Viable State Research Articles

Regrowth of Escherichia coli in environmental waters after chlorine disinfection: shifts in viability and culturability.

Bacterial regrowth after water/wastewater disinfection poses severe risks to public health. However, regrowth studies under realistic water conditions that might critically affect bacterial regrowth are scarce. This study aimed to assess for the first time the regrowth of Escherichia coli (E. coli) in terms of its viability and culturability in environmental waters after chlorine disinfection, which is the most widely used disinfection method. Post-chlorination regrowth tests were conducted in 1) standard 0.85% NaCl solution, 2) river water receiving domestic wastewater effluents, and 3) river water that is fully recharged by domestic wastewater effluents. The multiplex detection of plate count and fluorescence-based viability test was adopted to quantify the culturable and viable E. coli to monitor the regrowth process. The results confirmed that chlorine treatment (0.2, 0.5 and 1.0 mg L-1 initial free chlorine) induced more than 99.95% of E. coli to enter a viable but non-culturable (VBNC) state and the reactivation of VBNC E. coli is presumably the major process of the regrowth. A second-order regrowth model well described the temporal shift of the survival ratio of culturable E. coli after the chlorination (R2: 0.73-1.00). The model application also revealed that the increase in initial chlorine concentration and chlorine dose limited the maximum regrowth rate and the maximum survival ratio, and the regrowth rate and percentage also changed with the water type. This study gives a better understanding of the potential regrowth after chlorine disinfection and highlights the need for investigating the detailed relation of the regrowth to environmental conditions such as major components of water matrices.

The Effect of pesticide usage on grape yeast

For the conventional vineyard, reducing the pesticide usage is the most important issue for converting to organic vineyard. In order to study the effect of pesticide usage on grape yeast, an investigation were designed in vintage 2020, ten vineyards (include two organic vineyards) with different treatment frequency index (TFI) were chosen, and the yeast population counted by two method: cultivate method and the direct epifluorescence technique (DEFT). The results show, the grape yeast from Moldova exist the viable but non-culturable (VBNC) state. In organic vineyards, less proportion yeast reach the VBNC, however the opposite conclusion draw from conventional vineyards. High TFI with high level VBNC yeast population, inversely, less culturable yeast. No matter in which yeast state, the treatment of powdery mildew is the most influential factor on yeast population.

Metabolic Characteristics and Markers in Viable But Nonculturable State of Pseudomonas Aeruginosa Induced by Chlorine Stress

Metabolic Characteristics and Markers in Viable But Nonculturable State of Pseudomonas Aeruginosa Induced by Chlorine Stress

Research progress in the Helicobacter pylori with viable non-culturable state.

Helicobacter pylori (H. pylori) is one of the most common pathogens in human beings and it is responsible for diseases such as chronic gastritis, peptic ulcer, and even gastric cancer. Studies in recent years have found that H. Pylori could transform from the normal spiral-shaped bacillary form into the coccoid form and enter a viable but non-culturable (VBNC) state, which may pose a potential threat to public health. In this state,the morphological structure and physiological characteristics of H. Pylori have changed. It can maintain the metabolic activity but protein expression is decreased. And the H. Pylori in this state cannot grow in the culture medium. Conditions such as environmental factors, antibiotics, and inhibitors can induce H. Pylori to enter the VBNC state, but it is still not known whether H. pylori in the VBNC state can reactivate or not. Based on the cell membrane integrity and metabolic activity of H. pylori in the VBNC state, it can be detected by classical methods including direct microscopy of live bacteria and molecular biological methods such as reverse transcription-polymerase chain reaction. H. pylori in the VBNC state has been detected in water source and biological media. It has been also found that H. pylori can enter the VBNC state in artificially contaminated food, which poses challenges to public health and food safety. Therefore, it is of great significance to study the change pattern and detection methods of H. pylori in the VBNC state for the prevention and control of H. pylori in the VBNC state. It is valuable to further study the underlying mechanisms of H. pylori in the VBNC state.

Bancor Comes of Age: A Case for an Indian Bitcoin Reserve

John Maynard Keynes proposed the concept of ‘Bancor’ in 1940 as a supranational currency that would serve as the international reserve currency. The concept did not take off at the time, despite the underlying need to liberate the international system from the hegemonic tendencies of a national currency serving as a global medium of exchange. The emergence of Bitcoin makes it possible to revive the idea of a de-nationalised global medium of exchange. This article examines the feasibility of such an idea by examining a viable state policy for adoption and use in the international realm.

The Role of RelA and SpoT on ppGpp Production, Stress Response, Growth Regulation, and Pathogenicity in Xanthomonas campestris pv. campestris.

ABSTRACTThe alarmone ppGpp plays an important role in the survival of bacteria by triggering the stringent response when exposed to environmental stress. Although Xanthomonas campestris pv. campestris (Xcc), which causes black rot disease in crucifers, is a representative species of Gram-negative phytopathogenic bacteria, relatively little is known regarding the factors influencing the stringent response in this species. However, previous studies in other Gram-negative bacteria have indicated that RelA and SpoT play a critical role in ppGpp synthesis. The current study found that these proteins also had an important role in Xcc, with a ΔrelAΔspoT double mutant being unable to produce ppGpp, resulting in changes to phenotype including reduced production of exopolysaccharides (EPS), exoenzymes, and biofilm, as well the loss of swarming motility and pathogenicity. The ppGpp-deficient mutant also exhibited greater sensitivity to environment stress, being almost incapable of growth on modified minimal medium (mMM) and having a much greater propensity to enter the viable but nonculturable (VBNC) state in response to oligotrophic conditions (0.85% NaCl). These findings much advance our understanding of the role of ppGpp in the biology of Xcc and could have important implications for more effective management of this important pathogen.IMPORTANCE Xanthomonas campestris pv. campestris (Xcc) is a typical seedborne phytopathogenic bacterium that causes large economic losses worldwide, and this is the first original research article to investigate the role of ppGpp in this important species. Here, we revealed the function of RelA and SpoT in ppGpp production, physiology, pathogenicity, and stress resistance in Xcc. Most intriguingly, we found that ppGpp levels and downstream ppGpp-dependent phenotypes were mediated predominantly by SpoT, with RelA having only a supplementary role. Taken together, the results of the current study provide new insight into the role of ppGpp in the biology of Xcc, which could also have important implications for the role of ppGpp in the survival and pathogenicity of other pathogenic bacteria.

The Impact of Protease during Recovery from Viable but Non-Culturable (VBNC) State in Vibrio cholerae.

Vibrio cholerae can survive cold stress by entering into a viable but non-culturable (VBNC) state, and resuscitation can be induced either by temperature upshift only or the addition of an anti-dormancy stimulant such as resuscitation-promoting factors (Rpfs) at suitable temperature. In this study, the role of proteinase K was analyzed as an Rpf in V. cholerae. A VBNC state was induced in V. cholerae AN59 in artificial seawater (ASW) media at 4 °C, and recovery could be achieved in filtered VBNC microcosm, called spent ASW media, merely by a temperature upshift to 37 °C. The resuscitation ability of spent ASW was further enhanced by the addition of proteinase K. The mode of action of proteinase K was investigated by comparing its effect on the growth of the VBNC and culturable state of V. cholerae in ASW and spent ASW media. The presence of proteinase K allowed culturable cells to grow faster in ASW by reducing the generation time. However, this effect of proteinase K was more pronounced in stressed VBNC cells. Moreover, proteinase K-supplemented spent ASW could also accelerate the transition of VBNC into recovered cells followed by rapid growth. Additionally, we found that dead bacterial cells were the substrate on which proteinase K acts to support high growth in spent ASW. So, the conclusion is that the proteinase K could efficiently promote the recovery and growth of dormant VBNC cells at higher temperatures by decreasing the duration of the initial lag phase required for transitioning from the VBNC to recovery state and increasing the growth rate of these recovered cells.

Extracellular pH decline introduced by high pressure carbon dioxide is a main factor inducing bacteria to enter viable but non-culturable state

High pressure carbon dioxide (HPCD) has been used in food processing as a non-thermal pasteurization technology. However, the potential of HPCD to induce viable but non-culturable (VBNC) cells limits its application. The objective of this study was to explore the roles of extracellular pH of 3 (pHex3) and high pressure (HP) of HPCD during VBNC induction and the underlying molecular mechanism. By using the model organism Escherichia coli O157:H7, we found that the combined effects of pHex3 and HP could mimic the effect of HPCD for VBNC induction. Further investigation of the individual effect of pHex3 and HP on VBNC induction revealed that pHex3 could induce a higher proportion of VBNC cells with a slower induction rate compared with HPCD, whereas HP was unable to induce VBNC formation. Notably, the cells treated by pHex3 and HPCD had similar morphological changes, and VBNC cells induced by pHex3 and HPCD had similar stress resistance characteristics. These results strongly indicated that pHex3 introduced by HPCD was a main factor for VBNC induction. Additionally, we found that HP played the role in accelerating VBNC formation in the process of HPCD treatment. Transcriptomic analysis revealed 85, 263 and 529 differentially expressed genes (DEGs) for HP-, pHex3- and HPCD-treated cells compared with untreated ones. 59 DEGs shared by pHex3 and HPCD treatment might be responsible for VBNC induction, and they were mainly involved in cellular transport and localization.

Characterization in the icosahedral phase of the system Al63Cu25Fe12

The present work aimed to characterize the microstructure of the icosahedral phase (ɸ-quasicrystalline phase) of the system with stoichiometric composition of the quasicrystal Al63Cu25Fe12. The ternary alloy with nominal composition of Al63Cu25Fe12 was processed by Mechanical Alloying (MA) as a viable solid state processing method for producing various metastable and stable quasicrystalline phases. The structural characterization of the obtained samples was performed by X-ray diffraction (XRD) and Scanning Electron Microscopy (SEM), while the elemental composition of the chemical elements Al, Fe and Cu were determined by the technique of X-ray spectroscopy by dispersive energy (EDS). According to the results of XRD, the diffraction patterns of Al63Cu25Fe12 showed the presence of β-Al (Fe, Cu) and λ-Al13Fe4 phases coexist with the thermodynamic ɸ-phase quasicrystalline. Finally, elemental analysis indicates that during alloy synthesis there is little variation of the ideal composition. The results indicate that alloys with high percentage of icosahedral phase can be obtained by casting in the air.

MODEL UNJURAN GUNA TENAGA DALAM SEKTOR PERINDUSTRIAN DI NEGERI PAHANG: SATU KAJIAN KES

Pahang is a state that is constantly intensifying development efforts to ensure the well-being of its people in achieving the target of becoming a viable state by 2050. However, the Covid-19 pandemic crisis has threatened the well-being of the population, especially the negative impact on the socio-economic landscape. As a result, almost all economic and social sectors were closed and has resulted in workers experiencing wage reductions, layoffs, and consequently rising unemployment rates. Therefore, Pahang state government needs to optimize all the opportunities available, among them is throughout efficient and effective management of employment resources. This study is a case study under the topic of employment resource management for the Pahang state government. The main objective of this study is to support Pahang state government in identifying the projected source of labor for the year 2020-2025, especially in the industrial sector. Apart from that, this study also seeks to help the Pahang state government by identifying factors related to the development of the industrial sector in line with the increase in employment resources. To realize the objectives of the study, secondary data were fully used in the data analysis. The results of the data analysis have projected that employment resources in the state of Pahang will increase approximately by 5,000 people a year and will reach a total of 1.77 million by 2025. The total labor force also increased from 748,000 in 2021 to 767,300 people in 2025 with Labor Force Participation Rate equivalent to 63.31%. At the same time the projection also records the total external labor force increasing to 42,400 people although at the same time the total working population increased to 733,600 people. The study also proposes employment projections in the industrial sector from 2021 to 2025. It is hoped that various initiatives that are implemented by various stakeholders to the industrial sector in Pahang could offers innovative employment opportunities to various sections of society to enhance socio-economic growth and spread the wellbeing to the society.

A size and space structured model of tumor growth describes a key role for protumor immune cells in breaking equilibrium states in tumorigenesis.

Switching from the healthy stage to the uncontrolled development of tumors relies on complicated mechanisms and the activation of antagonistic immune responses, that can ultimately favor the tumor growth. We introduce here a mathematical model intended to describe the interactions between the immune system and tumors. The model is based on partial differential equations, describing the displacement of immune cells subjected to both diffusion and chemotactic mechanisms, the strength of which is driven by the development of the tumors. The model takes into account the dual nature of the immune response, with the activation of both antitumor and protumor mechanisms. The competition between these antagonistic effects leads to either equilibrium or escape phases, which reproduces features of tumor development observed in experimental and clinical settings. Next, we consider on numerical grounds the efficacy of treatments: the numerical study brings out interesting hints on immunotherapy strategies, concerning the role of the administered dose, the role of the administration time and the interest in combining treatments acting on different aspects of the immune response. Such mathematical model can shed light on the conditions where the tumor can be maintained in a viable state and also provide useful hints for personalized, efficient, therapeutic strategies, boosting the antitumor immune response, and reducing the protumor actions.

Maximizing precision in saturation-limited absorption measurements

Quantum fluctuations in the intensity of an optical probe is noise which limits measurement precision in absorption spectroscopy. Increased probe power can offer greater precision, however, this strategy is often constrained by sample saturation. Here, we analyse measurement precision for a generalised absorption model in which we account for saturation and explore its effect on both classical and quantum probe performance. We present a classical probe-sample optimisation strategy to maximise precision and find that optimal probe powers always fall within the saturation regime. We apply our optimisation strategy to two examples, high-precision Doppler broadened thermometry and an absorption spectroscopy measurement of Chlorophyll A. We derive a limit on the maximum precision gained from using a non-classical probe and find a strategy capable of saturating this bound. We evaluate amplitude-squeezed light as a viable experimental probe state and find it capable of providing precision that reaches to within > 85% of the ultimate quantum limit with currently available technology.

Biofilm formation under high shear stress increases resilience to chemical and mechanical challenges

The effect that the hydrodynamic conditions under which biofilms are formed has on their persistence is still unknown. This study assessed the behaviour of Pseudomonas fluorescens biofilms, formed on stainless steel under different shear stress () conditions (1, 2 and 4 Pa), to chemical (benzalkonium chloride – BAC, glutaraldehyde – GLUT and sodium hypochlorite – SHC) and mechanical (20 Pa) treatments (alone and combined). The biofilms formed under different showed different structural characteristics. Those formed under a higher were invariably more tolerant to chemical and mechanical stresses. SHC was the biocide which caused the highest biofilm killing and removal, followed by BAC. The sequential exposure to biocides and mechanical stress was found to be insufficient for effective biofilm control. A basal layer containing biofilm cells mostly in a viable state remained on the surface of the cylinders, particularly for the 2 and 4 Pa-generated biofilms.

Enumeration of Viable Non-Culturable Vibrio cholerae Using Droplet Digital PCR Combined With Propidium Monoazide Treatment.

Many bacterial species, including Vibrio cholerae (the pathogen that causes cholera), enter a physiologically viable but non-culturable (VBNC) state at low temperature or in conditions of low nutrition; this is a survival strategy to resist environmental stress. Identification, detection, and differentiation of VBNC cells and nonviable cells are essential for both microbiological study and disease surveillance/control. Enumeration of VBNC cells requires an accurate method. Traditional counting methods do not allow quantification of VBNC cells because they are not culturable. Morphology-based counting cannot distinguish between live and dead cells. A bacterial cell possesses one copy of the chromosome. Hence, counting single-copy genes on the chromosome is a suitable approach to count bacterial cells. In this study, we developed quantitative PCR-based methods, including real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR), to enumerate VBNC V. cholerae cells by counting the numbers of single-copy genes in samples during VBNC-state development. Propidium monoazide (PMA) treatment was incorporated to distinguish dead cells from viable cells. Both PCR methods could be used to quantify the number of DNA copies/mL and determine the proportion of dead cells (when PMA was used). The methods produced comparable counts using three single-copy genes (VC1376, thyA, and recA). However, ddPCR showed greater accuracy and sensitivity than qPCR. ddPCR also allows direct counting without the need to establish a standard curve. Our study develops a PMA-ddPCR method as a new tool to quantify VBNC cells of V. cholerae. The method can be extended to other bacterial species.

Roles of virulence regulator ToxR in viable but non-culturable formation by controlling reactive oxygen species resistance in pathogen Vibrio alginolyticus

Under adverse circumstances, bacteria enter the viable but non-culturable (VBNC) state, a dormancy-like state for survival. The altered gene regulation underlying the entry of the VBNC state has not yet been well elucidated. Here, we reported that a subpopulation of cells (23.8 %) in Vibrio alginolyticus cultures enters the VBNC state in response to nutrient limitation at alkaline pH. The proteolysis of pivotal virulence regulator ToxR at these conditions is associated with VBNC formation. Meantime, ToxR abrogation impaired the mobility and the expression of virulence-associated genes, resulting in attenuated virulence in V. alginolyticus. RNA-seq and ChIP-seq analyses of the cells grown in VBNC-inducing conditions revealed that ToxR directly controls the expression of ∼8 genes including ahpC and dps involved in reactive oxygen species (ROS) resistance. ToxR binds to the promoter regions of kdgR, ppiC, ahpC, and dps and further controls their respective expression under oxidative stress conditions. The cells with impaired ToxR accumulated detrimental intracellular ROS. Moreover, these genes contribute to bacterial culturability as their in-frame deletion strains exhibiting severely decreased plate counts and the complementary strain showed rescued viability. Collectively, this study revealed the role of ToxR in switching on the VBNC state by sensing unfavorable environmental signals such as endogenous ROS (hydrogen peroxide, H2O2) in V. alginolyticus and provided mechanistic insights into Vibrio lifestyle adaptation in the marine environment.

Identification of Type II Toxin-Antitoxin Loci in Levilactobacillus brevis.

Levilactobacillus brevis are present in various environments, such as beer, fermented foods, silage, and animal host. Like other lactic acid bacteria, L. brevis might adopt the viable but nonculturable (VBNC) state under unfavorable conditions. The toxin-antitoxin (TA) system, known to regulate cell growth in response to environmental stresses, is found to control the dynamic of the VBNC state. Here, we investigate the type II TA locus prevalence and compare the TA diversity in L. brevis genomes. Using the TAfinder software, we identified a total of 273 putative type II TA loci in 110 replicons of 21 completely sequenced genomes. Genome size does not appear to correlate with the amount of putative type II TA in L. brevis. Besides, type II TA loci are distributed differently among the chromosomes and plasmids. The most prevalent toxin domain is MazF-like in the chromosomes, and RelE/RelE-like in the plasmids; while for antitoxin, Xre-like and Phd-like domains are the most common in the chromosomes and plasmids, respectively. We also observed a unique GNAT-like/ArsR-like TA pair that presents only in the L. brevis chromosome. Detection of 273 putative type II TA loci in 21 complete genomes of Levilactobacillus brevis.

A novel procedure in combination of genomic sequencing, flow cytometry and routine culturing for confirmation of beer spoilage caused by Pediococcus damnosus in viable but nonculturable state

Spoilage bacteria had been shown to form viable but nonculturable (VBNC) state maintaining food spoilage capability. In this study, a novel procedure was used to confirm a beer spoilage case caused by a Pediococcus damnosus strain in the VBNC state. Firstly, flow cytometry, routine culturing and PMA-PCR methods were used to identify approximately 103 cells/ml VBNC cells in the spoiled beer sample based on the difference between CFU and viable cell numbers. Secondly, genomic sequencing showed all acquired scaffolds were identical to the genome of P. damnosus with no existence of other species or isolates. In addition, VBNC cells were obtained in both simulation conditions, including beer low temperature storage and subculturing. MRS agar supplemented with catalase was found to resuscitate VBNC cells. Normal, VBNC and resuscitated cells showed similar level of beer spoilage capability. As concluded, a novel procedure, in combination of genomic sequencing, flow cytometry and routine culturing was used to confirm VBNC cells in spoiled beer sample, providing direct evidence on the beer spoilage caused by VBNC P. damnosus cells, and will aid in further study on VBNC state in food industry so that more evidence on food safety problem caused by VBNC microbes will be shown.

Adaptations of Vibrio parahaemolyticus to Stress During Environmental Survival, Host Colonization, and Infection.

Vibrio parahaemolyticus (Vp) is an aquatic Gram-negative bacterium that may infect humans and cause gastroenteritis and wound infections. The first pandemic of Vp associated infection was caused by the serovar O3:K6 and epidemics caused by the other serovars are increasingly reported. The two major virulence factors, thermostable direct hemolysin (TDH) and/or TDH-related hemolysin (TRH), are associated with hemolysis and cytotoxicity. Vp strains lacking tdh and/or trh are avirulent and able to colonize in the human gut and cause infection using other unknown factors. This pathogen is well adapted to survive in the environment and human host using several genetic mechanisms. The presence of prophages in Vp contributes to the emergence of pathogenic strains from the marine environment. Vp has two putative type-III and type-VI secretion systems (T3SS and T6SS, respectively) located on both the chromosomes. T3SS play a crucial role during the infection process by causing cytotoxicity and enterotoxicity. T6SS contribute to adhesion, virulence associated with interbacterial competition in the gut milieu. Due to differential expression, type III secretion system 2 (encoded on chromosome-2, T3SS2) and other genes are activated and transcribed by interaction with bile salts within the host. Chromosome-1 encoded T6SS1 has been predominantly identified in clinical isolates. Acquisition of genomic islands by horizontal gene transfer provides enhanced tolerance of Vp toward several antibiotics and heavy metals. Vp consists of evolutionarily conserved targets of GTPases and kinases. Expression of these genes is responsible for the survival of Vp in the host and biochemical changes during its survival. Advanced genomic analysis has revealed that various genes are encoded in Vp pathogenicity island that control and expression of virulence in the host. In the environment, the biofilm gene expression has been positively correlated to tolerance toward aerobic, anaerobic, and micro-aerobic conditions. The genetic similarity analysis of toxin/antitoxin systems of Escherichia coli with VP genome has shown a function that could induce a viable non-culturable state by preventing cell division. A better interpretation of the Vp virulence and other mechanisms that support its environmental fitness are important for diagnosis, treatment, prevention and spread of infections. This review identifies some of the common regulatory pathways of Vp in response to different stresses that influence its survival, gut colonization and virulence.

The change in heat inactivation of Escherichia coli O157:H7 after entering into the viable but non-culturable state in salted fish, Hypophthalmichthys molitrix

Many species of non-sporulating bacteria including Escherichia coli can enter into a viable but non-culturable (VBNC) state under stress conditions. In this study, the change in thermal resistance of E. coli O157:H7 after entering into the VBNC state in salted silver carp, Hypophthalmichthys molitrix, was investigated. E. coli O157:H7 was inoculated on the fish control group (TF) and on those fish with 30% NaCl (TF + 30% NaCl) at room temperature. Culturability of bacteria was determined using routine culture and colony counting on Sorbitol MacConkey agar. When bacteria were non-cultivable, the RT-PCR of 16S rRNA gene (including direct extraction and purification of RNA, DNase I treatment for removing DNA contamination, cDNA synthesis and electrophoresis of PCR products of cDNA) was used to detect VBNC E. coli O157:H7. Also, cultivable and VBNC E. coli O157:H7 were individually heat-treated at 55, 62 and 70 °C for 5 min. The samples were cooled and after 24 h, the thermal resistance of bacteria was determined through viability detection using RT-PCR of 16S rRNA gene. The culturability of bacteria was kept in fish treatment but they were non-cultivable in fish under 30% NaCl after 5 days. The positive expression of 16S rRNA in all studied treatments indicated the entering of E. coli O157:H7 into the VBNC state in fish treatment under 30% NaCl. Moreover, the RT-PCR of 16S rRNA gene showed that only VBNC forms of E. coli O157:H7 showed viability at 62 °C for 5 min which indicated the increased resistant of VBNC bacteria to the thermal inactivation. The ability of E. coli O157:H7 to enter into VBNC state in salted fish and also the increase of its thermal resistance suggest that VBNC E. coli O157: H7 can be considered as critical threat to public health and food safety.

The change in heat inactivation of Escherichia coli O157:H7 after entering into the viable but non-culturable state in salted fish, Hypophthalmichthys molitrix

The change in heat inactivation of Escherichia coli O157:H7 after entering into the viable but non-culturable state in salted fish, Hypophthalmichthys molitrix