated on cherry tomatoes immersed in 3000 ppm NaB (pH 2.0) at 21 °C for 3 min was 4.99 ± 0.57 and 4.08 ± 0.65 log CFU/g, which was significantly higher (p < 0.05) than the treatments of 200 ppm free chlorine at pH 6.5. Conversely, the reduction of L. monocytogenes on tomatoes by 3000 ppm NaB (4.88 ± 0.73 log CFU/g) was similar (p > 0.05) to 200 ppm chlorine. Furthermore, the reduction of bacterial cocktails on tomatoes by 3000 ppm NaB at pH 2.0 was not affected after adding 1% tomato puree, and bacteria were not detected in NaB washing solutions with and without 1% tomato puree and on following un-inoculated tomatoes. This study showed that acidified NaB solution may be used as an alternative post-harvest wash of produce.