Search Results (40)

Foodborne illness caused by bacterial pathogens is a global health concern and results in millions of infections annually. Therefore, food products typically undergo several processing stages, including sanitation steps, before being distributed in an attempt to remove pathogens. However, many sanitation methods have compounding effects on the color, texture, flavor, and nutritional quality of the product or do not effectively reduce the pathogens that food can be exposed to. Some bacterial pathogens particularly possess traits and tactics that make them even more difficult to mitigate such as biofilm formation. Non-thermal plasma sanitation techniques, including plasma-treated water (PTW), have proven to be promising methods that significantly reduce pathogenic bacteria that food is exposed to. Published work reveals that PTW can effectively mitigate both gram-positive and gram-negative bacterial biofilms. This study presents a novel analysis of the differences in antimicrobial effects of PTW treatment between biofilm-forming gram-positive bacteria, commonly associated with foodborne illness, that are sporulating (Bacillus cereus) and non-sporulating (Listeria monocytogenes). After treatment with PTW, the results suggest the following hypotheses: (1) that the non-sporulating species experiences less membrane damage but a greater reduction in metabolic activity, leading to a possible viable but non-culturable (VBNC) state, and (2) that the sporulating species undergoes spore formation, which may subsequently convert into vegetative cells over time. PTW treatment on gram-positive bacterial biofilms that persist in food processing environments proves to be effective in reducing the proliferating abilities of the bacteria. However, the variance in PTW’s effects on metabolic activity and cell vitality between sporulating and non-sporulating species suggest that other survival tactics might be induced. This analysis further informs the application of PTW in food processing as an effective sanitation method. Full article

Non-thermal processing encompasses a range of emerging food technologies, including high-pressure processing (HPP), pulsed electric field (PEF), cold atmospheric plasma (CAP), high-pressure carbon dioxide (HPCD), and ultrasound (US). Unlike traditional thermal processing or chemical preservatives, these methods offer advantages such as lower energy consumption, enhanced environmental sustainability, and effective microbial inactivation, thereby extending food shelf life. Moreover, they can better preserve the nutritional integrity, color, flavor, and texture of food products. However, a critical concern associated with non-thermal processing is its potential to induce microorganisms into a viable but nonculturable (VBNC) state. These VBNC cells evade detection via conventional culturing techniques and may remain metabolically active and retain virulence, posing hidden food safety risks. Despite these implications, comprehensive reviews addressing the induction of a VBNC state by non-thermal treatments remain limited. This review systematically summarizes the microbial inactivation effects and mechanisms of non-thermal processing techniques, the VBNC state, and their associated hazards. This review aims to support technological innovation and sustainable advancement in non-thermal food processing. Full article

If lettuce is contaminated in the field, Shiga toxin-producing E. coli (STEC) O157:H7 can survive through the distribution chain. Prolonged cold storage during transportation may impact pathogen physiology, affecting subsequent stress survival and virulence. Greenhouse-grown Romaine lettuce, inoculated with three STEC O157:H7 strains, was harvested after 24 h and stored at 2 °C for 5 d following 4 h at harvest temperature (9 °C or 17 °C). Culturable, persister, and viable but non-culturable (VBNC) cells were quantified. Virulence was evaluated using Galleria mellonella and acid tolerance at pH 2.5 and tolerance to 20–25 ppm free chlorine were quantified. Colder harvest temperature (9 °C) before cold storage led to greater transformation of STEC O157:H7 into dormant states and decreased virulence in most cases. Increasing length of cold storage led to decreased virulence and acid tolerance of STEC O157:H7 on lettuce, while having no significant effect on chlorine tolerance. These findings highlight that entry of STEC O157:H7 into dormant states during harvest and transportation at cold temperatures leads to decreased stress tolerance and virulence with increasing cold storage. Changes in STEC O157:H7 physiology on lettuce during cold storage can be integrated into risk assessment tools for producers, which can assist in identifying practices that minimize risk of STEC O157:H7 from consumption of lettuce. Full article

This descriptive review summarizes the most recent findings on the induction and distribution of viable non-culturable (VBNC) Listeria monocytogenes in food production conditions and food. The aim was to obtain information on the factors that favor the transition to the VBNC state in L. monocytogenes; its resuscitation capacity; and, according to scientific articles published since 2020, how food contamination by the bacterium in a VBNC state can be prevented. The methods used for VBNC L. monocytogenes detection were also reviewed. A few studies reported the presence of VBNC L. monocytogenes in food, in which fresh produce and chicken meat were considered. Different physicochemical stresses such as exposure to disinfectants with diverse actions and essential oils, desiccation, low temperatures, absence of nutrients, high NaCl and iron concentrations, and low pH adjusted with acetic acid were reported to induce the VBNC state in L. monocytogenes. The VBNC forms of L. monocytogenes were able to regain growth and virulence. This could pose a safety risk that cannot be revealed by the standard culture-dependent methods recommended for L. monocytogenes detection. Therefore, the presence in food and food production plants of VBNC L. monocytogenes should be prevented by the appropriate use of hurdles and cleaning/disinfection procedures. The opportunity to harmonize VBNC cell detection methods for regular use in food safety evaluation also emerged. Full article

Effective antimicrobial and biofilm control strategies require an understanding of the differential effects of antimicrobial agents on the viability and culturability of microbial cells. A viable but non-culturable (VBNC) state, a survival strategy of non-spore-forming bacteria in response to adverse conditions, poses a significant challenge for public health and food safety. In the present study, we investigated the antimicrobial and antibiofilm effects of nisin and gallium (III) nitrate hydrate against the Gram-positive strain Lactiplantibacillus plantarum subsp. plantarum DSM 20174 and the Gram-negative strain Pseudomonas fluorescens ATCC 13525, respectively. Both strains were chosen as model systems for their relevance to food and clinical settings. Culture-based methods and flow cytometry (FCM) were used to evaluate the culturability and viability of both planktonic and sessile cells, providing insights into their physiological response to antimicrobial treatment-induced stress at different concentrations (100, 250, 350, and 500 ppm). The findings highlight the strain-specific action of nisin on L. plantarum and the promising antibiofilm effects of Ga (III) against P. fluorescens. This study underscores the promising potential of FCM as a powerful tool for high-throughput analyses of antimicrobial efficacy, providing valuable insights into developing targeted biofilm control strategies for food safety and clinical applications. Full article

Phenol and its chlorinated derivatives are introduced into the environment with wastewater effluents from various industries, becoming toxic pollutants. Phenol-degrading bacteria are important objects of research; among them, representatives of the genus Rhodoccocus are often highlighted as promising. Strain 7Ba was isolated by enrichment culture. A new isolate was characterized using culturing, biochemistry, high-throughput sequencing, microscopy (including electron microscopy), and functional genome analysis. Rhodococcus erythropolis strain 7Ba is able to grow on phenol and chlorophenols without losing its properties during long-term storage. It was shown that strain 7Ba is able to form viable but nonculturable (VBNC) forms during long-term storage under nutrient limitation, preserving both cell viability and the ability to degrade phenols. The ultrastructural organization of the vegetative forms of cells and VBNC forms was characterized. The following distinctive features were found: modifications (thickening) of cell membranes, cell size reduction, nucleoid condensation. Functional analysis of the genome showed the presence of genes for the degradation of alkanes, and two branches of the β-ketoadipate pathway for the degradation of aromatic compounds. Also, the genome of strain 7Ba contains several copies of Rpf (resuscitation promoting factor) genes, a resuscitation factor of resting bacterial forms. The new isolate strain 7Ba is a promising biotechnological agent that can not only utilize toxic aromatic compounds but also remain viable during long-term storage. For this reason, its further application as an agent for bioremediation can be successful under changing conditions of climate and given the deficiency of nutrient compounds in nature. Minor biostimulation will allow the strain to recover its metabolic activity and effectively degrade pollution. Full article

In this study, the performance of quantitative PCR, combined or not with propidium monoazide (PMA), to recover Salmonella from peanut products after different storage times was evaluated. The samples were inoculated with 5–6 log cfu g⁻¹ of Salmonella Typhimurium ATCC 14028 and stored at 28 °C for up to 540 d. The correlation between the threshold cycle number (Ct) and the colony-forming units (cfu) was obtained by a standard curve, which showed a linear correlation (R² = 0.97). The highest counts were recovered by qPCR (p < 0.05); however, it quantified both viable and non-viable cells. For roasted peanuts, a significant difference (p < 0.05) between qPCR-PMA and the culture method was verified only for samples stored for 30 d, i.e., 2.8 versus 4.0 log cfu g⁻¹. Further, there was no VBNC status in the roasted peanuts, even after long-term exposure to desiccation stress. For peanut-based products, after 540 d, only paçoca showed a significant difference (p < 0.05) among the three methods evaluated. In peanut brittle, qPCR-PMA detected 1.5 log cfu g⁻¹, while, in the culture method, Salmonella was recovered in 1 g. The pathogen was below the detection limit in pé-de-moça either by plate count or qPCR-PMA. Therefore, qPCR-PMA shows potential for use in quantifying Salmonella in peanut products. Full article

The general features of the shift to a dormant state in mycobacterial species include several phenotypic changes, reduced metabolic activities, and increased resistance to host and environmental stress conditions. In this study, we aimed to provide novel insights into the viability state and morphological changes in dormant M. smegmatis that contribute to its long-term survival under starvation or hypoxia. To this end, we conducted assays to evaluate cell viability, morphological changes and gene expression. During starvation, M. smegmatis exhibited a reduction in cell length, the presence of viable but non-culturable (VBNC) cells and the formation of anucleated small cells, potentially due to a phenomenon known as reductive cell division. Under hypoxia, a novel population of pleomorphic mycobacteria with a rough surface before the cells reached nonreplicating persistence 1 (NRP1) was identified. This population exhibited VBNC-like behaviour, with a loss of cell wall rigidity and the presence of lipid-body-like structures. Based on dosR and hspX expression, we suggest that M. smegmatis encounters reductive stress conditions during starvation, while lipid storage may induce oxidative stress during hypoxia. These insights into the heterogeneous populations presented here could offer valuable opportunities for developing new therapeutic strategies to control dormant mycobacterial populations. Full article

The purpose of the present study was to investigate the mechanism of action of our newly developed green sanitizer formulation comprising a natural phenolic compound, gallic acid (GA), strengthened by the Generally Recognized as Safe (GRAS) materials hydrogen peroxide (H₂O₂) and DL-lactic acid (LA). Combining 8 mM GA with 1 mM H₂O₂ resulted in an abundant generation of reactive oxygen species (ROS) and a bactericidal effect towards Gram-negative (Escherichia coli, Pseudomonas syringae, and Pectobacterium brasiliense) and Gram-positive (Bacillus subtilis) bacteria (4 to 8 log CFU mL⁻¹ reduction). However, the exposure to this dual formulation (DF) caused only a modest 0.7 log CFU mL⁻¹ reduction in the Gram-positive L. innocua population. Amending the DF with 20 mM LA to yield a triple formulation (TF) resulted in the efficient synergistic control of L. innocua proliferation without increasing ROS production. Despite the inability to grow on plates (>7 log CFU mL⁻¹ population reduction), the TF-exposed L. innocua maintained high intracellular ATP pools and stable membrane integrity. The response of L. innocua to TF could be qualified as a “viable but nonculturable” (VBNC) phenomenon, while with the other species tested this formulation caused cell death. This research system may offer a platform for exploring the VBNC phenomenon, a critical food safety topic. Full article

The viable but non-culturable (VBNC) state is a survival strategy for many foodborne pathogens under adverse conditions. Yersinia enterocolitica (Y. enterocolitica) as a kind of primary foodborne pathogen, and it is crucial to investigate its survival strategies and potential risks in the food chain. In this study, the effectiveness of ultraviolet (UV) irradiation and chlorine treatment in disinfecting the foodborne pathogen Y. enterocolitica was investigated. The results indicated that both UV irradiation and chlorine treatment can induce the VBNC state in Y. enterocolitica. The bacteria completely lost culturability after being treated with 25 mg/L of NaClO for 30 min and a UV dose of 100 mJ/cm². The number of culturable and viable cells were detected using plate counting and a combination of fluorescein and propidium iodide (live/dead cells). Further research found that these VBNC cells exhibited reduced intracellular Adenosine Triphosphate (ATP) levels, and increased levels of reactive oxygen species (ROS) compared to non-induced cells. Morphologically, the cells changed from a rod shape to a shorter, coccobacillary shape with small vacuoles forming at the edges, indicating structural changes. Both condition-induced VBNC-state cells were able to resuscitate in tryptic soy broth (TSB) medium supplemented with Tween 80, sodium pyruvate, and glucose. These findings contribute to a better understanding of the survival mechanisms of Y. enterocolitica in the environment and are of significant importance for the development of effective disinfection strategies. Full article

While confronted with unfavorable growth conditions, bacteria may transform into the dormant state, such as viable but nonculturable (VBNC) state, which is a reversible state characterized by low metabolic activity and lack of division. These dormant cells can be reactivated through the influence of the resuscitation promoting factor (Rpf) family, which are classified as autocrine growth factors and possess peptidoglycan hydrolase activities. To date, with the significant resuscitation or growth promotion ability of Rpf, it has been extensively applied to increasing bacterial diversity and isolating functional microbial species. This review provides a comprehensive analysis of the distribution, mode of action, and functional mechanisms of Rpf proteins in various bacterial species. The aim is to create opportunities for decoding microbial communities and extracting microbial resources from real samples across different research fields. Full article

Conventional disinfection techniques, relying on a single disinfection step, often fail to directly eliminate microorganisms, instead causing them to enter a viable but nonculturable (VBNC) state. However, microorganisms in the VBNC state retain metabolic activity and can reactivate under suitable conditions, representing a “hidden source of contamination” that threatens drinking water safety. This study fundamentally assessed the feasibility of combined disinfection methods by integrating UV₂₅₄ with disinfectant (NaClO, PAA, and PDS) for inactivating Pseudomonas aeruginosa (P. aeruginosa), an opportunistic pathogen that has been widely detected in water supply systems. The number of culturable cells was determined using the heterotrophic plate counting (HPC) method, and the number of VBNC cells was quantified using our recently developed qPCR approach. Quantitative analyses showed that combined disinfection methods can effectively reduce both culturable and VBNC cells by several orders of magnitude compared to a single disinfection step. Notably, VBNC P. aeruginosa, after 30 min of UV/NaCIO treatment, was below the detection limit (3.191 log CFU/mL) of PMA-qPCR. The reactivation experiment also confirmed that VBNC P. aeruginosa did not reactivate for 16 h after 30 min of UV/NaClO treatment under controlled laboratory conditions. The higher disinfection capacity of combined methods can be attributed to the generation of reactive radicals. This study highlighted combined disinfection as a promising approach for the inactivation of bacteria in the VBNC state, yet further studies are needed before an application can be considered for minimizing VBNC reactivation in city utility water processing or high-risk building water distribution systems. Full article

The bacterium Erwinia amylovora causes fire blight and continues to threaten global commercial apple and pear production. Conventional microbiology techniques cannot accurately determine the presence of live pathogen cells in fire blight cankers. Several factors may prevent E. amylovora from growing on solid culture media, including competing microbiota and the release of bacterial-growth-inhibitory compounds by plant material during sample processing. We previously developed a canker processing methodology and a chip-based viability digital PCR (v-dPCR) assay using propidium monoazide (PMA) to bypass these obstacles. However, sample analysis was still time-consuming and physically demanding. In this work, we improved the previous protocol using an automatic tissue homogenizer and transferred the chip-based v-dPCR to the BioRad QX200 droplet dPCR (ddPCR) platform. The improved sample processing method allowed the simultaneous, fast, and effortless processing of up to six samples. Moreover, the transferred v-ddPCR protocol was compatible with the same PMA treatment and showed a similar dynamic range, from 7.2 × 10² to 7.6 × 10⁷ cells mL⁻¹, as the previous v-dPCR. Finally, the improved protocol allowed, for the first time, the detection of E. amylovora viable but nonculturable (VBNC) cells in cankers and bark tissues surrounding cankers. Our v-ddPCR assay will enable new ways to evaluate resistant pome fruit tree germplasm, further dissect the E. amylovora life cycle, and elucidate E. amylovora physiology, epidemiology, and new options for canker management. Full article

In harsh environments, bacteria often enter a viable but nonculturable (VBNC) state, which cannot be detected using heterotrophic plate counting (HPC). Importantly, VBNC bacteria can potentially resuscitate under favorable conditions, posing a risk to drinking water safety. This study introduces an innovative approach, combining improved quantitative polymerase chain reaction (qPCR) with propidium monoazide (PMA) dye and HPC to accurately quantify VBNC Pseudomonas aeruginosa (P. aeruginosa). The method was applied to assess the ability of various disinfection techniques to induce P. aeruginosa into the VBNC state. Different disinfection methods, including ultraviolet radiation (UV), sodium hypochlorite (NaClO), and peracetic acid (PAA), significantly reduced bacterial culturability (>99.9%), with the majority entering the VBNC state. Notably, under favorable conditions, UV-induced VBNC cells were resuscitated faster than those induced by NaClO. VBNC P. aeruginosa exhibited relatively high intracellular adenosine triphosphate (ATP) levels, indicating ongoing metabolic activity. Scanning electron microscopy (SEM) reveals that some bacteria maintained cellular integrity for UV and PAA treatment, while evident membrane disruption was observed after NaClO disinfection. This study represents a significant advancement in quantitatively detecting VBNC state P. aeruginosa, contributing to an accurate assessment of microbial inactivation during drinking water disinfection. Full article

Many bacteria have the ability to survive in challenging environments; however, they cannot all grow on standard culture media, a phenomenon known as the viable but non-culturable (VBNC) state. Bacteria commonly enter the VBNC state under nutrient-poor environments or under stressful conditions. This review explores the concept of the VBNC state, providing insights into the beneficial bacteria known to employ this strategy. The investigation covers different chemical and physical factors that can induce the latency state, cell features, and gene expression observed in cells in the VBNC state. The review also covers the significance and applications of beneficial bacteria, methods of evaluating bacterial viability, the ability of bacteria to persist in environments associated with higher organisms, and the factors that facilitate the return to the culturable state. Knowledge about beneficial bacteria capable of entering the VBNC state remains limited; however, beneficial bacteria in this state could face adverse environmental conditions and return to a culturable state when the conditions become suitable and continue to exert their beneficial effects. Likewise, this unique feature positions them as potential candidates for healthcare applications, such as the use of probiotic bacteria to enhance human health, applications in industrial microbiology for the production of prebiotics and functional foods, and in the beer and wine industry. Moreover, their use in formulations to increase crop yields and for bacterial bioremediation offers an alternative pathway to harness their beneficial attributes. Full article