Viable but non-culturable state formation and resuscitation of different antibiotic-resistant Escherichia coli induced by UV/chlorine.

The presence of "viable but nonculturable" (VBNC) state and bacterial antibiotic resistance (BAR) both pose significant threats to the safety of drinking water. However, limited data was available that explicitly addressed the contribution of bacterial VBNC state in the maintenance and propagation of BAR. Here, the VBNC state induction and resuscitation of two antibiotic-resistant Escherichia coli K12 strains, one carrying multidrug-resistant plasmid (RP4 E. coli) and the other with chromosomal mutation (RIF E. coli) were characterized by subjecting them to different doses of UV/chlorine. The results illustrated that the induction, resuscitation, and associated mechanisms of VBNC ARB exhibit variations based on resistance determinants. RP4 E. coli exhibited a higher susceptibility to enter VBNC state compared to the RIF E. coli., and most VBNC state and resuscitated RP4 E. coli retained original antibiotic resistance. While, reverse mutation in the rpoB gene was observed in VBNC state and recovered RIF E. coli strains induced by high doses of UV/chlorine treatment, leading to the loss of rifampicin resistance. According to RT-qPCR results, ARGs conferring efflux pumps appeared to play a more significant role in the VBNC state formation of RP4 E. coli and the down-regulation of rpoS gene enhanced the speed at which this plasmid-carrying ARB entered into the dormant state. As to RIF E. coli, the induction of VBNC state was supposed to be regulated by the combination of general stress response, SOS response, stringent response, and TA system. Above all, this study highlights that ARB could become VBNC state during UV/chlorine treatments and retain, in some cases, their ability to spread ARGs. Importantly, compared with chromosomal mutation-mediated ARB, both VBNC and resuscitated state ARB that carries multidrug-resistant plasmids poses more serious health risks. Our study provides insights into the relationship between the VBNC state and the propagation of BAR in drinking water systems.

Repeatability of Noninvasive Break-Up Time Measurements using Keratograph Oculus 3.

AIM: The primary aim of this study is to evaluate the repeatability of noninvasive break-up time (NIBUT) measurement by keratograph when it is determined from one, two or three partial measurements, and to recommend a suitable methodology for practice. Another goal is to verify that repeated measurements do not affect the measured value. MATERIAL AND METHODS: Thirty-eight healthy volunteers (30 women and 8 men) aged between 19 and 50 years old were included in the study, in which only one eye of each volunteer was measured. The study was designed as a prospective one. Each subject adapted to the local conditions of the laboratory for 15 minutes and subsequently underwent two series of NIBUT measurements (test, retest) on an OCULUS 3 Keratograph. The minimum time interval between the two series was 10 minutes, in which each series contained three partial measurements approximately 3 three measurements in the given series. Repeatability was assessed by a Bland-Altman analysis and expressed as a repeatability coefficient. In every case, only the time of the first break-up of the tear film was monitored. RESULTS: The statistical analysis did not show statistically significant differences both between partial measurements of NIBUT in the individual series (p = 0.92, p = 0.81) and when comparing all six measurements (p = 0.95). The mean values of the partial measurements ranged from 13.6 s to 14.4 s. The repeatability coefficients were found to be 15.0 s, 12.1 s and 10.0 s for methodologies A, B and C, respectively. A supplementary analysis for 12 eyes with low NIBUT (< 10 s) showed statistically significantly better repeatability in this group, with coefficients of 7.0 s (methodology A), 6.0 s (B) and 4.6 s (C). CONCLUSION: Determination of NIBUT from three consecutive measurements (with a sufficient interval of ideally a few minutes) significantly improves repeatability. Such repeated NIBUT measurements do not have a significant effect on the measured value. The mentioned methodology for measuring NIBUT on a keratograph can be recommended for use in practice.

Bithiophene and coumestan derivatives from Eclipta prostrata (L.) L. and their hepatoprotective activity.

One new bithiophene derivative, 5-(but-3-en-1-yn-1-yl)-5'-(methoxymethyl)-2,2'-bithiophene (1), along with twelve known compounds, senecioester (2), tiglinsaureester (3), 5-acetoxymethyl-2'-(but-3-en-1-yn-1-yl)-2,5'-bithiophene (4), 5-(4-isovaleroyloxybut-1-ynyl)-2,2'-bithiophene (5), 5-hydroxymethyl-(2,5':2',5'')-terthienyl tiglate (6), 5-hydroxymethyl-(2,5':2',5'')-terthienyl agelate (7), 5- hydroxymethyl-2,5':2',5''-terthiophene dimethylacrylate (8), 5-methoxymethyl-2,2':5',2''-terthiophene (9), α-terthiophene (10), 1,3,8,9-tetrahydroxycoumestan 3-sulfate (11), demethylwedelolactone (12), and wedelolactone (13) were isolated from the methanol extract of aerial parts of Eclipta prostrata (L.) L. All isolated compounds were evaluated for the protective ability on the HepG2 cells. At the concentration of 100 µM, compounds 11-13 showed the highest hepatoprotective effects, with HepG2 cell viability ranging from 38.68% to 48.54%. Bithiophenes showed higher hepatoprotective cell viability than terthiophenes.

Increased flushing frequency of a model plumbing system initially promoted the formation of viable but non culturable cells but ultimately reduced the concentration of culturable and total Legionella DNA.

Legionella is the causative agent of Legionnaires' disease, and its prevalence in potable water is a significant public health issue. Water stagnation within buildings increases the risk of Legionella. However, there are limited studies investigating how stagnation arising through intermittent usage affects Legionella proliferation and the studies that are available do not consider viable but non culturable (VBNC) Legionella. This study used a model plumbing system to examine how intermittent water stagnation affects both VBNC and culturable Legionella. The model plumbing system contained a water tank supplying two biofilm reactors. The model was initially left stagnant for ≈5 months (147 days), after which one reactor was flushed daily, and the other weekly. Biofilm coupons, and water samples were collected for analysis at days 0, 14 and 28. These samples were analysed for culturable and VBNC Legionella, free-living amoebae, and heterotrophic bacteria. After 28 days, once-a-day flushing significantly (p < 0.001) reduced the amount of biofilm-associated culturable Legionella (1.5 log10 reduction) compared with weekly flushing. However, higher counts of biofilm-associated VBNC Legionella (1 log10 higher) were recovered from the reactor with once-a-day flushing compared with weekly flushing. Likewise, once-a-day flushing increased the population of biofilm-associated Vermamoeba vermiformis (approximately 3 log10 higher) compared with weekly flushing, which indicated a positive relationship between VBNC Legionella and V. vermiformis. This is the first study to investigate the influence of stagnation on VBNC Legionella under environmental conditions. Overall, this study showed that a reduction in water stagnation decreased culturable Legionella but not VBNC Legionella.

Real-time PCR methods for identification and stability monitoring of Bifidobacterium longum subsp. longum UABl-14 during shelf life.

Bifidobacterium longum subsp. longum UABl-14™ is an important probiotic strain that was found to support digestive health. Here we present the development and validation of real-time PCR methods for strain-specific identification and enumeration of this important strain. The identification method was evaluated for specificity using 22 target samples and 30 non-target samples. All target samples successfully amplified, while no amplification was observed from any non-target samples including other B. longum strains. The identification method was evaluated for sensitivity using three DNA dilution series and the limit of detection was 2 pg. of DNA. Coupled with a viability dye, the method was further validated for quantitative use to enumerate viable cells of UABl-14. The viability dye treatment (PMAxx) was optimized, and a final concentration of 50 µM was found as an effective concentration to inactivate DNA in dead cells from reacting in PCR. The reaction efficiency, linear dynamic range, repeatability, and reproducibility were also evaluated. The reaction efficiency was determined to be 97.2, 95.2, and 95.0% with R2 values of 99%, in three replicates. The linear dynamic range was 1.3 × 102 to 1.3 × 105 genomes. The relative standard deviation (RSD%) for repeatability ranged from 0.03 to 2.80, and for reproducibility ranged from 0.04 to 2.18. The ability of the validated enumeration method to monitor cell counts during shelf life was evaluated by determining the viable counts and total counts of strain UABl-14 in 18 multi-strain finished products. The viable counts were lower than label claims in seven products tested post-expiration and were higher than label claims in products tested pre-expiration, with a slight decrease in viable counts below label claim in three samples that were tested 2-3 months pre-expiration. Interestingly, the total counts of strain UABl-14 were consistently higher than label claims in all 18 products. Thus, the method enables strain-specific stability monitoring in finished products during shelf life, which can be difficult or impossible to achieve using the standard plate count method. The validated methods allow for simultaneous and cost-effective identification and enumeration of strain UABl-14 and represent an advancement in the quality control and quality assurance of probiotics.

Corrigendum: Phenotypic and transcriptional characterization of F. tularensis LVS during transition into a viable but non-culturable state.

[This corrects the article DOI: 10.3389/fmicb.2024.1347488.].

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.

Quantification and cultivation of Helicobacter pylori (H. pylori) from various urban water environments: A comprehensive analysis of precondition methods and sample characteristics.

Substantial evidence suggests that all types of water, such as drinking water, wastewater, surface water, and groundwater, can be potential sources of Helicobacter pylori (H. pylori) infection. Thus, it is critical to thoroughly investigate all possible preconditioning methods to enhance the recovery of H. pylori, improve the reproducibility of subsequent detection, and optimize the suitability for various water types and different detection purposes. In this study, we proposed and evaluated five distinct preconditioning methods for treating water samples collected from multiple urban water environments, aiming to maximize the quantitative qPCR readouts and achieve effective selective cultivation. According to the experimental results, when using the qPCR technique to examine WWTP influent, effluent, septic tank, and wetland water samples, the significance of having a preliminary cleaning step becomes more evident as it can profoundly influence qPCR detection results. In contrast, the simple, straightforward membrane filtration method could perform best when isolating and culturing H. pylori from all water samples. Upon examining the cultivation and qPCR results obtained from groundwater samples, the presence of infectious H. pylori (potentially other pathogens) in aquifers must represent a pressing environmental emergency demanding immediate attention. Furthermore, we believe groundwater can be used as a medium to reflect the H. pylori prevalence in a highly populated community due to its straightforward analytical matrix, consistent detection performance, and minimal interferences from human activities, temperature, precipitation, and other environmental fluctuations.

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.

Nonribosomal Peptide Synthetase Specific Genome Amplification Using Rolling Circle Amplification for Targeted Gene Sequencing.

Next-generation sequencing has transformed the acquisition of vast amounts of genomic information, including the rapid identification of target gene sequences in metagenomic databases. However, dominant species can sometimes hinder the detection of rare bacterial species. Therefore, a highly sensitive amplification technique that can selectively amplify bacterial genomes containing target genes of interest was developed in this study. The rolling circle amplification (RCA) method can initiate amplification from a single locus using a specific single primer to amplify a specific whole genome. A mixed cell suspension was prepared using Pseudomonas fluorescens ATCC17400 (targeting nonribosomal peptide synthetase [NRPS]) and Escherichia coli (non-target), and a specific primer designed for the NRPS was used for the RCA reaction. The resulting RCA product (RCP) amplified only the Pseudomonas genome. The NRPS was successfully amplified using RCP as a template from even five cells, indicating that the single-priming RCA technique can specifically enrich the target genome using gene-specific primers. Ultimately, this specific genome RCA technique was applied to metagenomes extracted from sponge-associated bacteria, and NRPS sequences were successfully obtained from an unknown sponge-associated bacterium. Therefore, this method could be effective for accessing species-specific sequences of NRPS in unknown bacteria, including viable but non-culturable bacteria.

Biocompatible plasma-treated liquids: A sustainable approach for decontaminating gastrointestinal-infection causing pathogens.

Nosocomial infections are a serious threat and difficult to cure due to rising antibiotic resistance in pathogens and biofilms. Direct exposure to cold atmospheric plasma (CAP) has been widely employed in numerous biological research endeavors. Nonetheless, plasma-treated liquids (PTLs) formulated with physiological solutions may offer additional benefits such as enhanced portability, and biocompatibility. Additionally, CAP-infused long-lived reactive oxygen and nitrogen species (RONS) such as nitrite (NO2-), nitrate (NO3-), and hydrogen peroxide (H2O2) can synergistically induce their antibacterial activity. Herein, we investigated those argon-plasma jet-treated liquids, including Ringer's lactate (RL), phosphate-buffered saline (PBS), and physiological saline, have significant antibacterial activity against nosocomial/gastrointestinal-causing pathogens, which might be due to ROS-mediated lipid peroxidation. Combining the conventional culture-based method with propidium iodide monoazide quantitative PCR (PMAxx™-qPCR) indicated that PTLs induce a minimal viable but non-culturable (VBNC) state and moderately affect culturable counts. Specifically, the PTL exposure resulted in pathogenicity dysfunction via controlling T3SS-related effector genes of S. enterica. Overall, this study provides insights into the effectiveness of PTLs for inducing ROS-mediated damage, controlling the virulence of diarrheagenic bacteria, and modulating homeostatic genes.

Induction of Dormancy in Cryptococcus neoformans In Vitro: The HypNOS Protocol.

Cryptococcus neoformans is the second major cause of death in patients with HIV. During a latent infection, this pathogenic fungus survives in the host for years without causing symptoms of active disease. Upon favorable conditions, such as immunosuppression due to HIV infection, or other conditions (steroid use or organ transplantation), the yeast may reactivate and cause active cryptococcosis. Hence, dormancy is an important phase in the pathogenesis of C. neoformans. Additionally, C. neoformans also persists during antifungal treatment and causes disease recurrence, which is a major medical problem, especially in low- and middle-income countries. To survive in the host, yeast cells must react to the stresses they are exposed to and generate a cellular response that is favorable for yeast survival. A prominent strategy used by C. neoformans to combat challenging surroundings is dormancy, which may translate into a viable, but nonculturable phenotype (VBNC). This chapter describes an in vitro protocol to generate and characterize dormant Cryptococci.

Exploration of genes associated with induction of the viable but non-culturable state of Campylobacter jejuni.

Campylobacter jejuni is known to enter a viable but non-culturable (VBNC) state when exposed to environmental stresses. Microarray and quantitative real-time polymerase chain reaction (qPCR) analyses were performed to elucidate the genes related to the induction of the VBNC state. The C. jejuni NCTC11168 strain was cultured under low-temperature or high-osmotic stress conditions to induce the VBNC state. mRNA expression in the VBNC state was investigated using microarray analysis, and the gene encoding peptidoglycan-associated lipoprotein, Pal, was selected as the internal control gene using qPCR analysis and software. The three genes showing particularly large increases in mRNA expression, cj1500, cj1254, and cj1040, were involved in respiration, DNA repair, and transporters, respectively. However, formate dehydrogenase encoded by cj1500 showed decreased activity in the VBNC state. Taken together, C. jejuni actively changed its mRNA expression during induction of the VBNC state, and protein activities did not always match the mRNA expression levels.

Flow cytometry: Unravelling the real antimicrobial and antibiofilm efficacy of natural bioactive compounds.

Flow cytometry (FCM) provides unique information on bacterial viability and physiology, allowing a real-time early warning antimicrobial and antibiofilm monitoring system for preventing the spread risk of foodborne disease. The present work used a combined culture-based and FCM approach to assess the in vitro efficacy of essential oils (EOs) from condiment plants commonly used in Mediterranean Europe (i.e., thyme EO, oregano EO, basil EO, and lemon EO) against planktonic and sessile cells of food-pathogenic Listeria monocytogenes 56 LY, and contaminant and alterative species Escherichia coli ATCC 25922 and Pseudomonas fluorescens ATCC 13525. Evaluation of the bacterial response to the increasing concentrations of natural compounds posed FCM as a crucial technique for the quantification of the live/dead, and viable but non-culturable (VBNC) cells when antimicrobial agents exert no real bactericidal action. Furthermore, the FCM results displayed higher numbers of viable bacteria expressed as Active Fluorescent Units (AFUs) with a greater level of repeatability compared with outcomes of the plate-count method. Overall, accurate counting of viable microbial cells is a critically important parameter in food microbiology, and flow cytometry provides an innovative approach with high-throughput potential for applications in the food industry as "flow microbiology".

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.

Comparative evaluation of insertion characteristics of PLMA using different techniques in the pediatric age group.

Background and Aims: The laryngeal mask airway ProSeal (PLMA) insertion should be easy, fast, and atraumatic. Most studies have been done on adults who cannot be considered as the reflection of pediatric patients. In this study, we compared the first attempt success rate of three techniques of PLMA insertion: introducer, 90° rotation, and pharyngoscopy technique in the pediatric population. Material and Methods: In this prospective comparative randomized study, a total of 135 patients of American Society of Anesthesiology grade I and II, aged three to eleven years, with normal airways scheduled for elective surgery, were randomly allocated into three groups: introducer, 90° rotation, and pharyngoscopy group. Parameters evaluated were: first attempt insertion success rate, insertion time, ease of insertion score, hemodynamic parameters, oropharyngeal seal pressure, manipulations, PLMA blood staining, postoperative sore throat, and hoarseness. Results: First attempt insertion success rate was higher in the 90° rotation (97.78%) and pharyngoscopy (97.78%) group as compared to the introducer group (93.33%). But the result was not statistically significant. PLMA insertion time was the least in the rotation group, followed by the pharyngoscopy and introducer group (P < 0.0001). Mean arterial pressure and heart rate were significantly raised in the pharyngoscopy versus rotation group and the introducer versus 90° rotation group after PLMA insertion. Oropharyngeal seal pressure was significantly higher in the introducer as compared to the rotation group (P = 0.007). Conclusion: All three techniques had a high first-attempt insertion success rate. As the rotation technique had the best result in insertion time and hemodynamic response, it may be considered a good alternative to pharyngoscopy and introducer technique in pediatric patients of age three to eleven years with a normal airway.

Persistence and viable but non-culturable state induced by streptomycin in Erwinia amylovora.

Persister cell and viable but non-culturable (VBNC) state of bacteria are survival strategies against antibiotics and various environmental stresses, respectively, but they tend to be ignored in agriculture fields, even though bacteria can regain their abilities to survive and produce disease once those stresses disappear. This study was carried out to determine whether persister cell and VBNC state in Erwinia amylovora are present after exposures to streptomycin, the length of their persistence, and the steps needed to decrease the inoculum. Persister cells were observed using biphasic killed growth curve for 4-8 h when the late stationary phase cells of E. amylovora were cultured in liquid medium containing streptomycin. This state was maintained for up to 12 h based on the colony forming units (CFUs) of the colonies that grew on the mannitol glutamate yeast extract (MGY) medium after streptomycin was removed. The CFUs on the MGY medium were lower than the total count determined using the LIVE/DEAD Kit, suggesting that persister cells and VBNC state might co-exist for up to 12 h after exposure to streptomycin. However, after 12 h, E. amylovora cells did not continue to grow on the medium for 9 days, suggesting that they entered a VBNC state at that time and remained in a persistent state. In addition, based on the Redox Sensor Green staining method, the presence of both states was confirmed for up to 12 h, and only then did the VBNC state became apparent. Furthermore, persister cells were observed for up to 24 h, and damaged cells reduced when E. amylovora cells were culture in distilled water with streptomycin, indicating that the uptake of lower nutrients in E. amylovora led to prolonged persister cells and VBNC state, which are more likely to survive after streptomycin treatments. The addition of sucrose and oxytetracycline to distilled water containing streptomycin reduced persister cells than other sources did. Thus, to inhibit the spread of fire blight, management techniques must consider the hazards of using streptomycin treatments that induce dormancy, such as persister cells and VBNC state, beyond the development of resistant strain.

Bacteriostasis of nisin against planktonic and biofilm bacteria: Its mechanism and application.

Food safety incidents caused by bacterial contamination have always been one of the public safety issues of social concern. Planktonic cells, viable but non-culturable (VBNC) cells, and biofilm cells of bacteria can coexist in food or food processing, posing more serious challenges to public health and safety by increasing bacterial survival and difficulty in detection. As a non-toxic, no side effect, and highly effective bacteriostatic substance, nisin has received wide attention from researchers. In this review, we summarized the species and biosynthesis of nisin, the effects of nisin alone or in combination with other treatments on planktonic and biofilm cells, and its applications in the fields of food, feed, and medicine by consulting numerous studies. Meanwhile, the mechanism of nisin on planktonic and biofilm cells was proposed based on existing researches. Nisin not only has antibacterial activity against most G+ bacteria but also exhibits a bacteriostatic effect on G- bacteria when combined with other antibacterial treatments. In addition to planktonic cells, nisin also has significant effects on bacterial cells in biofilms by changing the thickness, density, and composition of biofilms. Based on the three action processes of nisin on biofilms, we summarized the changes of bacteria in biofilms, including the causes of bacterial death and the formation of the VBNC state. We consider that research on the relationship between nisin and VBNC state should be strengthened.

Triglyceride-glucose index is capable of identifying metabolically obese, normal-weight older individuals.

BACKGROUND: The concept of metabolically obese, normal weight (MONW) has emerged to describe individuals with a normal body mass index (BMI) who are at a relatively high risk of chronic diseases. However, BMI itself is a suboptimal index for the assessment of the health risks associated with visceral fat. The triglyceride-glucose (TyG) index is considered to be a reliable and cost-effective marker of insulin resistance. Therefore, in the present study, we aimed to determine the TyG index cut-off values that could be used to define MONW in older people and to determine the usefulness of these values for the prediction of chronic diseases. METHODS: A total of 4,721 participants in the Korea National Health and Nutritional Examination Survey who were ≥ 60 years of age and did not have underweight or obesity were included. MONW was defined using the criteria for metabolic syndrome (MS), and the TyG index was calculated on the basis of the fasting plasma triglyceride and glucose concentrations. Chronic diseases, including T2DM, hypertension, and non-alcoholic fatty liver disease (NAFLD), were diagnosed. RESULTS: The prevalence of MS increased from the lowest to the highest TyG index tertile. The cut-off values of the TyG index for MONW were calculated as 8.88 and 8.80 for males and females, respectively. MONW, defined using these cut-off values, was associated with high odds ratios for NAFLD, T2DM, and hypertension in both males and females. CONCLUSIONS: The TyG index cut-off values calculated in the present study can be used to discriminate individuals with MONW from other older individuals without obesity and to predict the risk of chronic diseases. These findings show that the TyG index is an effective and cost-efficient method of assessing the risk of chronic diseases in people with MONW.