Analysis Of Bactericidal Effect Of Five Disinfectants On Common Bacteria In Duck Farm

Duck farming occupies an important position in my country's agricultural economic structure.With the development of the breeding industry,the traditional duck farming model in rural free-range farming has gradually changed to intensive farming.Due to the high density of intensive duck farming,the environmental sanitation of large-scale duck farms is not up to standard,the water pollution cannot be separated,the manure is degraded by natural precipitation,the dampness of the duck house site and the poor ventilation are easy to cause bacterial diseases.Farms bring huge economic losses.In order to prevent and control bacterial diseases,duck farms usually use a large number of disinfectants for environmental disinfection.Choosing a single disinfectant type and reusing it will not only cause environmental pollution,but also cause bacteria to be highly susceptible to drug resistance,thereby reducing the disinfectant's Sterilization effect.In this study,the total number of colonies in the environment(air,feces,litter and drinking water)of a duck farm in Guizhou Province was measured,and the 16S r DNA high-throughput sequencing method was used to analyze the microbial diversity and abundance of feces,litter and drinking water.The structure and composition of the environmental microbes in the duck farm;choose five disinfectants on the market(povidone iodine,benzalkonium bromide,benzalkonium chloride,compound glutaraldehyde,glutaraldehyde decyl bromide),Measure the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of five common bacteria(Escherichiacoli,Riemerella anatipestifer,Pasteurella multocida,Salmonella enteriditis,Staphylococcus)in the duck farm;and design 15 pairs of disinfection The specific primers of the agent resistance-related genes are amplified by PCR to analyze the relevant gene fragments,analyze the situation of the five pathogenic bacteria carrying the disinfectant resistance gene,and master the resistance of the bacteria to the disinfectant.Five disinfectants were prepared to conduct on-site disinfection tests on the duck farm,and the actual disinfection effects of these five disinfectants before and after disinfection on the duck farm were observed.Formulate disinfection procedures based on the above research results,and provide references for the implementation of disinfection measures in duck farms.1.Detection and analysis of environmental microorganisms in a feeding duck farm in Guizhou Province:In order to understand the diversity and abundance of microorganisms in duck farm,air,feces,pad and drinking water of duck farm were collected for the total bacterial colony determination.Specific primers were designed for the V4?V5 hypervariable region of the16Sr DNA gene of bacteria,and PCR amplification and sequencing were performed for the collected feces,pad and drinking water samples.Results:The total number of air colony was3.79×10~4CFU/m~3,feces colony was 2.67×10~7CFU/g,pad colony was 3.7×10~6CFU/g,and drinking water colony was 12 CFU/m L.Based on the high-throughput sequencing results of16S r DNA,a total of 406145 effective sequences and 336727 optimized sequences were obtained from 9 samples of duck feces,pad and drinking water.The average sequencing reading length was between 319?529 bp.At 97%similarity level,the total number of effective OTUs was 4375,and the total number was 24.In the community composition,41phyla,44 classes,82 orders,137 families,311 genera and 250 species were identified.At the bacterial level,the potential animal pathogenic bacteria such as Reebacter,Escherichia,Salmonella,streptococcus and Staphylococcus were detected.2.The germicidal efficacy of five disinfectants against common bacteria in duck farm and the gene detection and analysis of disinfectant resistance:Choose five kinds of disinfectant(povidone iodine,benzalkonium bromide,benzalkonium chloride,compound glutaraldehyde,glutaraldehyde decyl bromide),the five kinds of common bacteria isolated from the duck farm(Escherichiacoli,Riemerella anatipestifer,Pasteurella multocida,Salmonella enteriditis,Staphylococcus)for the minimum inhibition concentration(MIC)and minimum bactericidal concentration(MBC)determination,preparation of the five kinds of disinfectant and minimum bactericidal concentration of Escherichia coli suspension quantitative germicidal test,observe the five kinds of disinfectant effect on Escherichia coli sterilization the shortest time.Specific primers for 15 disinfectant resistance genes(ade G,ade J,fab I,amv A,ade T1,ade T2,abe D,abe M,ade B,car O,qac E?1,qac A/B,qac C,qac G,qac J)were designed and analyzed by PCR amplification for carrying disinfectant resistance genes in these 5 pathogens.Results:The MICs/MBCs of povidone iodine was 1:4 for Escherichia coli,Salmonella and Staphylococcus,and 1:8 for Riemerella anatipestifer and Pasteurella multocida.The MICs/MBCs of benzalkonium bromide against these 5 pathogens were 1:200 and 1:25 respectively.The MICs/MBCs of benzyl trimethylammonium chloride against these five pathogens were 1:192 and 1:960 respectively.The MICs/MBCs of compound glutaraldehyde against these 5 pathogens were 1:1200 and1:600 respectively.The MICs/MBCs of glutaraldehyde decamethylammonium bromide against these five pathogens were 1:1000 and 1:500 respectively.Compound glutaraldehyde can kill Escherichia coli with MBC for 1 min.The killing rate of Escherichia coli reached100%after 3min under the action of MBC.The killing rate of benzalmonium bromide on Escherichia coli reached 100%after 5 min of MBC.Bacterial disinfectant gene qac E?1 has been detected in 1 strain of Escherichia coli,1 strain of Staphylococcus and 1 strain of Riemerella anatipestifer,with a positive rate of 30%(3/10).The other 14 disinfectant genes have not been detected.3.Five kinds of disinfectant were used to disinfect the duck farm:Respectively formulate benzalkonium bromide,minimum bactericidal concentration on charting the top three ammonium chloride,compound glutaraldehyde and bromide 4 kinds of disinfectant glutaraldehyde decyl armour,fog line,spraying disinfection method for stabling duck farm leak air,floor,chute,egg box and truck wheels on-site disinfect,formulate povidone iodine,minimum bactericidal concentration of duck feet skin daub,determine the total number of colonies before and after disinfection,5 kinds of disinfectant for the death of bacteria rate calculation.Results:Glutaraldehyde decamethylammonium bromide had the highest extinction rate(34.85%).The bacterial death rate of compound glutaraldehyde was 96.78%.Glutaraldehyde decamethylammonium bromide had the highest extinction rate of 74.35%.The bacterial extinction rate of compound glutaraldehyde was 81.31%.The bacterial death rate of compound glutaraldehyde was 87.5%.The extinction rate of povidone iodine on duck foot skin bacteria was 72.45%.Conclusion:1.The number of air bacterial colonies in the duck farm was 3.79×10~4CFU/m~3,exceeding NY/T388-1999 environmental quality standard limit(25000 CFU/m~3).The total number of bacterial colonies in drinking water is 12 CFU/m L,which conforms to GB5749-2006 sanitary standard of drinking water(100 CFU/m L).The total number of fecal colonies was 2.67×10~7CFU/g,exceeding the national standard for fecal treatment(1×10~6CFU/g).The total number of pad colonies was 3.7×10~6CFU/g,which exceeded the national standard of clean soil value(1×10~4CFU/g).2.The diversity of bacterial community structure in duck farm(feces,bedding and drinking water)was successfully detected based on 16S r DNA high-throughput sequencing,and comprehensive and in-depth bacterial community information was obtained.The total number of effective sequences obtained from the 9 samples was 406145,and the total number of optimized sequences was 336727.The average sequencing read length was between319?529 bp.At 97%similarity level,the number of effective OTUs produced was 4375,covering bacterial flora of 41 phylums,44 classes,82 orders,137 families,311 genera and250 species.3.At a dilution ratio of 1:4,Povidone iodine solution had a good bactericidal effect for at least 3min,At the dilution ratio of 1:25,benzalkonium bromide solution showed good bactericidal effect at least 5 min.At the dilution ratio of 1:960,benzyl trimethylammonium chloride solution showed good bactericidal effect at least 3 min.At the dilution ratio of 1:600,the compound glutaraldehyde solution showed better bactericidal effect at least 1 min.At the dilution ratio of 1:500,glutaraldehyde decamethylammonium bromide solution showed good bactericidal effect at least 3 min.4.The qac E?1 disinfectant resistance gene was detected in Escherichia coli,Staphylococcus and Riemerella anatipestifer,and the positive rate was 30%(3/10).5.In the field disinfection test of duck farms by preparing five disinfectants with minimum sterilization concentration,compound glutaraldehyde has the highest rate of bacterial death on leaky floor,egg frame,and transport vehicle wheels,and glutaraldehyde decamyl bromide The rate of bacteria in the air and feed trough of the duckhouse is the highest,and povidone-iodine has a good killing effect on the bacteria at the foot of the duck.