Gaseous hydrogen peroxide (VH2O2) is becoming an increasingly important low-temperature sterilization modality for medical devices and is being positioned as a sustainable alternative to using ethylene oxide that sterilizes approximately 50% of medical devices globally. This review examines VH2O2 from a comparative sustainable process capability perspective and explores existing operational performance and limitations to overcome for scale optimisation including parametric release considerations. A PRISMA style review of the literature indicates that VH2O2 technology is microbiologically effective and industrially promising. Best published studies have improved our understanding of key determinants such as pressure, humidity, temperature, concentration, condensation behaviour, material compatibility, and biological indicator response. However, critical process parameters and their interactions have not been statistically defined that limits optimisation including for full industrial scale cycle implementation. Additionally, continuous process data generated during sterilization cycles is not used for operational assurance. Machine learning (ML) is examined as a complementary approach to address sterilization science opportunities. Evidence from related sterilization modalities shows that ML can reduce experimental burden by decreasing the number of experimental runs required, improving cycle output anomaly detection, supporting optimisation, and enhancing use of continuous sensor data. These findings highlight that sustainable VH2O2 can potentially be further advanced through ML enabled optimisation as a reliable, data-driven, industrial-scale terminal sterilization modality for global deployment.
Biological containment strategies are widely used to reduce possible risks associated with genetically modified microorganisms (GMMs). In the biosafety literature, the performance of these systems is often benchmarked against a maximum escape frequency of one cell per 108 cells. This value is commonly attributed to the USA National Institutes of Health Guidelines for Research Involving Recombinant DNA. However, the guideline refers specifically to laboratory certification of certain host-vector systems and does not define an acceptable escape frequency for applications outside controlled laboratory environments. Despite this limited scope, the 10-8 criterion has been repeatedly cited in research articles and reviews as a general biosafety standard for GMMs. At the same time, quantitative data on escape frequency and survival of genetically modified microorganisms under realistic environmental conditions remain scarce. This lack of empirical evidence complicates environmental risk assessment and can hinder regulatory approval and technology transfer for applications intended to operate beyond the laboratory. Here, we clarify the origin and scope of the 10-8 escape criterion and discuss why it should not be interpreted as a universal biosafety standard. We argue that experimentally validated measurements of escape and survival under application-relevant conditions are urgently needed to support evidence-based biosafety assessment and the responsible development and deployment of GMMs.
AIMS:Marine-derived Aspergillus species are prolific producers of bioactive secondary metabolites, yet the majority of their biosynthetic gene clusters (BGCs) remain silent. This study aimed to integrate genome mining with High-throughput Elicitor Screening (HiTES) to unlock the metabolic potential of Aspergillus sp. WHUF0304 and identify elicitors that promote the accumulation of previously undetected metabolites. METHODS AND RESULTS:A high-quality genome of Aspergillus sp. WHUF0304 was assembled and annotated using multiple functional databases, revealing substantial secondary metabolic potential. antiSMASH analysis identified diverse BGCs, including NRPS/indole-related clusters potentially associated with indole diketopiperazine biosynthesis. A HiTES-inspired elicitor screening strategy was then applied to evaluate 42 small molecules for their ability to alter the metabolite profile of this strain. Among the tested elicitors, fluconazole was identified as the optimal inducer, triggering the production of several indole diketopiperazine-related differential metabolites. Subsequent activity-guided isolation led to the identification of a bioactive indole diketopiperazine dimer, cristatumin E, which exhibited antibacterial activity against Escherichia coli and Bacillus subtilis with minimum inhibitory concentrations (MICs) of 32 µg mL-1 and 256 µg mL-1, respectively. CONCLUSIONS:These findings demonstrate that integrating genomic and functional approaches effectively activates silent BGCs in marine fungi. The fluconazole-associated accumulation and subsequent isolation of cristatumin E, a bioactive indole diketopiperazine dimer, highlight the potential of elicitor-mediated activation to expand the detectable metabolite profile of Aspergillus sp. WHUF0304.
AIMS:A vaginal microbiome dominated by Lactobacillus crispatus is associated with positive reproductive and sexual health outcomes, yet intra-individual genetic diversity within this species remains largely unexplored. This study characterised inter- and intra-individual genomic variation in L. crispatus strains isolated through a citizen science initiative and assessed implications for multi-strain probiotic development. METHODS AND RESULTS:Fifty-three women participated in this citizen science project. Self-sampling resulted in 48 shotgun metagenomes. Twenty-two participants isolated their own L. crispatus strains using selective enrichment and LAMP-based species confirmation, resulting in 53 whole-genome-sequenced isolates. L. crispatus dominated 20 of 48 metagenomes (50.1-99.6% relative abundance). Pangenome analysis revealed 3 456 gene families, of which 43.7% were core and 56.3% accessory. A 14-kb plasmid harbouring a Fic-domain toxin-antitoxin protein, but devoid of antimicrobial resistance genes, was present in 44 of 53 strains. Strains from the same individual clustered closely together yet harboured 1-123 gene differences. Intra-individual variation was observed in the pullulanase type I gene required for glycogen degradation: 40 strains were predicted to grow on glycogen, six showed genetic disruptions with unknown consequences, and seven were predicted to lack this ability entirely. Variation within individuals was also found for bacteriocin classes and CRISPR-Cas genes. CONCLUSIONS:Substantial functional diversity exists within L. crispatus, even among strains from the same individual, supporting the rationale for multi-strain vaginal probiotics. This citizen science approach enabled discovery of host-specific adaptations while ensuring participant ownership of their strains.
AIM:The yellow catfish, Pelteobagrus fulvidraco, is a major aquaculture species in China, but its intensive farming is threatened by bacterial diseases, particularly those caused by Edwardsiella ictaluri. The overuse of antibiotics to control these infections has led to concerns about resistance and environmental safety. This study aimed to isolate and characterize a novel antagonistic bacterial strain as a potential probiotic for sustainable disease management in P. fulvidraco aquaculture. METHODS AND RESULTS:In this study, a novel antagonistic bacterial strain, designated K, was isolated from soil samples collected on the East Lake Campus of Zhejiang A & F University. Through a polyphasic taxonomic approach, including morphological observation, physiological and biochemical assays, and 16S rDNA gene phylogenetic analysis, strain K was identified as Bacillus stratosphericus. The strain exhibited significant antagonistic activity against E. ictaluri, Citrobacter freundii, and Photobacterium damselae subsp. damselae. Optimal growth was observed at pH 6.4, 28°C, and 1.2% NaCl, indicating good adaptation to typical aquaculture conditions. Moreover, the strain showed broad susceptibility to commonly used antibiotics, supporting its potential safety as a probiotic candidate. Antagonistic substances extracted from the culture supernatant remained stable under various pH and temperature conditions, though their activity was affected by metal ions. In a challenge experiment, immersion in a suspension of B. stratosphericus K significantly enhanced the survival rate of P. fulvidraco following challenge with E. ictaluri. CONCLUSIONS:Bacillus stratosphericus K was found to be a promising probiotic candidate with significant protective effects against E. ictaluri infection in P. fulvidraco aquaculture, highlighting its potential for sustainable disease management.
AIMS:Antibiotic resistant bacteria (ARB) are high-risk pathogens of interest in the environment. Treatment wetlands are effective in reducing microbial contamination, but little is known of their ability to remove ARB such as methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant enterococci (VRE), and low-level ceftriaxone resistant Escherichia coli (LLCR). METHODS AND RESULTS:We measured putative MRSA, VRE, and LLCR E. coli from water column and sediments in five distinct sites within the Banklick Creek Wetland (BCW). Using a two-tier approach, we validated antibiotic resistance of putative ARB and authenticated genetic identity. We determined isolate resistance to clinically relevant antibiotics and screened for multidrug resistance (MDR). For LLCR E. coli, high degrees of antibiotic resistance (≥ 95%) and genetic identity verification (≥ 96%) were confirmed, but none of the putative VRE and MRSA isolates were confirmed genetically. LLCR E. coli from both matrices were most frequently resistant to beta-lactam antibiotics and least frequently to carbapenems. MDR E. coli isolates were significantly more prevalent at the wetland outlet (33%) compared to the inlet (19%). CONCLUSIONS:We observed limited removal of ARB, but an increase in the proportion of MDR isolates in the later treatment stages is concerning, as it suggests that the BCW may be acting as a reservoir and a possible conduit for evolution of MDR pathogens.
AIM:Lower respiratory tract infections frequently involve polymicrobial disease and antimicrobial-resistant pathogens, yet conventional culture may provide an incomplete and delayed microbiological picture. This study evaluated the complementary contribution of multiplex molecular testing and conventional culture in a high-AMR cohort in Egypt. METHODS:This retrospective cross-sectional study included 133 specimens collected at Souad Kafafi University Hospital, Egypt, during 2022-2023: 98 sputum records tested using the BioFire FilmArray Pneumonia Plus Panel and conventional culture with VITEK 2 susceptibility testing, and 35 blood-culture records evaluated using routine culture and BCID2 Panel. Paired molecular and culture results, descriptive analyses and multivariable logistic regression were performed. RESULTS:Among 133 specimen records, at least one pathogen was recorded at 58.6% (single-organism detection, 26.3%; mixed detection, 32.3%). Gram-negative bacteria predominated among organism-category detections (47.1%), followed by viruses (27.5%) and Gram-positive bacteria (25.5%). At least one AMR marker was detected in 51 records (38.3%), with blaCTX-M and blaNDM being the most frequent (each 27.8%). Among 98 paired sputum specimens, BioFire detected at least one target in 71.4%, compared with 51.0% by culture; the specimen-level PPA for any-target detection was 84.0%. Any degree of organism-level bacterial agreement was observed in 41.8%, while 58.2% showed complete discordance. Among 72 AMR-marker-positive observations with a matched cultured isolate and relevant susceptibility result, 64 demonstrated expected resistant phenotype (88.9% positive agreement). Both carbapenemases and Gram-negative detection were independently associated with phenotypic resistance. CONCLUSIONS:Integration of multiplex PCR and conventional culture expanded pathogen detection and provided valuable AMR profiling in resource-limited, high-resistance settings.
AIM:The bioinoculant properties of a newly identified ammonium releasing novel strain of Actinomycetota- Microbacterium bengalense sp. nov. GB16_1_BI (Accession number: SRX9280401) on the microbiome structure of rice rhizosphere was assessed. METHODS AND RESULT:GB16_1_BI may inhibit most bacteria present in the rice rhizosphere as well as encouraged the growth of rare bacteria specific to the waterlogged rice rhizosphere. The genome sequence as well as untargeted metabolome analyses of GB16_1_BI showed abundance of secondary metabolites with probable antimicrobial activity. Amplicon sequencing of the 16S rRNA V3-V4 region from the rhizosphere of the black rice showed inhibition of most bacteria by GB16_1_BI. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt2) analysis showed increased abundance in the marker genes for nitrogen cycling (nifH, nrfA and nrt) but not for nifD or nifK which was also reflected in the ANOSIM analysis in the OTUs of the N-fixing bacteria. Higher abundance of the nitrogen fixing methanotrophs-Methylosinus and Methylocystis in inoculated plants also led to study of the marker genes for methane metabolism. CONCLUSION:Microbes dwelling in the rhizosphere contributes to the biogeochemical cycle by transforming unavailable minerals and by retaining nutrients for its growth which get released after death for plant adsorption. However, not all microbes contribute positively to plant growth. Microbes compete with plants for nutrients, cause disease or produce harmful greenhouse gases. Hence, GB16_1_BI could influence plant growth predominantly by suppressing microbes and encouraged niche-specific microbes specifically involved in nitrogen cycling.
AIMS:To determine whether hypobaric hypoxia-induced alterations of the intestinal microbiota contribute to impaired exercise performance and intestinal dysfunction under simulated high-altitude conditions. METHODS AND RESULTS:A mouse model of exhaustive exercise under simulated 4500 m hypobaric hypoxia was used to assess exercise performance, oxidative stress, intestinal barrier integrity, and gut microbial composition. To evaluate the contribution of the intestinal microbiota, fecal microbiota transplantation (FMT) was performed using microbiota from hypoxia-exposed or normoxic donor mice into antibiotic-treated recipients before exhaustive exercise under hypobaric hypoxia. Compared with normoxic controls, hypoxia-exposed mice exhibited reduced endurance, increased oxidative stress, impaired intestinal barrier function, decreased Ruminococcus abundance, and significant alterations in both α- and β-diversity. Recipient mice receiving microbiota from hypoxia-exposed donors developed similar phenotypes, including greater oxidative stress, reduced barrier protein expression, and microbial community profiles resembling those of hypoxic donors. Comparisons with microbiota-depleted controls further indicated that gut microbial alterations contributed to, but were not sufficient alone to cause, impaired exercise performance in the absence of the hypoxic environment. CONCLUSIONS:Hypobaric hypoxia rapidly remodels the intestinal microbiota, and these microbial alterations contribute to oxidative stress, intestinal barrier dysfunction, and reduced exercise capacity under hypoxic conditions. FMT supports a contributory role of the intestinal microbiota in hypoxia-associated physiological dysfunction while indicating that hypoxic stress remains necessary for the full fatigue phenotype to develop. These findings support further investigation of microbiota-targeted strategies to mitigate exercise impairment during hypobaric hypoxia.
Loop-mediated isothermal amplification (LAMP) integrated with CRISPR-Cas systems has emerged as a promising molecular diagnostic platform for the rapid detection of microbial pathogens. By combining the efficient nucleic acid amplification of LAMP with the sequence-specific recognition capability of CRISPR-Cas effectors, these platforms offer potential advantages in analytical sensitivity, specificity, operational simplicity, and field applicability. In this review, we summarize the principles, assay formats, and recent advances of LAMP-CRISPR technologies for detecting a broad spectrum of microbial pathogens, including bacterial, viral, fungal, and parasitic agents across clinical, veterinary, food safety, environmental, and agricultural applications. Representative studies are compared with attention to pathogen type, sample matrix, assay design, CRISPR-Cas system, readout format, analytical performance, and practical application. We further discuss major technical challenges that continue to hinder practical implementation, particularly complex sample pretreatment, workflow integration, carry-over contamination, reagent stability, multiplexing capability, and platform standardization. Attention is given to sample pretreatment and system-level integration, including current extraction and rapid lysis strategies, closed-tube reactions, portable readouts, and microfluidic or cartridge-based formats, which may support simplified "sample-in, answer-out" diagnostic workflows. Finally, we outline future directions for improving matrix-adapted sample processing, assay robustness, standardized validation, large-scale evaluation, and field deployment. This review provides a structured overview of current LAMP-CRISPR platforms and highlights key technological considerations for translating rapid microbial pathogen detection from laboratory research to real-world applications.
AIMS:The emergence of antimicrobial resistance requires alternative antimicrobial strategies capable of controlling bacterial growth while minimizing the risk of resistance selection. This study aimed to evaluate whether combinations of natural bioactive compounds, particularly carvacrol and thymol, could provide bactericidal activity without detectable early resistance induction under short-term experimental conditions, while maintaining fibroblast biocompatibility. METHODS AND RESULTS:Phenolic monoterpenes, carvacrol and thymol, the aromatic aldehyde trans-cinnamaldehyde, t-CNM, and the phenylpropanoid eugenol were evaluated individually and in binary combinations against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 reference strains. Checkerboard assays identified carvacrol-thymol as the most consistent synergistic combination, reducing the concentrations required for bacterial inhibition. Based on MIC and MBC determinations, this combination showed bactericidal activity at reduced concentrations compared with the individual compounds. Under 96 h subinhibitory exposure, the carvacrol thymol combination did not produce a detectable progressive increase in MIC values in the tested strains. In sequential exposure assays using E. coli, susceptibility to carvacrol and thymol was maintained after subsequent ciprofloxacin exposure, as indicated by stable or reduced MIC values. Cytotoxicity assays using human dermal fibroblasts indicated that the effective carvacrol-thymol concentrations preserved cell viability under the tested conditions. CONCLUSIONS:These findings indicate that carvacrol-thymol synergy provides bactericidal activity without detectable early resistance induction in the short-term exposure model used here. Although further studies using clinical and multidrug-resistant isolates, longer serial passaging, and mechanistic assays are required, the results support the potential of natural bioactive combinations as candidates for alternative antimicrobial strategies.
AIMS:The convergence of multidrug resistance and hypervirulence in Klebsiella pneumoniae (MDR-HvKp) has narrowed treatment options. Despite growing interest in phage-antibiotic synergy (PAS), this study evaluates the underexplored combinatorial effects of phage and antibiotics, including drug-specific interactions and sequence dependency, against the biofilm-forming MDR-HvKp clinical strain. METHODS AND RESULTS:A T5-like Klebsiella bacteriophage, Round, within the genus Webervirus, was therapeutically and genomically characterized. A biofilm-forming clinical strain, Kleb_134, was used to evaluate in vitro phage-antibiotic interactions with meropenem, colistin, and tigecycline in planktonic and biofilm models.In planktonic assays, phage combinations with meropenem and colistin resulted in a multi-log CFU reduction compared to monotherapies, whereas reduced efficacy was observed with tigecycline. In biofilm assays, pre-phage treatment followed by antibiotic exposure demonstrated the strongest biofilm reduction. Drug-specific and sequence-dependent effects were evident. Meropenem-phage combinations reduced biofilm biomass by 2.85-fold (high phage titre) and 3.8-fold (low phage titre), while colistin-phage combinations achieved reductions of 8.4-fold (high phage titre) and 2.8-fold (low phage titre). CONCLUSIONS:Sequential phage-antibiotic treatment was effective against MDR-HvKp biofilms, with pre-phage exposure enhancing antibiotic access through biofilm disruption. The bacteriostatic nature of tigecycline reduced efficacy by affecting phage replication. These findings highlight the importance of treatment sequence and antibiotic selection, and extend existing knowledge in optimizing therapeutic outcomes in MDR-HvKp infections.
Driven by industrial policies and market expansion, China has formed the world's largest medicinal plant cultivation industry. However, sustainable herbal production is seriously hindered by continuous monoculture obstacles (CMOs), which arise from prolonged single-species successive cultivation and manifest as comprehensive soil degradation featuring autotoxic allelochemical buildup, unbalanced rhizosphere microbiota, impaired soil physicochemical conditions, and frequent soil-borne pathogen outbreaks. Such adverse alterations lower soil enzyme activity, disrupt microbial community homeostasis, and severely impair both the yield and bioactive constituent quality of medicinal plants. Synthetic microbial communities (SynComs), experimentally assembled from defined microbial isolates via precise cultivation and controlled assembly, exhibit promising potential to regulate plant metabolism, optimize nutrient cycling and boost plant stress tolerance, and thus may serve as a feasible strategy to mitigate medicinal plant CMOs. This review took rhizosphere microecology as its core research perspective and summarized CMOs' status, SynComs' significance, construction methods, and their mechanisms in reshaping rhizosphere microecology-focusing on root exudates, nutrient cycling and microbiome dynamics. SynComs adjusted root exudates, participate in carbon/nitrogen/phosphorus cycling, enhance stress/pathogen resistance, and strengthen root immunity via signaling pathways (e.g. jasmonic acid). Existing experimental evidence indicated SynComs may partially repair plant-microbe communication networks impaired under continuous monoculture. Future research should prioritize innovative SynCom designs with keystone microbes, improved assembly techniques, and deeper rhizosphere mechanistic studies to advance sustainable medicinal plant production.
BACKGROUND:Sudden infant death syndrome (SIDS) and sudden unexplained death in childhood are associated with prone positioning, yet the biological mechanism remains unclear. We assessed whether prone position increased bacterial titres in the anterior nares and whether this effect is amplified in the presence of upper respiratory tract infection (URTI). METHOD:This prospective cross-over study required adults, as substitutes for infants, to self-collect nasal swabs at two time points, when experiencing an URTI and when healthy. Swab eluates were cultured aerobically and anaerobically on blood agar for total bacterial counts. DNA and RNA were extracted for detection of respiratory viruses. A multivariate analysis accounted for gender, position sequence, breathing mechanism, and smoking. The study consisted of 100 adult (>18 years) participants. The final cohort consisted of the 28 participants who contracted an URTI during the study; of these, 5 did not provide asymptomatic samples. RESULTS:When asymptomatic, prone positioning resulted in increased bacterial titres relative to supine under anaerobic (P = 0.0101) and aerobic (P = 0.0101) culture conditions. When participants were symptomatic, this effect tended to be exacerbated with significantly higher titres observed in the prone position under anaerobic (P = 0.0001) and aerobic (P = 0.0003) conditions. The relationships between position and bacterial titres remained significant after adjusting for potential confounders (P < 0.0001). CONCLUSION:We found a significant association between prone positioning and increased bacterial titres in the anterior nares, particularly in the presence of a suspected viral illness. These data strengthen the results of previous studies and provide further support for the bacterial hypothesis of SIDS.
AIMS:To evaluate the antimicrobial susceptibility profiles of established and emerging cosmetic preservatives against selected skin-resident microorganisms and to assess their irritation and phototoxicity potential using in vitro methods. METHODS AND RESULTS:Methylparaben, propylparaben, phenoxyethanol, ethylhexylglycerin (EHG), and selected commercial preservative blends were tested against two bacterial species, Cutibacterium acnes and Staphylococcus epidermidis, and two fungal species, Malassezia furfur and Candida parapsilosis. Minimum inhibitory and bactericidal or fungicidal concentrations were determined. Irritation potential was assessed using the Short Time Exposure assay (STE; OECD TG 491) and the Hen's Egg Test-Chorioallantoic Membrane (HET-CAM), while phototoxicity was evaluated using the 3T3 Neutral Red Uptake assay (3T3-NRU; OECD TG 432). EHG showed the broadest antimicrobial activity, including against C. acnes, but also exhibited moderate potential for irritation, possibly owing to its surfactant properties. Phenoxyethanol was highly active against fungi and exerted a bacteriostatic effect on S. epidermidis. Parabens displayed selective antifungal activity but no antibacterial activity at the concentrations tested. All preservatives were classified as non-phototoxic, and no IC50 was reached in 3T3 cells under the tested conditions (concentrations ≤ 100 μg/mL). CONCLUSIONS:Most preservatives showed relatively favorable profiles against the selected skin-resident microorganisms, although species-specific antimicrobial activity and irritation potential were observed. Early comparative screening may support the selection of effective preservatives with lower biological aggressiveness before evaluation in finished formulations, challenge testing, and clinical trials.
AIMS:The requirement for alternative wastewater treatment methods continues to increase as current systems are overwhelmed, leading to accumulation of both microbial and chemical pollutants. The purpose of this study was to evaluate and compare the potential of two distinct PAWs, The bubble spark discharge (BSD) and dielectric barrier discharge diffuser (DBDD), for both microbial inactivation and decolourization of model organic contaminants in wastewater. The potential mechanisms associated with each PAW was explored using ROS and RNS quantification and chemical scavenger assays. METHODS AND RESULTS:Two direct plasma-in-liquid systems generating PAW in situ via microbubble discharge were evaluated: BSD and DBDD, tested for antimicrobial activity against relevant potential wastewater pathogens including Escherichia coli and Salmonella enterica. Their ability to degrade several water-soluble dyes (crystal violet, methylene blue, and Congo red) as relevant surrogates for environmental contaminants of industrial origin, was also assessed. The DBDD achieved the most rapid inactivation of all tested planktonic bacteria within 90 s, while the BSD took up to 5 min. The bactericidal activity of the DBDD was likely due to a greater production of short-lived species, such as O₂⁻ and ¹O₂, which specific scavenger assays (l-histidine and Tiron) confirmed were essential for the antimicrobial activity, while the longer-lived H2O2 contributed significantly to the antibacterial activity of the BSD system. CONCLUSIONS:These findings suggest that PAW could be used as a rapid, on-demand advanced oxidation process for the tertiary stage of wastewater treatment, with potential to reduce reliance on conventional UV or chlorine disinfection while targeting both chemical contaminants and microbial bioburdens.
AIMS:To investigate the role of multispecies drain biofilms in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity and persistence on contact surfaces used in meat processing plants. METHODS AND SESULTS:Environmental drain biofilms from pork (n = 12) and beef (n = 22) processing plants were grown with and without SARS-CoV-2 on stainless steel (S.S.), polyvinyl chloride (PVC), tile, and galvanized steel (G.S.) at 7°C for 5 days. Biofilm biomass, viral RNA persistence, and viral infectivity were quantified and compared with controls. Surface type significantly influenced viral RNA persistence with PVC, tile, and particularly G.S. supporting increased SARS-CoV-2 RNA persistence relative to virus controls (P < .001), while S.S. consistently showed reduced viral RNA persistence (P < .01). Plaque assays demonstrated that SARS-CoV-2 remained viable on all surfaces after 5 days. Beef drain biofilms on tile and G.S. exhibited two to four-fold higher infectivity than corresponding virus controls (P < .001), while infectivity was reduced in pork drain biofilms on PVC and tile. Viral RNA persistence and infectivity were not consistently correlated, indicating that detection of viral RNA within biofilm matrices does not necessarily reflect infectious virus. SARS-CoV-2 exposure also altered biofilm development in a surface and origin-dependent manner, increasing biomass of pork drain biofilms on PVC and tile (>2-fold; P < .01) while reducing growth of beef drain biofilms on S.S. and G.S. (0.9-1.8 log reductions; P < .01). CONCLUSIONS:Drain biofilms may act as transient reservoirs for SARS-CoV-2, with viral persistence governed by the combined effects of surface properties, biofilm structure, and microbial origin.
AIMS:Campylobacter jejuni and Campylobacter coli are leading causes of foodborne illnesses, including human enteritis and diarrhea, in many countries. The prevalence of antibiotic-resistant Campylobacter is increasing in both developing and developed countries, with fluoroquinolone-resistant Campylobacter recognized as a priority pathogen among antibiotic-resistant bacteria. Surveillance of Campylobacter antimicrobial resistance (AMR) is conducted worldwide because of its public health importance. In this study, we characterized the antimicrobial resistance patterns and genomic diversity of C. jejuni and C. coli strains isolated from food handlers in Japan. METHODS AND RESULTS:Surveillance was conducted on 106 Campylobacter isolates from food handlers between 2008 and 2023. Species identification by 16S rRNA sequencing, antimicrobial susceptibility testing, and whole-genome sequencing of selected isolates were performed to characterize phenotypic and genotypic features, compare AMR profiles, and assess phylogeny. Multilocus sequence typing (MLST) identified 36 sequence types (STs), including three novel STs and 10 clonal complexes (CCs). Among C. jejuni, CC21 (20.6%) was the most prevalent, followed by CC353 (2.8%). In C. coli, CC828 (4.7%) was the most prevalent. The AMR profiles and resistance determinants were as follows: quinolones (68.2%); mutations in the quinolone resistance-determining region (gyrA T86I); tetracyclines (40.2%); acquisition of tet(O) and macrolides (2.8%); and mutations in the 23S rRNA gene (A2075G). ST4526 (8.4%), reported only in Japan, was the most prevalent; however, it has not been detected since 2021, suggesting recent ST diversification. CONCLUSIONS:This study characterized the AMR profiles and genetic backgrounds of non-clinical human-derived Campylobacter strains isolated from food handlers.
AIMS:Precise characterization of Escherichia coli isolates plays a crucial role in the treatment and prevention of diseases in animal production. The classical diagnostic approach to define pathotypes of E. coli relies on detection of virulence genes. However, by targeting a limited set of genetic markers, routine Polymerase Chain Reaction (PCR)-based approaches may hinder the detection of atypical pathogenic isolates, especially in bacteria with high genomic plasticity. METHODS AND RESULTS:Genomic approaches were used to characterize a hybrid ETEC/ExPEC E. coli strain isolated from lambs submitted for necropsy over the course of six months, during an investigation of persistent neonatal mortality. Most lambs submitted for necropsy showed lesions suggestive of a septicemia. Routine PCR analysis detected genes encoding two ETEC-associated toxins but none of the ExPEC-associated virulence genes commonly targeted in routine diagnostics. Whole-genome sequencing (WGS) revealed a diverse set of virulence genes, consistent with the hybrid nature of the strain. Genes encoding toxins were located on plasmids, while ExPEC-associated virulence factors were found to be chromosomally encoded. Genomic analyses revealed rapid antimicrobial resistance evolution, driven by plasmid acquisition. CONCLUSIONS:This case highlights how horizontal gene transfer promotes the development of hybrid pathotype and facilitates resistance genes acquisition, compromising traditional diagnostic approaches and treatment. ExPEC strains are particularly difficult to identify due to their diverse and ill-defined virulence markers, which are not always targeted by standard genotyping tests. Emergence of atypical E. coli strains such as those with hybrid pathotypes reinforces the need for more comprehensive methods such as WGS in veterinary diagnostics.
OBJECTIVES:To investigate the antimicrobial resistance, β-lactamase genes, biofilm formation, and genomic characteristics of Klebsiella species isolated from diseased dogs in southeastern Brazil. METHODS:Fifty-five Klebsiella isolates from dogs with clinical infections were identified by MALDI‒TOF MS. Antimicrobial susceptibility was assessed by disk diffusion. ESBL and carbapenemase genes were detected by PCR. Biofilm formation was evaluated using a microtiter plate assay. The only blaKPC-positive isolate underwent whole-genome sequencing and phylogenetic analysis with publicly available Brazilian genomes. RESULTS:Klebsiella pneumoniae was the predominant species (74.5%), followed by K. variicola (20%). Multidrug resistance was observed in 36.4% of the K. pneumoniae isolates. High resistance rates of the isolates were observed for fluoroquinolones, cephalosporins, and sulfonamides, whereas chloramphenicol showed moderate susceptibility. The most frequent β-lactamase genes were blaTEM-1 (34.5%) and blaCTX-M-15 (30.9%). A single isolate (1.8%) harbored blaKPC. No hypervirulence-associated genes were detected. All the isolates formed biofilms, predominantly with weak to moderate adhesion. Whole-genome sequencing of the blaKPC-positive isolate revealed K. pneumoniae ST11 (CG340) carrying multiple resistance genes, including blaKPC-2, and clustering with human-associated genomes within a high-risk lineage. CONCLUSIONS:Multidrug-resistant K. pneumoniae, including a high-risk ST11 blaKPC-2-producing clone, was identified in diseased dogs, supporting the need to investigate companion animals as possible reservoirs of clinically relevant antimicrobial resistance determinants. The combination of multidrug resistance and biofilm formation highlights the importance of continued surveillance and prudent antimicrobial use within One Health.
