A New High-throughput Real-time PCR Assay for the Screening of Multiple Antimicrobial Resistance Genes in Broiler Fecal Samples from China

Objective Antimicrobial resistance(AMR)has become a global concern and is especially severe in China.To effectively and reliably provide AMR data,we developed a new high-throughput real-time PCR assay based on microfluidic dynamic technology,and screened multiple AMR genes in broiler fecal samples.Methods A high-throughput real-time PCR system with an new designed integrated fluidic circuit assay were performed AMR gene detection.A total of 273 broiler fecal samples collected from two geographically separated farms were screened AMR genes.Results The new assay with limits of detection ranging from 40.9 to 8,000 copies/reaction.The sensitivity rate,specificity rate,positive predictive value,negative predictive value and correct indices were 99.30%,98.08%,95.31%,99.79%,and 0.9755,respectively.Utilizing this assay,we demonstrate that AMR genes are widely spread,with positive detection rates ranging from 0 to 97.07%in 273 broiler fecal samples.bla CTX-M,bla TEM,mcr-1,fex A,cfr,optr A,and int I1 showed over 80%prevalence.The dissemination of AMR genes was distinct between the two farms.Conclusions We successfully established a new high-throughput real-time PCR assay applicable to AMR gene surveillance from fecal samples.The widespread existence of AMR genes detected in broiler farms highlights the current and severe problem of AMR.

Viral metagenomics reveals diverse viruses in the fecal samples of children with diarrhea

Diarrhea is the third leading cause of death in developing countries in children under the age of five.About half a million children die of diarrhea every year,most of which in developing countries.Viruses are the main pathogen of diarrhea.In China,the fecal virome of children with diarrhea has been rarely studied.Using an unbiased viral metagenomics approach,we analyzed the fecal virome in children with diarrhea.Many DNA or RNA viruses associated with diarrhea identified in those fecal samples were mainly from six families of Adenoviridae,Astroviridae,Caliciviridae,Parvoviridae,Picornaviridae,and Reoviridae.Among them,the family of Caliciviridae accounts for the largest proportion of 78.42%,following with Adenoviridae(8.94%)and Picornaviridae(8.36%).In addition to those diarrhea-related viruses that have already been confirmed to cause human diarrhea,the viruses not associated with diarrhea were also identified including anellovirus and picobirnavirus.This study increased our understanding of diarrheic children fecal virome and provided valuable information for the prevention and treatment of viral diarrhea in this area.

Fecal metabolomic profiles: A comparative study of patients with colorectal cancer vs adenomatous polyps

BACKGROUND Colorectal cancer(CRC),the third most common cause of death in both males and females worldwide,shows a positive response to therapy and usually a better prognosis when detected at an early stage.However,the survival rate declines when the diagnosis is late and the tumor spreads to other organs.Currently,the measures widely used in the clinic are fecal occult blood test and evaluation of serum tumor markers,but the lack of sensitivity and specificity of these markers restricts their use for CRC diagnosis.Due to its high sensitivity and precision,colonoscopy is currently the gold-standard screening technique for CRC,but it is a costly and invasive procedure.Therefore,the implementation of custom-made methodologies including those with minimal invasiveness,protection,and reproducibility is highly desirable.With regard to other screening methods,the screening of fecal samples has several benefits,and metabolomics is a successful method to classify the metabolite shift in living systems as a reaction to pathophysiological influences,genetic modifications,and environmental factors.AIM To characterize the variation groups and potentially recognize some diagnostic markers,we compared with healthy controls(HCs)the fecal nuclear magnetic resonance(NMR)metabolomic profiles of patients with CRC or adenomatous polyposis(AP).METHODS Proton nuclear magnetic resonance spectroscopy was used in combination with multivariate and univariate statistical approaches,to define the fecal metabolic profiles of 32 CRC patients,16 AP patients,and 38 HCs well matched in age,sex,and body mass index.RESULTS NMR metabolomic analyses revealed that fecal sample profiles differed among CRC patients,AP patients,and HCs,and some discriminatory metabolites including acetate,butyrate,propionate,3-hydroxyphenylacetic acid,valine,tyrosine and leucine were identified.CONCLUSION In conclusion,we are confident that our data can be a forerunner for future studies on CRC management,especially the diagnosis and evaluation of the effectiveness of treatments.

Diurnal variation in fecal concentrations of acid-detergent insoluble ash and alkaline-peroxide lignin from cattle fed bermudagrass hays of varying nutrient content

Background： The effect of time of fecal sampling on the accuracy of acid-detergent insoluble ash （ADIA） and alkaline-peroxide lignin （APL） for the prediction of fecal output （FO） in cattle was evaluated. Eight ruminally cannulated cows （594 _＋ 35.5 kg） were allocated randomly to 4 bermudagrass [Cynodon dactylon （L.） Pers.] hay diets markedly different in crude protein concentration （79-164 g/kg） with 2 replicates per diet for 3 periods. Cows were offered hay individually at 20 g DM/kg of body weight daily in equal feedings at 08：00 and 16：00 h for a 10-d adaptation period followed by 5-d of total fecal collection. Fecal grab samples also were taken each day during the fecal collection period at 06：00, 12：00, 18：00, and 24：00 h either directly from the rectum or from freshly voided feces. Samples were composited within cow and time across the 5 d total fecal collection period. Additionally, forage, ort, and fecal samples were analyzed for concentrations of APL and ADIA. Results： Fecal concentrations of ADIA and APL were not affected by sampling time （P 〉 0.22）, even though diet affected （P 〈 0.01） fecal ADIA and APL concentrations. There were no diet x sampling time interactions （P ≥ 0.60）. Estimates of FO and dry matter digestibility （DMD） from ADIA and APL were not affected （P 〉 0.16） by sampling time or the diet x sampling time interaction （P 〉 0.74）. Estimates of FO and DMD from markers from different sampling times or all different combinations of sampling time were not different （P 〉 0.72） from those of total collection among internal markers. Conclusion： Little variation in concentrations of ADIA and APL in daily fecal excretion across time increases flexibility in fecal grab sampling schedules for predicting FO and DMD.

A network meta-analysis on comparison of invasive and non-invasive sampling methods to characterize intestinal microbiota of birds

Birds maintain complex and intimate associations with a diverse community of microbes in their intestine.Multiple invasive and non-invasive sampling methods are used to characterize these communities to answer a multitude of eco-evolutionary questions related to host-gut microbiome symbioses.However,the comparability of these invasive and non-invasive sampling methods is sparse with contradicting findings.Through performing a network meta-analysis for 13 published bird gut microbiome studies,here we attempt to investigate the comparability of these invasive and non-invasive sampling methods.The two most used non-invasive sampling methods(cloacal swabs and fecal samples)showed significantly different results in alpha diversity and taxonomic relative abundances compared to invasive samples.Overall,non-invasive samples showed decreased alpha diversity compared to intestinal samples,but the alpha diversities of fecal samples were more comparable to the intestinal samples.On the contrary,the cloacal swabs characterized significantly lower alpha diversities than in intestinal samples,but the taxonomic relative abundances acquired from cloacal swabs were similar to the intestinal samples.Phylogenetic status,diet,and domestication degree of host birds also influenced the differences in microbiota characterization between invasive and non-invasive samples.Our results indicate a general pattern in microbiota differences among intestinal mucosal and non-invasive samples across multiple bird taxa,while highlighting the importance of evaluating the appropriateness of the microbiome sampling methods used to answer specific research questions.The overall results also suggest the potential importance of using both fecal and cloacal swab sampling together to properly characterize bird microbiomes.

Comparative analysis of two nonlethal methods for the study of the gut bacterial communities in wild lizards

Fecal samples or cloacal swabs are preferred over lethal dissections to study vertebrate gut microbiota for ethical reasons,but it remains unclear which nonlethal methods provide more accurate information about gut microbiota.We compared the bacterial communities of three gastrointestinal tract(GIT)segments,that is,stomach,small intestine(midgut),and rectum(hindgut)with the bacterial communities of the cloaca and feces in the mesquite lizard Sceloporus grammicus.The hindgut had the highest taxonomic and functional alpha diversity,followed by midgut and feces,whereas the stomach and cloaca showed the lowest diversities.The taxonomic assemblages of the GIT segments at the phylum level were strongly correlated with those retrieved from feces and cloacal swabs(rs>0.84 in all cases).The turnover ratio of Amplicon Sequence Variants(ASVs)between midgut and hindgut and the feces was lower than the ratio between these segments and the cloaca.More than half of the core-ASVs in the midgut(24 of 32)and hindgut(58 of 97)were also found in feces,while less than 5 were found in the cloaca.At the ASVs level,however,the structure of the bacterial communities of the midgut and hindgut were similar to those detected in feces and cloaca.Our findings suggest that fecal samples and cloacal swabs of spiny lizards provide a good approximation of the taxonomic assemblages and beta diversity of midgut and hindgut microbiota,while feces better represent the bacterial communities of the intestinal segments at a single nucleotide variation level than cloacal swabs.