Salmonella enterica biofilm is capable of VBNC state formation and virulence gene expression during low temperature food storage.

Dry surface biofilm of Salmonella and Cronobacter sakazakii: a real concern for the low moisture food industry.

Viable but Nonculturable Listeria monocytogenes May Resuscitate After Transfer From Stainless Steel Biofilms to Vacuum-Packed Smoked Herring.

Bioelectrochemical detection of viable but non-culturable bacterial cells on screen-printed carbon electrodes.

The effects and physiological phenotypic changes in Escherichia coli O157:H7 induced into VBNC state by hydrogen peroxide silver ion treatment in biofilms.

ClO₂-induced VBNC state in Enterococcus faecalis poses a health risk in drinking water systems.

A substantial proportion of symptomatic patients with urinary tract infections (UTIs) have negative or low-count urine cultures despite clinically compatible presentations. Biofilm-associated bacterial aggregation and the presence of viable bacteria not recovered by standard culture may contribute to this diagnostic gap. Flow cytometry (FACS) can detect these viable cells, but culturability remains essential for species identification and antimicrobial susceptibility testing. We evaluated whether low-concentration dithiothreitol (DTT) can safely disperse urinary biofilm-associated aggregates and improve bacterial recovery from urine samples. Seventy-two clinical urine samples were processed in parallel (untreated vs 0.1% DTT-treated). Viability was quantified using SYTO9/PI-based flow cytometry with bead-normalized bacterial counts. Culturability was assessed on chromogenic media. Comparative analyses included paired statistics, Bland-Altman agreement, and correlation between viable and culturable fractions. DTT (0.1%) did not alter bacterial viability (P = 0.47), confirming its non-toxicity at this concentration. In contrast, DTT significantly increased CFU recovery (median: 10 to 5.5 × 10³ CFU/mL; P < 0.0001), converting several previously negative or low-count samples into positive cultures. Agreement between viable and culturable fractions improved after DTT treatment, primarily through reduced systematic bias in Bland-Altman analysis, while correlation coefficients remained similar (r: 0.44 vs 0.45). A persistent discrepancy between FACS-derived viable counts and culture-based counts suggests the presence of viable bacterial populations not fully recovered by standard culture conditions, findings that are consistent with but do not directly demonstrate the presence of VBNC subpopulations. Low-dose DTT is a safe and effective pre-analytical treatment that markedly improves culture sensitivity without compromising bacterial viability. These findings support further investigation of DTT-assisted processing as a potential strategy to reduce culture underestimation in urinary diagnostics and to better characterize viable bacterial populations not detected by standard culture methods.IMPORTANCEStandard urine culture may fail to detect the full spectrum of bacteria present in urinary tract infections, particularly when microorganisms are embedded within mucus, biofilm-like aggregates, or enter a viable but non-culturable state. These hidden bacterial populations may contribute to persistent symptoms or recurrent infections while remaining undetected by routine culture methods. Our findings suggest that pre-treating urine samples with a low concentration of dithiothreitol may help disperse biofilm-like aggregates while preserving bacterial viability. When combined with flow cytometry, this approach may allow a more comprehensive estimation of the viable bacterial fraction and, in some cases, may increase the likelihood of bacterial recovery by culture in samples that would otherwise appear negative. Because the procedure relies on inexpensive reagents and techniques compatible with routine laboratory workflows, it may represent a practical adjunct to conventional diagnostics. Further studies are needed to confirm its clinical impact.

The rise in global consumption of fresh vegetables is a response to their nutrient-dense composition and low caloric content—key factors for optimising human metabolic health. This study evaluated the Quantitative Microbial Risk Assessment (QMRA) of Helicobacter pylori and enteric pathogens in fresh vegetables within the central highlands of Peru. The research integrated conventional microbiology, qPCR, and Monte Carlo simulations. The results revealed a high prevalence of Escherichia coli (83.7%), with a heterogeneous distribution where Huancayo presented the highest prevalence (95.5%) and Chupaca the lowest (68.2%). In contrast, pathogens such as H. pylori and Campylobacter jejuni showed marginal prevalences of 2.33% and 3.49%, respectively, with detections restricted to leafy and root vegetables at specific points of sale. Although biochemical tests indicated the presumptive presence of Helicobacter pylori, the qPCR results were negative, possibly due to the bacteria’s viable but non-culturable (VBNC) state. The QMRA model showed a highly skewed annual infection risk distribution, with E. coli presenting the highest risk: median Pann = 1.000 and 84.3% of simulations exceeding the WHO tolerable threshold of 10−4. For Salmonella Typhimurium and Shigella flexneri, 22.4% and 9.1% of simulations exceeded the same threshold, respectively. The results underscore the urgent need to implement traceability programs and improve agricultural practices across the evaluated provinces.

• Untreated wood supports higher bacterial diversity and richer communities. • S. epidermidis survives on untreated wood; P. aeruginosa loses culturability. • Wood type and surface treatments can strategically shape surface microbiomes. • Findings motivate the development of hygienic and probiotic wood material designs. Wood, beyond its structural and aesthetic qualities, exhibits antibacterial activity that may influence microbial persistence and transmission. However, bacterial adherence to wood, particularly in interior materials, remains understudied. Understanding the dynamics of bacterial adhesion, survival, and transmission requires a combination of laboratory and field research, especially given the variability of microbial diversity under real-use conditions. This two-part study has examined bacterial survival on untreated and treated wood surfaces. First, in vitro experiments assessed the adherence and viability of two model bacteria, Staphylococcus epidermidis and Pseudomonas aeruginosa . Secondly, public exposure trials evaluated bacterial colonization under real-world conditions using contact plates for sampling and 16S rRNA sequencing to analyze microbial diversity, including Shannon index and ASV richness. Metrics were assessed via linear mixed models. In vitro, P. aeruginosa cell counts declined rapidly across all materials, with viability staining suggesting many cells entered a viable but non-culturable state. In contrast, S. epidermidis showed enhanced survival on untreated wood, particularly on pine. Public trials mirrored these findings: untreated wood supported higher species richness, colony-forming units, and bacterial viability, whereas treated surfaces consistently exhibited lower microbial diversity and Shannon index values. Overall, these results demonstrate that untreated wood fosters more diverse and resilient microbial communities than treated wood, suggesting that surface treatment alters wood’s ecological role in shaping bacterial persistence. These findings highlight the importance of material selection in environments where microbial interactions impact hygiene, durability, and human health.

Early antiseizure medication tapering in 2-18-year-olds with single parenchymal neurocysticercosis based on risk assessment: A prospective intervention study.

Development and evaluation of a PMA-ddPCR for accurate detection of viable hypervirulent Klebsiella pneumoniae.

This study investigated the bactericidal effect and examined the associated cellular damage of low temperature plasma (LTP) combined with slightly acidic electrolyzed water (SAEW) against Listeria monocytogenes. Single-factor experiments were conducted to assess the bactericidal efficacy under individual treatment conditions, followed by the evaluation of three different combination sequences. An orthogonal experimental design was performed to optimize the key parameters, and the optimal treatment conditions were determined as LTP at 45 W with an electrode spacing of 1 mm for 2 min, combined with SAEW at an available chlorine concentration (ACC) of 30 mg/L. Under these conditions, confocal laser scanning microscopy (CLSM) with SYTO 9/PI staining confirmed that the combined treatment caused cell death, as indicated by loss of membrane integrity in treated cells. A resuscitation assay further ruled out the viable but non-culturable (VBNC) state, as no bacterial growth was detected after 48 h of enrichment. The leakage of intracellular proteins and nucleic acids was measured using the Coomassie Brilliant Blue method combined with a microplate reader, and changes in cellular morphology were observed by scanning electron microscopy (SEM). The results demonstrated that SAEW+LTP treatment exerted a distinct effect, significantly disrupting bacterial cell membrane integrity, inducing the leakage of intracellular contents, and causing obvious morphological damage to the bacterial cells. In conclusion, the combined treatment of LTP and SAEW significantly improved the bactericidal efficiency against L. monocytogenes, which may be due to the combined disruptive effects on membrane integrity and subsequent structural and functional damage to the cells. Future investigations are needed to unravel the precise mechanisms, establish the efficacy against a wider panel of strains, and explore the potential for practical application in food matrices.

This study systematically compared the induction and resuscitation characteristics of the viable but non-culturable (VBNC) state in Klebsiella pneumoniae FY170-1 following sublethal exposure to sodium hypochlorite (NaClO) or glutaraldehyde (GA). Treatment with 30 mg/L NaClO or 60 mg/L GA for 60 min reduced culturability to below the detection limit (<1 CFU/mL). However, CTC staining showed that 50.80% and 63.44% of cells, respectively, retained respiratory activity, while SYTO 9/PI staining indicated that membrane integrity was largely preserved, consistent with induction of the VBNC state. Scanning electron microscopy revealed distinct morphological alterations in the two groups. NaClO-induced VBNC cells showed surface depressions and wrinkling, consistent with oxidative damage, whereas GA-induced cells exhibited filamentous and net-like surface structures, consistent with aldehyde-mediated cross-linking. Among the tested additives, sodium succinate showed the strongest resuscitation-promoting effect under the experimental conditions, with OD600 increasing after approximately 2 h of incubation. Post-resuscitation analysis further revealed marked differences between the two VBNC states. In resuscitated NaClO-induced VBNC cells, ATP partially recovered, but reactive oxygen species remained elevated and catalase activity showed little recovery. In contrast, resuscitated GA-induced VBNC cells exhibited lower ATP recovery but more rapid normalization of ROS and better recovery of oxidative stress-related parameters. Total protein analysis and SDS-PAGE further supported distinct patterns of protein-level alteration between the two treatments. Overall, these findings suggest that NaClO and GA induce phenotypically distinct VBNC states in K. pneumoniae, with different recovery behaviors and stress response profiles. Sodium succinate was identified as the most effective recovery-promoting additive under the tested conditions. These results highlight the risk of underestimating bacterial survival when culturability is used as the sole indicator of disinfection efficacy and support the need for more comprehensive viability assessment.

Aeromonas hydrophila is a major seafood-borne pathogen capable of persisting under preservative-associated stress by entering a viable but non-culturable (VBNC) state, thereby evading culture-based detection. Here, untargeted metabolomics was applied as the primary analytical approach to elucidate metabolic reprogramming during VBNC formation under seafood-relevant preservation conditions. Cells were incubated at 4 °C for 30 days in sodium benzoate-supplemented saline, comparing 0.85% NaCl (culturable condition) and 4% NaCl (VBNC-inducing condition), with sampling every 6 days. Under 4% NaCl with sodium benzoate, culturability declined from 6.18 log CFU/mL at day 0 to undetectable levels by day 30, while cell viability was retained, confirming VBNC induction. UHPLC-ESI-QTOF-MS profiling detected over 893 intracellular metabolic features, of which 518 metabolites were significantly altered between VBNC and culturable states at day 30. Principal component analysis revealed clear, time-dependent metabolic divergence, with the VBNC trajectory explaining 34.4% (PC1) and 11.5% (PC2) of total variance. Pathway enrichment analysis demonstrated significant remodeling of alanine, aspartate and glutamate metabolism (8/28 hits, FDR = 5.7 × 10-4); arginine biosynthesis (5/14 hits, FDR = 5.44 × 10-3); purine metabolism (10/70 hits, FDR = 8.34 × 10-3); and pyrimidine metabolism (7/39 hits, FDR = 1.35 × 10-2), indicating nitrogen conservation and metabolic downshifting. A robust biomarker panel, including depleted cyclic AMP, aminoadipic acid, hypotaurine, O6-CM-dG, and betaine, and enriched urocanic acid, pipecolic acid, proline, azelaic acid, and orcinol perfectly discriminated VBNC from culturable cells. These findings demonstrate that sodium benzoate-based preservation can induce a metabolically reprogrammed VBNC state in A. hydrophila, highlighting a hidden food safety risk beyond culture-based assessment.

Cronobacter spp. is an opportunistic foodborne pathogen responsible for life-threatening infections such as meningitis and necrotizing enterocolitis in neonates, but also for various complications in elderly and immunocompromised people. Environmental monitoring in powdered milk manufacturing plants shows that Cronobacter can persist in production environments for long periods of time. The aim of this study is to develop a method to assess the viability of Cronobacter cells after implementation of stress encountered in industries to determine whether this bacterium can persist in processing environments in a dormant state known as Viable But Non-Cultivable (VBNC) state, making it undetectable by conventional enumeration methods. We developed two detection systems specific to the genus Cronobacter based on quantitative PCR (qPCR) and Droplet Digital PCR (ddPCR) in combination with a PMAxx™ (Propidium Monoazide) treatment and agar enumeration to assess the viability of detected cells. Despite a better sensitivity for ddPCR, qPCR-PMAxx™ was more suitable for VBNC detection, as it effectively differentiates viable from dead cells. Desiccation at 58°C and 20% relative humidity led to a significant reduction in culturable bacteria, with a drop from 6.82 to 2.58 log copies of genome per coupon over 48h, while qPCR-PMAxx™ revealed that the proportion of VBNC cells represents approximately 1/100 of the total population of viable bacteria (V). Similarly, treatment of Cronobacter biofilms with a peracetic acid containing disinfectant induced the VBNC state in Cronobacter. The ability of this bacterium to enter the VBNC state may explain its long-term survival in processing plants and highlights the need for appropriate detection methods for effective environmental monitoring plans.

Thermal processing of functional beverages often drives probiotics into a viable but non-culturable (VBNC) state, causing severe underestimation by conventional colony-forming unit (CFU) assays. To develop and assess an anaerobic, catalase-assisted resuscitation approach that restores culturability of heat-stressed Lacticaseibacillus rhamnosus GG and aligns CFU counts with actual viable populations. Dairy beverages (95 °C, 30 s; 2 × 106 cells/mL, pH 3.9) containing encapsulated L. rhamnosus GG were incubated at 37 °C for 16 h in Rogosa/MRS broth supplemented with 50-100 U/mL catalase under anaerobic conditions prior to plating. Recovery was benchmarked against flow cytometry (active fluorescent units, AFU), while pH and glucose monitoring confirmed resuscitation rather than growth. Method performance was examined across three production batches (single-laboratory study) and further evaluated in a multi-laboratory setting. Direct plating yielded no CFU, whereas flow cytometry detected 6.34 ± 0.04 log10 AFU/g. After resuscitation, CFU recovered to 6.05 ± 0.05 log10/g (CFU/AFU ratio = 0.86 ± 0.03; p = 0.12). Omitting catalase or anaerobiosis reduced recovery by > 1.9 log10 CFU/g. CFU restoration occurred without acidification or glucose depletion, confirming true resuscitation. Single-laboratory reproducibility showed CV < 2%; inter-laboratory reproducibility was <15% RSD. Application to heat-treated milk (pH 6.85) yielded 1.74 × 106 CFU/g (CV 15.9%, n = 4), supporting method suitability across other dairy beverages. The catalase-assisted, anaerobic resuscitation developed protocol provides a reproducible workflow for probiotic enumeration in heat-treated beverages. The method is compatible with AOAC/ISO standards, addresses underestimation of counts, and supports adoption into quality control (QC) laboratories for accurate probiotic labelling. Heat-stressed L. rhamnosus GG cells recovered culturability when incubated with catalase under anaerobic conditions, restoring CFU counts to AFU-equivalent levels and confirming true resuscitation rather than growth.

Sustainable food sanitation and preservation technologies are highly needed to meet the increasing demand for healthy and safe food. In this work, we investigated the effects of a recently developed antimicrobial triple formulation (TF) comprised of a natural polyphenol gallic acid amended with the generally recognized as safe materials hydrogen peroxide and DL-lactic acid, on a gram-positive bacterium Listeria innocua during its exponential and stationary growth phases. Our findings revealed the physiological changes associated with the application of this material, as observed using single-cell flow cytometry, photometric assays, as well as confocal and scanning electron microscopy. Both exponential- and stationary-phase L. innocua cells lost culturability after incubation with TF, but their cellular responses were different. After 30 min of contact with the TF, the nonculturable, stationary-phase cells maintained membrane integrity, membrane potential, stable respiratory electron-transport chains, and high levels of intracellular ATP. These phenotypic features, characteristic of the viable but nonculturable (VBNC) adaptation strategy, appear to be associated with upregulation of the stress-response factor sigB. In contrast, in TF-treated exponential-phase cells, membrane integrity was severely compromised and membrane potential showed hyperpolarization, suggesting sublethal injury accompanied by downregulation of sigB. Furthermore, the TF treatment enhanced the formation of extracellular vesicles in L. innocua and the deposition of extracellular polymeric substances in stationary-phase cells. The overall findings of this work demonstrate that sensitivity of Listeria cells to nature-based antimicrobials depends on their growth phase and, therefore, modulating their growth-phase-associated physiological status may counteract their VBNC defense strategy and improve biocidal efficacy.IMPORTANCEIn this work, we examined the efficacy of our novel, nature-based green sanitizer as a means to improve microbiological food safety without health and environmental risks, using gram-positive Listeria innocua as a model organism. L. innocua serves as a surrogate of the foodborne pathogen L. monocytogenes for evaluating sanitation efficacy and may also be involved in virulence and multidrug resistance transfer between the Listeria species. We demonstrate that the sanitizer effect depends on the physiological state of bacterial cells, which is affected by their growth phase. The treatment caused membrane damage to L. innocua in its exponential phase but induced the viable but nonculturable state during its stationary phase. Thus, this study demonstrates a way to overcome bacterial defense mechanisms and improve sanitizer efficacy by modulating the growth-phase-associated physiological status of targeted bacteria.

The viable but non-culturable (VBNC) state is a stress adaptation strategy in bacteria, characterized by the loss of culturability and reduced metabolism, which may complicate both diagnosis and treatment. This review describes the potential transition of Staphylococcus aureus into a VBNC state during intramammary infections, and in dairy-associated food products. It further distinguishes VBNC cells from persister cells, another non-proliferative stress adaptation in bacterial cells. We propose that the resuscitation window, in terms of both the intensity and duration of stress, can serve as a valuable indicator offering insight into the reactivation process following transformation into the VBNC state. Current pasteurization and other food processing techniques may not be sufficient in addressing the VBNC state, posing a potential risk of foodborne illnesses following undetected contamination. This necessitates the optimization of advanced diagnostic tools, for use in complex matrices, including milk and other dairy products, to effectively estimate VBNC cells, prevent hidden contamination in milk and other dairy products, and safeguard both public and animal health.

Introduction/Objective The aim of the study was to develop a technological approach for the production, storage, and preparation of strawberry microplants, facilitating their simultaneous acclimatization before planting in a greenhouse when favorable temperature and lighting conditions are established. Methods The initial material for the experiment was obtained via clonal micropropagation. Preparation, accumulation, and preservation of strawberry plants in vitro for delayed acclimatization were performed by periodically repeating the procedure of removing roots and leaves from previously rooted microplants and transferring them in the form of vegetating buds onto a fresh nutrient medium for further rehabilitation. The mass acclimatization of microplants was carried out under substrate-free flow-through hydroponic conditions, following which they were transplanted into a greenhouse in early spring. Results The survival rate of microplants during the periodically repeated procedures of removing roots and leaves from previously obtained microplants reached 100% for all studied cultivars. Active leaf and root recovery began on days 3–7 and 10–14, respectively. A 1-month acclimatization period on flow-through hydroponics allowed for a 2–2.5-fold increase in microplant height, a 2-fold increase in root length, and an almost 3-fold increase in weight. Discussion The repetitive removal of microplant roots and leaves, with their further transplantation on fresh nutrient medium as vegetating buds for subsequent rehabilitation during periods of unfavorable environmental conditions, does not reduce the viability of microplants, allowing the preservation of planting material without loss until acclimatization is advisable. Acclimatization using flow-through hydroponics enables the elimination of fungal pathogenesis that occurs during the use of solid substrates. This protocol also increases the predictability of acclimatization outcomes and ensures successful transplantation into a greenhouse. Conclusion The developed alternative strategy of strawberry clonal micropropagation allows prediction of the output volume of acclimatized microplants throughout a calendar year with a high degree of accuracy. The estimated quantity of such material can be compactly stored in a lighted tissue culture room, significantly saving time and resources. Microplants are well preserved until a favorable moment for acclimatization in a viable state as vegetating buds. After the rehabilitation cycles are complete, the microplants can be simultaneously transferred for acclimatization on flow-through hydroponics.

Ciprofloxacin and sulfadiazine at environmentally relevant concentrations induce a viable but non-culturable state in Escherichia coli.