Human Fecal Samples Research Articles

End-point diagnostics of Giardia duodenalis assemblages A and B by combining RPA with CRISPR/Cas12a from human fecal samples.

Giardia duodenalis is a common enteric protozoan parasite that is categorized into eight assemblages (A-H). In particular, assemblages A and B are zoonotic, capable of infecting both humans and animals worldwide, resulting in significant economic losses and public health challenges in epidemic regions. Thus, the development of rapid, accurate and non-laboratory-based diagnostic methods for infected animals is crucial for the effective prevention and control of giardiasis. Recent advancements in clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein (Cas12a) systems allow promising avenues for nucleic acid detection, characterized by their high flexibility, sensitivity and specificity. Combined recombinase polymerase amplification and CRISPR/Cas12a systems were combined and used as end-point diagnostic methods (termed REPORT) to detect G. duodenalis assemblage A and B. The diagnostic results can be observed by fluorescence readouts with the naked eye under blue light or colorimetric signals using a lateral flow strip (LFS). The limit of detection (LOD) of the REPORT‑based G. duodenalis assemblage A detection was 2.04 CFU/ml and 10 trophozoites per gram (TPG), and the LOD of assemblage B was 1.1 CFU/ml and 10 cysts per gram (CPG). The REPORT‑based G. duodenalis assemblage A and assemblage B detection methods have strong specificity and no cross-reactivity with other assemblages of G. duodenalis or common enteric parasitic protozoa and have excellent performance in clinical sample detection. This study presents a novel strategy for the direct identification of G. duodenalis assemblages A and B, requiring neither highly trained personnel nor costly specialized equipment.

Rapid and multiple visual detection of Fasciola hepatica in feces via recombinase polymerase amplification integrated with CRISPR/Cas12a technology

Fasciola hepatica is a foodborne zoonotic parasite causing significant economic losses and impacting human and livestock health in resource-limited regions. We developed a rapid, reliable, and sensitive detection method combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a, allowing visualization with the naked eye or a fluorescence reader. Multiple visual methods were used to analyze the assay results. Fluorescence signals were collected using a fluorescence reader or observed under UV or blue light. Lateral flow strips (LFS) were used for visual detection. Among seven primer pairs and three CRISPR RNA (crRNA) screened, F1/R1 and crRNA3 were optimal. The Cas12a reaction buffer was optimized with 50 mM Tris-HCl and 80 mM NaCl, with an RPA reaction time of 20 min. The assay showed high specificity and excellent sensitivity for F. hepatica, detecting 0.122 copies/μL with fluorescence and 8.6 copies/μL with LFS. Testing of 143 sheep and 43 human fecal samples showed 98.39 % consistency with qPCR and nested PCR, with prevalence rates of 52.45 % and 18.6 % in sheep and humans, respectively. Our assay offers substantial potential for point-of-care testing in resource-limited areas, addressing the need for rapid and accurate diagnosis of F. hepatica.

Complete genome sequence of two Christensenella minuta strains CIP 112228 and CIP 112229, isolated from human fecal samples.

Christensenella minuta is one of the representative bacterial species of the human gut microbiome. We report the complete genome sequence of two strains, Christensenella minuta CIP 112228 and CIP 112229, isolated from two healthy volunteers.

In vivo manipulation of human gut Bacteroides fitness by abiotic oligosaccharides.

Synthetic glycans (SGs) containing glycosidic linkages and structures not identified in nature offer a means for deliberately altering microbial community properties. Here pools of SG oligosaccharides were generated via polymerization of monosaccharides and screened for their ability to increase saccharolytic Bacteroides in ex vivo cultures of human fecal samples. A lead SG preparation was orally administered to gnotobiotic mice harboring a consortium of 56 cultured, phylogenetically diverse human gut bacteria and fed a Western diet. The abundances of 3 of 15 Bacteroides strains increased, most prominently B. intestinalis. Underlying mechanisms were characterized by analyzing in vivo expression of the carbohydrate utilization machinery, using retrievable microscopic paramagnetic particles with bound SG oligosaccharides and assaying SG degradation by individual purified B. intestinalis glycoside hydrolases. The results reveal that SGs can selectively co-opt carbohydrate utilization machinery in different human gut Bacteroides and demonstrate a means for identifying artificial carbohydrate structures for targeted bacterial manipulation.

Next-generation IgA-SEQ allows for high-throughput, anaerobic, and metagenomic assessment of IgA-coated bacteria

BackgroundThe intestinal microbiota plays a significant role in maintaining systemic and intestinal homeostasis, but can also influence diseases such as inflammatory bowel disease (IBD) and cancer. Certain bacterial species within the intestinal tract can chronically activate the immune system, leading to low-grade intestinal inflammation. As a result, plasma cells produce high levels of secretory antigen-specific immunoglobulin A (IgA), which coats the immunostimulatory bacteria. This IgA immune response against intestinal bacteria may be associated with the maintenance of homeostasis and health, as well as disease. Unraveling this dichotomy and identifying the immunostimulatory bacteria is crucial for understanding the relationship between the intestinal microbiota and the immune system, and their role in health and disease.IgA-SEQ technology has successfully identified immunostimulatory, IgA-coated bacteria from fecal material. However, the original technology is time-consuming and has limited downstream applications. In this study, we aimed to develop a next-generation, high-throughput, magnet-based sorting approach (ng-IgA-SEQ) to overcome the limitations of the original IgA-SEQ protocol.ResultsWe show, in various settings of complexity ranging from simple bacterial mixtures to human fecal samples, that our magnetic 96-well plate-based ng-IgA-SEQ protocol is highly efficient at sorting and identifying IgA-coated bacteria in a high-throughput and time efficient manner. Furthermore, we performed a comparative analysis between different IgA-SEQ protocols, highlighting that the original FACS-based IgA-SEQ approach overlooks certain nuances of IgA-coated bacteria, due to the low yield of sorted bacteria. Additionally, magnetic-based ng-IgA-SEQ allows for novel downstream applications. Firstly, as a proof-of-concept, we performed metagenomic shotgun sequencing on 10 human fecal samples to identify IgA-coated bacterial strains and associated pathways and CAZymes. Secondly, we successfully isolated and cultured IgA-coated bacteria by performing the isolation protocol under anaerobic conditions.ConclusionsOur magnetic 96-well plate-based high-throughput next-generation IgA-SEQ technology efficiently identifies a great number of IgA-coated bacteria from fecal samples. This paves the way for analyzing large cohorts as well as novel downstream applications, including shotgun metagenomic sequencing, culturomics, and various functional assays. These downstream applications are essential to unravel the role of immunostimulatory bacteria in health and disease.-iBMnzqwvJXR12FS2s7VEFVideo

Escherichia coli Occurrence and Antimicrobial Resistance in a Swine Slaughtering Process.

The swine production chain can be a reservoir of antimicrobial-resistant Escherichia coli, which transfers resistance genes to other bacteria, serving as an important biomarker in the One Health approach. This study aimed to identify the frequency and antimicrobial resistance profile of E. coli in the swine production chain, assess the presence of extended-spectrum beta-lactamases (ESBL), and compare resistance profiles across different sample types. A total of 622 samples of swine carcasses from various points of the slaughter process (n = 400), swine feces (n = 100), commercial cuts (n = 45), environment (n = 67), and feces from employees (n = 10) of a pig slaughterhouse certified by the Federal Inspection Service, located in São Paulo state, Brazil, were collected. A total of 1260 E. coli isolates were obtained from the samples, with 73.6% of the samples testing positive. The agar disk diffusion test was performed with 10 different classes of antimicrobials. To confirm the production of ESBLs, the isolates were submitted to a double-disk synergism test using cefotaxime, ceftazidime, and amoxicillin with clavulanic acid. Of the total isolates, 80.71% were multidrug resistant. All ESBL-producing isolates were multidrug resistant and resistant to amoxicillin, tetracycline, and chloramphenicol. Isolates from human feces samples had less chance of being multidrug resistant than samples from other sources. The diversity of resistance profiles was verified in the samples, not clustering according to the sources, except for human feces isolates that clustered, evidencing lower antimicrobial resistance variability of these samples. Antimicrobial resistance is significantly present in the pork production chain, necessitating a comprehensive multidisciplinary approach to effectively mitigate risks within the One Health framework.

Emergence of blaOXA-181-bearing tigecycline-resistant Klebsiella aerogenes in China.

We report the isolation of a blaOXA-181-positive, tigecycline-resistant Klebsiella aerogenes strain KA04 from a Chinese inpatient's fecal sample. Species identification was performed using MALDI-TOF MS. The antibiotic susceptibilities were assessed via the broth microdilution method. To elucidate the transmission and genetic structure of the blaOXA-181 gene, conjugation assays and whole-genome sequencing (WGS) were performed. KA04 displayed resistance to carbapenems, quinolones, piperacillin/tazobactam and tigecycline. Through WGS and conjugation experiments, it was possible to confirm blaOXA-181 and qnrS1 genes causing antibiotic resistance were located on a 51-kb IncX3 type mobile plasmid, blaOXA-181 gene could be successfully transferred into E. coli EC600 at a conjugation frequency of 1.1 × 10- 4. tet(A) gene was located on both the chromosome and non-transmissible IncFIB(K) plasmid. This is a tigecycline-resistant K. aerogenes harboring blaOXA-181 isolate from human fecal sample, highlighting a significant public health concern. Further comprehensive surveillance is needed.

High-quality draft genome of Lactiplantibacillus plantarum strain APC2688 isolated from human feces.

Lactiplantibacillus plantarum is a commensal bacterial species often found within the human gut. To expand our knowledge about this human-associated taxon with potential probiotic properties, here we present the draft genome sequence of Lactiplantibacillus plantarum strain APC2688, isolated from a human fecal sample.

Molecular Diagnosis and Typing of Cryptosporidium spp. Species in Human Stools with Diarrhea.

This study was conducted to molecularly identify and classify Cryptosporidium spp. in fecal samples (n=150) from patients with diarrhea received at the microbiology laboratory of a private hospital in Denizli. In this study, the positivity of Cryptosporidium spp. in fecal samples was investigated using direct microscopy, Kinyoun's acid-fast staining method, and Nested polymerase chain reaction (PCR) techniques. Positive PCR products were sequenced. In the examined fecal samples of patients with diarrhea, no parasites were detected through direct microscopic examination. Using the Kinyoun acid-fast staining method, Cryptosporidium spp. was identified in 2.7% (n=4) of the samples, while Nested PCR detected it in 4.67% (n=7) of the samples. The four positive samples were sequenced using primers that amplify the 18S rRNA gene region. The sequencing results identified the isolates as C. parvum. Cryptosporidiosis is an important public health issue as it is a zoonotic disease caused by the Cryptosporidium parasite that can be transmitted from animals to humans. This study focuses on the molecular characterization of Cryptosporidium species detected in human fecal samples, which is significant for understanding which specific strains or species are involved in human infections. According to the findings, it is recommended that control measures be implemented to reduce the risk of exposure to Cryptosporidium in both humans and animals in Türkiye.

Cryptosporidium species and subtypes in Norway: Predominance of C. parvum and emergence of C. mortiferum

PCR-based diagnostics has revealed the previously largely unknown Cryptosporidium transmission and infections in high-income countries. This study aimed to determine domestic and imported subtypes of Cryptosporidium species in Norway, evaluate their demographic distribution, and identify potential small outbreaks. Cryptosporidium-positive human faecal samples were obtained from six medical microbiology laboratories between February 2022 and January 2024, together with 22 Cryptosporidium-positive animal samples. Species and subtypes were identified by sequencing PCR products from gp60 and SSU rRNA genes. Most cryptosporidiosis cases occurred during late summer/early autumn, primarily in children and young adults. Of 550 human samples, 359 were successfully characterized molecularly (65%), revealing infection with 10 different Cryptosporidium species. C. parvum occurred in 245 (68%) human isolates with IIa and IId being major allele families, with distinct regional distribution patterns of common subtypes. A kindergarten outbreak with 5 cases was due to C. parvum IIaA14G1R1. C. mortiferum was identified in 33 (9.2%) human cases of which 24 were known to be of domestic origin, making it the second most common species in human autochthonous cases in Norway. All C. mortiferum isolates were of the same genotype; XIVaA20G2T1, including 13 cases from a suspected small outbreak in Trøndelag. C. hominis occurred in 68 typed cases (19%), but mostly in infections acquired abroad, with allele families Ib and If occurring most often. In conclusion, this study of recent Cryptosporidium spp. and subtypes in Norway, highlights the predominance of C. parvum and the emergence of C. mortiferum among autochthonous cases.

B-172 Correlation of Microbiome Sequencing Data and Quantified Short-Chain Fatty Acids by LC-MS/MS in IBD and Non-IBD Stool Samples

Abstract Background SCFAs are the primary metabolites produced by bacterial fermentation of dietary fiber and undigested proteins. They play a crucial role in various metabolic pathways, such as regulating the host’s energy balance and anti-inflammatory processes. The most studied SCFAs are acetic acid, propionic acid and butyric acid, with an approximate molecular ratio of 60:20:20 in healthy adults. A significantly different distribution may indicate a bacterial dysbiosis. While most SCFAs have a positive impact on the host’s health, the impact of branched short-chain fatty acids (bSCFAs), such as isobutyric acid and isovaleric acid, are less well-known. In order to evaluate whether the determination of a broader SCFA panel holds additional diagnostic value, we have developed a sensitive and robust LC-MS/MS method to determine 11 C2 - C7 SCFAs in human fecal samples. Methods The LC-MS/MS method includes acetic acid (AA), propionic acid (PA), butyric acid (BA), valeric acid (VA), caproic acid (CA) and heptanoic acid (HA), as well as the isomers isobutyric acid (iBA), 2-methylbutyric acid (2-MBA), isovaleric acid (iVA), isocaproic acid (iCA) and 2-methylhexanoic acid (2-MHA). After suspending the stool samples in a stabilizing buffer, the tubes were centrifuged and 25 µl of the supernatant was mixed with an internal standard solution (10 isotopically labeled internal standards) and subsequently derivatized. After one hour, the reaction was quenched and samples were injected into an ACQUITY UPLC H-Class (Waters) with an injection cycle of 9.5 min. Analytes were measured in positive mode by ESI-MS/MS on a Xevo TQ-MS (Waters). For quantification, six calibrators and three controls covering the anticipated physiological ranges were used. Results Microbiome sequencing data identified a total of 193 species, most of which belong to the Firmicutes phylum. Next, Actinobacteria and Proteobacteria are in descending order of abundance. In our IBD samples, we see a significant dysbiosis of these ratios and orders in comparison to the screening patients. E.g. in one sample, no Bacteroidetes were found, but a high amount of Actinobacteria (40 %) were sequenced. Corresponding SCFA data detected an AA:PA:BA ratio of 76:11:10, as well as no VA, iCA, CA, 2-MHA and HA, whereas SCFA data from most screening patients showed an AA:PA:BA ratio of 60:20:20 (± 10:10:10). Conclusions After a fast and simple sample preparation and derivatization, the LC MS/MS method allows robust and sensitive measurement of all presented SCFAs from human feces samples. The preliminary analysis of the combined microbiome and SCFA data showed that the detected AA:PA:BA ratio difference could be a valid indicator for a dysbiosis or an altered microbiome, corroborating data from previous studies. While we could detect differences on bSCFAs and longer SCFAs between IBD and non-IBD patient samples in this pilot study, larger patient and control cohorts are needed in order to substantiate our initial promising findings and finally allow a robust evaluation of these parameters as diagnostic tool to monitor and determine the state of the patient microbiome.

A-087 Stability of fecal calprotectin extracted with BÜHLMANN CALEX® Cap

Abstract Background Calprotectin, a granulocyte-derived alarmin protein, is an established noninvasive biomarker of intestinal mucosal inflammation. Fecal calprotectin testing has become the gold standard for diagnosis of inflammatory bowel diseases. The aim of this work was to determine the stability of calprotectin in stool extracts prepared with the BÜHLMANN CALEX® Cap stool preparation device. Methods The analyte stability study was carried out using CALEX® Cap extracts of human fecal left-over samples. Extraction was performed for 24 hours at room temperature and samples were aliquoted and frozen at < -20°C. The baseline calprotectin concentration was measured from thawed aliquots, which were moved to target storage temperatures (2-8 °C, 18 °C, 28 °C, and 37 °C) for up to 22 days. Five stool samples covering the measuring range of the assay were analyzed with the BÜHLMANN fCAL® turbo in four replicates. Stability evaluations were performed according to CLSI guideline EP25-A1 and EP25Ed2. Results CALEX® Cap derived extracts stored refrigerated at 2-8°C for up to 20 days and at room temperature (18-28°C) for up to 6 days demonstrated deviations from baseline of less than 20%. CALEX® Cap derived extracts stored up to 15 days at 2-8°C and up to 2 days at room temperature (18-28°C) demonstrated deviations from baseline of less than 10%. Conclusions CALEX® Cap provides a stool collection method that stabilizes fecal calprotectin concentrations for an increased processing window. This allows significant timely separation between stool collection and the actual measurement of calprotectin. This specimen stabilization provides for accurate measurements for days after sample collection and thus allows for flexibility for diagnostic laboratories.

Azithromycin Resistance Patterns in Escherichia coli and Shigella before and after COVID-19, Kenya.

Escherichia coli and Shigella spp. are leading bacterial causes of acute diarrhea in sub-Saharan Africa and pose risks to global communities, travelers, and the US military. Increasing antimicrobial resistance (AMR) in those and other enteric pathogens creates treatment challenges for clinicians. Inappropriate use of antimicrobial drugs, such as azithromycin for viral respiratory infections, increased during the COVID-19 pandemic. We evaluated AMR trends of 116 E. coli and 109 Shigella spp. isolates obtained from 1,672 pre-COVID-19 (2017-2019) and 1,118 post-COVID-19 (2022-2023) human fecal samples from Kenya. Azithromycin resistance increased significantly from before to after COVID-19, from 6.3% to 40.4% (p = 0.001). Phenotypic AMR profiles from a subset of isolates were compared with genotypic AMR information derived from whole genome sequencing. The most common AMR gene detected was the macrolide mph(A) gene. This study highlights the need for continued AMR surveillance.

MetaExpertPro: A Computational Workflow for Metaproteomics Spectral Library Construction and Data-Independent Acquisition Mass Spectrometry Data Analysis

Analysis of large-scale data-independent acquisition mass spectrometry metaproteomics data remains a computational challenge. Here, we present a computational pipeline called metaExpertPro for metaproteomics data analysis. This pipeline encompasses spectral library generation using data-dependent acquisition MS, protein identification and quantification using data-independent acquisition mass spectrometry, functional and taxonomic annotation, as well as quantitative matrix generation for both microbiota and hosts. By integrating FragPipe and DIA-NN, metaExpertPro offers compatibility with both Orbitrap and timsTOF MS instruments. To evaluate the depth and accuracy of identification and quantification, we conducted extensive assessments using human fecal samples and benchmark tests. Performance tests conducted on human fecal samples indicated that metaExpertPro quantified an average of 45,000 peptides in a 60-min diaPASEF injection. Notably, metaExpertPro outperformed three existing software tools by characterizing a higher number of peptides and proteins. Importantly, metaExpertPro maintained a low factual false discovery rate of approximately 5% for protein groups across four benchmark tests. Applying a filter of five peptides per genus, metaExpertPro achieved relatively high accuracy (F-score=0.67-0.90) in genus diversity and showed a high correlation (rSpearman=0.73-0.82) between the measured and true genus relative abundance in benchmark tests. Additionally, the quantitative results at the protein, taxonomy, and function levels exhibited high reproducibility and consistency across the commonly adopted public human gut microbial protein databases IGC and UHGP. In a metaproteomic analysis of dyslipidemia patients, metaExpertPro revealed characteristic alterations in microbial functions and potential interactions between the microbiota and the host.

Highly accurate and sensitive absolute quantification of bacterial strains in human fecal samples

BackgroundNext-generation sequencing (NGS) approaches have revolutionized gut microbiome research and can provide strain-level resolution, but these techniques have limitations in that they are only semi-quantitative, suffer from high detection limits, and generate data that is compositional. The present study aimed to systematically compare quantitative PCR (qPCR) and droplet digital PCR (ddPCR) for the absolute quantification of Limosilactobacillus reuteri strains in human fecal samples and to develop an optimized protocol for the absolute quantification of bacterial strains in fecal samples.ResultsUsing strain-specific PCR primers for L. reuteri 17938, ddPCR showed slightly better reproducibility, but qPCR was almost as reproducible and showed comparable sensitivity (limit of detection [LOD] around 104 cells/g feces) and linearity (R2 > 0.98) when kit-based DNA isolation methods were used. qPCR further had a wider dynamic range and is cheaper and faster. Based on these findings, we conclude that qPCR has advantages over ddPCR for the absolute quantification of bacterial strains in fecal samples. We provide an optimized and easy-to-follow step-by-step protocol for the design of strain-specific qPCR assays, starting from primer design from genome sequences to the calibration of the PCR system. Validation of this protocol to design PCR assays for two L. reuteri strains, PB-W1 and DSM 20016 T, resulted in a highly accurate qPCR with a detection limit in spiked fecal samples of around 103 cells/g feces. Applying our strain-specific qPCR assays to fecal samples collected from human subjects who received live L. reuteri PB-W1 or DSM 20016 T during a human trial demonstrated a highly accurate quantification and sensitive detection of these two strains, with a much lower LOD and a broader dynamic range compared to NGS approaches (16S rRNA gene sequencing and whole metagenome sequencing).ConclusionsBased on our analyses, we consider qPCR with kit-based DNA extraction approaches the best approach to accurately quantify gut bacteria at the strain level in fecal samples. The provided step-by-step protocol will allow scientists to design highly sensitive strain-specific PCR systems for the accurate quantification of bacterial strains of not only L. reuteri but also other bacterial taxa in a broad range of applications and sample types.-oGjUmj5e8MXZDxyDjzcm-Video

Targeted isolation of Methanobrevibacter strains from fecal samples expands the cultivated human archaeome

Archaea are vital components of the human microbiome, yet their study within the gastrointestinal tract (GIT) is limited by the scarcity of cultured representatives. Our study presents a method for the targeted enrichment and isolation of methanogenic archaea from human fecal samples. The procedure combines methane breath testing, in silico metabolic modeling, media optimization, FACS, dilution series, and genomic sequencing through Nanopore technology. Additional analyzes include the co-cultured bacteriome, comparative genomics of archaeal genomes, functional comparisons, and structure-based protein function prediction of unknown differential traits. Successful establishment of stable archaeal cultures from 14 out of 16 fecal samples yielded nine previously uncultivated strains, eight of which are absent from a recent archaeome genome catalog. Comparative genomic and functional assessments of Methanobrevibacter smithii and Candidatus Methanobrevibacter intestini strains from individual donors revealed features potentially associated with gastrointestinal diseases. Our work broadens available archaeal representatives for GIT studies, and offers insights into Candidatus Methanobrevibacter intestini genomes’ adaptability in critical microbiome contexts.

The effect of oral probiotics in the last trimester on the human milk and infant gut microbiotas at six months postpartum: A randomized controlled trial

ObjectiveThe main aim of this study was to evaluate the effect of oral probiotics on the human milk microbiota and determine whether that influenced infant microbiota development. MethodsA total of 27 pregnant women were recruited; 14 were assigned to the probiotic group, and the rest were assigned to the control group. Their infants were likewise assigned to the probiotic group or the control group. Pregnant women in the probiotic group received probiotic supplementation from 32 weeks of gestation until delivery. Human milk samples and infant fecal samples were collected at 6 months after delivery, and 16S rRNA sequencing was used to analyze the composition of the human milk and infant gut microbiota (NCT06241222). ResultsIn the control group, bacterial microbiota were detected in 8 out of 13 milk samples, whereas in the probiotic group, only 6 out of 14 milk samples contained bacterial microbiota. We examined the composition of the human milk and infant gut microbiota in both the control and probiotic groups. Spearman correlation analysis revealed that various genera in human milk were correlated with the infant gut microbiota. The Linear discriminant analysis effect size (LEfSe) showed that 6 bacteria in the human milk microbiota in the control group were significantly more abundant than those in the probiotic group. Nine bacteria were significantly more abundant in the human milk microbiota in the probiotic group than the control group. According to the LEfSe results, 11 bacteria in the infant gut microbiota in the control group were significantly more abundant than those in the probiotic group. Fourteen bacteria were significantly more abundant in the infant gut microbiota in the probiotic group than in the control group. ConclusionThe infant gut microbiota at 6 months has a complicated relationship with the maternal human milk microbiota. Oral probiotic supplementation can change the composition of the human milk microbiota and the infant gut microbiota.

Development of a novel crAss-like phage detection method with a broad spectrum for microbial source tracking

CrAssphage has been recognized as the most abundant and human-specific bacteriophage in the human gut. Consequently, crAssphage has been used as a microbial source tracking (MST) marker to monitor human fecal contamination. Many crAss-like phages (CLPs) have been recently discovered, expanding the classification into the new order Crassvirales. This study aims to assess CLP prevalence in South Korea and develop a detection system for MST applications. Thirteen CLPs were identified in six human fecal samples and categorized into seven genera via metagenomic analysis. The major head protein (MHP) displayed increased sequence similarity within each genus. Eight PCR primer candidates, designed from MHP sequences, were evaluated in animal and human feces. CLPs were absent in animal feces except for those from raccoons, which hosted genera VI, VIIa, and VIIb. CLPs were detected in 91.52% (54/59) of humans, with genus VI (38 out of 59) showing the highest prevalence, nearly double that of p-crAssphage in genus I (22 out of 59). This study highlights genus VI as a potent MST marker, broadening the detection range for CLPs. Human-specific and selectively targeted MST markers can significantly impact hygiene regulations, lowering public health costs through their application in screening liver, sewage, wastewater, and various environmental samples.

Unveiling the Transmission Potential of Opisthorchis viverrini and Intestinal Helminths Along the Thailand-Laos Border in Thailand.

In pursuit of enhancing prevention efforts for the notably high endemic Opisthorchis viverrini infection in lower Mekong sub-region countries, particularly Thailand and Laos, this cross-sectional study investigated the transmission potential of O. viverrini and other intestinal helminths along the Thailand-Laos border in seven Thai villages. Human and pet faecal samples, Bithynia snails and cyprinid fish were analysed for helminth infections. Additionally, a questionnaire survey assessed relevant risk factors among the human population. Two groups of helminth infections were detected in humans: foodborne infections (FBIs) including O. viverrini, minute intestinal flukes (MIFs), and Taenia spp., and soil-transmitted infections (STIs) including hookworm and Strongyloides stercoralis, with prevalence rates of 7.4%, 0.5%, 2.5%, 0.5%, 2.5% and 3%, respectively. Smoking was identified as a risk factor for O. viverrini infection [adjusted odds ratios (ORa) = 3.12, 95% confidence intervals (CI): 1.33-7.30, p = 0.009] and FBIs (ORa = 2.47, 95% CI: 1.14-5.33, p = 0.022), while male was a risk factor for FBIs (ORa = 2.62, 95% CI: 1.16-5.94, p = 0.021). In dogs, hookworm, Toxocara spp., Spirometra mansoni, Trichuris vulpis and Hymenolepis diminuta were identified with prevalence rates of 35.6%, 8.1%, 8.1%, 1.2% and 1.2%, respectively. In cats, hookworm, Toxocara spp., S. mansoni, Strongyloides spp., Platynosomum fastosum, MIFs and H. diminuta were identified with prevalence rates of 50%, 17.9%, 10.7%, 7.1%, 3.6%, 3.6% and 3.6%, respectively. Bithynia snails showed 2% virgulate and 0.7% unknown cercariae infections, while among 19 cyprinid fish species, only unknown metacercariae were found. Our findings underscore the necessity of an integrated approach following the One Health concept to effectively combat these parasitic diseases while addressing human, animal and environmental health.

Antibacterial Resistance and Extended-Spectrum Beta-Lactamase (ESBL) Phenotypes in Enterobacteriaceae Isolated from Fecal Samples of Humans and Animals in Selected Local Government Areas of Nasarawa State, Nigeria

It is quite alarming the increasing rate of antibacterial resistance all over the world considering the public health threat and the re-emergence of multi-drug resistant Enterobacteriaceae. The aim of this study is Antibacterial resistance and phenotypic detection of Extended Spectrum Beta-Lactamase (ESBL) producing Enterobacteriaceae isolated from human and animal fecal samples in selected local government areas of Nasarawa state, Nigeria was carried out in the study. Hundred (100) samples comprising human and animal (goats, cattle, and chicken) were collected and 55 samples were multidrug resistant. A commercial biochemical kit (Eneterosystem 18R) was used for the isolation and identification of Enterobacteriaceae. Kirby Bauer Disk Diffusion Method was used for antibacterial susceptibility testing of Enterobacteriaceae isolates. The Double Disc Synergy Test (DDST) method was also used for the phenotypic confirmation test of Extended Spectrum Beta Lactamase (ESBL). The occurrence of multidrug-resistant isolates shows that Escherichia coli (100.00%) which is the highest, Proteus mirabilis (14.54%), Klebsiella pneumoniae, and Salmonella enterica (10.90%), while the occurrence of Shigella flexneri (9.09%) was the lowest. The Enterobacteriaceae isolates were more resistant to Cefuroxime, Cefexime, Amoxicillin Clavulanate, and Imipenem/Cilastatin with percentage resistance ranges from 66.6% - 100%. The occurrence of ESBL producers shows that Escherichia coli (60.00%) and Proteus mirabilis (62.5%) were high while Shigella flexneri (20.0%) had a low occurrence of ESBL. The sale and in-discriminate use of antibiotics without a prescription is an important regulatory issue in the abuse of antibiotics for both humans and animals. The Beta-Lactam and gentamycin antibiotics were not effective against the Multi-Drug Resistant (MDR) isolates and most of the isolates were ESBL producers.