Characteristics Of Bacteriophage Carrying ARGs And Transduction Mechanism In A Porcine-Derived Environment

Nowadays,antibiotic resistance has been thought as one of the major global health threats by the United Nations World Health Organization due to the over-use of antibiotics(including synthetic antimicrobial).Widespread spread of antibiotic resistance is usually caused by horizontal gene transfer(HGT)in bacterial populations,which is completed through conjugation,transformation and transduction process.Among the three processes,those mediated by plasmids through transformation and conjugation have been studied in detail.However,the contribution of bacteriophages has been largely overlooked.There is mounting evidence had suggested that phage probably be a potential reservoir and vector for ARG dissemination.On the other hand,in view of the close relationship between animalderived environment and human health,insight into the ARGs distribution characteristics in bacteriophages genome as well as formation mechanism of antibiotic resistance transmission through transduction from its upstream and downstream genetic environment from animalderived environmental will of great theoretical significance to control horizontal transduction transmission of ARGs in the future.Three large commercial pig farms in Guangdong province of China from eastern Guangdong(Huizhou),western Guangdong(Maoming)and northern Guangdong(Shaoguan)were slected as core sites to collect pregnant sow,boar,barren sow and piglet manure samples.The samples were then screened for 32 ARGs in nine antimicrobial categories to study ARG occurrence and persistence in bacteriaophage and bacteria metagenomes.Then,exploring the characteristics of bacteriophage carried ARGs at different types of feces under geographic and temporal factor.Results showed that bacteriophage DNA contained 35.5%of the target ARG types and sul l,blaTEM and ermB were found in 100%of the phage DNA samples.Gene ermB was the most abundant in both.In contrast,floR was the least.In order to better evaluate the ability of each ARGs to carry and spread through bacteriophage,the ratio value of the abundance of ARGs in bacteriophage and bacteria was firstly used in this study as an estimator of bacteriophage ability to transmit ARGs.The ratio for qnrA was the greatest and not ermB.In addition,fexA had the lowest ratio value and not floR.The geographical and temporal also do not affect the fixed hierarchy:qnrA>bla TEM～qnrS>sul1>ermB～floR>fexA.These results illustrate that ARGs abundance and detection rates used alone probably be not suitable for comprehensively judging the contribution of bacteriophage to the dissemination of antibiotic resistance.A more suitable model is the application of three indices as warning and monitoring tools for environmental ARG assessments in bacteriophages.We further closely linked the pig farm and the external receiving environment and selected the Huizhou pig farm water system as the core area.Metagenomic sequencing method was used to explore and clarify the possession characteristics of all bacteriophages carrying ARGs.The results showed that tetracycline-,macrolide-and aminoglycosamine resistant genes were the three most abundant antibiotics.To our surprise,colistin-resistant resistance gene mcr-1 was also positively detected.Further classified the ARGs according to their mechanisms of action,bacteriophages were more likely to carry major facilitator superfamily(MFS)genes,including floR,mef(A),mef(B)and erm(F)and genes encoding the ATP-binding cassette(ABC)transporter family,including the optrA,tet44,tetM,tetW,tetQ,and tetO.The most important was that they were all independent of location and environment.The results that evaluating the impact of wastewater treatment processes on bacteriophage carrying ARGs showed that aerobic processes increase the ratios value of tetW and tet44 and decreasing ratio value of β-lactam gene blaOXA-347.Insteresting,the treatment of facultative digestion(both anaerobic and aerobic microorganisms)finally brings ARGs abundance back to its original level,indicating that wastewater treatment did not significantly change the relative abundance of bacterial-or phage-associated ARGs.In all.these findings again indicate that phages not only could serve as important reservoir of ARG but also carry particular ARGs in swine feedlot wastewater,and this phenomenon is independent of the environment.Evidences have shown that prophages in bacterial host genome can provide additional functions to the host to regulate the host’s tolerance to the environment.Therefore,an indepth understanding of the distribution and diversity of prophage in the bacterial genome,the carrying characteristics of ARGs will provide insight into the mechanism of transductiontransmission resistance and provide new insights into transmission resistance.132 ETEC isolates were obtained and a total of 1105 potential prophages were identified,and distribution of the genome size showed three "overlapping" trends.The size of 14 kb account for the highest density(6.73%)of all prophage numbers.All 132 ETEC strains carrying 1206 ARGs and only 16 strains carrying 32 ARGs through prophages,with a very low detection rate of 2.65%(32/1206).Analysis of flanking sequences showed that almost all the ARGs could be grouped into two types:“blaTFM-1B" and“classic class 1 integron(IntI1)".They cooccurred with strictly conserved recombinase and transposon Tn3 family but with a difference:the "bla TEM-1B" prophages exhibited a classic Tn2 transposon structure with 100%sequence identity,whereas the "IntI1 type" co-occurred with the TnAs2 transposon with only 84%sequence identity.These results imply that ARGs might be pervasive in natural bacterial populations through transmission by transposable bacteriophages,suggesting that phages might contribute to a more rapid and stable horizontal transmission of ARGs.In general,although the specialized transduction mediated by temperate phage(in form of prophage)is the most common,the generalized transduction mediated by lytic phage also should not be ignored.Here,we targetes the lytic phages,and then focus on "transducing particle" in the specialized transduction process,and finaly explored the potential impact of lytic phages to the spread of blaCTX-M-65 and floR.A strain of T4-like lytic phage AH67C600_Q9 was successfully isolated and identified.In the study of its effect on the blaCTX-M-65 and floR transmission,it was found that under the same phage and bacterial titer,the carrying frequency of "transducing particle" to blaCTX-M-65 and floR was significantly different,suggesting that the genetic environment may affect the spread of resistant genes.NGS sequencing of phage genomes on analyzing the formation mechanism of blaCTX-M-65 and floR transmission is not a sufficient method.