Search Results (941)

CAD/CAM reinforced resin composites are increasingly used in restorative dentistry because of their favorable mechanical and esthetic properties. This in vitro study evaluated the effect of acidic exposure on surface properties and Streptococcus mutans biofilm formation on three CAD/CAM resin composites: a short-fiber-reinforced composite (SFRC), a woven glass fiber-reinforced composite (TRINIA; TR), and a resin nanoceramic-reinforced composite (Lava Ultimate; LU). Twenty-four specimens from each material were prepared and assessed for surface roughness and wettability using an optical profilometer and contact angle goniometer before and after 24 h of storage in neutral (pH 7) or acidic (pH 4) buffer solutions (n = 12 per group). Surface morphology was further examined using scanning electron microscopy (SEM). Biofilm formation was quantified using S. mutans colony-forming unit (CFU) counts, followed by confocal laser scanning microscopy (CLSM) for biofilm architecture and viability. At baseline, TR exhibited the highest roughness values (0.744 to 0.785 µm, p < 0.001), whereas no significant differences in contact angle were observed among materials. Acidic exposure significantly increased surface roughness and reduced contact angle values in all examined materials (p < 0.001). Compared to storage in neutral conditions, bacterial adhesion was significantly reduced in TR and LU stored in acid (p < 0.001), but SFRC showed no significant change (p = 0.079). Among specimens exposed to acidic conditions, TR exhibited the highest bacterial adhesion (3.24 log₁₀ CFU/mm²) relative to SFRC and LU (p < 0.001). CLSM analysis demonstrated denser biofilm accumulation on TR and LU compared with SFRC. These findings demonstrate that acidic exposure significantly affects the surface properties and bacterial adhesion behavior of CAD/CAM resin composites in a material-dependent manner. SFRC showed no statistically significant difference in S. mutans CFU/mm² between neutral and acidic storage conditions despite measurable surface changes, highlighting its potential suitability for clinical applications associated with acidic oral environments. Full article

The Enterobacteriaceae family is a significant source of foodborne pathogens and represents a severe threat to human and animal health. These bacteria can penetrate the dairy supply chain through direct contact with cattle and the livestock environment and can survive production processes. Escherichia coli (E. coli), one of the most diffuse bacteria in raw and processed milk, exposes consumers to the risk of contaminated milk. As a result of this exposition, several milk-borne illness outbreaks have been reported worldwide, underscoring the urgent need for effective detection and prevention measures. Conventional analysis methods are effective but have significant limitations, including the requirement of pre-treatment and pre-enrichment steps. Thus, the need for advanced detection techniques that can accurately identify these pathogens without pre-treatment steps is critical. In this work, a proof-of-concept biosensor based on a spoon-shaped optical biochip was developed to detect E. coli via surface plasmon resonance (SPR) phenomena and was combined with a polyclonal antibody layer against E. coli as a molecular recognition element (MRE). The proposed label-free biosensing strategy, achieved by exploiting simple SPR spoon-shaped biochips, exhibits a remarkable detection limit (6.8 colony-forming units, CFU/mL) and high specificity towards other interfering bacteria belonging to the Enterobacteriaceae family. In addition, tests on commercial milk samples were carried out, achieving recovery values of 95% and 102% for whole milk and infant milk, respectively. The proposed spoon-shaped biosensor enables label-free biosensing without the need for microfluidic systems. It provides a rapid response (10 min), paving the way for its use as a point-of-care test (POCT) in real-world settings. Full article

Background: Corallopyronin A (CorA) is a bacterial RNA polymerase inhibitor that binds a site distinct from rifamycins, but its activity across the ESKAPEE pathogen panel and its translational potential remain incompletely defined. Methods: CorA activity was evaluated against representative ESKAPEE pathogens using broth microdilution assays ± polymyxin B nonapeptide (PMBN). Activity was benchmarked against rifampin (Rif). Resistance, cross-resistance, serum activity, in vivo efficacy in the Galleria mellonella wax moth larva model, and biofilm disruption were assessed. Results: CorA inhibited Acinetobacter baumannii (minimal inhibitory concentration [MIC] = 16–32 µg/mL), including MDR isolates, with susceptibility enhanced 4–32-fold by efflux disruption or membrane permeabilization. In contrast, most other Gram-negative ESKAPEE pathogens required PMBN for activity, while Gram-positive organisms were intrinsically susceptible. Rif was consistently more potent than CorA across the panel. Rif-resistant A. baumannii and Klebsiella pneumoniae remained fully susceptible to CorA, confirming the absence of cross-resistance. Fractional inhibitory concentration (FIC) index analysis revealed pharmacological indifference between CorA and Rif, with no synergy or antagonism detected. CorA activity was abolished in 50% serum conditions and was not restored by PMBN, consistent with serum sequestration; no efficacy was observed in a G. mellonella infection model at doses up to 20 mg/kg despite Rif demonstrating significant protection. Notably, CorA reduced established A. baumannii biofilms by ∼3–4 log colony-forming units per mL (CFU/mL) across concentrations ≥4× MIC after 24 h of treatment. Conclusions: CorA exhibits selective activity against A. baumannii and retains efficacy against Rif-resistant strains. While high serum binding reduces in vitro activity and efficacy was not observed in the G. mellonella model at a lower dose than in other in vivo models, established in vivo activity against other pathogens demonstrates that serum binding does not preclude therapeutic utility. These findings highlight both the translational considerations for CorA against ESKAPEE pathogens and potential niches for its application, including filarial nematodes, biofilm-associated infections and combination strategies. Full article

Environmental contamination represents an important risk factor for the transmission of healthcare-associated pathogens in both human and veterinary medicine. In equine hospitals, open stable environments and high patient turnover may further increase the risk of microbial spread. The aim of this study was to evaluate the hygiene status of an equine referral hospital and to assess the effect of implementing standardized cleaning and disinfection procedures combined with targeted staff training on environmental microbial burden. Surface samples were collected using agar contact plates from predefined environmental surfaces in surgical areas, examination rooms, and stable units of an equine clinic. Sampling was conducted during three investigation periods: prior to intervention, after implementation of revised cleaning protocols and staff training, and during a later follow-up assessment. Colony-forming units (CFU) were quantified and categorized into an ordinal contamination scale (0–4) to enable standardized statistical analysis. Furthermore, delta values were calculated for every sampling period, describing the reduction in the environmental microbial burden. Pre- and post-cleaning microbial burdens were compared using nonparametric statistical methods. A total of 76 locations were sampled before and after cleaning and disinfection in sampling period 1 and 2; in sampling period 3, sample collection focused on sensitive surfaces, and 21 locations were sampled. Bacterial contamination varied considerably between functional areas, with the highest baseline contamination consistently observed in stable environments. Examination and surgical areas showed heterogeneous contamination patterns, particularly on high-touch surfaces, such as keyboards, switches, and door handles. In sampling periods 1 and 2, cleaning and disinfection significantly reduced microbial burden. The delta values in sampling period 2 were significantly lower than the delta values in both sampling period 1 and sampling period 3. These findings demonstrate that environmental contamination in equine hospitals is highly variable and influenced by surface type, clinical workflow, and proximity to patients. Structured hygiene interventions, including standardized cleaning protocols and continuous staff education, can significantly improve environmental hygiene and should be integrated into infection prevention programs in veterinary clinical facilities. Full article

The increasing demand for food products and the implementation of sustainable development principles have encouraged the search for technological solutions that can reduce food losses and the environmental burden of food processing. Milk and dairy products are nutrient-rich matrices, but they also provide favourable conditions for microbial growth. Therefore, ensuring microbial safety during cheese production, ripening, and storage is essential. This study aimed to evaluate the potential application of gaseous ozone as a low-residue and potentially more sustainable approach for controlling surface microbial contamination in semi-hard cheese during ripening or storage. Ozone is characterized by low cost, strong oxidative properties, antimicrobial activity, and rapid decomposition into oxygen without leaving persistent chemical residues. Semi-hard cheese samples were treated with gaseous ozone at a concentration of 4.84 ± 0.22 parts per million (ppm) for 10, 30, 60, 90, and 150 min. After treatment, the counts of aerobic microorganisms, yeasts, and moulds were determined, and changes in moisture, fat, and protein content were assessed. After only 10 min of ozonation, aerobic microorganism counts decreased from 2826 ± 1911 × 10⁴ to 275 ± 184 × 10⁴ colony-forming units per gram (CFU/g). In contrast, a reduction in yeast counts was observed only after a longer treatment duration of 60 min. No clear treatment-dependent changes were detected in mould counts or in total fat and protein contents. Cheese moisture content decreased significantly after 10 min of ozonation and continued to decline as the ozonation duration increased. The results suggest that gaseous ozone may be used as an additional microbial control approach for semi-hard cheese during ripening or storage. However, the findings only partially confirmed a significant effect of gaseous ozone on surface microorganisms and its neutrality with respect to product proximate composition. Full article

Mycobacterium abscessus subsp. massiliense (Mabs) is an emerging nontuberculous mycobacterium associated with difficult-to-treat infections due to intrinsic antimicrobial resistance, intracellular persistence, biofilm formation, and limited responsiveness to currently available therapeutic regimens. In this context, adjuvant strategies targeting iron-dependent metabolic pathways and metal homeostasis may enhance the efficacy of conventional antimicrobials. This study investigated deferoxamine (DFO), a clinically approved iron chelator, as a potential adjuvant against Mabs using integrated in vitro and in silico approaches. Cytocompatibility was assessed using an MTT assay in RAW 264.7 macrophages and a hemolysis assay in human erythrocytes. Antimicrobial activity was evaluated through minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays, while interactions with clarithromycin (CLA) and amikacin (AMK) were assessed using the checkerboard method. Effects on virulence-associated phenotypes were examined through biofilm formation assays and protein quantification in extracellular vesicle-enriched fractions. Intracellular activity and modulation of inflammatory mediator gene expression were investigated in Mabs-infected RAW 264.7 macrophages through colony-forming unit (CFU) recovery and reverse transcription quantitative polymerase chain reaction (qPCR). DFO exhibited low cytotoxicity and negligible hemolytic activity under the tested conditions. Direct antimicrobial testing revealed a predominantly bacteriostatic profile (MIC = 9.75 µg/mL; MBC > 10 mg/mL), whereas checkerboard analysis suggested a synergistic interaction with CLA (FICI = 0.047), which requires further confirmation by time-kill or CFU-based combination assays. Furthermore, DFO reduced biofilm biomass, decreased protein levels in vesicle-enriched fractions, lowered intracellular bacterial burden, and modulated cytokine gene expression in infected macrophages. Molecular docking, ADME/Tox, and PASS analyses generated exploratory hypotheses regarding potential molecular interactions and pharmacological properties. Overall, these findings support DFO as a promising experimental adjuvant candidate for further investigation against Mabs, particularly in combination with clarithromycin. However, confirmation of a putative iron-restriction-associated mechanism and its translational relevance will require validation in additional clinical isolates, iron-rescue experiments, mature biofilm models, and in vivo studies. Full article

Consumers are looking for plant-based drinks that provide natural colour and bioactive compounds. Microgreens can be used as a source of pigments and phenolics for such beverages. This study developed a beetroot–tarragon microgreen beverage using hydroalcoholic extracts obtained with a green extraction approach and examined its stability during refrigerated storage. The drink was evaluated for proximate composition, water activity, colour parameters (CIE L*a*b*), microbiological quality and antioxidant activity by the ABTS radical cation decolorization assay (ABTS) over 15 days at 4 ± 2 °C. The beverage showed low fat and energy content and water activity values close to 1.00, so microbiological safety relied on pasteurization and cold storage. Escherichia coli and Staphylococcus aureus were not detected, while total aerobic mesophilic counts reached 10⁴–10⁵ colony-forming units per gram (CFU/g), with slightly lower values in samples containing tarragon. Colour measurements indicated betalain loss and colour fading in the beetroot drink, whereas the reduction in E* was more than 80 percentage points lower in the beetroot–tarragon beverage than in the beetroot-only drink, indicating a strong protective effect of tarragon microgreens on colour stability. For the mixed beetroot–tarragon beverage, mean TEAC increased by about 37% between day 1 and day 10 of refrigerated storage. These results indicate that beetroot and tarragon microgreen extracts can be used to formulate refrigerated plant-based beverages with acceptable colour, microbiological safety and antioxidant capacity. Full article

Bacterial infections pose a serious threat to human health, and antibiotics remain the first-line therapeutic agents in clinical practice. However, the vast majority of antibiotics lack the ability to penetrate cell membranes, which severely limits the number of clinically available options for treating intracellular bacterial infections. Developing efficient intracellular antibiotic delivery strategies is therefore of considerable clinical significance, both for reducing antibiotic dosage and for expanding the repertoire of drugs applicable to intracellular infections. To address this challenge, we constructed a protein-based delivery platform mediated by a cell-penetrating miniprotein for efficient intracellular antibiotic delivery. In this system, bovine serum albumin (BSA), which possesses broad antibiotic-binding capability, was employed as the drug carrier, while the cell-penetrating miniprotein ZF5.3, which is capable of endosomal escape, served as the transmembrane delivery mediator. ZF5.3 was conjugated to BSA via a bioorthogonal reaction, and ceftriaxone (CRO) was selected as the model antibiotic to construct a nanoscale delivery system. The binding interaction between CRO and BSA was characterized using UV-Vis, HPLC, and molecular docking techniques. The assembly of the ZF5.3–BSA delivery platform was confirmed by UV-Vis absorption spectroscopy and gel electrophoresis. Intracellular delivery efficiency was evaluated by confocal fluorescence imaging and flow cytometry, and the results demonstrated that ZF5.3 conjugation enhanced intracellular protein delivery efficiency by over 5-fold. Fluorescence co-localization analysis revealed that ZF5.3-mediated cargo is mainly distributed in the cytoplasm and does not completely co-localize with lysosomal markers, suggesting its ability to effectively escape from lysosomes. An intracellular infection model using Staphylococcus aureus was established. Colony-forming unit (CFU) counting experiments confirmed that the delivery system significantly enhanced the intracellular antibacterial activity of ceftriaxone. CCK8 cytotoxicity assays confirmed that the system is non-toxic to cells. Full article

Urinary tract infections (UTIs), particularly catheter-associated UTIs (CAUTIs), represent a significant clinical problem due to the predominance of Gram-negative uropathogens, their ability to form biofilms, and the increasing prevalence of antimicrobial resistance, which together reduce the effectiveness of conventional antibiotic therapy. This study aimed to evaluate the antimicrobial activity of selected natural plant-derived compounds against clinical Gram-negative uropathogens isolated from CAUTIs. The antibacterial effects of gallic acid, quercetin-3-D-glucuronide, and apigenin were assessed against Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis, including both ciprofloxacin-susceptible and -resistant strains. Antimicrobial activity was determined using the broth microdilution method, followed by quantitative assessment of bacterial viability based on colony-forming unit (CFU) enumeration. Gallic acid exhibited the strongest concentration-dependent inhibitory activity, reducing bacterial viability by up to 2–3 log₁₀ CFU across all tested species. Quercetin-3-D-glucuronide demonstrated moderate antibacterial effects with a predominantly bacteriostatic profile, resulting in a partial but consistent reduction in CFU counts. In contrast, apigenin showed only weak effects on bacterial viability under the applied experimental conditions. None of the tested compounds achieved complete bacterial eradication. These findings indicate that gallic acid and quercetin-3-D-glucuronide possess inhibitory activity against Gram-negative uropathogens, including antibiotic-resistant strains, supporting their potential use as adjunctive agents targeting bacterial persistence in UTIs rather than as standalone antimicrobials. In the present study, the viability of planktonic bacterial cells was assessed; however, future studies should focus on evaluating the direct impact of the tested compounds on biofilm structure and biofilm formation dynamics. Full article

Achieving in situ and time-resolved monitoring of microbial metabolites without disrupting the microbial growth environment remains a key challenge in electrochemical biosensing. Herein, we propose a self-healing bilayer hydrogel-based solid-state electrochemical sensing platform for the in situ, time-resolved analysis of purine metabolites produced by Escherichia coli (E. coli). This platform integrates an upper Agar culture module and a lower borax-crosslinked poly(vinyl alcohol) (PVA) detection module, forming a contiguous structure that allows metabolites (e.g., guanine, xanthine, hypoxanthine) to migrate across the solid–solid interface for sensitive electrochemical detection. The detection layer exhibits excellent ionic conductivity; when coupled with its robust structural self-healing capacity, the platform achieved a detection limit of 0.05 µM for guanine. For E. coli detection, a linear response range of 1.1 × 10⁶ to 9.5 × 10⁶ CFU·mL⁻¹ (R² = 0.9974) was obtained, and relative standard deviations (RSDs) of less than 2.34% even after two weeks of storage. Leveraging this integrated design, the platform enables continuous, label-free tracking of bacterial metabolic dynamics throughout all growth phases. Notably, it detects metabolic transition points earlier than traditional plate counting methods and accurately evaluates antibiotic inhibition trends, with results consistent with colony-forming unit (CFU) analysis. This integrated culture–detection architecture thus provides a versatile strategy for functional microbial analysis and rapid antimicrobial susceptibility testing. Full article

Background/Objectives: Denture disinfection is a crucial step in reducing microbial colonization and the risk of denture stomatitis, as well as contributing to patient health and denture longevity; thus, it was obligatory to select an effective cleanser without undesirable impact on properties of acrylic denture base material. This study aimed to assess the synergistic effect of white vinegar and sodium bicarbonate (WVSB) mixture on Candida albicans by means of colony forming unit (CFU) and adhesion assays, as well as the surface roughness and flexural strength of heat-cured acrylic denture base material after being immersed in the WVSB mixture. Methods: In total, 200 specimens of heat-cured acrylic resin were prepared: 50 per each test, 5 per each group. They were divided into ten groups; distilled water (negative control), a Corega denture cleanser tablet soaked for 5 min (positive control), and four concentrations (2%, 3%, 4% and 5%) of WVSB mixture were made and examined for (5 and 10 min) immersion durations. Statistical analysis was performed by using Welch’s ANOVA alongside Games–Howell post hoc tests for CFU assay and one-way ANOVA along with Tukey HSD post hoc tests for remaining tests. A p < 0.05 was considered significant in all experiments. Results: The results for the CFU, adhesion and surface roughness tests showed that the WVSB mixture demonstrated a statistically significant difference in most test groups compared to the negative control group, while the flexural strength test showed a statistically non-significant difference. Conclusions: The WVSB mixture showed concentration and time-dependent antifungal effects against C. albicans, with increased surface roughness and no negative effect on the flexural strength of heat-cure acrylic. Full article

Background: Oral candidiasis is an opportunistic infection caused by Candida albicans (C. albicans), highlighting the need to evaluate candidate substances that ensure both antifungal efficacy and mucosal safety. This study aimed to assess the potential of Sambucus williamsii var. coreana (S. williamsii var. coreana) extract as a naturally derived antifungal agent for topical oral application by investigating its antifungal activity against C. albicans and its cytotoxicity in human keratinocyte (HaCaT) cells. Methods: S. williamsii var. coreana was extracted with 70% ethanol, concentrated, and freeze-dried. The extract was prepared at concentrations of 1–40 mg/mL and applied under 6 h and 24 h exposure conditions. Antifungal activity against cultured C. albicans was evaluated using colony-forming unit (CFU) analysis, while cytotoxicity in HaCaT cells was assessed via the Water-soluble Tetrazolium Salt-1 assay after incubation at 37 °C in 5% CO₂ for 2 h. Statistical significance was analyzed using Student’s t-test, ANOVA, and Tukey’s HSD test (p < 0.05). Results: The S. williamsii var. coreana extract exhibited concentration- and time-dependent antifungal activity. A 99.99% inhibition of C. albicans was observed at 5 mg/mL. No detectable CFUs were observed at 30 mg/mL after 6 h and at 10 mg/mL after 24 h. HaCaT cell viability decreased in a concentration-dependent manner, with the half-maximal inhibitory concentration determined to be 10 mg/mL. Conclusions: The extract of S. williamsii var. coreana exhibited concentration- and time-dependent in vitro antifungal activity against C. albicans. However, the concentration associated with no detectable CFUs overlapped with the cytotoxic concentration range in HaCaT cells, indicating that further studies are required to define an appropriate concentration range for potential oral application. Full article

Honey maturity is increasingly discussed in relation to product integrity, fair trade, and the classification of immature honey production as a form of adulteration. This narrative critical review examines honey maturity using evidence from peer-reviewed microbiological, physicochemical, and metabolomic studies, combined with an analysis of international and European regulatory frameworks, including Codex Alimentarius CXS 12-1981, Council Directive 2001/110/EC, Regulation (EC) No 178/2002, and Regulation (EC) No 852/2004. Particular attention is given to the interpretation of osmophilic yeast counts, water activity ($a_w$), moisture content, comb cell capping, fermentation, and technological dehumidification. The reviewed evidence indicates that osmophilic yeasts are natural components of honey and that their presence, expressed as colony-forming units per gram (CFU/g), should not be treated as an independent non-compliance criterion in the absence of active fermentation. Existing honey standards define compositional and quality requirements, including moisture, hydroxymethylfurfural, enzymatic activity, and absence of fermentation or effervescence, but do not establish a honey-specific CFU/g limit for yeasts. On this basis, the review formulates a functional maturity assessment framework integrating $a_w$, moisture, enzymatic indicators, and metabolomic biomarkers. The proposed framework is presented as a conceptual model derived from the synthesis of the existing literature and requiring further multilaboratory validation prior to adoption in official control practice. This approach may improve proportionality in honey quality assessment and reduce the risk of misclassifying microbiologically stable honeys as immature or adulterated. Full article

Background/Objectives: Methods of smoking have evolved over the years, including heated tobacco products. The impact of exposure to traditional tobacco smoke and heated/electronic tobacco products (IQOS) on biofilm formation has not been previously compared in vitro. Aims and objectives: The present study aimed to evaluate the impact of tobacco and electronic smoking on microbial biofilm formation on dental hard tissues. Materials and Methods: Thirty premolars were randomly assigned to six groups (n = 10 per group) according to tissue type and smoking exposure: Six experimental groups were defined: Group 1, non-exposed enamel; Group 2, enamel subjected to conventional cigarette smoke (CS); Group 3, enamel subjected to heated tobacco (HT); Group 4, non-exposed cementum; Group 5, cementum subjected to conventional cigarette smoke; and Group 6, cementum exposed to heated tobacco. Enamel and root discs were then immersed in 2 mL of an adjusted, standardized bacterial suspension of Streptococcus mutans (S. mutans) to allow bacterial biofilm adhesion after incubation for 48 h at 37 °C. The mean colony-forming unit (CFU) count was calculated, and the surface topography and roughness were assessed using scanning electron microscopy and ImageJ software with the SurfCharJ plugin, respectively. Results: Conventional cigarette smoking showed significantly higher S. mutans adhesion on the enamel and root discs compared with IQOS and control groups. Both IQOS and cigarette smoking increased roughness on enamel and root versus the control group, and cigarette smoking produced significantly higher roughness on the enamel surface when compared to IQOS; however, there were no significant differences in the roughness between the two smoking methods on the root surface. SEM analysis showed the most extensive enamel and root microtopography change in IQOS smoking. Conclusions: Aerosols from heated tobacco products (IQOS) alter the surface topography and roughness of enamel and root, while traditional cigarette smoking significantly increases bacterial colonization. Further in vivo studies are warranted to simulate the dynamic nature of the oral cavity. Full article

In Europe, travel-associated Legionnaires’ disease (TALD) cases require timely environmental investigations to support risk assessment, rapid control measures, and prompt reporting of investigation findings to the European Legionnaires’ Disease Surveillance Network (ELDSNet). This study evaluated TALD-related environmental investigations conducted during 2025 and early 2026 in Crete, Greece, following notifications through ELDSNet. Overall, 30 notifications corresponded to 24 unique confirmed TALD cases with illness onset in 2025 and 24 implicated hotels, with some cases involving stays in multiple hotels and Regional Units and clusters identified in some implicated hotels. The investigation framework combined microbiological, physicochemical, and operational data, focusing on delays from symptom onset, notification, sampling, and laboratory reporting. Overall, 516 environmental samples were collected, of which 503 yielded valid analytical results. Among the 503 samples analyzed, Legionella spp. were detected at ≥50 colony-forming units per liter (CFU/L) in 127 samples (25.25%). This included 123 samples positive for L. pneumophila (24.45%), of which 31 were serogroup 1 (6.16%). Concentrations exceeding the 1000 CFU/L threshold were recorded in 53 samples (10.54%). Operational indicators varied, with median values of 31.0 days for reporting delay (RD), 14.5 days from notification to first sampling (TTF), 47.5 days from symptom onset to first sampling (TDS), and 67.0 days from symptom onset to first laboratory result (OELR). The findings underscore the necessity to document response delays, enhance inspector capacity and cross-regional coordination, and integrate microbiological results with operational indicators. This integration is crucial for facilitating earlier environmental risk assessments, expediting reporting, and implementing more effective TALD public health interventions. Full article