Viable But Nonculturable Research Articles

Dark repair of sunlight-inactivated tetracycline-resistant bacteria: Mechanisms and important role of bacteria in viable but non-culturable state

The spread of antibiotic resistant bacteria (ARB) in the environment poses a potential threat to human health, and the reactivation of inactivated ARB accelerated the spread of ARB. However, little is known about the reactivation of sunlight-inactivated ARB in natural waters. In this study, the reactivation of sunlight-inactivated ARB in dark conditions was investigated with tetracycline-resistant E. coli (Tc-AR E. coli) as a representative. Results showed that sunlight-inactivated Tc-AR E. coli underwent dark repair to regain tetracycline resistance with dark repair ratios increasing from (0.124 ± 0.012)‱ within 24 h dark treatment to (0.891 ± 0.033)‱ within 48 h. The presence of Suwannee River fulvic acid (SRFA) promoted the reactivation of sunlight-inactivated Tc-AR E. coli and tetracycline inhibited their reactivation. The reactivation of sunlight-inactivated Tc-AR E. coli is mainly attributed to the repair of the tetracycline-specific efflux pump in the cell membrane. Tc-AR E. coli in a viable but non-culturable (VBNC) state was observed and dominated the reactivation as the inactivated ARB remain present in the dark for more than 20 h. These results explained the reason for distribution difference of Tc-ARB at different depths in natural waters, which are of great significance for understanding the environmental behavior of ARB.

Occurrence and survival of Campylobacter spp. in dairy matrices investigated by viability qPCR

The aim of this study was to investigate the survival of Campylobacter jejuni in dairy matrices by viability qPCR. Survival in UHT milk was examined first and the cheese outbreak strain, C. jejuni 11 218 showed enhanced tendency to become viable but nonculturable (VBNC). It survived for long at different pH values, in raw and fermented milk and cheese. Viability qPCR was also used to examine the occurrence of viable Campylobacter spp. in raw milk, detecting the bacteria in 40.9% of 25 mL samples negative on culture. Results showed that viability qPCR is valuable in estimating Campylobacter risk in dairy products.

Interactive impact of Myrothecium verrucaria and MnO2 on fertilizer quality and biological safety of anaerobic digestate

Low contents of the active compound and high pathogens counts are considered the major factors hampering land application of anaerobic digestate. Therefore, the role of Myrothecium verrucaria and MnO2 treatment in enhancing fertilizer quality and biological safety of digestate for land application was explored. Higher contents of humic acid (8.9 g/L) with a higher degree of humification index and oxygen-containing functional groups was observed in the digestate receiving Myrothecium verrucaria coupled with MnO2 treatment. Moreover, the viable and culturable count of S. faecalis, S. typhi, C. perfringens, and E. coli pathogens in the digestate decreased approximately to 12.50%, 41.70%, 18.87%, 50.00% and 25.97%, 64.44%, 37.51%, 75.27%, respectively after treatment with Myrothecium verrucaria and MnO2. However, the count of pathogens in the VBNC (viable but not culturable) state increased from 3.75%-9.44% to 28.25%–54.01% after treatment. This study provided a novel strategy to enhance the fertilizer quality and biological safety of anaerobic digestate for land application.

Applying RNA-Seq to investigate the transition into a Viable But Non Culturable State (VBNC) for the intracellular pathogen, Francisella tularensis LVS.

Many species of bacteria, under conditions of stress or nutrient limitation, enter a state of dormancy referred to as viable but non-culturable (VBNC). VBNC bacteria persist in the environment, are difficult to detect and identify by many standard laboratory methods and can be altered in their susceptibility to antibiotics. Entry into the VBNC state is often accompanied by morphological changes; however, the mechanisms underlying this are poorly understood. Francisella transitions rapidly and spontaneously to a VBNC state, and therefore has the potential to be an excellent model organism for the study of this phenomenon. To investigate the transcriptome of VBNC F. tularensis LVS we extracted RNA from culturable and VBNC cells and carried out an RNA-Seq analysis using both Long-read Nanopore and Illumina Sequencing. Differentially expressed genes (DEGs) were identified using a DE-Seq pipeline. Over 300 genes were significantly upregulated and ~100 genes were down regulated in VBNC cells. Amongst the upregulated genes were some involved in the transport of metals or small molecules, and we have also identified putative transcriptional regulators that may be master controllers of the VBNC process. Our data represents the first transcriptomic analysis of F. tularensis LVS as it transitions into the VBNC state. Identifying genes that are involved in this transition is a critical first step in understanding the mechanisms that drive F. tularensis LVS into the VBNC state and will help us identify factors that allow the resuscitation of these bacteria. This will have important implications for understanding both environmental persistence and pathogenicity of Francisella species. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence)

Characterization of two predicted lipid metabolism genes (FTL_1569 and FTL_1570) in the intracellular pathogen, Francisella tularensis LVS.

Many species of bacteria enter a viable but non-culturable (VBNC) state when exposed to stressful conditions. The VBNC state acts as an adaptive strategy that allows for long-term survival of bacteria in unfavorable environments. VBNC bacteria are difficult to detect in the environment due to an inability to grow on nutrient agar. Although the ability to enter the VBNC state may be advantageous for bacteria, it poses a risk to human health. Francisella tularensis, transitions rapidly and spontaneously to the VBNC state, giving it the potential to be an excellent model organism for the study of this phenomenon. Microscopic observations reveal that changes in cell morphology are coincident with the VBNC state. To characterize these morphological changes, fluorescence microscopy imaging using membrane lipid staining has been used in the live vaccine strain of F. tularensis. Microscopy imaging suggested that lipid metabolism may be important for the transition into the VBNC state, and the change in morphology. To investigate further, genetic deletion mutants have been constructed from two predicted lipid metabolism genes, FTL_1569 (phosphoglycolate phosphatase) and FTL_1570 (phospholipase D family protein). Gentamicin protection assays with immune cells and red blood cells have been conducted to investigate the role of these genes in virulence of F. tularensis. Our preliminary results suggest that lipid metabolism may play roles in both persistence and in invasion in F. tularensis. (This work was supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence).

Roles of viable but non-culturable state in the survival of Campylobacter jejuni.

Despite being considered fragile and fastidious, Campylobacter jejuni is the most prevalent cause of foodborne bacterial gastroenteritis, and chicken meat is considered the main vehicle of transmission to humans. This agent can survive adverse conditions in the form of biofilms, but extreme stress (nutritional, oxidative and thermal) promotes the acquisition of a state called viable but not culturable (VBNC). The emergence of this pathogen worldwide and the recent international requirements in its control instigated us to qualitatively and quantitatively estimate the time required for the acquisition of the VBNC form in 27 strains of C. jejuni, characterize morphological aspects, determine its adaptive and invasive potential and perform comparative metabolomic evaluation. Extreme stress promoted the complete acquisition of the VBNC form in a mean time of 26 days. Starting from an average initial count of 7.8 log CFU/mL, the first four days determined the greatest average reduction of the culturable form of 3.2 log CFU/mL. The scanning and transmission image analyses showed a transition from the typical viable form (VT) to the VBNC form, with initial acquisition of the straight rod shape, followed by loss of the flagella and subdivision into two to 11 imperfect cocci arranged in a chain and rich in cellular content, until their individual release. RT-PCR identified the presence of ciaB and p19 transcripts in the 27 cultivable C. jejuni strains, a character maintained in the VBNC form only for p19 and in 59.3% (16/27) of the VBNC strains for the ciaB gene. The average inoculation of 1.8 log CFU/mL of C. jejuni VBNC into primary chicken embryo hepatocyte cells promoted the occurrence of apoptosis processes significantly after 24 hours of contact by one of the strains tested. In C. jejuni VBNC, we detected higher expression of metabolites linked to protective and adaptation mechanisms and of volatile organic precursor compounds indicative of metabolism interruption. The oscillations in the time of acquisition of the VBNC form together with the presence of transcripts for ciaB and p19, the identification of cell lysis and metabolites that ensure the maintenance of the pathogen alert to the fact that C. jejuni VBNC remains virulent and adapted to stress, which makes evident the potential danger of this latent form, which is not detectable by official methodologies.

Effect of preliminary stresses on the induction of viable but non-culturable Escherichia coli O157:H7 NCTC 12900 and Staphylococcus aureus ATCC 6538

As a novel non-thermal pasteurization technology, high pressure carbon dioxide (HPCD) has been used in food processing. However, it could induce microorganisms into a viable but nonculturable (VBNC) state, posing a potential risk to food safety and public health. In this study, we attempted to investigate the effect of various preliminary stresses including cold, heat, osmosis, acidity and oxidation on HPCD-induced VBNC formation. The results indicated that there was no effect of preliminary stresses on VBNC Staphylococcus aureus induction. However, heat, acidity and long-term (24 h) cultivation preadaptation could significantly increase the VBNC E. coli production induced by HPCD. Transcriptome analysis revealed that genes involved in ATP production were significantly decreased in these three stress-treated cells, and further ATP levels determination revealed that the ATP levels of the cell were significantly decreased after heat, acidity and long-term cultivation preadaptation, implying the decrease of ATP level caused by these stresses might be the reason for increasing VBNC production. To further study the relationship between ATP level and VBNC cell ratios after preadaptation. We artificially decreased the ATP levels, and detect their VBNC ratios after HPCD treatment. We found that with the ATP concentration decreasing after exposure to carbonyl cyanide m-chlorophenyl hydrazine (CCCP), the VBNC ratios were increased after HPCD treatment, indicating that the ATP contents were highly negatively correlated with VBNC ratios. This study proved that the preadaptation of heat, acidity and long-term cultivation could promote VBNC induction by decreasing the intracellular ATP level. In general, the obtained result gave the instruction about the storage environment for food materials, helped to further develop and optimize the HPCD processing to prevent VBNC formation and accelerate the development of HPCD technology in food industry.

Helicobacter pylori Dormant States Are Affected by Vitamin C.

Helicobacter pylori colonizes human gastric mucosa, overcoming stressful conditions and entering in a dormant state. This study evaluated: (i) H. pylori's physiological changes from active to viable-but-non-culturable (VBNC) and persister (AP) states, establishing times/conditions; (ii) the ability of vitamin C to interfere with dormancy generation/resuscitation. A dormant state was induced in clinical MDR H. pylori 10A/13 by: nutrient starvation (for VBNC generation), incubating in an unenriched medium (Brucella broth) or saline solution (SS), and (for AP generation) treatment with 10xMIC amoxicillin (AMX). The samples were monitored after 24, 48, and 72 h, 8-14 days by OD600, CFUs/mL, Live/Dead staining, and an MTT viability test. Afterwards, vitamin C was added to the H. pylori suspension before/after the generation of dormant states, and monitoring took place at 24, 48, and 72 h. The VBNC state was generated after 8 days in SS, and the AP state in AMX for 48 h. Vitamin C reduced its entry into a VBNC state. In AP cells, Vitamin C delayed entry, decreasing viable coccal cells and increasing bacillary/U-shaped bacteria. Vitamin C increased resuscitation (60%) in the VBNC state and reduced the aggregates of the AP state. Vitamin C reduced the incidence of dormant states, promoting the resuscitation rate. Pretreatment with Vitamin C could favor the selection of microbial vegetative forms that are more susceptible to H. pylori therapeutical schemes.

Current Perspectives on Viable but Non-Culturable Foodborne Pathogenic Bacteria: A Review.

Foodborne diseases caused by foodborne pathogens pose risks to food safety. Effective detection and efficient inactivation of pathogenic bacteria has always been a research hotspot in the field of food safety. Complicating these goals, bacteria can be induced to adopt a viable but non-culturable (VBNC) state under adverse external environmental stresses. When in the VBNC state, pathogens cannot form visible colonies during traditional culture but remain metabolically active and toxic. The resulting false negative results in growth-related assays can jeopardize food safety. This review summarizes the latest research on VBNC foodborne pathogens, including induction conditions, detection methods, mechanism of VBNC formation, and possible control strategies. It is hoped that this review can provide ideas and methods for future research on VBNC foodborne pathogenic bacteria.

Transcriptional profiling of Xanthomonas campestris pv. campestris in viable but nonculturable state

BackgroundXanthomonas campestris pv. campestris (Xcc) is an important seed-borne plant pathogenic bacteria that can cause a serious threat to cruciferous crops. Bacteria can enter into the viable but non-culturable (VBNC) state under stress conditions, and cause potential risks to agricultural production because the VBNC bacterial cells will evade culture-based detection. However, little is known about the mechanism of VBNC. Our previous study showed that Xcc could be induced into VBNC state by copper ion (Cu2+).ResultsHere, RNA-seq was performed to explore the mechanism of VBNC state. The results indicated that expression profiling was changed dramatically in the different VBNC stages (0 d, 1 d, 2 d and 10 d). Moreover, metabolism related pathways were enriched according to COG, GO and KEGG analysis of differentially expressed genes (DEGs). The DEGs associated with cell motility were down-regulated, whereas pathogenicity related genes were up-regulated. This study revealed that the high expression of genes related to stress response could trigger the active cells to VBNC state, while the genes involved in transcription and translation category, as well as transport and metabolism category, were ascribed to maintaining the VBNC state.ConclusionThis study summarized not only the related pathways that might trigger and maintain VBNC state, but also the expression profiling of genes in different survival state of bacteria under stress. It provided a new kind of gene expression profile and new ideas for studying VBNC state mechanism in X. campestris pv. campestris.

Glyoxylate cycle maintains the metabolic homeostasis of Pseudomonas aeruginosa in viable but nonculturable state induced by chlorine stress

Bacteria enter a viable but non-culturable (VBNC) state with low metabolic activity to cope with environmental stress (e.g., chlorine disinfection). Elucidating the mechanism and key pathway of VBNC bacteria maintaining low metabolic competence is of great significance to realize their effective control and reduce their environmental and health risks. This study discovered that the glyoxylate cycle is a key metabolic pathway for VBNC bacteria, but not for culturable bacteria. And blocking the glyoxylate cycle pathway inhibited the reactivation and led to the death of VBNC bacteria. The main mechanisms involved the breakdown of material and energy metabolism and the antioxidant system. Gas chromatography-tandem mass spectrometry analysis showed that blocking the glyoxylate cycle led to a disruption of carbohydrate metabolism and fatty acid catabolism in VBNC bacteria. As a result, the energy metabolism system of VBNC bacteria collapsed and the abundance of energy metabolites (ATP, NAD+ and NADP+) decreased significantly. Moreover, the decrease in the level of quorum sensing signaling molecules (quinolinone and N-Butanoyl-D-homoserine lactone) inhibited the synthesis of extracellular polymeric substances (EPSs) and biofilm formation. And the downregulation of glycerophospholipid metabolic competence increased the permeability of cell membranes, leading to the entry of large amounts of hypochlorous acid (HClO) into the bacteria. In addition, the down-regulation of nucleotide metabolism, glutathione metabolism, and the reduction of antioxidant enzyme content resulted in the inability to scavenge reactive oxygen species (ROS) generated by chlorine stress. The large production of ROS and the reduction of antioxidants together led to the breakdown of the antioxidant system of VBNC bacteria. In short, the glyoxylate cycle is the key metabolism pathway of VBNC bacteria for stress resistance and maintaining cellular metabolic balance, and targeting the glyoxylate cycle represents an attractive strategy for developing new and efficient disinfection methods for the control of VBNC bacteria.

Gene expression analysis during the conversion from a viable but nonculturable to culturable state in Vibrio cholerae

We previously reported that Vibrio cholerae in a viable but non-culturable (VBNC) state can be converted to a culturable state by treatment with catalase. This finding enabled us to develop an assay system to observe the time course of the conversion from VBNC to culturable in V. cholerae. VBNC cells began to convert to culturable cells as early as 2 h after catalase supplementation. Gene expression in VBNC cells during catalase treatment was analyzed using RNA microarray. Many ribosomal DNA genes were stimulated 6 h post catalase exposure, suggesting that the conversion-driving signal started prior to 6 h. Focusing on the period prior to cell proliferation, we found that 16 genes might be involved in the conversion mechanism in V. cholerae, and they showed enhanced expression at 2 h and 4 h after catalase addition. These upregulated genes included phage shock proteins (pspA, B, and C), alternative sigma factor (rpoE) and its negative regulator (rseA), cobW C terminal domain-containing protein, damage-inducible helicase (dinG), cholerae toxin secretion protein epsM, HTH-type transcription regulator (iscR), mechanosensitive ion channel family protein, anthranilate synthase component I, fructose-specific IIBC component, molybdenum import ATP-binding protein (modC), LysE family translocator, putative organic hydroperoxide resistance protein, and a hypothetical protein. This study identified genes involved in the catalase-induced conversion of V. cholerae VBNC cells to a culturable state and provided valuable insights into the mechanisms involved in the conversion process.

Role of Resuscitation Promoting Factor-like Protein from Nocardiopsis halophila.

Resuscitation promoting factors (Rpf), a class of proteins secreted by gram-positive bacteria including actinobacteria, promote the resuscitation of dormant bacteria and spore germination. Here, we describe the reconstitution of the resuscitation promoting activity of the Rpf protein from Nocardiopsis halophila CGMCC 4.1195Tin vitro and in vivo. The Rpf protein was expressed in the host Escherichia coli BL21 codon plus (DE3) and was confirmed to have a significant resuscitation effect on the viable but non-culturable (VBNC) N. halophila. Subsequently, the rpf gene of N. halophila was knocked out. We found that the growth rate of the mutant strain (Δrpf) was slower than that of the wild strain, and the former produced significantly shorter spores than the wild-type strain. Our results confirmed the activity of the Rpf protein in N. halophila to promote dormant bacteria resuscitation. This study will lay the foundation for the application of the Rpf protein from N. halophila to exploit actinomycetes resources.

Quantification and antibiotic resistance risk assessment of chlorination-residual viable/VBNC Escherichia coli and Enterococcus in on-site hospital wastewater treatment system

On-site hospital wastewater treatment system widely applying chlorination has been regarded as an important barrier to curb the dissemination of antibiotic resistance. Chlorination-residual viable and viable but non-culturable (VBNC) bacteria probably lead to overestimate the effect of disinfection, while their antibiotic resistance risks imported from hospital effluents to municipal pipe network may be ignored. In this study, we quantified viable/VBNC Escherichia coli and Enterococcus in chlorination of an on-site hospital wastewater treatment system and assessed their antibiotic resistance risks. The numbers of viable/VBNC Escherichia coli and Enterococcus in raw wastewater were detected as high as 5.76–6.34/5.76–6.33 and 5.44–5.76/5.44–5.75 log10(cells/mL). Meanwhile, high proportions of antibiotic-resistant Escherichia coli and Enterococcus to culturable Escherichia coli and Enterococcus were observed, especially carrying ampicillin resistance (22.25–41.70 % and 28.09–54.05 %). Chlorination could remove 0.44–1.88‐/0.43–1.88- and 0.29–1.29‐/0.28–1.28-log of viable/VBNC and complete culturable Escherichia coli and Enterococcus, but cause antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) to be released outside cells, and possibly further enhance the antibiotic resistance of viable bacteria. Low detections of antibiotics suggested that the occurrence of antibiotic-resistant bacteria (ARB) may not be accompanied by the corresponding antibiotics. Different sampling months had some impacts on above results, while the results were basically stable at different sampling times of hospital daily working period. The high release rates (11.26–13.02 and 11.59–12.98 log10(cells/h)) and cumulative amounts (15.41–16.12 and 15.75–16.14 log10(cells)) of chlorination-residual viable/VBNC Escherichia coli and Enterococcus indirectly assessed the potential risks of bacterial antibiotic resistance entering municipal pipe network. Additionally, the contributions from the corresponding antibiotic ceftazidime, ciprofloxacin, and vancomycin with the cumulative amounts of 2.57–4.85, 5.73–7.50, and 5.21–7.14 kg should also be taken seriously. Residual chlorine could serve as an important signal indicator for the risk assessment.

Extended lag phase indicates the dormancy of biphenyl degrading Rhodococcus biphenylivorans TG9 under heat stress

Microbial remediation is a green and sustainable technology, but harsh environmental conditions could lead to microbial dormancy, such as entering a viable but non-culturable (VBNC) state. However, the evidence of VBNC is controversial and limited. In this study, heat stress (60 °C), one of the leading challenges for mesophilic degrading bacteria, was mimicked to investigate the physiological response of Rhodococcus biphenylivorans TG9. After 2 h of heat stress, the culturable TG9 cell count decreased from 108 cells/mL to undetectable while the viable cell count was still 105 cells/mL. The biphenyl degradation efficiency of stressed TG9 dropped by 50% compared to that of cells at logarithmic phase. During heat stress, the respiratory activity of TG9 declined dramatically while the intracellular ATP level initially increased and then decreased. Notably, the corresponding indicators recovered when restored to 30 °C. These characteristics were in consistent with bacteria entering into VBNC state. Furthermore, fluorescence activated cell sorting together with single cell as seed culture detection verified the unculturability and viability of VBNC state of TG9 cells. Also, we found that single cells in VBNC state could resuscitate and regrowth with significantly extended lag phase (LP). Our results highlight the potential of TG9 for microbial remediation and hint LP duration as an indicator for survival state of bacteria.

Identification of determinants for entering into a viable but nonculturable state in Vibrio alginolyticus by Tn-seq.

The viable but nonculturable (VBNC) state is a dormant state of nonsporulating bacteria that enhances survival in adverse environments. Systematic genome-wide research on the genetic basis of VBNC formation is warranted. In this study, we demonstrated that the marine bacterium Vibrio alginolyticus lost culturability but remained viable and entered into the VBNC state when exposed to low nutrient concentrations for prolonged periods of time. Using transposon-insertion sequencing (Tn-seq), we identified 635 determinants governing the formation of the VBNC state, including 322 genes with defective effects on VBNC formation and 313 genes contributing to entry into the VBNC state. Tn-seq analysis revealed that genes involved in various metabolic pathways were shown to have an inhibitory effect on VBNC formation, while genes related to chemotaxis or folate biosynthesis promoted entry into the VBNC state. Moreover, the effects of these genes on the formation of VBNC were validated with the growth of deletion mutants of eight selected genes under nutrient-limited conditions. Interestingly, fleQ and pyrI were identified as essential for entry into the VBNC state, and they affected the formation of the VBNC state independent of RpoE or ToxR regulation. Collectively, these results provide new insights into the mechanism of VBNC formation. KEY POINTS: • Vibrio alginolyticushas the ability to enter into the VBNC state under low nutrient conditions at lowtemperature. • The 635 determinants for entry into the VBNC state were systematically identified bytransposon-insertion sequencing. • PyrI and FleQ were validated to play significant roles in the formation of the VBNC state.

Induction of Viable but Non-Culturable State in Clinically Relevant Staphylococci and Their Detection with Bacteriophage K

Prosthetic joint infections are frequently associated with biofilm formation and the presence of viable but non-culturable (VBNC) bacteria. Conventional sample culturing remains the gold standard for microbiological diagnosis. However, VBNC bacteria lack the ability to grow on routine culture medium, leading to culture-negative results. Bacteriophages are viruses that specifically recognize and infect bacteria. In this study, we wanted to determine if bacteriophages could be used to detect VBNC bacteria. Four staphylococcal strains were cultured for biofilm formation and transferred to low-nutrient media with different gentamycin concentrations for VBNC state induction. VBNC bacteria were confirmed with the BacLightTM viability kit staining. Suspensions of live, dead, and VBNC bacteria were incubated with bacteriophage K and assessed in a qPCR for their detection. The VBNC state was successfully induced 8 to 19 days after incubation under stressful conditions. In total, 6.1 to 23.9% of bacteria were confirmed alive while not growing on conventional culturing media. During the qPCR assay, live bacterial suspensions showed a substantial increase in phage DNA. No detection was observed in dead bacteria or phage non-susceptible E. coli suspensions. However, a reduction in phage DNA in VBNC bacterial suspensions was observed, which confirmed the detection was successful based on the adsorption of phages.

Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR.

Legionella pneumophila is a waterborne pathogen and, as the causative agent of Legionnaires' disease, a significant public health concern. Exposure to environmental stresses, and disinfection treatments, promotes the formation of resistant and potentially infectious viable but non-culturable (VBNC) Legionella. The management of engineered water systems to prevent Legionnaires' disease is hindered by the presence of VBNC Legionella that cannot be detected using the standard culture (ISO11731:2017-05) and quantitative polymerase reaction (ISO/TS12869:2019) methods. This study describes a novel method to quantify VBNC Legionella from environmental water samples using a "viability based flow cytometry-cell sorting and qPCR" (VFC + qPCR) assay. This protocol was then validated by quantifying the VBNC Legionella genomic load from hospital water samples. The VBNC cells were unable to be cultured on Buffered Charcoal Yeast Extract (BCYE) agar; however, their viability was confirmed through their ATP activity and ability to infect amoeba hosts. Subsequently, an assessment of the ISO11731:2017-05 pre-treatment procedure demonstrated that acid or heat treatment cause underestimation of alive Legionella population. Our results showed that these pre-treatment procedures induce culturable cells to enter a VBNC state. This may explain the observed insensitivity and lack of reproducibility often observed with the Legionella culture method. This study represents the first time that flow cytometry-cell sorting in conjunction with a qPCR assay has been used as a rapid and direct method to quantify VBNC Legionella from environmental sources. This will significantly improve future research evaluating Legionella risk management approaches for the control of Legionnaires' disease.

Detection of Unculturable Fungi in Soil Regions of Aziz Awa in Sulaimani province

Unculturable and non-sporulting fungi represent a great challenge instudying biotrophic, endotrophic and mycorrhizal fungal groups. In thisstudy collection of soil sample from region Aziz Awa in sulaimaniprovince and using culture-dependent and culture in-dependenttechniques for cultivation of unculturable fungi for the purpose ofexpanding studies on fungal biodiversity in soil. Sabouraud DextroseAgar with supplement as pyridoxine by replicating master plate fromhigher dilution of soil three tinny colonies (less than 1mm in diameter)had grown on medium after incubation for 5-7 weeks at 280C but had notshown growth when replicated on traditional mycological media (PDA,Czapack Dox Agar and Sabouraud Dextrose Agar) were selected,purified, studied culturally and microscopically and identified bymolecular methods. Four specific primer sets (NSIF/ITS4r, NSIF/ LRIF,EF4F/ITS4r and EF60F/ITS4r) were used to amplify partial sequences offungal r RNA gene included ITS sequences. The partial sequences ofthree clones were aligned through the BLASTN phylogenetic analysis inNCBI were available at GenBank database and revealed higher scoresand identities with Fusarium sp, Penicillium sp and Uncultured soilfungus clone. These results led us to consider the clones as viable butnon culturable (VBNC) soil fungi like the common phenomenon in somebacterial species.

Direct ferrous sulfate exposure facilitates the VBNC state formation rather than ferroptosis in Listeria monocytogenes

Listeria monocytogenes frequently causes Listeriosis in humans and animals. In present study, we discovered that in the presence of FeSO4, L. monocytogenes became viable but non-culturable (VBNC), and remained virulent to Caenorhabditis elegans. The killing assay indicated that these VBNC cells kept sensitive to tetracycline, differing from dormant cells. Transcriptomic analysis revealed more gene transcription occurrence in the VBNC cells compared to dormant cells, involving stress response and ribosome binding. No ferroptosis hallmarks were observed in the VBNC cells, whereas the application of either intracellular Fe2+ chelator or the ferroptosis inhibitor arrested the formation of VBNC state by FeSO4, as well as by Benzakonium chloride or Haz-Tab. This implicated the universal involvement of intracellular Fe2+ and other cascades related to ferroptosis in the formation of VBNC state in L. monocytogenes. Taken together, we discovered an iron-induced VBNC state in L. monocytogenes, and provided clues to further understanding their potential risks.