Search Results (1,985)

The development of high-throughput, sensitive and portable strategies for detecting foodborne pathogens is urgently needed in food safety, especially during an outbreak. Herein, an ultrasensitive suspension array was constructed by designing photonic crystal microsphere (PCM)-assisted loop-mediated isothermal amplification (LAMP) for Staphylococcus aureus detection. The PCM-LAMP suspension array integrated the optical signal enhancement capability of the biomimetic microporous three-dimensional PCM surface with the thousand-fold signal amplification of LAMP. The biomimetic PCMs displayed a periodic dielectric nanostructure and enhanced the fluorescence intensity of the LAMP reaction, leading to high sensitivity. The PCM-LAMP suspension array allowed sensitive detection of the target DNA of S. aureus without long-term culture. Under optimal conditions, the limit of detection for S. aureus genomic DNA reached as low as 0.18 fM, and the assay exhibited excellent specificity against other bacteria. Furthermore, trace target DNA in food samples was accurately quantified, demonstrating its potential for practical applications. Therefore, the developed PCM-LAMP suspension array holds great promise for ultrasensitive and rapid detection of foodborne pathogens. Full article

In order to assess the spreading of tick-borne bacteria and protozoa in a protected area largely frequented by people and in which numerous domestic and wild animals live, molecular analyses were carried out in ticks collected from fallow deer (Dama dama) to detect Anaplasma phagocytophilum, Borrelia sp., Coxiella burnetii, Francisella tularensis, Hepatozoon sp., and piroplasms. A total of 148 tick pools, for a total of 475 ticks collected from fallow deer and identified as female adult Ixodes ricinus, were submitted to DNA extraction and different PCR assays. One hundred and two (68.92%) pools were positive for one or more pathogens: three (2.02%) for C. burnetii, 21 (14.19%) for Borrelia sp., 35 (23.64%) for piroplasms, and 87 (58.78%) for A. phagocytophilum. All tick pools were negative for F. tularensis and Hepatozoon sp. Sixty-seven (45.27%) pools were positive for only one investigated pathogen, whereas in 35 (23.64%) pools DNA of two or more pathogens were found. Sequencing analyses identified 28 piroplasm amplicons as Theileria sp. OT3 and seven amplicons as possible Theileria cervi. Sequencing of the 21 Borrelia amplicons identified six samples as B. miyamotoi and eight as B. lusitaniae, whereas seven amplicons had 100% homology with a Borrelia sp. found in France and 99.37% with a B. theileri strain detected in Zambia. Monitoring tick-borne pathogens in ticks is pivotal to assess the spread of these microorganisms, the evolution of their epidemiology, and the risk of infections for animals and humans. Full article

Background/Objectives: Enterococcus spp. are important indicators of AMR and potential opportunistic pathogens. Urban green spaces, frequented by dogs and humans, may serve as reservoirs for resistant bacteria. This study assessed the occurrence, AMR profiles, and virulence traits of Enterococcus spp. in dog feces from urban green spaces in Porto (Portugal). Methods: In December 2023 and May 2024, 240 dog fecal samples were collected from 12 urban green spaces across Porto. Enterococcus spp. were isolated using selective culture, identified to species level, and tested for antimicrobial susceptibility following CLSI guidelines. PCR screening was performed for resistance genes (vanA, vanB, erm(A/B/C), vatD/E, tet(M/O/L/K)) and virulence genes (gelE, ace). Environmental and socioeconomic features, including vegetation density (NDVI), presence of water features, and neighborhood deprivation (EDI), were recorded to explore associations with bacterial occurrence and traits. Results: Thirty-two isolates were recovered, mainly E. faecium (n = 9) and E. faecalis (n = 7). High resistance rates were observed to tetracycline (56.3%) and quinupristin/dalfopristin (37.5%), with lower rates for vancomycin, teicoplanin, and ciprofloxacin (3.1%), and imipenem (6.3%). Tet(M) was the most prevalent resistance gene (40.6%), and gelE and ace were frequently detected, often co-occurring with resistance determinants. Distribution of resistance and virulence genes varied across green spaces, with widely used parks showing more isolates. Vegetation density and water features were not directly associated with bacterial recovery. Conclusions: Dog feces in urban green spaces contribute to localized AMR hotspots, acting as potential reservoirs of resistant and potentially pathogenic Enterococcus spp. These findings highlight the importance of One Health strategies for urban sanitation and AMR surveillance. Full article

Background/Objectives: The frequent use of antibiotics has led to increasing drug resistance in Aeromonas hydrophila; therefore, there is an urgent need to develop novel antimicrobial agents to prevent and control bacterial diseases in aquaculture. Allicin E (ALE) is derived from garlic (Allium sativum L.), a plant extensively used in traditional medicine for treating infections. This study aimed to evaluate the potential of ALE against A. hydrophila, a major aquaculture pathogen, by investigating its antibacterial efficacy, mechanisms of action, and in vivo protective effects. Methods: The minimum inhibitory and bactericidal concentrations (MIC/MBC) were determined by broth microdilution. Antibacterial mechanisms were investigated through ROS detection, electron microscopy, fluorescent staining, and content leakage measurement. In vivo efficacy was evaluated in Carassius auratus gibelio by monitoring survival rates and bacterial loads, analyzing immune and antioxidant biomarkers, and histopathological analysis after A. hydrophila challenge. Results: ALE exhibited potent antibacterial activity (MIC = MBC = 8 μg/mL), achieving complete bacterial elimination within 1 h and showing a low resistance propensity. Mechanistically, ALE induced ROS accumulation, causing oxidative damage that disrupted membrane integrity and facilitated the leakage of cellular contents. In vivo, ALE significantly enhanced fish survival, reduced bacterial loads, modulated inflammatory cytokines, boosted antioxidant enzyme activities (SOD and CAT), and alleviated tissue damage. Conclusions: ALE possesses potent in vitro antibacterial activity and exerts an inhibitory effect on bacteria-induced inflammatory responses, effectively combating A. hydrophila through a multi-target mechanism and enhancing host resistance. Full article

The implementation of stringent regulatory policies for foodborne pathogens necessitates ultra-sensitive analytical methods. Digital detection, characterized by absolute quantification and tolerance to complex matrices, serves as a robust approach for food safety monitoring. This review summarizes recent advances in digital detection for foodborne pathogens, including nucleic acid amplification-based platforms such as droplet digital PCR and digital isothermal amplification, as well as emerging preamplification-free approaches based on enzyme-mediated signal conversion, functional nanomaterials, and microfluidic devices. We also profile the applications of digital detection technologies for achieving highly specific and accurate detection of foodborne pathogens and discuss their capabilities in viable bacteria quantification, antimicrobial resistance analysis, and multiplex detection. We finally discuss emerging trends, including partition-free digital detection and artificial intelligence-assisted analysis. These advances are expected to promote the development of intelligent and data-driven food safety surveillance strategies. Full article

Antibiotics, antibiotic-resistant bacteria (ARB), and antibiotic resistance genes (ARGs) have emerged as critical environmental contaminants posing serious ecological and public health concerns. The widespread occurrence and proliferation of ARB and ARGs in wastewater treatment plants (WWTPs) and reclaimed wastewater (RWW) used for irrigation represent major pathways for their dissemination into the environment. Current knowledge indicates that ARGs from the environmental resistome can be transferred among diverse microbial communities, including clinically relevant human pathogens. Numerous studies have also linked the expansion of the environmental resistome to anthropogenic activities. Therefore, preventing and mitigating the spread of antibiotic resistance in the environment requires a deeper understanding of how resistance genes evolve, transfer, and persist across ecological compartments. This review synthesizes the current state of knowledge on the occurrence, prevalence, and detection of antibiotics, ARB, and ARGs in various environmental matrices, providing essential insights for developing preventive strategies and promoting the sustainable management of ecosystems. Full article

Background/Objectives: Municipal wastewater treatment plants (WWTPs) act as reservoirs for antibiotic-resistant bacteria, which pose a threat to global public health. In this study, we used whole-genome sequencing (WGS) to characterize antibiotic resistance genes (ARGs) and their association with mobile genetic elements (MGEs) in five multidrug-resistant (MDR) Escherichia coli isolates from dewatered sludge cake samples collected from a municipal WWTP in Cheongju, Republic of Korea. Methods: Susceptibility to nine antibiotics was evaluated via disk diffusion assay. Among the isolates exhibiting multidrug resistance (MDR) to three or more antibiotic classes, five isolates were randomly selected for whole-genome sequencing using the Illumina NovaSeqX platform. Additionally, we compared the genomic structures of five WWTP isolates with 35 environmental E. coli isolates from South Korea deposited in the NCBI pathogen database. ARGs and MGEs, including plasmids, integrons, and insertion sequences (ISs), were detected in the genome assemblies. Results: ARGs were differentially distributed between chromosomal and plasmid-derived contigs. Efflux pump-related genes were predominantly located on the chromosome across all isolates, whereas several beta-lactamase genes (e.g., bla_TEM-30 and bla_TEM-33), fluoroquinolone, and tetracycline resistance genes were localized on putative plasmid contigs. Furthermore, we characterized specific MGEs associated with these ARGs, including a class 1 integron gene cassette (dfrA17–aadA5–qacEΔ1–sul1) and an IS-mediated module (mph(A)–mrx–IS6100). Core-genome multilocus sequence typing (cgMLST) revealed that these MDR isolates represented diverse genetic lineages rather than a single clonal cluster. Conclusions: The results from this study highlight the necessity of enhanced post-treatment management of wastewater byproducts and WGS-based surveillance to mitigate the environmental spread of MDR bacteria. Full article

Aquaponic systems are increasingly recognized as sustainable technologies for integrated fish and vegetable production. However, concerns remain regarding the potential internalization of human pathogens into vegetables grown in these systems. This study assessed the risk of pathogen internalization in lettuce leaves grown in aquaponic systems with Nile tilapia challenged with Escherichia coli or Vibrio cholerae. The system comprised nine fish tanks, eighteen hydroponic pipes, and eighty-one lettuce plants, with tanks assigned to three treatments. Samples of water, fish gut, fish blood, and lettuce leaves were collected. Microbiological analyses included selective culture, biochemical assays, and molecular identification. Although colonies consistent with E. coli and V. cholerae were recovered on selective media, molecular sequencing identified other bacterial species, including Aeromonas sp., Aeromonas caviae, Aeromonas veronii, Enterobacter hormaechei, and Citrobacter freundii. The findings indicate that conventional culture-based methods may produce false-positive results and highlight the importance of molecular confirmation. Notably, pathogenic bacteria associated with tilapia were detected and appeared capable of disseminating through the system and internalizing into lettuce tissues. This result highlights the need for biosecurity measures, contamination monitoring, and the combined use of conventional and molecular diagnostic tools to ensure accurate pathogen detection and compliance with international food safety standards. Full article

Background/Objectives: Antimicrobial resistance (AMR) in companion animals is an emerging public health threat due to zoonotic potential and limited therapeutic options. Dogs with otitis externa may harbor multidrug-resistant (MDR) bacteria, including Escherichia coli (E. coli), Proteus mirabilis (P. mirabilis), and Enterobacter cloacae complex (E. cloacae complex), some producing extended-spectrum beta-lactamase (ESBL) or AmpC β-lactamases. This study aimed to assess the prevalence, AMR patterns, MDR occurrence, β-lactamase production, and co-infection profiles of these pathogens in canine otitis externa. Methods: Ear canal samples were collected from 592 dogs presenting clinical signs of otitis externa, with one sample per dog included in the analysis. Samples were collected from veterinary clinics in Timiș County, Romania, from 2022 to 2025. Samples were cultured on blood agar and MacConkey agar, followed by biochemical testing and MALDI-TOF mass spectrometry for bacterial identification. Antimicrobial susceptibility testing against 15 agents across six classes was performed using the VITEK^® 2 system. MDR and β-lactamase production (ESBL, AmpC) were determined according to CLSI 2018 veterinary guidelines. Co-isolation with bacterial and fungal species were recorded. Results: E. coli, P. mirabilis, and E. cloacae complex were isolated in 9.12%, 6.25%, and 1.2% of cases, respectively. E. coli exhibited the highest resistance to aminoglycosides (tobramycin 72.2%, gentamicin 61.1%) and full susceptibility to carbapenems. P. mirabilis showed the highest resistance to ampicillin (54%) and trimethoprim + sulfamethoxazole (46%), with complete susceptibility to carbapenems and fluoroquinolones. E. cloacae complex displayed universal resistance to cephalosporins but remained susceptible to non-cephalosporin β-lactams (piperacillin–tazobactam), carbapenems and aminoglycosides. MDR prevalence was 35.2% for E. coli, 18.9% for P. mirabilis, and 14.3% for the E. cloacae complex. ESBL production was detected in 13% of E. coli and 8.1% of P. mirabilis isolates, while all E. cloacae complex isolates were AmpC-positive. Co-isolations were common, primarily involving Staphylococcus pseudintermedius (S. pseudintermedius) and Malassezia pachydermatis (M. pachydermatis). Conclusions: MDR and β-lactamase-producing bacteria were identified in dogs with otitis externa, emphasizing the importance of routine antimicrobial susceptibility testing, targeted therapy based on local resistance profiles, and continuous AMR surveillance to prevent treatment failure and mitigate zoonotic risk. Full article

The bovine milk microbiome is a complex and dynamic microbial ecosystem, comprising both commensal and pathogenic bacteria. Its composition is shaped by endogenous factors, including udder physiology, lactation stage, and health status, particularly mastitis, as well as by exogenous factors, such as housing conditions, farm infrastructure, milking practices, and post-milking processing. Mastitis not only alters milk quality but also induces persistent dysbiosis that may persist even after clinical recovery, highlighting the need for continuous microbiome monitoring to ensure milk safety. Advances in molecular and metagenomic techniques have enabled the detection of microbial taxa that are difficult to identify using traditional culture-based methods. However, challenges remain due to low microbial biomass, reagent contamination, and the inability to distinguish live from dead bacteria, all of which complicate accurate characterization. Environmental contamination from skin, air, and equipment, along with microbial shifts during transport, storage, pasteurization, and product separation, further modulate microbial communities. While mastitis-related changes in milk microbiota have been extensively studied, the effects of other bovine diseases and systemic health conditions remain largely unexplored, constituting a critical knowledge gap. Understanding the factors that shape milk microbial communities is essential for ensuring dairy product safety, optimizing herd management, and developing microbiome-based innovations in milk production. Full article

Sesame is an oilseed crop threatened by a phyllody disease associated with the presence of phytoplasmas, which can reduce yields by up to 80%. The molecular identification of these bacteria in crops located in Western Iran was achieved from samples showing symptoms of diverse intensity and types. For biological characterization, the pathogen was also dodder-transmitted to periwinkle plants. After nucleic acid extraction and nested PCR using phytoplasma-specific primer pairs amplifying part of the 16S rRNA gene, it was possible to amplify DNA fragments from both symptomatic sesame samples and dodder-inoculated periwinkle plants. The virtual RFLP pattern from the 16S rRNA gene sequences using iPhyClassifier indicated the presence of phytoplasma strains in 16SrII-D and 16SrIX-C subgroups. The identity percentage values of the obtained amplified sequences corroborated by the phylogenetic analysis identified them as ‘Candidatus Phytoplasma australasiaticum’ and ‘Ca. P. phoenicium’, respectively. The two phytoplasma strains were detected in different sesame samples collected in the same field. The coexistence of two phytoplasmas may influence the observed differences in disease severity and suggests appropriate management strategies, since diverse insect vectors were reported alongside diverse phytoplasmas associated with this disease. Moreover, the widespread disease presence strongly suggests the breeding of resistant varieties. Full article

Urinary tract infections (UTIs) are the most frequent infections in hospitalized and outpatient settings, where Escherichia coli is the predominant pathogen. Conventional diagnostic and antimicrobial susceptibility testing (AST) methods are time-consuming, often requiring 48 h, leading to empirical antibiotic therapy and contributing to antimicrobial resistance (AMR). FASTinov^® developed a rapid phenotypic method that enables AST directly from urine samples within two hours using flow cytometry. In this study, 154 inoculated urine samples were analyzed to evaluate the performance of two diagnostic panels: FASTgramneg for Gram-negative bacteria and FASTgrampos for Gram-positive bacteria. Data analysis was performed using bioFAST^® software (version 3.0), providing results in accordance with EUCAST guidelines. The FASTgramneg panel allows detection of resistance mechanisms, including extended-spectrum β-lactamases (ESBLs), and screening of AmpC β-lactamases and carbapenemases; the FASTgrampos panel additionally determines the minimal inhibitory concentration (MIC) of vancomycin for Staphylococcus aureus. Overall agreement with conventional AST methods was 97.5% for Gram-negative bacteria and 95.0% for Gram-positive bacteria. All resistance mechanisms were correctly identified with no false positives. The essential agreement for vancomycin’s MIC was 95.2%, with a BIAS of +14.3%. Reproducibility was 99.5% for FASTgramneg and 95.0% for FASTgrampos. These results demonstrate that the FASTinov^® kit significantly reduces turnaround time while maintaining high accuracy, supporting improved UTI management and antimicrobial stewardship. Full article

To address the issues of operational complexity, long duration association, and reliance on specialized equipment with existing detection methods for Vibrio parahaemolyticus, this study established a rapid detection method for V. parahaemolyticus in exported aquatic products based on the domestically developed Enzymatic Recombinase Amplification (ERA) technology. To target the thermolabile hemolysin gene (tlh) and the iron-regulated virulence regulatory protein gene (irgB) of V. parahaemolyticus, highly specific ERA primers and probes were designed and screened. Two detection platforms, a colorimetric method and a fluorescent method, were developed. Method validation results showed that this detection system achieved specific amplification for all 30 tested V. parahaemolyticus strains, with no cross-reactivity observed with 30 other common foodborne pathogenic bacteria. The detection sensitivity for both the fluorescent and colorimetric methods reached 10⁻¹ ng/μL, with a minimum detection limit of 10 CFU/25 g for artificially contaminated samples. The entire detection process, including sample preparation, requires only approximately 20 min—significantly faster than traditional culture (24–72 h) or even conventional PCR methods. Collaborative validation across five independent laboratories confirmed excellent reproducibility, with inter-laboratory agreement yielding a Kappa coefficient of 0.98. The ERA method operates at a low, constant temperature (37–39 °C), eliminating the need for thermal cyclers. When combined with portable isothermal amplification devices and visual (colorimetric) readout, it offers a distinct advantage in terms of speed, cost-effectiveness, and suitability for resource-limited or field settings compared to existing PCR-based or culture-based platforms. This method is simple to operate, rapid, sensitive, and highly suitable for on-site application, providing a reliable and practical technical solution for the rapid screening and risk monitoring of V. parahaemolyticus in exported aquatic products. Full article

Colorectal cancer (CRC) is the third most common cancer globally and the second leading cause of cancer-related deaths. While intestinal microbiota dysbiosis is linked to CRC, the direct role of intratumoral bacteria in metastasis remains poorly understood. In this study, we isolated pathogenic bacteria from CRC tumor tissues, identified as Hungatella hathewayi (H. hathewayi), through the 16S rRNA gene and whole-genome sequencing. We developed specific primers (P48/P52) and polyclonal antibodies for detecting H. hathewayi in samples. Using quantitative real-time PCR (qPCR), we found significant enrichment of H. hathewayi in fecal samples from CRC patients compared to healthy controls, with mean fold changes of 137-fold and 142-fold for primers P48 and P52, respectively. Analysis of tissue samples revealed that H. hathewayi abundance was higher in CRC tumor tissues compared to normal tissues, with mean fold changes of 2.90 for P48 and 3.97 for P52. Fluorescence in situ hybridization (FISH), immunofluorescence (IF), and immunohistochemistry (IHC) confirmed its spatial distribution within tumor tissues. In vitro assays using CRC cell lines demonstrated that H. hathewayi-derived succinate upregulates HIF-1α and SUCNR1 expression and promotes cell metastasis by inducing epithelial–mesenchymal transition (EMT). Collectively, these findings identify H. hathewayi as a novel pro-metastatic bacterium and a potential non-invasive biomarker for CRC diagnosis, providing direct evidence for the role of intratumoral bacteria in CRC progression. Full article

Molecular point-of-care testing (POCT) for respiratory infections has undergone remarkable advancement over the past two decades, driven by technological innovation and urgent clinical needs highlighted by the COVID-19 pandemic. This comprehensive systematic review was conducted following PRISMA 2020 guidelines, synthesizing evidence from 254 peer-reviewed studies published between 2006 and 2026, with detailed analysis of the 30 most relevant papers selected through a rigorous four-stage screening process. The review examines the evolution of molecular POCT technologies, including reverse transcription polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and CRISPR-based detection systems. Key findings demonstrate that modern molecular POCT platforms achieve diagnostic performance comparable to laboratory-based testing, with sensitivities ranging from 88% to 100% and specificities from 98% to 100%, while delivering results in 15 to 80 min. These technologies enable rapid, accurate detection of major respiratory pathogens, including SARS-CoV-2, influenza A/B, respiratory syncytial virus (RSV), and atypical bacteria. The integration of microfluidic systems, portable devices, and smartphone-based analysis has expanded access to testing in resource-limited settings, emergency departments, and wearable platforms. This review provides critical insights for clinicians, researchers, and policymakers regarding the current state, clinical applications, and future directions of molecular POCT for respiratory infections. Full article