| Pig breeding is an important industry in China’s animal husbandry.The high incidence rate and mortality of post-weaning diarrhea(PWD)in piglets has brought huge economic losses to the pig industry.The factors that cause PWD are complex and varied,but they are all related to the imbalance of intestinal microflora.In addition,96% of the functional pathways in the macro genome directory of human intestinal microflora also exist in the gene directory of porcine intestinal microflora.Therefore,the intestinal microflora of weaned piglets has attracted extensive attention.In recent years,the study of viral macrogenomics has revealed the virus community in human intestinal environment.Phages belonging to prokaryotic viruses dominate and play an important role in maintaining human health.However,Studies on intestinal microorganisms and their correlation with diarrhea in weaned piglets mainly focus on bacterial components,but no reports have been found on the intestinal phageome in weaned piglets.Phages and their encoded lytic enzyme genes are considered to be a promising alternative to antibiotics.Therefore,pig intestinal phageome may be a potential library of biological agents.In addition,due to the widespread use of antibiotics in pig farming,the pig farming industry is considered to be one of the potential transmission hotspots of antibiotic resistance genes(ARGs).As an important mobile transfer element,phages can promote the spread of ARGs in bacterial communities through lysogenic transformation or horizontal gene transfer.However,the distribution of phages related ARGs in the gut of weaned piglets and the pathways and drivers of their transduction are unknown.In summary,it is of great significance to reveal the intestinal phage community of weaned piglets and explore the relationship between intestinal phages and diarrhea as well as ARGs.This study selects natural cases of stress diarrhea in weaned piglets to identify and select healthy(4)and diarrhea weaned piglets(4)through the score of piglets’ feces and the observation of intestinal tissue pathology.The contents of ileum(small intestine),cecum(large intestine)and fresh feces(rectal contents)of piglets were collected as study samples(24 in total).The total DNA and virus DNA in the samples were extracted separately for metagenomic sequencing,the following research results were obtained:First,the sequencing results of all piglet enterovirus genome was analyzed,a total of 50756 virus sequences were identified,of which up to 99.88% did not match the virus sequence in the NCBI-NT virus database,indicating that the piglet enterovirus group sequence was highly novel.The annotated enteroviruses are mainly composed of Caudovirales,Microviridae and Smacoviridae virus contigs.The analysis of the abundance and diversity of virus in different samples showed that there were significant differences in the enrichment and diversity of virus species in the ileum,caecum and fecal samples of healthy piglets.In contrast,diarrhea changed the distribution and diversity of virus groups in different intestinal samples of piglets.Second,the relationship between intestinal phages and intestinal microflora in healthy and diarrhea weaned piglets was analyzed.The results showed that the abundance of intestinal Microviridae contigs was significantly higher than that of Caudovirales(P < 0.01)in healthy and diarrhea piglets.Compared with healthy piglets,the abundance of Caudovirales contigs in diarrhea piglet samples(especially feces samples)increased.The composition and species diversity of phage community in ileum,caecum and fecal samples of healthy piglets are very different.Diarrhea led to changes in the species diversity of phages in these three intestinal sites,and the community structure tended to be more similar.In addition,the intestinal microflora and intestinal phage community of piglets changed in parallel.However,the abundance and diversity of phage and the abundance and diversity of bacterial flora were significantly inversely correlated under the condition of health and diarrhea in piglets(P < 0.05).Among them,the phage community with significant correlation is mainly reflected in the Caudovirales taxonomy.However,the bacterial communities with significant relationship include Prevotellaceae,Lactobacillaceae,Clostridiaceae,Lachnospiraceae,Rumococcaceae,Streptococcidae,Enterobacteriaceae and Bacteroidaceae,which indicates that the change in the correlation between intestinal phage and bacterial communities in diarrhea piglets is limited to specific taxonomy.In the result of phage-host prediction,it was found that the host matched by higher abundance phages was mainly some common pathogenic bacteria,and diarrhea caused changes in the abundance of these phages in different intestinal sites.In the sequencing results of intestinal microflora of piglets,the relative abundance of these bacteria and corresponding phages changed in the same way.In addition,compared with healthy piglets,the diversity of polyvalent phage species in the feces of diarrhea piglets is significantly higher(P <0.05).The matched host bacteria tend to be common pathogenic bacteria,such as Campylobacter,Streptococcus,Staphylococcus,Enterococcus,Klebsiella pneumoniae and Salmonella,among which Campylobacter and Salmonella are common pathogenic bacteria for diarrhea.In conclusion,these results show that piglet diarrhea is related to intestinal phages,which play an important role in maintaining piglet intestinal ecological health.Third,the intestinal phage-related ARGs of piglets and explored the potential pathway and driving factors of phage-mediated ARGs transfer were analyzed.Through comparison with CARD database,112 ARGs were screened from 59,955 phage genes.These phage-related ARGs mainly come from the caecum contents of piglets,and the abundance of phage-related ARGs in fecal samples of diarrhea piglets is significantly higher(P < 0.05)than that of healthy piglets.According to Spearman correlation analysis,11 ARGs were significantly positively correlated with Siphoviridae,Podoviridae,Myoviridae and Microviridae,respectively.In addition,303 MEGs homologous genes were found in 59,955 phage genes through comparison and screening.The number and relative abundance of transposase genes are the highest,followed by recombinase and integrase genes.Consistent with the distribution characteristics of phage-associated ARGs,phage-associated MGE gene mainly comes from cecum samples,and the abundance of MGE gene in fecal samples of diarrhea piglets is significantly higher(P < 0.05)than that of healthy piglets.It is noteworthy that a total of 18 phage contigs(including a polyvalent phage contig)carry both ARGs and MGE genes and are all annotated as streptococcal phages,and there is a significant positive correlation between these ARGs and MGE genes,indicating that the co-location of ARGs and MGE genes in the phage is related to specific phage host(Streptococcus).Importantly,the ARGs sequence fragments encoded by phage genomes and the sequences of multiple mobile elements such as plasmids,ICEs or transposons in various bacteria(including Streptococcus)in the NCBI nucleotide data showed high collinearity.In addition,the phage-associated ARG(ANT(9)-Ia)expression strains in vitro showed resistance to antibiotics,which proved that the piglet intestinal phage carried ARG with real resistance function.The above results show that piglet intestinal phage group is an important storage and mobile carrier of ARGs.The phage-associated MGE gene is an important factor affecting the maintenance and transfer of ARGs.In addition,diarrhea caused a significant increase in the abundance of phages carrying ARGs and MGEs genes in piglet feces,and a significant increase in the diversity of polyvalent phages(phages with a wide host range),which would contribute to the transfection and wider distribution of ARGs in bacterial communities.It is worth noting that the predicted host spectrum of polyvalent phages in diarrheal feces tends to be potential intestinal pathogenic bacteria,which greatly increases the risk of intestinal pathogens acquiring ARGs.Finally,a total of 75 phage lysin genes were screened from the predicted genes of weaned piglet enterovirus.Based on the identified multivalent phage sequence,a novel and predicted gene of Streptococcus phage lysin(Lys33795)was screened.After the lysin sequence was synthesized,the vector construction and protein expression were successfully completed.The lytic spectrum,p H stability,temperature stability,in vitro bactericidal activity and biofilm scavenging ability of the lysin were determined.The results showed that Lys33795 had a wide spectrum of cleavage,not only had cleavage activity against Streptococcus suis,but also targeted to cleave Streptococcus lactis,Streptococcus equi subsp.zooepidemicus and Streptococcus equi subsp.equi.Importantly,Lys33795 showed high bactericidal activity against multidrug resistant Streptococcus suis and was not easy to induce resistance.In addition,Lys33795 can maintain its lyase activity in a wide range of p H(p H = 3 ～ 10)and temperature(4 ～ 45°C),and can significantly remove the mature biofilm formed by Streptococcus suis and Streptococcus equi cultured for 72 h.These results will help Lys33795 become a backup clinical drug against multi-drug resistant Streptococcus suis infection.In conclusion,this study has greatly expanded and enriched our understanding of the intestinal virome(especially the phageome)of weaned piglets.It provides a reference for further revealing the succession of intestinal phages in piglets under normal and diarrhea conditions,and provides a basis for the prevention and control of ARGs pollution in pig farms.And it provides a new idea for the micro-ecological prevention and control of piglet intestinal diseases and the mining of new phages and antimicrobial proteins of multidrug resistant bacteria. |
PDF Full Text Request