Viable But Nonculturable Research Articles

The effect of some disinfectants in inducing the viable but nonculturable (VBNC) state of some pathogenic bacteria

The effect of some disinfectants in inducing the viable but nonculturable (VBNC) state of some pathogenic bacteria

The DUF348 domains of resuscitation promoting factor 2 play important roles in the enzymatic and biological activities in Rhodococcus erythropolis KB1.

Rhodococcus erythropolis KB1 is a member of the Actinomycetota and a petroleum-degrading bacterium, isolated from soil contaminated with petroleum products. The resuscitation-promoting factors (Rpf) widely exist among Actinomycetota, which revive the viable but nonculturable (VBNC) state cells and facilitate growth of normal cells. The Rpf2 of the R. erythropolis KB1 is the most complex Rpf protein, which consists of the conserved Rpf domain, one G5 domain and three DUF348 domains. The protein demonstrates muralytic activity and growth-promoting and resuscitation effect, but the exact roles of these DUF348 domains in the enzymic and biological activities remain unclear. In this paper, the recombinant plasmids containing rpf2 genes with different DUF348 domain deletion were constructed and expressed in Escherichia coli. The enzymatic and biological activities of the mutated Rpf2 proteins were examined. The results showed that the enzymatic activities of the mutated Rpf2 proteins with 1, 2, and 3 DUF348 deletion decreased by 26.27%, 38.17%, and 42.56% respectively when compared with that of the wild-type Rpf2. A negative correlation between the number of DUF348 deletions and the growth-promoting and resuscitation effect on R.erythropolis KB1 cells were also observed. The muralytic activities of the mutated Rpf2 proteins showed stability at the temperature range of 20°C to 40°C, but showed sharp declines at 50°C, with the activity dropping by 50.07% to 90.06%, and complete loss at 70°C and 80°C, underscoring importance of the DUF348 in thermal stability of the Rpf2. Zn2+ and Mn2+ slightly enhanced the muralytic activity, while Mg2+, Ca2+ and Co2+ had negligible effects. These findings offered significant insights into mechanism of the Rpf action, emphasizing the critical role of the DUF348 domain.

Intracellular ATP concentration is a key regulator of bacterial cell fate.

ATP, most widely known as the primary energy source for numerous cellular processes, also exhibits the characteristics of a biological hydrotrope. The viable but nonculturable (VBNC) and persister states are two prevalent dormant phenotypes employed by bacteria to survive challenging environments, both of which are associated with low metabolic activity. Here, we investigate the intracellular ATP concentration of individual VBNC and persister cells using a sensitive ATP biosensor QUEEN-7μ and reveal that both types of cells possess a lower intracellular ATP concentration than culturable and sensitive cells, although there is a certain overlap in the intracellular ATP concentrations between antibiotic-sensitive cells and persisters. Moreover, we successfully separated VBNC cells from culturable cells using fluorescence-activated cell sorting based on the intracellular ATP concentration threshold of 12.5 µM. Using an enriched VBNC cell population, we confirm that the precipitation of proteins involved in key biological processes promotes VBNC cell formation. Notably, using green light-illuminated proteorhodopsin (PR), we demonstrate that VBNC cells can be effectively resuscitated by elevating their intracellular ATP concentration. These findings highlight the crucial role of intracellular ATP concentration in the regulation of bacterial cell fate and provide new insights into the formation of VBNC and persister cells.IMPORTANCEThe viable but nonculturable (VBNC) and persister states are two dormant phenotypes employed by bacteria to counter stressful conditions and play a crucial role in chronic and recurrent bacterial infections. However, the lack of precise detection methods poses significant threats to public health. Our study reveals lower intracellular ATP concentrations in these states and establishes an ATP threshold for distinguishing VBNC from culturable cells. Remarkably, we revive VBNC cells by elevating their intracellular ATP levels. This echoes recent eukaryotic studies where modulating metabolism impacts outcomes like osteoarthritis treatment and lifespan extension in Caenorhabditis elegans. Our findings underscore the crucial role of intracellular ATP levels in governing bacterial fate, emphasizing ATP manipulation as a potential strategy to steer bacterial behavior.

Novel Populations of Mycobacterium smegmatis Under Hypoxia and Starvation: Some Insights on Cell Viability and Morphological Changes.

The general features of the shift to a dormant state in mycobacterial species include several phenotypic changes, reduced metabolic activities, and increased resistance to host and environmental stress conditions. In this study, we aimed to provide novel insights into the viability state and morphological changes in dormant M. smegmatis that contribute to its long-term survival under starvation or hypoxia. To this end, we conducted assays to evaluate cell viability, morphological changes and gene expression. During starvation, M. smegmatis exhibited a reduction in cell length, the presence of viable but non-culturable (VBNC) cells and the formation of anucleated small cells, potentially due to a phenomenon known as reductive cell division. Under hypoxia, a novel population of pleomorphic mycobacteria with a rough surface before the cells reached nonreplicating persistence 1 (NRP1) was identified. This population exhibited VBNC-like behaviour, with a loss of cell wall rigidity and the presence of lipid-body-like structures. Based on dosR and hspX expression, we suggest that M. smegmatis encounters reductive stress conditions during starvation, while lipid storage may induce oxidative stress during hypoxia. These insights into the heterogeneous populations presented here could offer valuable opportunities for developing new therapeutic strategies to control dormant mycobacterial populations.

VBNC Cronobacter sakazakii survives in macrophages by resisting oxidative stress and evading recognition by macrophages

Survival in host macrophages is an effective strategy for pathogenic bacterial transmission and pathogenesis. Our previous study found that viable but non-culturable (VBNC) Cronobacter Sakazakii (C. sakazakii) can survive in macrophages, but its survival mechanism is not clear. In this study, we investigated the possible mechanisms of VBNC C. sakazakii survival in macrophages in terms of environmental tolerance within macrophages and evasion of macrophages recognition. The results revealed that VBNC C. sakazakii survived under oxidative conditions at a higher rate than the culturable C. sakazakii. Moreover, the stringent response gene (relA and spoT) and the antioxidant-related genes (sodA, katG, and trxA) were up-regulated, indicating that VBNC C. sakazakii may regulate antioxidation through stringent response. On the other hand, compared with culturable C. sakazakii, VBNC C. sakazakii caused reduced response (Toll-like receptor 4) in macrophages, which was attributed to the suppression of biosynthesis of the lipopolysaccharides (LPS). Furthermore, we found that ellagic acid can reduce the survival rate of bacteria in macrophages by improving the immune TLR4 recognition ability of macrophages. In conclusion, VBNC C. sakazakii may survive in macrophages by regulating oxidative tolerance through stringent response and altering LPS synthesis to evade TLR4 recognition by macrophages, which suggests the pathogenic risk of VBNC C. sakazakii.

Comparison of three methods for generating the coccoid form of Helicobacter pylori and proteomic analysis

BackgroundHelicobacter pylori changes from spiral to coccoid depending on the host state, environmental factors, and surrounding microbial communities. The coccoid form of H. pylori still maintains its complete cellular structure, retains virulence genes, and thus plays a role in pathogenicity. To understand the coccoid form, it is crucial to establish the in vitro generation of the coccoid H. pylori. Although some conditions have been studied for the generation of the coccoid form, few studies have compared these conditions for coccoid generation. Here, we generated coccoid forms via three methods and compared the differences in morphology, viability, culturability, and protein expression.ResultsThe coccoid H. pylori was generated in vitro via three methods: a starvation method, a method using amoxicillin, and a method using the culture supernatant of Streptococcus mitis. The morphology and viability of the cells were examined by fluorescence microscopy after staining with SYTO9 and propidium iodide. The culturability of H. pylori was examined by counting colony-forming units on chocolate agar plates. In the starvation group, no colonies formed after 7 days, but viable coccoids were continuously observed. In the amoxicillin-treated group, the culturability decreased rapidly after 12 h, and showed a viable but non culturable (VBNC) state after the third day. Most cells treated with S. mitis supernatant changed to coccoid forms after 7 days, but colonies were continuously formed, probably due to living spiral forms. We performed proteomics to analyse the differences in protein profiles between the spiral and coccoid forms and protein profiles among the coccoid forms generated by the three methods.ConclusionAmoxicillin treatment changed H. pylori to VBNC cells faster than starvation. Treatment with the S. mitis supernatant prolonged the culturability of H. pylori, suggesting that the S. mitis supernatant may contain substances that support spiral form maintenance. Proteomic analysis revealed that the expression of proteins differed between the spiral form and coccoid form of H. pylori, and this variation was observed among the coccoid forms produced via three different methods. The proteins in the coccoid forms produced by the three methods differed from each other, but common proteins were also observed among them.

Significance of Viable But Non-culturable (VBNC) State in Vibrios and Other Pathogenic Bacteria: Induction, Detection and the Role of Resuscitation Promoting Factors (Rpf).

Still, it remains a debate after four decades of research on surviving cells, several bacterial species were naturally inducted and found to exist in a viable but non-culturable (VBNC) state, an adaptive strategy executed by most bacterial species under different stressful conditions. VBNC state are generally attributed when the cells lose its culturability on standard culture media, diminish in conventional detection methods, but retaining its viability, virulence and antibiotic resistance over a period of years and may poses a risk to marine animals as well as public health and food safety. In this present review, we mainly focus the VBNC state of Vibrios and other human bacterial pathogens. Exposure to several factors like nutrient depletion, temperature fluctuation, changes in salinity and oxidative stress, antibiotic and other chemical stress can induce the cells to VBNC state. The transcriptomic and proteomic changes during VBNC, modification in detection techniques and the most significant role of Rpf in conversion of VBNC into culturable cells. Altogether, detection of unculturable VBNC forms has significant importance, since it may not only regain its culturability, but also reactivate its putative virulence determinants causing serious outbreaks and illness to the individual.

Biofilm formation dynamics in long-distance water conveyance pipelines: Impacts of nutrient levels and metal stress

Biofilm formation in long-distance water conveyance pipelines poses significant risks to water quality, particularly under varying nutrient levels and heavy metal stress. However, the impacts of pipeline material on biofilm formation dynamics under different raw water conditions remain elusive. This study investigated the effects of nutrient availability and Fe-Mn stress on biofilm development, structural stability, bacterial community composition, and the occurrence of viable but non-culturable (VBNC) bacteria. Using reactors with different nutrient conditions, we observed that increased nutrient levels promote biofilm growth but lead to greater instability, heightening the risk of secondary contamination. Notably, nutrient escalation beyond a critical threshold had a diminishing impact on biofilm community composition. Additionally, Fe-Mn stress, while initially enhancing microbial adhesion and metabolic activity, ultimately inhibited biofilm formation over time and increases the prevalence of VBNC bacteria, particularly on stainless steel (SS) surfaces. Our findings also highlighted the importance of material selection for pipelines, with polyvinyl chloride (PVC) showing reduced biofilm formation compared to SS, making it a more suitable option for transporting raw water in environments with high metal content. Dispersal limitation determined the bacterial community assembly during the biofilm formation, accounting for 64.53–90.67 % of the variability in different scenarios. These insights offer valuable guidance for managing biofilm-related issues in water distribution systems, emphasizing the need for careful control of nutrient levels and material choice to ensure water safety over long distances.

Aquatic environment drives the emergence of cell wall-deficient dormant forms in Listeria

Stressed bacteria can enter a dormant viable but non-culturable (VBNC) state. VBNC pathogens pose an increased health risk as they are undetectable by growth-based techniques and can wake up back into a virulent state. Although widespread in bacteria, the mechanisms governing this phenotypic switch remain elusive. Here, we investigate the VBNC state transition in the human pathogen Listeria monocytogenes. We show that bacteria starved in mineral water become VBNC by converting into osmotically stable cell wall-deficient coccoid forms, a phenomenon that occurs in other Listeria species. We reveal the bacterial stress response regulator SigB and the autolysin NamA as major actors of VBNC state transition. We lastly show that VBNC Listeria revert to a walled and virulent state after passage in chicken embryos. Our study provides more detail on the VBNC state transition mechanisms, revealing wall-free bacteria naturally arising in aquatic environments as a potential survival strategy in hypoosmotic and oligotrophic conditions.

Global transcriptional analysis for molecular responses of Alicyclobacillus acidoterrestris spores in drinking water after low- and medium-pressure ultraviolet irradiation

Ultraviolet (UV) irradiation can effectively disinfect water contaminated with pathogens. However, the biological mechanisms of inactivation by different types of UV irradiation are unknown. The present study investigated the inactivation mechanisms of Alicyclobacillus acidoterrestris spores in water by low-pressure UV (LPUV) and medium-pressure UV (MPUV) using a quasi-collimated beam apparatus. Global transcriptomic data obtained by RNA-seq revealed 291 shared differentially expressed genes (DEGs) that damaged DNA, reduced biofilm formation, and had other reactions. The individual downregulated DEGs (n = 123) mainly related to cell motility, membrane transport, and metabolism were induced by LPUV, and in turn contributed to energy-saving and metabolic activity inhibition, forcing bacteria into a viable but non-culturable (VBNC) state. The individual upregulated DEGs (n = 244) following MPUV treatment were mainly enriched in cell motility, membrane transport, metabolism, DNA replication and repair, and spore germination pathways. This results in high-energy consumption, severe damage to genetic material, and enhanced spore germination accelerated cell death. Additionally, hub genes in the protein-protein interaction network were mainly involved in transcription and translation. These findings contribute to the comprehensive understanding of the inactivation mechanisms of different types of UV irradiation, and will improve applications of UV disinfection in the treatment of water.

Propidium monoazide (PMA) qPCR assay compared to the plate count method for quantifying the growth of Salmonella enterica serotypes in vacuum-packaged turkey breast combined with a mathematical modeling approach

This study compares the plate count (PC) and the Propidium Monoazide-quantitative Polymerase Chain Reaction (PMA-qPCR) methods to assess the growth of a cocktail of three serotypes of Salmonella enterica (Heidelberg, Typhimurium, and Enteritidis) in cooked, sliced, and vacuum-packaged turkey breast (STB) under isothermal storage temperatures (8 °C–20 °C), using predictive models. Standard curves were developed for PMA-qPCR, demonstrating high efficiency (101%) and sensitivity, with quantification limits ranging from 1 to 2 log10 CFU/g for all temperatures studied. Comparative analysis revealed a significant correlation (R2 = 0.99; 95% CI) between the PC and PMA-qPCR methods; however, the agreement analysis indicated a mean difference (Bias) of −0.11 log10 CFU/g (p < 0.05), suggesting underestimation by the PC method. This indicates the presence of stressed or viable but nonculturable (VBNC) cells, detectable by PMA-qPCR but not by PC. The Baranyi and Roberts model showed a good ability to describe the behavior of S. enterica cocktail in STB for PC and PMA-qPCR data under all isothermal conditions. The exponential secondary model more accurately represented the temperature dependence of the maximum specific growth rate compared to the Ratkowsky square root model, with R2 values ≥ 0.984 and RMSE values ≤ 0.011 for both methods. These results suggest that combining PMA-qPCR with predictive modeling allows for a more accurate prediction of S. enterica growth, compared to PC method. In the event of cold chain disruptions of meat products, the use of PMA-qPCR method allow the quantification of VBNC cells, that can still pose a health risk to consumers, especially in ready-to-eat products.

Study of chlorine disinfection on the formation and resuscitation of Viable but nonculturable (VBNC) bacteria in drinking water distribution systems

The presence of Viable but nonculturable (VBNC) bacteria in drinking water distribution systems(DWDS)which can recover and regain pathogenicity under certain conditions, leads to a serious underestimation of the number of microorganisms in the water and consequently microbiological risks. This study investigated E. coli's biofilm integrity and metabolic activity post‑chlorine disinfection and during resuscitation in a Circulating-flow Dynamic biofilm Contactor (CDC) reactor. Chlorine rapidly induced the VBNC state, with concentrations ≥0.3 mg/L causing all respiratory-active bacteria to transition. Higher chlorine levels damaged cell biofilms, reducing intact cell counts by 95 % and depleting ATP. VBNC bacteria in biofilms quickly regained culturability, utilizing available nutrients and ATP. Within 1–4 days, culturable bacteria in the biofilm increased from 2.29 to 4.95 log CFU/mL, significantly faster than in water. This growth corresponded with rising ATP levels, though slower than in aqueous environments. Signaling molecules (C6, C8, C12, C14) regulated gene expression during resuscitation, aiding bacterial recovery and proliferation. Chlorine disinfection disrupted intercellular communication, altering VBNC bacteria's physiological and metabolic characteristics. These findings highlight the complex dynamics of VBNC bacteria in water systems, their resuscitation potential, and the importance of biofilms in bacterial recovery, emphasizing the need for comprehensive water treatment strategies.

Induction of Viable but Non-culturable (VBNC) State in Shigella flexneri Under Osmotic and Nutritional Stresses at Low Temperature

Background: Viable but non-culturable (VBNC) is a specific physiological state in which living bacteria lose the ability to grow and form colonies on conventional bacterial growth media; however, these cells remain metabolically active. Objectives: Considering the importance of the pathogenicity of Shigella flexneri and the possibility of its transmission through contaminated water and food, we aimed to study the possibility of this bacterium entering the VBNC state under osmotic and nutritional stresses at low temperature (4°C). Methods: Shigella flexneri was inoculated in distilled water and NaCl solutions with concentrations of 0, 5, 10, 15, 20, 25, and 30% at 4°C. The cultivability of the bacteria was checked daily. After observing the loss of cultivability, entry into the VBNC state was assessed using RT-PCR, flow cytometry, and fluorescence microscopy. Results: After cultivation of S. flexneri in different concentrations of salt solution, the time of non-cultivability was observed at concentrations of 0, 5, 10, 15, 20, 25, and 30%, respectively, on days 19, 20, 21, 22, 22, 24, and 24. Expression results of ipaH and ipaD genes using RT-PCR, along with the presence of live cells detected by flow cytometry and fluorescent staining, indicated that the cells had entered the VBNC state. Conclusions: Shigella flexneri can survive and enter the VBNC state under nutritional and osmotic stresses at low temperatures (4°C). In this state, although the bacterium is alive, it cannot be detected by conventional culture-dependent methods. Due to the potential risk of bacterial recovery, conventional techniques may be insufficient to detect the presence of pathogens in the VBNC state when evaluating water and food.

Preliminary Study of the Characterization of the Viable but Noncultivable State of Yersinia enterocolitica Induced by Chloride and UV Irradiation.

The viable but non-culturable (VBNC) state is a survival strategy for many foodborne pathogens under adverse conditions. Yersinia enterocolitica (Y. enterocolitica) as a kind of primary foodborne pathogen, and it is crucial to investigate its survival strategies and potential risks in the food chain. In this study, the effectiveness of ultraviolet (UV) irradiation and chlorine treatment in disinfecting the foodborne pathogen Y. enterocolitica was investigated. The results indicated that both UV irradiation and chlorine treatment can induce the VBNC state in Y. enterocolitica. The bacteria completely lost culturability after being treated with 25 mg/L of NaClO for 30 min and a UV dose of 100 mJ/cm². The number of culturable and viable cells were detected using plate counting and a combination of fluorescein and propidium iodide (live/dead cells). Further research found that these VBNC cells exhibited reduced intracellular Adenosine Triphosphate (ATP) levels, and increased levels of reactive oxygen species (ROS) compared to non-induced cells. Morphologically, the cells changed from a rod shape to a shorter, coccobacillary shape with small vacuoles forming at the edges, indicating structural changes. Both condition-induced VBNC-state cells were able to resuscitate in tryptic soy broth (TSB) medium supplemented with Tween 80, sodium pyruvate, and glucose. These findings contribute to a better understanding of the survival mechanisms of Y. enterocolitica in the environment and are of significant importance for the development of effective disinfection strategies.

Dormancy of pathogenic bacteria in the fresh produce supply chain

The increased consumption of fresh produce in the last decades has led to an escalation in the number of associated foodborne outbreaks. Various stressful conditions along the fresh produce supply chain can induce bacteria to enter a dormancy state, such as the persistence state and the viable but nonculturable (VBNC) state. Bacteria can develop an increased tolerance to a range of environmental stresses in the dormancy state and return to their normal growing state when conditions become favorable, leading to foodborne illnesses. This review will explore how bacteria contaminate fresh produce along the supply chain and the conditions that can promote the formation of dormant cells. Additionally, various options for detecting and eradicating dormant bacterial cells in the fresh produce supply chain will be discussed.

Induction and comparative resuscitation of viable but nonculturable state on Vibrio parahaemolyticus serotypes O3:K6 and O1:K25.

Vibrio parahaemolyticus, an important food-borne pathogens found to be associated with seafoods and marine environs. It has been a topic of debate for many decades that most pathogens are known to enter a viable but nonculturable (VBNC) state under cold temperature and nutrient limited conditions. The present study examined the time required for the induction of VBNC state and the revival strategies of both the endemic O3:K6 and O1:K25 sporadic strains of V. parahaemolyticus. The results revealed that V. parahaemolyticus survived even after 55 days of incubation in nutrient starved media such as phosphate buffered saline (PBS) and Coastal Water (CW) and could be recovered by temperature upshift method, and compared the resuscitation using Dulbecco's Modified Eagle Medium (DMEM), sheep blood serum, chitin flakes with live Artemia salina, and the results suggests that chitin plays a significant role in regulating the VBNC state. It was also confirmed by Confocal Laser Scanning Microscopy (CLSM) and Scanning Electron Microscope (SEM) analysis that VBNC cells can alter their morphology to coccoid forms in order to survive in most extreme nutrient limited environment. Further data on the promoting factors and the exact mechanism that resuscitate VBNC V. parahaemolyticus in cold natural environments and frozen foods are needed to perform a robust risk assessment.

Copper single-atom catalysts for broad-spectrum antibiotic-resistant bacteria (ARBs) antimicrobial: Activation of peroxides and mechanism of ARBs inactivation

Antibiotic-resistant bacteria (ARBs) have been widely detected in wastewater and become a potential threat to human health. This work found that low-load single-atom copper (0.1 wt%) anchored on g-C3N4 (SA-Cu/g-C3N4) exhibited excellent ability to activate H2O2 and inactivate ARBs during the photo-Fenton process. The presence of SA-Cu/g-C3N4 (0.4 mg/mL) and H2O2 (0.1 mM) effectively inactivated ARBs. More than 99.9999 % (6-log) of methicillin-resistant Staphylococcus aureus (MRSA), and carbapenem-resistant Acinetobacter baumannii (CRAB) could be inactivated within 5 min. Extended-spectrum β-lactamase-producing pathogenic Escherichia coli (ESBL-E) and vancomycin-resistant Enterococcus faecium (VRE) were killed within 10 and 30 min, respectively. In addition, more than 5-log of these ARBs were killed within 60 min in real wastewater. Furthermore, D2O-labeling with Raman spectroscopy revealed that SA-Cu/g-C3N4 completely suppressed the viable but nonculturable (VBNC) state and reactivation of bacteria. Electron paramagnetic resonance spectroscopy results demonstrated that g-C3N4 mainly produced 1O2, while SA-Cu/g-C3N4 simultaneously produced both 1O2 and •OH. The •OH and 1O2 cause lipid peroxidation damage to the cell membrane, resulting in the death of the bacteria. These findings highlight that the SA-Cu/g-C3N4 catalyst is a promising photo-Fenton catalyst for the inactivation of ARBs in wastewater.

The viable but non-culturable (VBNC) status of Shewanella putrefaciens (S. putrefaciens) with thermosonication (TS) treatment

Although thermosonication (TS) treatment has been widely used in food sterilization, the viable but non‐culturable (VBNC) of bacteria with TS treatment has still concerned potential food safety and public health. The molecular mechanism of VBNC status of bacteria with TS treatment is not clearly known. Therefore, in this study, we used Shewanella putrefaciens, which was a common putrefactive bacteria in aquatic products, to study the VBNC state of bacteria with TS treatment. Firstly, our results revealed that S. putrefaciens still could enter the VBNC state after TS treatments: 50 kHz, 300 W, 30 min ultrasonic treatment and 70 °C heating; Subsequently, we found the VBNC state of S. putrefaciens can resist the damage of TS treatment, such as cell wall break, DNA degradation, etc; Finally, four-dimensional data-independent acquisition-based proteomics showed that under VBNC state, S. putrefaciens upregulated functional proteins to resist TS treatment, such as: ribosomal proteins to accelerate the synthesis of stress proteins to counteract TS treatments, ornithine decarboxylase SpeF and MraY to repair TS treatment-induced damage, etc. Meanwhile, S. putrefaciens downregulates metabolic and transport functional proteins such as dehydrogenase to reduce the metabolism. Importantly, among those proteins, the ribosomal transcriptional regulatory protein family, such as rpsB, etc, may be the key proteins for S. putrefaciens entering VBNC state. This finding can provide some new strategies for preventing VBNC status of bacteria with TS treatment, such as: inhibition of key proteins, etc.

Enhanced biodegradation of polychlorinated biphenyls by co-cultivation of resuscitated strains with unique advantages

The investigation into viable but non-culturable (VBNC) bacteria through the implementation of resuscitation promoting factors (Rpfs) has broadened the potential sources for isolating strains capable of degrading polychlorinated biphenyls (PCBs). Nonetheless, there has been limited research on the efficacy of resuscitated strains and the potential improvement of their performance through co-cultivation. In this work, the PCB degradation potential of resuscitated strains, specifically Pseudomonas sp. HR1 and Achromobacter sp. HR2, as well as their co-cultures, was investigated. Of particular importance was the comparative analysis between the optimal co-culture and individual strains regarding their ability to degrade PCB homologs and mineralize intermediate metabolites. The results suggested that the resuscitated strains HR1 and HR2 demonstrated robust growth and effective degradation of Aroclor 1242. The co-culture CO13, with an optimal HR1 to HR2 ratio of 1:3, exhibited a remarkable improvement in PCB degradation and intermediate metabolite mineralization compared to individual strains. Analysis of functional genes and degradation metabolites revealed that both the individual strains and co-culture CO13 degraded PCBs via the HOPDA-benzoate pathway, then mineralized through protocatechuate meta- and ortho-cleavage pathways, as well as the catechol ortho-cleavage pathway. This study represents the first documentation of the improved PCB degradation through the co-cultivation of resuscitated strains, which highlights the great promise of these resuscitated strains and their co-cultures as effective bio-inoculants for enhanced bioremediation.

Resting for viability: Gordonia polyisoprenivorans ZM27, a robust generalist for petroleum bioremediation under hypersaline stress

The intrinsic issue associated with the application of microbes for practical pollution remediation involves maintaining the expected activity of engaged strains or consortiums as effectively as that noted under laboratory conditions. Faced with various stress factors, degraders with dormancy ability are more likely to survive and exhibit degradation activity. In this study, a hydrocarbonoclastic and halotolerant strain, Gordonia polyisoprenivorans ZM27, was isolated via stimulation with resuscitation-promoting factor (Rpf). Long-term exposure to dual stresses of 10% NaCl and starvation induced ZM27 to enter a viable but nonculturable (VBNC)-like state, and ZM27 cells could be resuscitated upon Rpf stimulation. Notable changes in both morphological and physiological characteristics between VBNC-like ZM27 cells and resuscitated cells confirmed the response to Rpf and their robust resistance against harsh environments. Whole-genome sequencing and analysis indicated ZM27 could be a generalist degrader with dormancy ability. Subsequently, VBNC-like ZM27 was applied in a soil microcosm experiment to investigate the practical application potential under harsh conditions. VBNC-like ZM27 combined with Rpf stimulation exhibited the most effective biodegradation performance, and the initial n-hexadecane content (1000 mg kg−1) decreased by 63.29% after 14-day incubation. Based on 16S rRNA amplicon sequencing and analysis, Gordonia exhibited a positive response to Rpf stimulation. The relative abundance of genus Gordonia was negatively correlated with that of Alcanivorax, a genus of obligate hydrocarbon degrader with the greatest abundance during soil incubation. Based on the degradation profile and community analysis, generalist Gordonia may be more efficient in hydrocarbon degradation than specialist Alcanivorax under harsh conditions. The characteristics of ZM27, including its sustainable culturability under long-term stress, response to Rpf and robust performance in soil microcosms, are valuable for the remediation of petroleum pollution under stressful conditions. Our work validated the importance of dormancy and highlighted the underestimated role of low-activity degraders in petroleum remediation.