Viable Bacteria Research Articles

Nonconventional Imaging for Viable Bacteria Detection: A Review.

The first attempts of bacteria observation started with the use of glass lenses to generate magnified images of specimens. This technique is constrained by the principal limit to the resolution of any optical system. Besides optical microscopy, other imaging techniques emerged to reveal more levels of details. The more the achievable resolution, the more complex the imaging systems, and at the same time, the more potentially cell-killing or DNA-damaging they may become. This article provides a state of the art of nonconventional sensor techniques that have been used in applications related to bacteria imaging, for the purpose of comparing the information they provide and determine their suitability or find out if their combination can yield new results without compromising the ability to keep the cells alive.

Evaluation of the probiotic ability of Cellulolytic Bacillus subtilis SBMP4 (FSP20) isolated from waste-dump site in FIIRO, Lagos Nigeria

BACKGROUND: Current invention in the field of biotechnology includes research into the probiotic potentials of enzyme-producing bacteria species. OBJECTIVE: This study evaluated the probiotic ability of cellulolytic Bacillus subtilis SBMP4 isolated from waste dump site. METHODS: Isolated Bacillus species was screened for cellulolytic ability on carboxyl methylcellulose agar (CMCA), and hydrolysis efficiency was calculated. Probiotic ability of cellulolytic Bacillus species was carried out as follows: pathogenic potentials, bacteriocin producing ability, antibiotic susceptibility testing, gastric acid, and bile tolerance. Molecular identification was done by means of 16S rRNA gene sequencing, blast search analysis and phylogenetic analysis. RESULTS: Zone of clearance of 30 ± 2.08 mm on CMCA with hydrolysis efficiency of 114 % depicted the isolate’s cellulolytic potential. Bacillus subtilis SBMP4 showed γ-hemolytic phenotype with negative lecithinase activity. Antibacterial activity of bacteriocin observed against Staphylococcus aureus ATCC 25923 reference culture, had maximum inhibition zone of 14±1.0 mm at 72 hour of incubation. Substantial susceptibility towards 9 out of 10 commonly used antibiotics further depicted the isolate’s probiotic ability. Bacillus subtilis SBMP4 exhibited good gastric acid tolerance (pH 2.5) of 5.857 Log cycle/ml and good tolerance to 5 % and 10 % bile salt concentrations with 6.134 and 6.301 Log cycle/ml of viable bacteria cells. Molecular identification of the selected bacteria species revealed it to be Bacillus subtilis SBMP4 with RefSeq accession number NR_118383, showing maximum sequence similarity of 98%. CONCLUSION: Cellulolytic Bacillus subtilis SBMP4 possesses good probiotic ability and has the potentials of being applied as additive in livestock feeding to enhance overall productivity. KEYWORDS: Cellulolytic, Probiotic, CelA gene, Bacteriocin, Bacillus subtilis

Viability of Lactobacillus acidophilus in acidophilus milk during frozen storage and its potential to lower cholesterol: an In vivo study.

The viability and functional activity of lactic acid bacteria (LAB), such as Lactobacillus acidophilus in a fermented milk product is important. One of the functional activities of the LAB in fermented milk is the ability of the LAB to positively impact the human health. This study aimed to determine the effect of frozen storage on the fermented milk, namely acidophilus milk, based on the nutritional milk quality, the viability of L. acidophilus, and its potential to lower cholesterol levels in Wistar rats. The parameters measured was including milk quality (mainly pH, protein content, lactic acid levels, and syneresis), L. acidophilus bacterial viability in frozen storage (for 1, 2, and 3months), and some biological assays to evaluate the potential of milk in lowering blood cholesterol levels in Wistar rats. The result of this study suggests that acidophilus milk quality can be maintained in frozen storage for two months, and it had lactic acid levels of 1.07%, pH of 4.08, and protein levels of 3.33%. Giving acidophilus milk to Wistar rats for 15days could reduce the cholesterol level continuously until 30days of treatment. Therefore, this study proves that acidophilus milk quality can be maintained very well in frozen storage, and its functional properties to lower the cholesterol level of Wistar rats can be achieved after two weeks of consumption.

Tenacibaculum maritimum can boost inflammation in Dicentrarchus labrax upon peritoneal injection but cannot trigger tenacibaculosis disease.

Despite being a bacterial pathogen with devastating consequences, Tenacibaculum maritimum's pathogenesis is not fully understood. The aim of the present study was to elucidate if different inoculation routes (intraperitoneal - i.p - injection and bath challenge - known to induce mortality) can induce tenacibaculosis (i.e., using the same T. maritimum inoculum), as well as evaluate the short-term immune response of European sea bass (D. labrax). Additionally, the host response against i.p. injection of extracellular products (ECPs) was also studied. Fish were i.p. challenged with 5.5 × 105 CFU mL-1 of T. maritimum cells with or without ECPs (BECPs and BWO, respectively), ECPs alone or marine broth (mock). Another group of fish was bath-challenged with 5.5 × 105 CFU mL-1 to confirm the virulence of the bacterial inoculum. Undisturbed specimens were used as controls. The severity of both challenges was determined by following percentage survival. Blood, liver and head-kidney samples were collected at 0, 3, 6, 24 and 48h post-challenge for assessing immune parameters, oxidative stress and gene expression. Total and differential peritoneal cell counts were performed. The presence of viable bacteria in the blood and peritoneal cavity was studied. Symptoms of tenacibaculosis, such as skin/fin abrasions, were only observed in the bath-challenged fish, where 0% survival was recorded, whereas 100% survival was observed after i.p. injection of the same bacterial inoculum. An increase in total leukocyte numbers in the peritoneal cavity was observed 3h post-injection of BECPs when compared to the other treatments. Blood total leukocytes, lymphocytes, and thrombocyte numbers dropped after the challenge, mainly in fish challenged with BECPs. At 48h post-challenge, bactericidal activity in the plasma increased in fish injected with bacteria (with and without ECPs). The same tendency was seen for some of the oxidative stress parameters. The increased expression of il1β, il6, il8, and hamp1 in fish challenged with ECPs and BECPs suggests a more exacerbated pro-inflammatory response in the head-kidney against these inocula. The infection trial and the observed immune responses showed that the infection route is a determinant factor regarding T. maritimum-induced pathogenesis in European sea bass.

Ameliorated Viability of Lactic Acid Bacteria in Fruit Juice Isolated from Indigenous Dahi with Prebiotics (Asparagus falcatus and Zingiber officinale)

Dahi is a fermented milk product containing probiotic lactic acid bacteria. This study aimed to isolate, identify, and characterize lactic acid-producing bacteria from native Dahi and evaluate their viability in orange juice using natural prebiotics. Dahi samples were obtained from local shops in Chattogram and Bogura, Bangladesh. Lactic acid-producing bacteria were isolated using MRS (de Mann Rogosa and Sharpe) medium. The isolated bacteria were identified through colony morphology, biochemical tests, and probiotic characteristics. Molecular identification was performed using polymerase chain reaction (PCR) targeting conserved 16S rDNA regions. Isolates of the genus Lactobacillus and Lactococcus lactis sp. Lactis were confirmed and used to develop probiotic orange juice. Prebiotics (Asparagus falcatus and Zingiber officinale) were added to the juice to support probiotic growth. The inoculated cell’s viability and the juice’s physicochemical parameters were evaluated during fermentation (48 hours) and storage (28 days). All fruit juice samples showed a mean number of viable cells of at least 1×105 CFU/mL during the 48-hour fermentation and 28-day storage in the refrigerator. Using natural prebiotics positively affected the survival of lactic acid bacteria, as demonstrated by bacterial colony growth on Petri dishes. Developing probiotic fruit juice enriched with prebiotics could be an effective alternative for individuals allergic or intolerant to milk-based products. Incorporating lactic acid bacteria from native Dahi into orange juice, combined with natural prebiotics, resulted in viable probiotic cells throughout fermentation and storage.

A Copper-Based Coating for the Control of Airborne Viable Bacteria in a Prison Environment

Infections in confined environments can spread by direct contact, contaminated surfaces, and airborne transmission. This is critical in prison facilities, where cleaning and sanitary conditions are inadequate. An alternative is the development of antimicrobial surfaces. A new antimicrobial coating was developed by incorporating copper microparticles into a standard commercial paint, aiming to reduce the concentration of bacteria on surfaces by granting antimicrobial properties to surfaces. The copper additive comprised Cu2Cl(OH)3 deposited on polyhedral zeolite. The efficacy of this coating was evaluated in detention cells in a police station, which are temporary prisons and inherently dirty environments. The experiment compared a cell painted with the copper additive coating and a control cell with the standard paint. Viable coliforms were measured on different surfaces and in the air for five months under normal usage. Bacterial load was reduced by ca. 68% by the copper-amended paint on cement surfaces. Surprisingly, airborne viable coliforms were reduced by ca. 87% in the detention cell treated with the copper coating. This research highlights the potential of antimicrobial coatings in controlling the spread of infections through contact with contaminated surfaces and emphasizes the significant reduction in airborne bacterial load. It is especially relevant for controlling infections where sanitization is limited but can be extended to other built environments, such as healthcare facilities.

Optimization of a gelatin/carboxymethylcellulose-based probiotic microcapsule and its application in preventing dextran sodium sulfate-induced colitis in mice.

Oral administration of probiotics has demonstrated substantial potential in alleviating colitis. However, most of the ingested microorganisms struggle to survive the harsh conditions of the gastrointestinal tract, leading to decreased efficacy. In the present study, using double emulsification (W1/O/W2) and complex coacervation methods, we developed a gelatin/carboxymethyl cellulose (CMC)-based probiotic microcapsule and analyzed the efficacy of encapsulated probiotics in preventing dextran sodium sulfate (DSS)-induced colitis in mice. Our results reveal that nearly 90% of the encapsulated probiotics remained viable after 30-day storage at 4°C and approximately 38.1% of viable bacteria (4.0 × 108 cfu/g) survived after 4-h simulated gastrointestinal digestion. In a DSS-induced colitis model, pretreatment with probiotics exerted significant protective effects, with the bacterial microcapsule-treated group having superior outcomes to those of the bacterial suspension plus empty carrier group. Probiotic treatments, especially those administered in the encapsulated form, significantly increased fecal short-chain fatty acid contents, and altered the intestinal microbial composition. The family Muribaculaceae, dominant bacteria in the mouse gut, may be the key microorganism involved in the BM regulation process. Our study presents an alternative approach to treating colitis using probiotics. PRACTICAL APPLICATION: The encapsuled probiotic showed remarkable storage stability at 4°C, maintained good vitality after simulated digestion, and gained superior outcomes in preventing colitis. Our results offer an alternative approach for the probiotic preparations aiming to prevent the intestinal inflammation.

Effects of Tooth Desensitizers on Streptococcus mutans Biofilm Formation Using a Modified Robbins Device Flow Cell System.

This study aimed to assess the antibiofilm effects of dentin desensitizers using a modified Robbins device flow cell system. The test desensitizers were Saforide, Caredyne Shield, and Clinpro White Varnish. Standardized dentin specimens were prepared from human single-rooted premolars, treated with one of the materials, and mounted on the modified Robbins device flow cell system. Streptococcus mutans biofilms were developed for 24 h at 37 °C under anaerobic conditions. Scanning electron microscopy, fluorescence confocal laser scanning microscopy, viable and total cell counts, acid production, and gene expression analyses were performed. A wavelength-dispersive X-ray spectroscopy electron probe microanalyzer was used to analyze the ion incorporations. Clinpro White Varnish showed the greatest inhibition, suggesting its suppression of bacterial adherence and transcription of genes related to biofilm formation. Saforide reduced only the number of viable bacteria, but other results showed no significant difference. The antibiofilm effects of Caredyne Shield were limited. The uptake of ions released from a material into dentin varies depending on the element. Clinpro White Varnish is effective for the short-term treatment of tooth sensitivity due to dentin demineralization. It prioritizes remineralization by supplying calcium and fluoride ions while resisting biofilm formation.

Evaluating Bacterial Viability in Faecal Microbiota Transplantation: A Comparative Analysis of InVitro Cultivation and Membrane Integrity Methods.

Faecal microbiota transplantation (FMT) is a developing therapy for disorders related to gut dysbiosis. Despite its growing application, standardised protocols for FMT filtrate preparation and quality assessment remain undeveloped. The viability of bacteria in the filtrate is crucial for FMT's efficacy and for validating protocol execution. We compared two methods-in vitro cultivation and membrane integrity assessment-for their accuracy, reproducibility and clinical applicability in measuring bacterial viability in frozen FMT stool filtrate. Bacterial viability in stool filtrate was evaluated using (i) membrane integrity through fluorescent DNA staining with SYTO9 and propidium iodide, followed by flow cytometry and (ii) culturable bacteria counts (colony-forming units, CFU) under aerobic or anaerobic conditions. Using different types of samples (pure bacterial culture, stool of germ-free and conventionally bred mice, native and heat-treated human stool), we refined the bacterial DNA staining protocol integrated with flow cytometry for assessment of bacterial viability in frozen human stool samples. Both the membrane integrity-based and cultivation-based methods exhibited significant variability in bacterial viability across different FMT filtrates, without correlation. The cultivation-based method showed a mean coefficient of variance of 30.3%, ranging from 7.4% to 60.1%. Conversely, the membrane integrity approach yielded more reproducible results, with a mean coefficient of variance for viable cells of 6.4% ranging from 0.2% to 18.2%. Bacterial viability assessment in stool filtrate using the membrane integrity method offers robust and precise data, making it a suitable option for faecal material evaluation in FMT. In contrast, the cultivation-dependent methods produce inconsistent outcomes.

A-277 Viability of multi-drug resistant organisms in a fecal collection and transport system stored at ultra-low temperature

Abstract Background Epidemiological studies are constantly investigating new, easier ways to collect and store patient specimens that allow for flexible testing windows and long-term storage as well as accurate quantification of organisms of interest. The FecalSwabTM system (Copan, Brescia, Italy) is intended for the collection and preservation of rectal and fecal specimens. This system includes a preservation medium which serves to maintain the viability of enteric pathogens during transport to a clinical laboratory for up to 72 hours at 4oC. The objective of this study was to assess the continued viability of multi-drug resistant organisms (MDROs) suspended in the FecalSwab sample format when frozen at -80°C and the impact of freeze-thaw cycles. Methods A total of 40 organisms were chosen spanning methicillin-resistant Staphylococcus aureus (MRSA, n=10), carbapenem-resistant Pseudomonas aeruginosa (CRPA, n=5), carbapenem-resistant Enterobacterales (CRE, n=5), vancomycin-resistant Enterobacterales (VRE, n=10) and extended-spectrum beta-lactamase containing Enterobacterales (ESBL, n=10). Each organism was spiked into FecalSwab at standardized concentrations. At each time point, FecalSwab media was plated onto blood agar and colonies were quantified. Each of the 40 organisms were assessed one-week post-freeze and over the course of freeze-thaw cycles at days 3 and 5. The survival of each MDRO was evaluated by calculating a difference between the concentration of organisms recovered (CFU per 10uL media) at each time point and the concentration of organisms confirmed present at the time of inoculation. Percent recovery was calculated from this difference. Results One week after freezing, the percent recovery for MRSA, VRE, ESBL, CRE, and CRPA averaged to 102%, 201%, 17.8%, 7.6%, and 0.8%, respectively (Kruskal Wallis p < 0.001), with significant differences noted between gram positive and gram negative organisms (Kruskal Wallis p <0.001). Conclusions Epidemiological studies interested in MDRO surveillance rely on methodologies that allow for accurate quantification of viable organisms. In this study, we’ve evaluated the viability of several multi-drug resistant organisms when frozen at -80°C in the FecalSwab. The FecalSwab system is intended for the collection of rectal swab and fecal specimens and to preserve the viability of enteric pathogenic bacteria for testing in the clinical laboratory. Although it was not specifically designed to preserve MDROs, it performed well with MRSA and VRE, but less well with CRE, CRPA and ESBL organisms. When using the FecalSwab system, the observed gram-specific differences may inform the suitability of this system depending on the organism(s) of interest.

A-165 Value Of Monocyte Distribution Width In Bacteremia Assessment In Emergency Department Patients

Abstract Background Monocyte distribution width (MDW) is an immune response biomarker and part of a CBC differential. MDW is derived from the distribution of monocyte volumes and elevated MDW values correlate with severe infections and sepsis in Emergency Department (ED) adult patients. Neutrophil to lymphocyte ratio (NLR) is predictive of infections and inflammatory stress. Evaluating these biomarkers could be valuable in bacteremia risk assessment. Bacteremia is the presence of viable bacteria in the bloodstream and is present in up to 20% of septic patients. Blood cultures establish pathogen identity but require culture time, potentially delaying appropriate treatment. Our goal was to characterize MDW and NLR clinical performance in ED patients with suspected bacteremia. Methods This was an observational cohort study of ED patients 18 years and older with CBC differential and blood cultures obtained. MDW values ≥20 is the published cut-off for increased infection risk. Clinical literature suggests that NLR ≥3 is abnormal. We assessed the diagnostic performance of MDW and NLR for positive blood cultures using SPSS Version 25. Results From 07/2021-09/2023, 9,400 blood cultures were ordered and 5174 patients had differential CBCs. The overall blood culture positivity rate was 14.4%. MDW and NLR performance for positive blood cultures is shown in Table 1. Conclusions Our analysis provides evidence that MDW and NLR may be useful for early recognition of bacteremia in ED patients. CBC differential results are reported sooner than culture results. MDW is directly reported, NLR can be readily calculated from the differential. Therefore both could contribute to earlier risk stratification and our findings suggest potentially distinct information from MDW and NLR. Combining MDW with other CBC parameters may provide additional diagnostic advantage. Further analysis will incorporate added clinical data for enhanced decision support in interpreting MDW values in the context of pending blood culture results.

Real-time fluorescence growth curves for viable bacteria quantification in foods

Here, for the first time, we used a membrane permeable fluorescent nucleic acid stain (SYBR Green) to trace the in-vivo DNA replication during bacterial binary fission. Such stain did not influence the growth of bacteria. Nor did the bacteria degrade the stain, enabling the fluorescent microplate reader to monitor sensitively the growth of the bacteria. Hence, a real-time fluorescence growth curve (RTFGC) method was put forward for the sensitive quantification of viable bacteria in foods. Using E. coli O157:H7 as a bacteria model, the RTFGC method could quantify bacteria within the range of 10 to 1 × 106 cfu/mL, with a linear correlation coefficient R2 of 0.997. It was found that melting curve was unique for a particular bacterial strain, which could be used for contamination identifications. Good practicability of the RTFGC in quantifying E. coli O157:H7 from tap water, juices, and milks was demonstrated.

Lack of genotoxicity and subchronic toxicity in safety assessment studies of Akkermansia muciniphila formulation

A powder formulation of viable Akkermansia muciniphila bacteria (AMUC) was evaluated in a 90-day repeated-dose toxicity study in rats and a battery of genotoxicity studies to evaluate AMUC as a food ingredient. All studies followed Organisation for Economic Co-operation and Development protocols (OECD TG 408, 471 473, 474). AMUC was administered to rats via gavage at 0, 500, 1000, and 2000 mg/kg body weight/day (equivalent to 0, 4.1 × 1010, 9.2 × 1010, and 1.64 × 1011 CFU/kg body weight/day). No mortality or treatment-related adverse effects were reported in any endpoints that were attributed to AMUC consumption. No bacterial translocation of viable A. muciniphila from the intestinal tract was found to the liver, mesenteric lymph nodes, or blood. The no-observed-adverse-effect level was concluded to be the highest dose tested (2000 mg/kg body weight/day), approximately 1.64 × 1011 CFU/kg body weight/day. AMUC (nonviable) was not mutagenic when examined in an in vitro bacterial reverse mutation assay and not clastogenic in an in vitro mammalian chromosomal aberration test. Viable AMUC was not genotoxic when evaluated in an in vivo mammalian cell micronucleus assay when administered at up to 1.64 ×1011 CFU/kg body weight/day. These results confirm that AMUC is not toxic under the conditions of these studies.

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.

Lag Plot: A Novel Method to Determine Viable Bacterial Cell Number via a Single OD Measurement.

Bactericidal activity is a valuable parameter which is considered and measured for antimicrobial compounds. The available standard protocol to evaluate bactericidal activity is based on the direct colony count. Colony counting requires serial dilution, plating, overnight incubation, and direct counting, which is time-and labor-intensive. In regard to eliminate direct plate count, novel techniques were developed based on the real-time growth monitoring which can come with some limitations and drawbacks. These drawbacks encourage us to develop a novel technique with simple procedure to determine viable bacterial cell count. In this procedure, real-time growth monitoring is not required. In fact, after an incubation time, the number of viable bacteria can be determined with a single OD measurement and through an equation. In this regard, four standard bacterial strains, including Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Bacillus subtilis (ATCC 23857), were cultured with descending inoculum densities (1.5 × 107 to 15CFU/mL) and growth curves were drawn. As expected, growth in the samples with lower inoculum densities recorded with longer lag phase. Also, a direct relation was observed between the recorded turbidities and initial cell counts. A logarithmic curve (lag plot) was obtained by plotting the OD, after 12-18h incubation, against initial cell counts. In all examined strains, R2 was calculated in the range of 0.96-0.99 which is acceptable value for coefficient of determination. Equation corresponding to the lag plot was obtained and called lag equation. This equation is applicable to calculate the number of viable cells in unknown samples simply by inoculation, incubation, and single OD measurement. Developed technique can be introduced as a simple substitution for labor- and time-intensive direct colony counting.

Profiling the biocontrol agents, nitrogen-fixing bacteria, and indole acetic acid-producing bacteria in anthill soil

Due to the adverse ecological impacts of prolonged and excessive use of agrochemicals, many researchers have called for the adoption of eco-friendly materials in farming. Consequently, the aim of this study is to profile the biocontrol agents, nitrogen-fixing, and indole-acetic-acid-producing bacteria present in anthill soil. Anthill soils and adjacent soils were collected, and their physicochemical properties were analysed using standard analytical methods. Viable bacteria were isolated and screened for plant growth-promoting (PGP) activity using standard biochemical, morphological, and bacteriological methods. The PGP capacity of the isolates was evaluated using standard protocol for nitrogen fixation and indole-acetic-acid production, while antagonistic effect against plant pathogens was evaluated using the disk diffusion assay. Results revealed that Pseudomonas aeruginosa, Klebsiella pneumoniae, and Bacillus sp. isolated from the anthill soil demonstrated PGP characteristics including IAA production and nitrogen fixation. Bacillus sp. exhibited zones of clearance with values of 20.00 ± 1.50 mm, 20.00 ± 1.75 mm, 17.00 ± 1.33 mm, and 17.00 ± 1.33 mm against Aspergillus niger, Trichoderma sp., Penicillium sp., and Fusarium sp., respectively. This study demonstrated that anthill soils contain beneficial microbes with the potential to stimulate plant growth and suppress soil-borne plant pathogens.

Lysozyme-Responsive Hydrogels of Chitosan-Streptomycin Conjugates for the On-Demand Release of Biofilm-Dispersing Enzymes for the Efficient Eradication of Oral Biofilms.

Hydrogels with controlled degradation and sustained antibiofilm activity are promising biomaterials for the treatment of oral infections such as periodontitis or caries. In this article, an in situ forming chitosan-streptomycin hydrogel is developed that can target established bacterial biofilms in response to lysozyme, an enzyme that is overexpressed in saliva during oral infections. When the new hydrogel is applied to simulated oral biofilms, the overexpressed lysozyme degrades the hydrogel and releases chitosan-streptomycin oligosaccharides that can eradicate the biofilm. This work has shown that the coupling of chitosan and streptomycin can have a synergistic effect and that the new hydrogel based on chitosan-streptomycin conjugate can effectively combat biofilms of E. coli, S. aureus, and P. aeruginosa formed in vitro achieving a significant reduction in the biomass of the biofilm and a substantial reduction in the population of viable bacteria in established biofilms. Finally, the CS-Str hydrogel loaded with biofilm-disrupting enzymes, in particular, DNase I and/or DspB, showed a significantly increased ability to reduce the biofilm biomass of P. aeruginosa and S. aureus (by over 84% and up to 92%, respectively), resulting in a drastic reduction in cell viability, which fell below 4% for P. aeruginosa and below 5% for S. aureus.

Enhancing Removable Partial Dentures Hygiene: Investigating Mucolytic Agents and Biocides for Disrupting Biofilms and Improving Antimicrobial Efficacy.

This study evaluates the antibiofilm action of 2.5 mg/mL peracetic acid (PA), 0.5 mg/mL cetylpyridinium chloride (CPC), and 160 mg/mL N-Acetylcysteine (NAC) against multispecies biofilm of Streptococcus mutans, Staphylococcus aureus, Candida albicans, and Candida glabrata, developed on surfaces of heat-polymerizing acrylic resin (AR) and cobaltchromium (Co-Cr) alloy. A multispecies biofilm was grown on the surface of AR and Co-Cr specimens (Ø 12×3mm). After biofilm maturation, the specimens were immersed in experimental solutions and evaluated through biofilm viability (CFU) (n&#61;9), biofilm metabolic activity (XTT) (n&#61;9), biofilm-covered areas (Live/Dead) (n&#61;2), effects on the extracellular polymeric substance (EPS) (n&#61;2) and biofilm morphology (n&#61;1). Data were analyzed by ANOVA and the Tukey post-test or Kruskal-Wallis followed by the Dunn post-test (α&#61;.05). Overall, all evaluated solutions impacted biofilm viability. PA presented wider activity by reducing CFU of all microorganisms on both surfaces, XTT (P<.001) and Live/Dead (P<.001). NAC had a notorious effect in reducing the viability of bacteria without affecting the yeasts. NAC reduced XTT on AR (P&#61;.006) and Co-Cr (P&#61;.003) but did not reduce the aggregated biofilm layer. CPC had distinct effect according to the surface, being most effective in reducing CFU on AR than the Co-Cr surface. However, it did not influence XTT, and the amount of residual aggregated biofilm. PA provided the greatest antibiofilm action, while CPC and NAC showed intermediate action. Nonetheless, no solution was able to completely remove the biofilm adhered to the surfaces of heat-polymerizing AR and Co-Cr alloy.

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.

Viable Bacteria Assay of Helicobacter pylori by RT-qPCR Measurement of cgt Gene Expression Levels: Establishment and Application of a New Method

To establish a viable bacteria assay for Helicobacter pylori (H. pylori) by assessing the cgt gene expression, and to develop accordingly a rapid and novel testing method for clinical precision treatment. Viable bacteria count was determined in bacterial cultures. The transcriptional expression level of cgt (hp0421), the conserved gene that encodes cholesterol-α-glucosyltransferase (CGT) in H. pylori, was measured by RT-PCR. The correlation between the number of colonies and cgt gene transcription expression was analyzed and the regression model was constructed. The linear range, sensitivity, and specificity of the new method were examined accordingly. The bactericidal action of clarithromycin was assessed using this method to verify the performance of the method in determining clinical bacterial drug resistance. The Ct values of cgt for H. pylori colony counts of 102, 104, 106, and 108 CFU/mL were 29.67±0.14, 23.37±0.36, 17.65±0.37, and 11.38±0.39, respectively. In the range of 101-108 CFU/mL, the regression equation for cgt gene expression and viable bacterial counts determined by RT-qPCR was y=-0.3501x+12.49, with the correlation coefficient being R 2=0.9992 and the sensitivity being 101 CFU/mL, showing no cross-reaction with 13 other bacteria. The lg values of live H. pylori bacteria treated with clarithromycin at 0, 5, 10, 20, and 40 μg/mL for 12 h were 2.57±0.02, 2.45±0.01, 2.19±0.02, 1.91±0.07, and 1.33±0.05, respectively. The corresponding cgt gene Ct values were 27.76±0.09, 28.37±0.24, 29.51±0.14, 30.11±0.12, and 31.66±0.11. By applying the cgt gene expression in the equation, the estimated counts of viable bacteria were found to be 2.73±0.03, 2.52±0.08, 2.11±0.05, 1.89±0.02, and 1.33±0.04, showing no significant difference in statistical analysis (P>0.05). The method for assessing viable bacteria account by evaluating cgt gene expression in H. pylori was successfully established, significantly reducing the time required to determine viable bacteria count and providing a new method for clinical viable bacteria testing.