Viable But Nonculturable Cells Research Articles

Nanowire-assisted electroporation via inducing cell destruction for inhibiting formation of VBNC bacteria: Comparison with chlorination

The induction of viable but nonculturable (VBNC) bacteria with cellular integrity and low metabolic activity by chemical disinfection causes a significant underestimation of potential microbiological risks in drinking water. Herein, a physical Co3O4 nanowire-assisted electroporation (NW-EP) was developed to induce cell damage via the locally enhanced electric field over nanowire tips, potentially achieving effective inhibition of VBNC cells as compared with chemical chlorination (Cl2). NW-EP enabled over 5-log removal of culturable cell for various G+/G- bacteria under voltage of 1.0 V and hydraulic retention time of 180 s, and with ∼3–6 times lower energy consumption than Cl2. NW-EP also achieved much higher removals (∼84.6 % and 89.5 %) of viable Bacillus cereus (G+) and Acinetobacter schindleri (G-) via generating unrecoverable pores on cell wall and reversible/irreversible pores on cell membrane than Cl2 (∼28.6 % and 41.1 %) with insignificant cell damage. The residual VBNC bacteria with cell wall damage and membrane pore resealing exhibited gradual inactivation by osmotic stress, leading to ∼99.8 % cell inactivation after 24 h storage (∼59.4 % for Cl2). Characterizations of cell membrane integrity and cell morphology revealed that osmotic stress promoted cell membrane damage for the gradual inactivation of VBNC cells during storage. The excellent adaptability of NW-EP for controlling VBNC cells in DI, tap and lake waters suggested its promising application potentials for drinking water, such as design of an external device on household taps.

Understanding the transition to viable but non-culturable state in Escherichia coli W3110: a comprehensive analysis of potential spectrochemical biomarkers

The viable but non-culturable (VBNC) state is considered a survival strategy employed by bacteria to endure stressful conditions, allowing them to stay alive. Bacteria in this state remain unnoticed in live cell counts as they cannot proliferate in standard culture media. VBNC cells pose a significant health risk because they retain their virulence and can revive when conditions normalize. Hence, it is crucial to develop fast, reliable, and cost-effective methods to detect bacteria in the VBNC state, particularly in the context of public health, food safety, and microbial control assessments. This research examined the biomolecular changes in Escherichia coli W3110 induced into the VBNC state in artificial seawater under three different stress conditions (temperature, metal, and antibiotic). Initially, confirmation of VBNC cells under various stresses was done using fluorescence microscopy and plate counts. Subsequently, lipid peroxidation was assessed through the TBARS assay, revealing a notable increase in peroxidation end-products in VBNC cells compared to controls. ATR-FTIR spectroscopy and chemomometrics were employed to analyze biomolecular changes, uncovering significant spectral differences in RNA, protein, and nucleic acid concentrations in VBNC cells compared to controls. Notably, RNA levels increased, while protein and nucleic acid amounts decreased. ROC analyses identified the 995 cm− 1 RNA band as a consistent marker across all studied stress conditions, suggesting its potential as a robust biomarker for detecting cells induced into the VBNC state under various stressors.

Deletion of codY impairs Staphylococcus epidermidis biofilm formation, generation of viable but non-culturable cells and stimulates cytokine production in human macrophages.

Introduction. Staphylococcus epidermidis biofilms are one of the major causes of bloodstream infections related to the use of medical devices. The diagnosis of these infections is challenging, delaying their treatment and resulting in increased morbidity and mortality rates. As such, it is urgent to characterize the mechanisms employed by this bacterium to endure antibiotic treatments and the response of the host immune system, to develop more effective therapeutic strategies. In several bacterial species, the gene codY was shown to encode a protein that regulates the expression of genes involved in biofilm formation and immune evasion. Additionally, in a previous study, our group generated evidence indicating that codY is involved in the emergence of viable but non-culturable (VBNC) cells in S. epidermidis.Gap statement/Hypothesis. As such, we hypothesized that the gene codY has have an important role in this bacterium virulence.Aim. This study aimed to assess, for the first time, the impact of the deletion of the gene codY in S. epidermidis virulence, namely, in antibiotic susceptibility, biofilm formation, VBNC state emergence and in vitro host immune system response.Methodology. Using an allelic replacement strategy, we constructed and then characterized an S. epidermidis strain lacking codY, in regards to biofilm and VBNC cell formation, susceptibility to antibiotics as well as their role in the interaction with human blood and plasma. Additionally, we investigate whether the codY gene can impact the activation of innate immune cells by evaluating the production of both pro- and anti-inflammatory cytokines by THP-1 macrophages.Results. We demonstrated that the deletion of the gene codY resulted in biofilms with less c.f.u. counts and fewer VBNC cells. Furthermore, we show that although WT and mutant cells were similarly internalized in vitro by human macrophages, a stronger cytokine response was elicited by the mutant in a toll-like receptor 4-dependent manner.Conclusion. Our results indicate that codY contributes to S. epidermidis virulence, which in turn may have an impact on our ability to manage the biofilm-associated infections caused by this bacterium.

The effects and cell phenotype changes in Escherichia coli O157:H7 induced into VBNC state by l-malic acid at low temperature

Under environmental stress conditions, microorganisms can form a viable but non-culturable (VBNC) state, which poses a potential risk to food safety and human health. l-Malic acid (L-MA) is widely found in low-temperature fruit and vegetable juices as a common natural organic acid as well as a food additive. However, there is no report on whether L-MA in food processing can promote the formation of VBNC states. In this study, we investigated the pattern of the L-MA-induced VBNC state of Escherichia coli O157:H7 as a model organism, as well as the characteristics at the cellular and molecular levels. We discovered that L-MA can induce the VBNC state in a significant number of E. coli cells, a process accelerated by low temperatures (4 °C). Our analysis revealed that VBNC cells induced by both L-MA and 4 °C resuscitated earlier than those subjected to either condition alone, indicating increased food safety risks. For survival, L-MA-induced VBNC E. coli cells became substantially smaller and exhibited a significant decrease in respiratory activity while maintaining the basic function of the cell membrane. These cellular changes were supported by molecular-level evidence, reinforcing the implications for food safety under these conditions. This study underscores the importance of recognizing the potential risks posed by L-MA and low temperatures in food preservation, particularly in terms of underestimating the presence of harmful bacteria in food products. The insights gained from this research could contribute to the development of more effective food safety measures, particularly in the handling and processing of fruit and vegetable juices.

Induction of Salmonella Enteritidis into a Viable but Nonculturable State by Cinnamaldehyde and Its Resuscitation.

Trans-cinnamaldehyde (TC), a typical plant-derived compound, has been widely used in the control of foodborne pathogen contamination. Nevertheless, the risk associated with the occurrence of viable but nonculturable (VBNC) bacteria induced by TC remains unclear. The results of this study showed that Salmonella Enteritidis (S. Enteritidis) entered the VBNC state after being induced by TC at a minimum inhibitory concentration of 312.5 μg/mL and survived for at least 22 days under TC treatment. Enhanced resistance was found against heat treatment (75°C, 30 s), antibiotics (i.e., ampicillin, ceftriaxone sodium, chloramphenicol), and hydrogen peroxide (3%) in VBNC S. Enteritidis. A synergistic effect against VBNC S. Enteritidis occurred when TC was combined with acid treatment, including lactic acid and acetic acid (pH = 3.5). VBNC and resuscitated S. Enteritidis by sodium pyruvate treatment (100 mM) were found to retain the infectious ability to Caco-2 cells. Relative expression levels of the stress-related genes relA, spoT, ppx, lon, katG, sodA, dnaK, and grpE were upregulated in VBNC S. Enteritidis. Accumulation of reactive oxygen species (ROS) and protein aggregates was observed in VBNC cells. Besides, the resuscitation of VBNC cells was accompanied with clearance of ROS and protein aggregates. In summary, this study presents a comprehensive characterization of stress tolerance and resuscitation of VBNC S. Enteritidis induced by cinnamaldehyde, and the results provide useful information for the development of effective control strategy against VBNC pathogenic bacteria in food production.

A Combination of UV and Disinfectant for Inactivating Viable but Nonculturable State Pseudomonas aeruginosa: Efficiency and Mechanisms

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.

Characterization of viable but nonculturable state of Campylobacter concisus

Campylobacter concisus is an opportunistic bacterial pathogen linked with a range of human diseases. The objective of this study was to investigate the viable but nonculturable (VBNC) state of the bacterium. To induce the VBNC state, C. concisus cells were maintained in sterilized phosphate-buffered saline at 4°C for three weeks. The VBNC cells were monitored using quantitative analysis by propidium monoazide (PMAxx) coupled with quantitative real-time PCR (PMAxx-qPCR), targeting the DNA gyrase subunit B gene. The results demonstrated that C. concisus ATCC 51562 entered the VBNC state in 15 days, while ATCC 51561 entered the VBNC state in 9 days. The viable cell counts, assessed by PMAxx-qPCR, consistently remained close to the initial level of 10 7 CFU ml −1 , indicating a substantial portion of the cell population had entered the VBNC state. Notably, morphological analysis revealed that the VBNC cells became coccoid and significantly smaller. The cells could be resuscitated through a temperature increase in the presence of a highly nutritious growth medium. In conclusion, under environmental stress, most C. concisus cells converted to the VBNC state. The VBNC state of C. concisus may be important for its environmental survival and spread, and the presence of VBNC forms should be considered in environmental and clinical monitoring.

Unveiling the distribution characteristics of rpf-like genes and indigenous resuscitation promoting factor production in PCB-contaminated soils

Resuscitation promoting factors (Rpfs), known for their anti-dormancy cytokine properties, have been extensively investigated in the medical field. Although the Rpf from Micrococcus luteus has been successfully utilized to resuscitate and stimulate microbial populations for the degradation of polychlorinated biphenyls (PCBs), the presence of indigenous Rpf homologs in PCB-contaminated soils has not been established. In this study, the distribution characteristics of rpf-like genes and indigenous strain capable of producing Rpf in PCB-contaminated soils were explored. The results revealed the widespread presence of Rpf-like domains and their associated genes, particularly in close association with heavy metals and PCBs. The rpf-like genes were predominantly found in Proteobacteria and displayed a positive correlation with genes involved in PCB degradation and viable but non-culturable (VBNC) formation. Notably, the recombinant Rpf-Ac protein derived from the indigenous strain Achromobacter sp. HR2 exhibited muralytic activity and demonstrated significant efficacy in resuscitating the growth of VBNC cells, while also stimulating the growth of normal cells. These findings shed light on the prevalent presence of Rpf homologs in PCB-contaminated soils and their potential to resuscitate functional populations in the VBNC state, thereby enhancing in situ bioremediation.

Development, evaluation and application of a polymerase spiral reaction (PSR) based assay on accurate detection of Vibrio parahaemolyticus viable cells and virulence factors from rice product

Vibrio parahaemolyticus is a predominate cause of microbiological food safety. This study aimed to develop a polymerase spiral reaction (PSR) based assay for rapid and visual detection on V. parahaemolyticus. Firstly, the specificity and conservativity of species-specific target tlh gene were determined. Secondly, targeting tlh and virulence factors tdh and trh, PSR assay was developed and optimized using constructed positive plasmid controls and standard strain. Visualized result determination using fluorescent dye was applied to avoid false positive issues. Thirdly, 44 strains were used for specificity evaluation. Fourthly, pure and mixed species artificial contamination systems were included to test its applicability. In addition, a propidium monoazide (PMA)-PSR assay was set up to determine viable but non-culturable (VBNC) cells. Target tlh gene acquired high specificity and PSR assay was optimized to carry out at 65ᵒC for 1 h. Detection limit of PSR assay in purified genomic DNA, plasmid and artificially contaminated food samples were 5.31–53.1 pg/μL, 100 copies, and 103 CFU/mL, respectively, demonstrating the sensitivity as 100-fold higher than regular PCR. With PMA-PSR assay, VBNC cells were accurately determined without the disruption of dead cells and food compositions. Thus, PSR is applicable in detection of V. parahaemolyticus viable cells and virulence factors.

Quantitative Detection of VBNC State Pseudomonas aeruginosa Contributing to Accurate Assessment of Microbial Inactivation in Drinking Water Disinfection

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.

Stress response of Salmonella Newport with various sequence types toward plasma-activated water: Viable but nonculturable state formation and outer membrane vesicle production

This study aims to investigate the response of Salmonella Newport to plasma-activated water (PAW), a novel disinfectant that attracts attention due to its broad-spectrum antimicrobial efficacy and eco-friendliness. In this work, we demonstrated that S. Newport of different sequence types (STs) could be induced into the viable but nonculturable (VBNC) state by PAW treatment. Notably, a remarkable 99.96% of S. Newport ST45 strain entered the VBNC state after a 12-min PAW treatment, which was the fastest observed among the five S. Newport STs (ST31, ST45, ST46, ST166, ST2364). Secretion of outer membrane vesicles was observed in ST45, suggesting a potential strategy against PAW treatment. Genes related to oxidative stress (sodA, katE, trxA), outer membrane proteins (ompA, ompC, ompD, ompF) and virulence (pagC, sipC, sopE2) were upregulated in the PAW-treated S. Newport, especially in ST45. A reduction of 38–65% in intracellular ATP level after PAW treatment was observed, indicating a contributor to the formation of the VBNC state. In addition, a rapid method for detecting the proportion of VBNC cells in food products based on pagC was established. This study contributes to understanding the formation mechanism of the VBNC state in S. Newport under PAW stress and offers insights for controlling microbial risks in the food industry.

Effect of RpoS on the survival, induction, resuscitation, morphology, and gene expression of viable but non-culturable Salmonella Enteritidis in powdered infant formula

Involvement of the transcriptional regulator RpoS in the persistence of viable but non-culturable (VBNC) state has been demonstrated in several species of bacteria. This study investigated the role of the RpoS in the formation and resuscitation of VBNC state in Salmonella enterica serovar Enteritidis CICC 21482 by measuring bacterial survival, morphology, physiological characteristics, and gene expression in wild-type (WT) and rpoS-deletion (ΔrpoS) strains during long-term storage in powdered infant formula (PIF). The ΔrpoS strain was produced by allelic exchange using a suicide plasmid. Bacteria were inoculated into PIF for 635-day storage. Survival, morphology, intracellular reactive oxygen species (ROS) levels and intercellular quorum sensing autoinducer-2 (AI-2) contents were regularly measured. Resuscitation assays were conducted after obtaining VBNC cells. Gene expression was measured using real-time quantitative polymerase chain reaction (qPCR). The results showed that RpoS and low temperature conditions were associated with enhanced culturability and recoverability of Salmonella Enteritidis after desiccation storage in low water activity (aw) PIF. In addition, the synthesis of intracellular ROS and intercellular quorum sensing AI-2 was regulated by RpoS, inducing the formation and resuscitation of VBNC cells. Gene expression of soxS, katG and relA was found strongly associated with RpoS. Due to the lack of RpoS factor, the ΔrpoS strain could not normally synthesize SoxS, catalase and (p)ppGpp, resulting in its early shift to the VBNC state. This study elucidates the role of rpoS in desiccation stress and the formation and resuscitation mechanism of VBNC cells under desiccation stress. It serves as the basis for preventing and controlling the recovery of pathogenic bacteria in VBNC state in low aw foods.

Viable but nonculturable (VBNC) state, an underestimated and controversial microbial survival strategy.

As a unique microbial response to adverse circumstances, the viable but nonculturable (VBNC) state is characterized by the loss of culturability of microbial cells on/in nutrient media that normally support their growth, while maintaining metabolic activity. These cells can resuscitate to a culturable state under suitable conditions. Given the intrinsic importance of the VBNC state and recent debates surrounding it, there is a need to redefine and standardize the term, and to address essential questions such as 'How to differentiate VBNC from other similar terms?' and 'How can VBNC cells be standardly and accurately determined?'. This opinion piece aims at contributing to an improved understanding of the VBNC state and promoting its proper handling as an underestimated and controversial microbial survival strategy.

The VBNC state: a fundamental survival strategy of Acinetobacter baumannii.

Currently, the viable but non-culturable (VBNC) state is an underappreciated niche for pathogenic bacteria which provides a continuous source for recurrent infections and transmission. We propose the VBNC state to be a global persistence mechanism used by various A. baumannii strains to cope with many stresses it is confronted with in the clinical environment and in the host. This requires a novel strategy to detect viable cells of this pathogen that is not only based on plating assays.

Metabolomic Differences between Viable but Nonculturable and Recovered Lacticaseibacillus paracasei Zhang.

The fermentation process can be affected when the starter culture enters the viable but nonculturable (VBNC) state. Therefore, it is of interest to investigate how VBNC cells change physiologically. Lacticaseibacillus (L.) paracasei Zhang is both a probiotic and a starter strain. This study aimed to investigate the metabolomic differences between VBNC and recovered L. paracasei Zhang cells. First, L. paracasei Zhang was induced to enter the VBNC state by keeping the cells in a liquid de Man-Rogosa-Sharpe (MRS) medium at 4 °C for 220 days. Flow cytometry was used to sort the induced VBNC cells, and three different types of culture media (MRS medium, skim milk with 1% yeast extract, and skim milk) were used for cell resuscitation. Cell growth responses in the three types of recovery media suggested that the liquid MRS medium was the most effective in reversing the VBNC state in L. paracasei Zhang. Metabolomics analysis revealed 25 differential metabolites from five main metabolite classes (amino acid, carbohydrate, lipid, vitamin, and purine and pyrimidine). The levels of L-cysteine, L-alanine, L-lysine, and L-arginine notably increased in the revived cells, while cellulose, alginose, and guanine significantly decreased. This study confirmed that VBNC cells had an altered physiology.

Resuscitation of viable but nonculturable bacteria promoted by ATP-mediated NAD+ synthesis

IntroductionEntry into the viable but nonculturable (VBNC) state is a survival strategy adopted by bacteria to survive harsh environment. Although VBNC cells still have metabolic activity, they lose the ability to form colonies on nonselective culture media. Thus, conventional bacterial detection methods, such as plate counting, are unable to detect the presence of VBNC cells. When the environmental conditions are appropriate, VBNC cells can initiate resuscitation, posing a great risk to the safety of public health. The study of the VBNC resuscitation mechanism could provide new insights into the prevention and control of VBNC resuscitation. ObjectivesUncovering the molecular mechanism of VBNC cell resuscitation by investigating the role of O-antigen ligase (RfaL) in inhibiting the resuscitation of Escherichia coli O157:H7 in the VBNC state. MethodsRfaL was screened and verified as a resuscitation inhibitor of VBNC Escherichia coli O157:H7 by detecting resuscitation curve and time-lapse microscopy. The mechanism of RfaL impacts VBNC E. coli resuscitation was investigated by detecting the single cell ATP content, metabolomic changes, NAD(H) content and new protein biosynthesis of WT and ΔrfaL at different stage of resuscitation. ResultsMutation of rfaL, which encoded an O-antigen ligase, markedly shortened the resuscitating lag phase. Further studies indicated that ΔrfaL VBNC cells contained higher ATP levels, and ATP consumption during the resuscitating lag phase was highly correlated with resuscitation efficiency. Metabolomic analysis revealed that ATP was utilized to activate the Handler and salvage pathways to synthesize NAD+, balancing redox reactions to recover cell activity and promote cell resuscitation. ConclusionOur findings revealed a strategy employed by VBNC cells for revival, that is, using residual ATP to primarily recover metabolic activity, driving cells to exit dormancy. The synthesis pathway of lipopolysaccharide (LPS) in rfaL null mutant was inhibited and could supply more ATP to synthesis NAD+ and promote resuscitation.

Involvement of Acquired Tobramycin Resistance in the Shift to the Viable but Non-Culturable State in Pseudomonas aeruginosa.

Persistent and viable but non-culturable (VBNC) Pseudomonas aeruginosa cells are mainly responsible for the recurrence and non-responsiveness to antibiotics of cystic fibrosis (CF) lung infections. The sub-inhibitory antibiotic concentrations found in the CF lung in between successive therapeutic cycles can trigger the entry into the VBNC state, albeit with a strain-specific pattern. Here, we analyzed the VBNC cell induction in the biofilms of two CF P. aeruginosa isolates, exposed to starvation with/without antibiotics, and investigated the putative genetic determinants involved. Total viable bacterial cells were quantified by the validated ecfX-targeting qPCR protocol and the VBNC cells were estimated as the difference between qPCR and cultural counts. The isolates were both subjected to whole genome sequencing, with attention focused on their carriage of aminoglycoside resistance genes and on identifying mutated toxin-antitoxin and quorum sensing systems. The obtained results suggest the variable contribution of different antibiotic resistance mechanisms to VBNC cell abundance, identifying a major contribution from tobramycin efflux, mediated by MexXY efflux pump overexpression. The genome analysis evidenced putative mutation hotspots, which deserve further investigation. Therefore, drug efflux could represent a crucial mechanism through which the VBNC state is entered and a potential target for anti-persistence strategies.

Modeling the survival of Campylobacter jejuni in raw milk considering the viable but non‐culturable cells (VBNC)

AbstractCampylobacter spp. cannot grow in raw milk, but it is able to transform into a viable but non‐culturable (VBNC) state enabling the survival in such harsh conditions. In this study, Campylobacter jejuni survival in raw milk was investigated taken into consideration colony‐forming units (CFUs) and VBNC cells. CFU from two different strains of C. jejuni (DSM 4688 and BfR‐CA‐18043) were enumerated at three temperatures (5°C, 8°C, and 12°C). In parallel, a viability real‐time PCR was conducted to quantify intact and putatively infectious units (IPIUs) (comprising CFU and VBNC bacteria). The data generated were used to model the viability of C. jejuni during raw milk storage. Here, a one‐step fitting approach was performed using parameter estimates from an intermediate two‐step fit as starting values to generate tertiary models. Different primary model equations (Trilinear and Weibull) were required to fit the CFU and the IPIU data. Strain‐specific linear secondary models were generated to analyze the effect of storage temperature on the maximum specific inactivation rate of the CFU data. The time of the first decimal reduction parameter of the IPIU models could be modeled by a strain‐independent linear secondary model. The developed tertiary models for CFU and IPIU differ significantly in their predictions, for example, for the time required for a one log10 reduction. Taken into consideration that VBNC could revert to a culturable state during the raw milk storage, our results underline the importance of considering IPIU and not only CFU to avoid underestimation of the survival of C. jejuni in raw milk.

Dysregulated NAD(H) homeostasis associated with ciprofloxacin tolerance in Escherichia coli investigated on a single-cell level with the Peredox [NADH:NAD+] biosensor.

Antibiotic persistence (subpopulation tolerance) occurs when a subpopulation of antibiotic sensitive cells survives prolonged exposure to a bactericidal concentration of an antibiotic, and is capable of regrowth once the antibiotic is removed. This phenomenon has been shown to contribute to prolonged treatment duration, infection recurrence, and accelerated development of genetic resistance. Currently, there are no biomarkers which would allow for segregation of these antibiotic-tolerant cells from the bulk population prior to antibiotic exposure, limiting research on this phenomenon to retrograde analyses. However, it has been previously shown that persisters often have a dysregulated intracellular redox homeostasis, warranting its investigation as a potential marker for antibiotic tolerance. Furthermore, it is currently unknown whether another antibiotic tolerant subpopulation - viable but non-culturable cells (VBNCs), are simply persisters with extreme lag phase, or are formed through separate pathways. VBNCs similarly to persisters remain viable following antibiotic exposure, however, are not capable of regrowth in standard conditions. In this article we employed an NADH:NAD+ biosensor (Peredox) to investigate NADH homeostasis of ciprofloxacin-tolerant E. coli cells on a single-cell level. [NADH:NAD+] was used as a proxy for measuring intracellular redox homeostasis and respiration rate. First, we demonstrated that ciprofloxacin exposure results in a high number of VBNCs, several orders of magnitude higher than persisters. However, we found no correlation in the frequencies of persister and VBNC subpopulations. Ciprofloxacin-tolerant cells (persisters & VBNCs) were actively undergoing respiration, although at a significantly lower rate on average when compared to the bulk population. We also noted significant heterogeneity on a single-cell level within the subpopulations, however were unable to segregate persisters from VBNCs based on these observations alone. Finally, we showed that in the highly-persistent strain of E. coli, E. coli HipQ, ciprofloxacin-tolerant cells have a significantly lower [NADH:NAD+] ratio than tolerant cells of its parental strain, providing further link between disturbed NADH homeostasis and antibiotic tolerance.

Comparative Analysis Using Raman Spectroscopy of the Cellular Constituents of Lacticaseibacillus paracasei Zhang in a Normal and Viable but Nonculturable State.

This study aimed to investigate the molecular composition of a viable but nonculturable (VBNC) state of a probiotic strain, Lacticaseibacillus paracasei Zhang (L. paracasei Zhang), using single-cell Raman spectroscopy (SCRS). Fluorescent microcopy with live/dead cell staining (propidium iodide and SYTO 9), plate counting, and scanning electron microscopy were used in combination to observe bacteria in an induced VBNC state. We induced the VBNC state by incubating the cells in de Man, Rogosa, and Sharpe broth (MRS) at 4 °C. Cells were sampled for subsequent analyses before VBNC induction, during it, and up to 220 days afterwards. We found that, after cold incubation for 220 days, the viable plate count was zero, but active cells could still be observed (as green fluorescent cells) under a fluorescence microscope, indicating that Lacticaseibacillus paracasei Zhang entered the VBNC state under these conditions. Scanning electron microscopy revealed the altered ultra-morphology of the VBNC cells, characterized by a shortened cell length and a wrinkled cell surface. Principal component analysis of the Raman spectra profiles revealed obvious differences in the intracellular biochemical constituents between normal and VBNC cells. Comparative analysis of the Raman spectra identified 12 main differential peaks between normal and VBNC cells, corresponding to carbohydrates, lipids, nucleic acids, and proteins. Our results suggested that there were obvious cellular structural intracellular macromolecular differences between normal and VBNC cells. During the induction of the VBNC state, the relative contents of carbohydrates (such as fructose), saturated fatty acids (such as palmitic acid), nucleic acid constituents, and some amino acids changed obviously, which could constitute a bacterial adaptive mechanism against adverse environmental conditions. Our study provides a theoretical basis for revealing the formation mechanism of a VBNC state in lactic acid bacteria.