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.

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.

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.

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.

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.

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.

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.

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.

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

Francisella tularensis is a gram-negative, intracellular pathogen which can cause serious, potentially fatal, illness in humans. Species of F. tularensis are found across the Northern Hemisphere and can infect a broad range of host species, including humans. Factors affecting the persistence of F. tularensis in the environment and its epidemiology are not well understood, however, the ability of F. tularensis to enter a viable but non-culturable state (VBNC) may be important. A broad range of bacteria, including many pathogens, have been observed to enter the VBNC state in response to stressful environmental conditions, such as nutrient limitation, osmotic or oxidative stress or low temperature. To investigate the transition into the VBNC state for F. tularensis, we analyzed the attenuated live vaccine strain, F. tularensis LVS grown under standard laboratory conditions. We found that F. tularensis LVS rapidly and spontaneously enters a VBNC state in broth culture at 37°C and that this transition coincides with morphological differentiation of the cells. The VBNC bacteria retained an ability to interact with both murine macrophages and human erythrocytes in in vitro assays and were insensitive to treatment with gentamicin. Finally, we present the first transcriptomic analysis of VBNC F. tularensis, which revealed clear differences in gene expression, and we identify sets of differentially regulated genes which are specific to the VBNC state. Identification of these VBNC specific genes will pave the way for future research aimed at dissecting the molecular mechanisms driving entry into the VBNC state.

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

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

Collaborative cytometric inter-laboratory ring test for probiotics quantification.

Introduction: Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. From this definition, accurate enumeration of probiotic products is a necessity. Nonetheless, this definition does not specify the methods for assessing such viability. Colony forming units is the de facto gold standard for enumerating viable in probiotic products. The notion of microbial viability has been anchored in the concept of cultivability, which refers to a cell's capacity to replicate and form colonies on agar media. However, there is a growing consensus that the term "viability" should not be exclusively tied to the ability to cultivate cells. For example, bacterial cells can exist in a Viable But Non-Culturable (VBNC) state, characterized by the maintenance of characteristics such as membrane integrity, enzymatic activity, pH gradients, and elevated levels of rRNA, despite losing the ability to form colonies. Methods: Herein we present the results of a collaborative inter-laboratory ring test for cytometric bacterial quantification. Specifically, membrane integrity fluorescence flow cytometry (FFC) method and the newer impedance flow cytometry (IFC) method have been used. Both methods interrogate single cells in solution for the presence of intact membranes. FFC exploits fluorochromes that reflect the presence or absence of an intact membrane. IFC probes membrane integrity in a label-free approach by detecting membrane-induced hindrances to the propagation of electricity. Results: A performance ring-test and comparison design on the FFC method showed that the method is robust against the exchange of equipment, procedures, materials, and operators. After initial method optimization with assessments of rehydration medium, wake-up duration, and phase shift gating on the individual strains, the IFC method showed good agreement with the FFC results. Specifically, we tested 6 distinct species of probiotic bacteria (3 Lactobacillus and 3 Bifidobacterium strains) finding good agreement between FFC and IFC results in terms of total and live cells. Discussion: Together, these results demonstrate that flow cytometry is a reliable, precise, and user-friendly culture-independent method for bacterial enumeration.

Detection of Viable but Non-Culturable (VBNC)-Campylobacter in the Environment of Broiler Farms: Innovative Insights Delivered by Propidium Monoazide (PMA)-v-qPCR Analysis.

Campylobacteriosis cases in humans are of global concern, with high prevalence rates in the poultry reservoir considered the most important source of infection. Research findings show Campylobacters' ability to enter a viable but non-culturable (VBNC) state, remaining "viable" but unable to grow on culture media. We explored the persistence of VBNC states in specific environments, particularly at broiler farms, as this state may lead to an underestimation of the present Campylobacter prevalence. For VBNC detection, a propidium monoazide PMA-dye viability qPCR (v-qPCR) was used in combination with cultivation methods. We examined samples collected from broiler farm barns and their surroundings, as well as chicken manure from experimental pens. In addition, the tenacity of culturable and VBNC-Campylobacter was studied in vitro in soil and water. In a total of three visits, Campylobacter was not detected either culturally or by v-qPCR (no Campylobacter DNA) in the environment of the broiler farms. In four visits, however, VBNC-Campylobacter were detected both inside and outside the barns. The overall prevalence in environmental samples was 15.9% for VBNC-Campylobacter, 62.2% for Campylobacter DNA, and 1.2% for culturable C. jejuni. In the experimental pens, no cultivable C. jejuni was detected in chicken manure after 24 h. Strikingly, "VBNC-Campylobacter" persisted even after 72 h. "VBNC-Campylobacter" were confirmed in barn surroundings and naturally contaminated chicken manure. Laboratory studies revealed that VBNC-Campylobacter can remain intact in soil for up to 28 days and in water for at least 63 days, depending on environmental conditions.

Analyzing the pressure resistant, sublethal injury and resuscitable viable but non-culturable state population of Escherichia coli, Staphylococcus aureus, Bacillus amyloliquefaciens and Lactiplantibacillus plantarum under high pressure processing.

This study aimed to analyze and reduce the pressure resistance (PR), sublethal injury (SLI), and viable but non-culturable (VBNC) populations during HPP. Escherichia coli, Staphylococcus aureus, Bacillus amyloliquefaciens and Lactiplantibacillus plantarum were selected for evaluation of PR, SLI and VBNC cell counts and proportions during HPP. The results revealed that the bactericidal efficiency against these strains gradually improved as the processing pressure increased. However, viable bacteria could still be detected, suggesting that there may involve the presence of resistant population that difficult to be killed or revived from SLI. Further detecting the quantity and proportion of PR, SLI and VBNC bacteria found that these state of cells were present during whole HPP treatment. Additionally, the more resistant a bacterial species was to high pressure, the fewer SLI and more resuscitable VBNC (RVBNC) populations it generated, and vice versa. Therefore, correlation analysis was also employed to make the relationship between log reduction, SLI and RVBNC population ratios clearer. The results demonstrated that the log reduction was highly positive correlation with SLI population ratios, and negative correlation with RVBNC population within our detected species at 500 MPa. Furthermore, CO2 and Nisin were employed to combined with HPP to reduce these survivors. Comparing with 233, 218, 241 and 259 MPa for HPP treatment, it took 37, 89, 135 and 229 MPa for HPP + CO2, and 189, 161, 199 and 292 MPa for HPP + Nisin to the first decimal reduction for E. coli, S.aureus, B. amyloliquefaciens and L. plantarum, respectively. The results indicated that HPP combined with CO2 or Nisin could significantly reduce the quantity of PR, SLI, and RVBNC cells during HPP, and provide better bactericidal effects. In conclusion, we quantified the presence of PR, SLI, and VBNC bacteria after high pressure treatment and investigate the effectiveness of HPP combined with CO2 or Nisin to enhance the inactivation of bacteria and reduce the occurrence of PR, SLI, and RVBNC bacteria.

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

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