Comparative analysis of nutritional quality and microbial contamination in camel milk: Evaluating safety and public health risks between camel milk obtained from street vendors and farms in Saudi Arabia.

OBJECTIVES: To evaluate the nutritional quality and microbial contamination of raw camel milk sourced from street vendors and compare it with milk obtained from farms, with a particular emphasis on pathogenic organisms. METHODS: Twenty samples were systematically collected from street vendors and farms between July 2022 and February 2023 and analyzed at King Fahad Medical Research Centre and the Pharmacy College, King Abdulaziz University, Jeddah, Saudi Arabia. The microbial analysis employed culture-dependent techniques for colony-forming unit analysis and isolation of microbial colonies from milk samples. Microbial identification utilized advanced methods, including VITEK-MS equipment and the MALDI-TOF technique. The chemical composition was analysed through enzyme-linked immunosorbent assays. RESULTS: The findings revealed significant differences in microbial loads, with milk sourced from street vendors exhibiting considerably higher microbial counts than farm-sourced milk, including pathogenic species like Staphylococcus aureus and Escherichia coli. The results indicated that camel milk from street vendors possessed a higher level of microbial contamination, suggesting potential health risks associated with its purchase and consumption from these sources. CONCLUSION: This study highlights the urgent need for stringent food safety practices in handling, selling, and distributing camel milk to reduce microbial risks to safe levels, thereby mitigating potential health hazards.

Endoprotein-activating DNAzyme assay for nucleic acid extraction- and amplification-free detection of viable pathogenic bacteria.

Pathogenic bacteria in food or environment, can pose threats to public health, highlighting the requirement of tools for rapid and accurate detection of viable pathogenic bacteria. Herein, we report a sequential endoprotein RNase H2-activating DNAzyme assay (termed epDNAzyme) that enables nucleic acid extraction- and amplification-free detection of viable Salmonella enterica (S. enterica). The direct detection allows for a rapid detection of viable S. enterica within 25 min. Besides, the assay, based on sequential reporting strategy, circumvents internal modifications in the DNAzyme's active domain and improve its catalytic activity. The multiple-turnover DNAzyme cutting and the enhanced catalytic activity of DNAzyme render the epDNAzyme assay to be highly sensitive, and enables the detection of 190 CFU/mL and 0.1% viable S. enterica. The assay has been utilized to detect S. enterica contamination in food and clinical samples, indicating its potential as a promising tool for monitoring pathogen-associated biosafety.

Microbial signatures predictive of short-term prognosis in severe pneumonia.

Objective: This retrospective cohort study aimed to investigate the composition and diversity of lung microbiota in patients with severe pneumonia and explore its association with short-term prognosis. Methods: A total of 301 patients diagnosed with severe pneumonia underwent bronchoalveolar lavage fluid metagenomic next-generation sequencing (mNGS) testing from February 2022 to January 2024. After applying exclusion criteria, 236 patients were included in the study. Baseline demographic and clinical characteristics were compared between survival and non-survival groups. Microbial composition and diversity were analyzed using alpha and beta diversity metrics. Additionally, LEfSe analysis and machine learning methods were employed to identify key pathogenic microorganism associated with short-term mortality. Microbial interaction modes were assessed through network co-occurrence analysis. Results: The overall 28-day mortality rate was 37.7% in severe pneumonia. Non-survival patients had a higher prevalence of hypertension and exhibited higher APACHE II and SOFA scores, higher procalcitonin (PCT), and shorter hospitalization duration. Microbial α and ß diversity analysis showed no significant differences between the two groups. However, distinct species diversity patterns were observed, with the non-survival group showing a higher abundance of Acinetobacter baumannii, Klebsiella pneumoniae, and Enterococcus faecium, while the survival group had a higher prevalence of Corynebacterium striatum and Enterobacter. LEfSe analysis identified 29 distinct terms, with 10 potential markers in the non-survival group, including Pseudomonas sp. and Enterococcus durans. Machine learning models selected 16 key pathogenic bacteria, such as Klebsiella pneumoniae, significantly contributing to predicting short-term mortality. Network co-occurrence analysis revealed greater complexity in the non-survival group compared to the survival group, with differences in central genera. Conclusion: Our study highlights the potential significance of lung microbiota composition in predicting short-term prognosis in severe pneumonia patients. Differences in microbial diversity and composition, along with distinct microbial interaction modes, may contribute to variations in short-term outcomes. Further research is warranted to elucidate the clinical implications and underlying mechanisms of these findings.

Lower fecal microbiota transplantation ameliorates ulcerative colitis by eliminating oral-derived Fusobacterium nucleatum and virulence factor.

BACKGROUND: Recently, the oral oncobacterium Fusobacterium nucleatum (F. nucleatum), has been linked with ulcerative colitis (UC). Here, we aim to investigate whether Fecal Microbiota Transplantation (FMT) can alleviate UC by restoring gut microbiota and eliminating oral-derived F. nucleatum and virulence factor fadA. METHOD: C57BL/6J mice were randomly divided into a healthy control group (HC), Dextran Sulfate Sodium group (DSS), oral inoculation group (OR), upper FMT group (UFMT), and lower FMT group (LFMT). Disease activity index, body weight, survival rate, and histopathological scores were used to measure the severity of colitis. The function of the intestinal mucosal barrier was evaluated by performing immunohistochemical staining of the tight junction protein Occludin. Real-time PCR was used to assess the relative abundance of the nusG gene and the virulence gene fadA. Cytokine levels were detected by ELISA. Full-length sequencing of 16S rRNA was used to analyze the changes and composition of gut microbiota. FINDINGS: Oral incubation of F. nucleatum further exacerbated the severity of colitis and gut dysbiosis. Peptostreptococcaceae, Enterococcaceae, and Escherichia coli were significantly enriched in OR mice. However, LFMT mice showed an obvious decrease in disease activity and were more effective in restoring gut microbiota and eliminating F. nucleatum than UFMT mice. Bacteroidota, Lachnospiraceae, and Prevotellaceae were mainly enriched bacteria in LFMT mice. In addition, Genera such as Lactobacillus, Allobaculum, and Bacteroidales were found negative correlation with TNF-α, IL-1ß, and IL-6. Genera like Romboutsia, Escherichia Shigella, Enterococcus, and Clostridium were found positively correlated with TNF-α, IL-1ß, and IL-6. CONCLUSIONS: Oral incubation of F. nucleatum further exacerbates the severity and dysbiosis in DSS-induced colitis mice. Besides, lower tract FMT can ameliorate colitis by restoring the gut microbiota diversity and eliminating F. nucleatum and virulence factor fadA.

Phage probe on RAFT polymer surface for rapid enumeration of E. coli K12.

This study presents a simple, fast, and sensitive label-free sensing assay for the precise enumeration of modeled pathogenic Escherichia coli K12 (E. coli K12) bacteria for the first time. The method employs the covalent binding bacteriophage technique on the surface of a reversible addition-fragmentation chain transfer (RAFT) polymer film. The Nyquist plots obtained from electrochemical impedance spectroscopy (EIS) identified the charge transfer resistance Rct was calculated from a suitable electrochemical circuit model through an evaluation of the relevant parameter after the immobilization of the bacteriophage and the binding of specific E. coli K12. The impedimetric biosensor reveals specific and reproducible detection with sensitivity in the linear working range of 104.2-107.0 CFU/mL, a limit of detection (LOD) of 101.3 CFU/mL, and a short response time of 15 min. The SERS response validates the surface roughness and interaction of the SERS-tag with E. coli K12-modified electrodes. Furthermore, the covalently immobilized active phage selectivity was proved against various non-targeting bacterial strains in the presence of targeted E.coli K12 with a result of 94 % specificity and 98 % sensitivity. Therefore, the developed phage-based electrode surface can be used as a disposable, label-free impedimetric biosensor for rapid and real-time monitoring of serum samples.

Understanding the Evolution and Transmission Dynamics of Antibiotic Resistance Genes: A Comprehensive Review.

Antibiotic resistance poses a formidable challenge to global public health, necessitating comprehensive understanding and strategic interventions. This review explores the evolution and transmission dynamics of antibiotic resistance genes, with a focus on Bangladesh. The indiscriminate use of antibiotics, compounded by substandard formulations and clinical misdiagnosis, fuels the emergence and spread of resistance in the country. Studies reveal high resistance rates among common pathogens, emphasizing the urgent need for targeted interventions and rational antibiotic use. Molecular assessments uncover a diverse array of antibiotic resistance genes in environmental reservoirs, highlighting the complex interplay between human activities and resistance dissemination. Horizontal gene transfer mechanisms, particularly plasmid-mediated conjugation, facilitate the exchange of resistance determinants among bacterial populations, driving the evolution of multidrug-resistant strains. The review discusses clinical implications, emphasizing the interconnectedness of environmental and clinical settings in resistance dynamics. Furthermore, bioinformatic and experimental evidence elucidates novel mechanisms of resistance gene transfer, underscoring the dynamic nature of resistance evolution. In conclusion, combating antibiotic resistance requires a multifaceted approach, integrating surveillance, stewardship, and innovative research to preserve the efficacy of antimicrobial agents and safeguard public health.

Direct targeting of host microtubule and actin cytoskeletons by a chlamydial pathogenic effector protein.

To propagate within a eukaryotic cell, pathogenic bacteria hijack and remodulate host cell functions. The Gram-negative obligate intracellular Chlamydiaceae, which pose a serious threat to human and animal health, attach to host cells and inject effector proteins that reprogram host cell machineries. Members of the conserved chlamydial TarP family have been characterized as major, early effectors that bind to and remodel the host actin cytoskeleton. We now describe a new function for the Chlamydia pneumoniae TarP member CPn0572, namely the ability to bind and alter the microtubule cytoskeleton. Thus, CPn0572 is unique in being the only prokaryotic protein that directly modulates both dynamic cytoskeletons of a eukaryotic cell. Ectopically expressed GFP-CPn0572 associates in a dose-independent manner with either cytoskeleton singly or simultaneously. In vitro, CPn0572 binds directly to microtubules. Expression of a microtubule-only CPn0572 variant resulted in the formation of an aberrantly thick, stabilized microtubule network. Intriguingly, during infection, secreted CPn0572 also co-localized with altered microtubules, suggesting that this protein also affects microtubule dynamics during infection. Our analysis points to a crosstalk between actin and microtubule cytoskeletons via chlamydial CPn0572.

Microbial community diversity analysis of kiwifruit pollen and identification of potential pathogens.

The kiwifruit industry typically uses commercial pollen for artificial pollination. However, during the collection of male flowers and pollen production, pollen can be easily contaminated by pathogenic bacteria that cause diseases such as canker and flower rot. Consequently, it is crucial to understand the structure of the pollen microbial community. This study employed Illumina high-throughput sequencing technology to analyze the fungal and bacterial composition in pollen samples from various regions in Shaanxi Province. Concurrently, potential pathogenic strains were isolated using traditional microbial isolation and cultivation techniques, and their molecular identification was performed through 16S rDNA sequence analysis. A tieback test was conducted on healthy branches to verify the pathogenicity of the strains. The results revealed a rich diversity of fungi and bacteria in kiwifruit pollen. At the phylum level, pollen fungi were mainly distributed in Ascomycota, and bacteria were mainly distributed in Proteobacteria and Firmicutes. The dominant fungal genera were Mycosphaerella, Aspergillus, and Cladosporium; the dominant bacterial genera were Weissella, Pantoea, Enterobacter, and Pseudomonas, respectively. Additionally, both Erwinia persicina and Pseudomonas fluorescens, isolated from pollen, exhibited high pathogenicity toward healthy kiwifruit branches. These findings contribute to a deeper understanding of the microbial diversity in commercial kiwifruit pollen used for mass pollination.

[Distribution and Drug Resistance Characteristics of Pathogenic Bacteria in the Elderly Population in China in 2021]. / 2021å¹´ä¸­å›½è€å¹´äººç¾¤æ„ŸæŸ“ç— åŽŸèŒåˆ†å¸ƒå’Œè€è¯æ€§ç‰¹å¾.

Objective: To study the distribution and drug resistance characteristics of pathogenic bacteria in the elderly population of China by collecting and analyzing the standardized case data on the pathogens of infections in elderly patients, and to facilitate the establishment of a standardized layered surveillance system for pathogenic bacteria in China. Methods: We collected the case data of elderly patients (≥65 years old) from 62 sentinel hospitals across the country in 2021. Then, we statistically analyzed the data by patient age, their geographical region, the distribution of pathogenic bacteria, and the drug resistance characteristics of main pathogens. Results: A total of 3468 cases from across the country were included in the study. The top three sources of patients were the intensive care unit (13.2%), the department of respiratory medicine (11.2%), and the department of general surgery (8.4%). The top three types of specimens were urine (25.5%), sputum (20.6%), and blood (18.7%). A total of 3468 strains of pathogens were isolated, among which, 78.9% were gram-negative bacteria and 21.1% were gram-positive bacteria. The top five types of bacteria were Escherichia coli (20.9%), Klebsiella pneumoniae (18.3%), Pseudomonas aeruginosa (11.2%), Staphylococcus aureus (9.0%), and Acinetobacter baumannii (7.0%). The isolation rates of common important drug-resistant bacteria were 38.0% for methicillin-resistant Staphylococcus aureus (MRSA), 68.7% for carbapenem-resistant Acinetobacter baumannii (CRAB), and 38.2% for carbapenem-resistant Pseudomonas aeruginosa (CRPA), 20.1% for carbapenem-resistant Klebsiella pneumoniae (CRKP), 5.2% for carbapenem-resistant Escherichia coli (CRECO), and 2.1% for vancomycin-resistant Enterococcus (VRE). There were differences in the isolation rates of CRAB and CRKP in clinical care in the elderly population in seven geographical regions of China (P<0.05). Klebsiella pneumoniae is the most important pathogen in the elderly population ≥85 years old, and the isolation rates of CRKP showed significant differences in different age groups (P<0.05). Conclusion: There are significant differences in the drug resistance of pathogenic bacteria in the elderly populations of different regions and age groups in China. Therefore, monitoring the distribution and drug resistance of pathogenic bacteria in the elderly population and formulating targeted treatment plans according to the characteristics of the specific regions and age groups are of great significance to the improvement in the treatment outcomes and prognosis of the elderly population.

Recent Advances in Bacterial Detection Using Surface-Enhanced Raman Scattering.

Rapid identification of microorganisms with a high sensitivity and selectivity is of great interest in many fields, primarily in clinical diagnosis, environmental monitoring, and the food industry. For over the past decades, a surface-enhanced Raman scattering (SERS)-based detection platform has been extensively used for bacterial detection, and the effort has been extended to clinical, environmental, and food samples. In contrast to other approaches, such as enzyme-linked immunosorbent assays and polymerase chain reaction, SERS exhibits outstanding advantages of rapid detection, being culture-free, low cost, high sensitivity, and lack of water interference. This review aims to cover the development of SERS-based methods for bacterial detection with an emphasis on the source of the signal, techniques used to improve the limit of detection and specificity, and the application of SERS in high-throughput settings and complex samples. The challenges and advancements with the implementation of artificial intelligence (AI) are also discussed.

Microbial Diversity and Screening for Potential Pathogens and Beneficial Bacteria of Five Jellyfish Species-Associated Microorganisms Based on 16S rRNA Sequencing.

Jellyfish, microorganisms, and the marine environment collectively shape a complex ecosystem. This study aimed to analyze the microbial communities associated with five jellyfish species, exploring their composition, diversity, and relationships. Microbial diversity among the species was assessed using 16S rRNA gene sequencing and QIIME analysis. Significant differences in bacterial composition were found, with distinct dominant taxa in each species: Mycoplasmataceae (99.21%) in Aurelia coerulea, Sphingomonadaceae (22.81%) in Cassiopea andromeda, Alphaproteobacteria_unclassified (family level) (64.09%) in Chrysaora quinquecirrha, Parcubacteria_unclassified (family level) (93.11%) in Phacellophora camtschatica, and Chlamydiaceae (35.05%) and Alphaproteobacteria_unclassified (family level) (38.73%) in Rhopilema esculentum. C. andromeda showed the highest diversity, while A. coerulea exhibited the lowest. Correlations among dominant genera varied, including a positive correlation between Parcubacteria_unclassified (genus level) and Chlamydiaceae_unclassified (genus level). Genes were enriched in metabolic pathways and ABC transporters. The most abundant potential pathogens at the phylum level were Proteobacteria, Tenericutes, Chlamydiae, and Epsilonbacteraeota. The differing microbial compositions are likely influenced by species and their habitats. Interactions between jellyfish and microorganisms, as well as among microorganisms, showed interdependency or antagonism. Most microbial gene functions focused on metabolic pathways, warranting further study on the relationship between pathogenic bacteria and these pathways.

Synergistic Antibacterial Effect of Eisenia bicyclis Extracts in Combination with Antibiotics against Fish Pathogenic Bacteria.

The aquaculture industry faces significant challenges due to bacterial infections caused by Edwardsiella tarda, Photobacterium damselae, and Vibrio harveyi. The extensive use of traditional antibiotics, has resulted in widespread antibiotic resistance. This study aimed to investigate the antibacterial potential of the brown seaweed Eisenia bicyclis, particularly its synergistic effects with antibiotics against these fish pathogenic bacteria. E. bicyclis were processed to obtain methanolic extracts and fractionated using different polar solvents. The antibacterial activities of these extracts and fractions were assessed through disc diffusion and minimum inhibitory concentration (MIC) assays. The study further evaluated the antibiotic susceptibility of the bacterial strains and the synergistic effects of the extracts combined with erythromycin and oxyteteracycline using the fractional inhibitory concentration index. Results showed that the ethyl acetate (EtOAc) fraction of E. bicyclis methanolic extract exhibited the highest antibacterial activity. The combination of the EtOAc fraction with erythromycin significantly enhanced its antibacterial efficacy against the tested strains. This synergistic effect was indicated by a notable reduction in MIC values, demonstrating the potential of E. bicyclis to enhance the effectiveness of traditional antibiotics. The findings suggest that E. bicyclis extracts, particularly the EtOAc fraction, could serve as a potent natural resource to counteract antibiotic resistance in aquaculture.

Enhancing Deer Sous Vide Meat Shelf Life and Safety with Eugenia caryophyllus Essential Oil against Salmonella enterica.

Modern lifestyles have increased the focus on food stability and human health due to evolving industrial goals and scientific advancements. Pathogenic microorganisms significantly challenge food quality, with Salmonella enterica and other planktonic cells capable of forming biofilms that make them more resistant to broad-spectrum antibiotics. This research examined the chemical composition and antibacterial and antibiofilm properties of the essential oil from Eugenia caryophyllus (ECEO) derived from dried fruits. GC-MS analyses identified eugenol as the dominant component at 82.7%. Additionally, the study aimed to extend the shelf life of sous vide deer meat by applying a plant essential oil and inoculating it with S. enterica for seven days at 4 °C. The essential oil demonstrated strong antibacterial activity against S. enterica. The ECEO showed significant antibiofilm activity, as indicated by the MBIC crystal violet test results. Data from MALDI-TOF MS analysis revealed that the ECEO altered the protein profiles of bacteria on glass and stainless-steel surfaces. Furthermore, the ECEO was found to have a beneficial antibacterial effect on S. enterica. In vacuum-packed sous vide red deer meat samples, the anti-Salmonella activity of the ECEO was slightly higher than that of the control samples. These findings underscore the potential of the ECEO's antibacterial and antibiofilm properties in food preservation and extending the shelf life of meat.

Structural idiosyncrasies of glycyl T-box riboswitches among pathogenic bacteria.

T-box riboswitches are widespread bacterial regulatory noncoding RNAs that directly interact with tRNAs and switch conformations to regulate the transcription or translation of genes related to amino acid metabolism. Recent studies in Bacilli have revealed the core mechanisms of T-boxes that enable multivalent, specific recognition of both the identity and aminoacylation status of the tRNA substrates. However, in-depth knowledge of a vast number of T-boxes in other bacterial species remains scarce, although a remarkable structural diversity particularly among pathogens, is apparent. In the present study, analysis of T-boxes that control the transcription of glycyl-tRNA synthetases from four prominent human pathogens revealed significant structural idiosyncrasies. Nonetheless, these diverse T-boxes maintain functional T-box:tRNAGly interactions both in vitro and in vivo. Probing analysis not only validated recent structural observations but also expanded our knowledge on the substantial diversities among T-boxes and suggest interesting distinctions from the canonical Bacilli T-boxes. Surprisingly, some glycyl T-boxes seem to redirect the T-box trajectory in the absence of recognizable K-turns or contain Stem II modules that are generally absent in glycyl T-boxes. These results consolidate the notion of lineage-specific diversification and elaboration of the T-box mechanism and corroborate the potential of T-boxes as promising species-specific RNA targets for next-generation antibacterial compounds.

Distribution Patterns and Antibiotic Resistance Profiles of Bacterial Pathogens Among Patients with Wound Infections in the Jiaxing Region from 2021 to 2023.

Purpose: To systematically assess the distribution and antimicrobial susceptibility of pathogens in wound infections, and analyze risk factors associated with multidrug resistance (MDR). Patients and Methods: Retrospectively analyzing Jiaxing-region medical records between January 2021 and December 2023, we identified a cohort of 461 wound infection patients. Cultures were grown on various agars, with bacteria identified via Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. The antimicrobial susceptibility of the organisms were conducted by VITEK 2 system, Kirby-Bauer disk diffusion method and Epsilometer test. Statistical Package for the Social Sciences (SPSS) version 22 was used for statistical analysis. Multivariable logistic regression models were developed to pinpoint risk factors for multidrug-resistant organism (MDRO) infections and predict occurrences. Results: From 461 patients, 549 bacterial pathogens were isolated, predominantly consisting of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Enterobacter cloacae, and Enterococcus faecalis. Vancomycin, linezolid, and tigecycline maintained their efficacy against Staphylococcus aureus and Enterococcus species, while Pseudomonas aeruginosa demonstrated sensitivity to aminoglycosides. Conversely, Escherichia coli exhibited high amoxicillin resistance (85.4%). More than half of the isolates were resistant to levofloxacin, ceftriaxone, cotrimoxazole, and gentamicin, with Acinetobacter baumannii strains showing considerable resistance (65.8-68.4%) to advanced cephalosporins and carbapenems. Within this group, 58 MDROs were detected, primarily originating from Burn Plastic Surgery, Emergency, and Intensive Care Unit (ICU) departments. Multivariate logistic regression identified hyperglycemia, hypoalbuminemia, surgery, extended hospitalization, and exposure to multiple antibiotic classes as independent risk factors for MDRO wound infections. Based on these findings, a predictive model for MDRO occurrence in wounds was constructed, which had a sensitivity of 0.627, specificity of 0.933, and an Area Under the Curve (AUC) of 0.838. Conclusion: Staphylococcus aureus and Pseudomonas aeruginosa dominated in wound infections with differential antibiotic resistance. Independent risk factors included hyperglycemia, hypoalbuminemia, surgery, extended hospitalization, and polyantibiotic use. We urge prioritizing culture, susceptibility tests, and personalized antibiotic strategies to address MDRO risks and improve wound infection management specificity and efficacy.

Effects of cadmium on the intestinal health of the snail Bradybaena ravida Benson.

The heavy metal cadmium (Cd) is a toxic and bioaccumulative metal that can be enriched in the tissues and organs of living organisms through the digestive tract. However, more research is needed to determine whether food-sourced Cd affects the homeostasis of host gut microflora. In this study, the snail Bradybaena ravida (Benson) was used as a model organism fed with mulberry leaves spiked with different concentrations of Cd (0, 0.052, 0.71, and 1.94 mg kg-1). By combining 16S rRNA high-throughput sequencing with biochemical characterization, it was found that there were increases in the overall microbial diversity and abundances of pathogenic bacteria such as Corynebacterium, Enterococcus, Aeromonas, and Rickettsia in the gut of B. ravida after exposure to Cd. However, the abundances of potential Cd-resistant microbes in the host's gut, including Sphingobacterium, Lactococcus, and Chryseobacterium, decreased with increasing Cd concentrations in the mulberry leaves. In addition, there was a significant reduction in activities of energy, nutrient metabolism, and antioxidant enzymes for gut microbiota of snails treated with high concentrations of Cd compared to those with low ones. These findings highlight the interaction of snail gut microbiota with Cd exposure, indicating the potential role of terrestrial animal gut microbiota in environmental monitoring through rapid recognition and response to environmental pollution.

LC-MS/MS-based phospholipid profiling of plant-pathogenic bacteria with tailored separation of methyl-branched species.

Plant-pathogenic bacteria are one of the major constraints on agricultural yield. In order to selectively treat these bacteria, it is essential to understand the molecular structure of their cell membrane. Previous studies have focused on analyzing hydrolyzed fatty acids (FA) due to the complexity of bacterial membrane lipids. These studies have highlighted the occurrence of branched-chain fatty acids (BCFA) alongside normal-chain fatty acids (NCFA) in many bacteria. As several FA are bound in the intact phospholipids of the bacterial membrane, the presence of isomeric FA complicates lipid analysis. Furthermore, commercially available reference standards do not fully cover potential lipid isomers. To address this issue, we have developed a reversed-phase high-performance liquid chromatography (RP-HPLC) method with tandem mass spectrometry (MS/MS) to analyze the phospholipids of various plant-pathogenic bacteria with a focus on BCFA containing phospholipids. The study revealed the separation of three isomeric phosphatidylethanolamines (PE) depending on the number of bound BCFA to NCFA. The validation of the retention order was based on available reference standards in combination with the analysis of hydrolyzed fatty acids through gas chromatography with mass spectrometry (GC/MS) after fractionation. Additionally, the transferability of the retention order to other major lipid classes, such as phosphatidylglycerols (PG) and cardiolipins (CL), was thoroughly examined. Using the information regarding the retention behavior, the phospholipid profile of six plant-pathogenic bacteria was structurally elucidated. Furthermore, the developed LC-MS/MS method was used to classify the plant-pathogenic bacteria based on the number of bound BCFA in the phospholipidome.

Highly accurate measurement of the relative abundance of oral pathogenic bacteria using colony-forming unit-based qPCR.

PURPOSE: Quantitative polymerase chain reaction (qPCR) has recently been employed to measure the number of bacterial cells by quantifying their DNA fragments. However, this method can yield inaccurate bacterial cell counts because the number of DNA fragments varies among different bacterial species. To resolve this issue, we developed a novel optimized qPCR method to quantify bacterial colony-forming units (CFUs), thereby ensuring a highly accurate count of bacterial cells. METHODS: To establish a new qPCR method for quantifying 6 oral bacteria namely, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Prevotella intermedia, Fusobacterium nucleatum, and Streptococcus mutans, the most appropriate primer-probe sets were selected based on sensitivity and specificity. To optimize the qPCR for predicting bacterial CFUs, standard curves were produced by plotting bacterial CFU against Ct values. To validate the accuracy of the predicted CFU values, a spiking study was conducted to calculate the recovery rates of the predicted CFUs to the true CFUs. To evaluate the reliability of the predicted CFU values, the consistency between the optimized qPCR method and shotgun metagenome sequencing (SMS) was assessed by comparing the relative abundance of the bacterial composition. RESULTS: For each bacterium, the selected primer-probe set amplified serial-diluted standard templates indicative of bacterial CFUs. The resultant Ct values and the corresponding bacterial CFU values were used to construct a standard curve, the linearity of which was determined by a coefficient of determination (r²) >0.99. The accuracy of the predicted CFU values was validated by recovery rates ranging from 95.1% to 106.8%. The reliability of the predicted CFUs was reflected by the consistency between the optimized qPCR and SMS, as demonstrated by a Spearman rank correlation coefficient (ρ) value of 1 for all 6 bacteria. CONCLUSIONS: The CFU-based qPCR quantification method provides highly accurate and reliable quantitation of oral pathogenic bacteria.

Endotypes of severe neutrophilic and eosinophilic asthma from multi-omics integration of U-BIOPRED sputum samples.

BACKGROUND: Clustering approaches using single omics platforms are increasingly used to characterise molecular phenotypes of eosinophilic and neutrophilic asthma. Effective integration of multi-omics platforms should lead towards greater refinement of asthma endotypes across molecular dimensions and indicate key targets for intervention or biomarker development. OBJECTIVES: To determine whether multi-omics integration of sputum leads to improved granularity of the molecular classification of severe asthma. METHODS: We analyzed six -omics data blocks-microarray transcriptomics, gene set variation analysis of microarray transcriptomics, SomaSCAN proteomics assay, shotgun proteomics, 16S microbiome sequencing, and shotgun metagenomic sequencing-from induced sputum samples of 57 severe asthma patients, 15 mild-moderate asthma patients, and 13 healthy volunteers in the U-BIOPRED European cohort. We used Monti consensus clustering algorithm for aggregation of clustering results and Similarity Network Fusion to integrate the 6 multi-omics datasets of the 72 asthmatics. RESULTS: Five stable omics-associated clusters were identified (OACs). OAC1 had the best lung function with the least number of severe asthmatics with sputum paucigranulocytic inflammation. OAC5 also had fewer severe asthma patients but the highest incidence of atopy and allergic rhinitis, with paucigranulocytic inflammation. OAC3 comprised only severe asthmatics with the highest sputum eosinophilia. OAC2 had the highest sputum neutrophilia followed by OAC4 with both clusters consisting of mostly severe asthma but with more ex/current smokers in OAC4. Compared to OAC4, there was higher incidence of nasal polyps, allergic rhinitis, and eczema in OAC2. OAC2 had microbial dysbiosis with abundant Moraxella catarrhalis and Haemophilus influenzae. OAC4 was associated with pathways linked to IL-22 cytokine activation, with the prediction of therapeutic response to anti-IL22 antibody therapy. CONCLUSION: Multi-omics analysis of sputum in asthma has defined with greater granularity the asthma endotypes linked to neutrophilic and eosinophilic inflammation. Modelling diverse types of high-dimensional interactions will contribute to a more comprehensive understanding of complex endotypes. KEY POINTS: Unsupervised clustering on sputum multi-omics of asthma subjects identified 3 out of 5 clusters with predominantly severe asthma. One severe asthma cluster was linked to type 2 inflammation and sputum eosinophilia while the other 2 clusters to sputum neutrophilia. One severe neutrophilic asthma cluster was linked to Moraxella catarrhalis and to a lesser extent Haemophilus influenzae while the second cluster to activation of IL-22.

MassARRAY: a high-throughput solution for rapid detection of foodborne pathogens in real-world settings.

Introduction: Bacterial foodborne pathogens pose a substantial global public health concern, prompting government agencies and public health organizations to establish food safety guidelines and regulations aimed at mitigating the risk of foodborne illness. The advent of DNA-based amplification coupled with mass spectrometry, known as MassARRAY analysis, has proven to be a highly precise, sensitive, high-throughput, and cost-effective method for bacterial detection. This study aimed to develop, validate, and evaluate a MassARRAY-based assay for the detection and identification of significant enteropathogenic bacteria. Methods: The MassARRAY-based assay was developed for the detection of 10 crucial bacterial foodborne pathogens, including Campylobacter coli, Campylobacter jejuni, Clostridium perfringens, Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Salmonella spp., Shigella spp., and Staphylococcus aureus. The assay was optimized using the reference gDNA (n = 19), followed by validation using gDNA (n = 85) of reference and laboratory isolates. Additionally, the evaluation of the assay's reaction using a mixture of gDNA from all nine targeted species was performed. The limit of detection of the developed MassARRAY-based assay was determined using bacterial cells. Moreover, the validation method for field samples was evaluated by comparing it with standard microbiological testing methods routinely analyzed. Results: The developed MassARRAY-based assay demonstrated 100% concordance with known bacterial pure cultures. The assay's reaction using a mixture of gDNA from all nine targeted species revealed the MassARRAY's capability to detect all targeted species in a single assay with the lowest concentration of 1 ng/µL of gDNA. The limits of detection of the assay range from 357 ± 101 to 282,000 ± 79,196 cells. Moreover, the validation of the assay in field samples revealed a 100% correlation between the data obtained from the standard microbiological method and the MassARRAY-based assay. Discussion: These findings suggested that the developed MassARRAY-based assay exhibited the excellence in high-throughput detection of foodborne bacterial pathogens with high accuracy, reliability, and potential applicability within real-world field samples.