| At present,there is little systematic analysis on antibiotic resistance of different poultry source of Salmonella and Carbapenem-resistant Escherichia coli（CREC）in our country.Therefore,cloacaal swabs from different poultry（broilers,laying hens,waterfowl）in Jiaodong area were collected in this study.Isolation and identification,drug resistance analysis,molecular typing and whole genome sequencing analysis were conducted to provide evidence for effective prevention and control of the spread of drug-resistant bacteria,and to explore the feasibility of combined application of bacteriophage and antibiotics to slow down bacterial resistance.In this study,Salmonella and Escherichia coli from different poultry sources were investigated by conventional detection methods,and the differential sites and genetic relationships among the strains were analyzed by whole genome sequencing technology combined with bioinformatics.The combination of bacteriophage and antibiotics was used to screen the combination with synergistic bacteriostatic effect in order to reduce the use of antibiotics and improve the bacteriostatic effect,so as to slow down the generation of multi-resistant salmonella bacteria.Specific research results are as follows:1.Main test results of Salmonella:（1）There was a certain difference in the isolation rate of Salmonella from different poultry in Jiaodong area.A total of 194 Salmonella strains（6.16%）were isolated from 3150 cloacal swabs of poultry.The isolation rates of Salmonella from broilers,laying hens and waterfowl were 5.04%,1.15%and 14.36%,respectively.（2）A total of 11 serotypes were identified from 194 Salmonella strains,mainly S.Enteritidis（38.14%）and S.Kentucky（21.65%）.There were more Salmonella serotypes in broilers and waterfowl（both 7 species）than in laying hens（3 species）,but S.Enteritidis was the main serotype in all of them.（3）Resistance analysis showed that:The 110 strains of Salmonella contained 43 types of drug resistance spectrum,and the situation of multiple drug resistance was not optimistic.The rate of multiple drug resistance was 73.64%,mainly 7 resistant strains,and no 10 resistant strains were isolated.The resistance to ampicillin,sulfamisoxazol and tetracycline was relatively serious（58.18%-77.27%）,and the meropenem was sensitive（100%）.Waterfowl strain（79.07%）had the most severe multi-drug resistance,followed by broilers（73.21%）and laying hens（54.55%）.There were significant differences in drug resistance to amoxicillin/clavulanate（P=0.031）and tetracycline（P=0.007）.ESBLs-producing strains are circulating and spreading,with a detection rate of 44.55%,and their multiple drug resistance（100%）is more serious than that of non-ESBLs-producing strains（52.46%）.The detection rates of bla CTX-M,bla TEM and bla OXA were 70.00%,51.82%and 26.36%,respectively,and no carbapenem resistance genes were detected in 110 Salmonella strains.（4）Multi-site sequence typing combined with serotype results showed that the isoserotype of Salmonella was mainly transmitted by cloning.The 49 strains of ESBLS-producing Salmonella contained seven ST types,mainly ST198（34.69%）and ST17（30.61%）.There was a strong correlation between the serotypes and ST types,with one ST type corresponding to one serotype.（5）Whole genome sequencing results showed that a total of 39 drug-resistant genes in 11categories were detected in 23 strains of ESBLS-producing Salmonella,each strain carried 2～21drug-resistant genes,and the detection rate of bla OXA-455,flo R,tet（A）,ARR-3 and other drug-resistant genes was relatively high.7 species of plasmid replicon were detected,with Inc HI2A and Inc HI2being the most prevalent.SNP analysis showed that 23 strains of Salmonella could be divided into three branches,and the strains with the same serotype and ST type gathered under the same branch,and the genetic relationship was close.2.Main test results of CREC:（1）There was a certain difference in the CREC isolation rate of different poultry in Jiaodong area.A total of 364 CREC strains（32.18%）were isolated from 1131 cloacal swabs.The isolation rates of broiler,laying hens and waterfowl were 55.56%,3.85%and 34.67%,respectively.（2）Drug resistance analysis showed that 104 strains of CREC contained a total of 40 types of drug resistance spectrum,with a multiple resistance rate of 100%,83.65%of strains with 8resistance or above,and the resistance rate to 13 other drugs was above 65.00%except for the low resistance rate to colistin（35.58%）.The drug resistance of broiler strains was the most serious,with10 resistant strains accounting for 39.66%.There were significant differences in drug resistance to flufenicol,cotrimoxazole and colistin in three types of poultry（P<0.01）.Only one carbapenem resistance gene,bla NDM（100%）,was detected in 104 CREC,and the detection rates of bla CTX-M,bla TEM and bla OXA in ESBLs resistance genes were 100%,79.81%and 82.70%,respectively.（3）The results of multi-locus sequence typing showed that 46 ST types were detected in 104NDM-CREC strains,and the dominant ST type was ST156（12/11.54%）.There was no obvious correlation between ST types and poultry species,most ST types were unique to certain types of poultry,and only ST746,ST156,ST10 and ST101 were shared by the three types of poultry.（4）Whole genome sequencing analysis showed that 52 drug-resistant genes of 12 classes were detected in 45 NDM-CREC strains,each strain carried 8-23 drug-resistant genes,and 4 bla NDMvariants were detected,dominated by Bland M-5（77.78%）.33 species of plasmid replicon were detected,Inc FIB,Inc HI2A and Inc HI2 were the most prevalent.SNP analysis showed that 45 strains of NDM-CREC were divided into four branches,and the difference of the same ST locus was smaller in the same poultry,and the genetic relationship was more similar.3.Main phage test results:（1）A total of 133（100%）bacteriophages were isolated using S.Enteritidis as host bacteria,and 43 bacteriophages were purified.（2）The phage SL-1 was observed by electron microscopy to belong to the order caudophages and the family Caudophages.Only S.Enteritidis had a good cracking ability,with a cracking rate of 55%,while other serotypes had weak cracking ability or no cracking ability.The optimal number of infection was 0.01,and the incubation period was within 10 min.The titer was stable when the temperature was 4～50℃and p H was 4～11.It can be seen that the phage cleavage spectrum is narrow,the incubation period is short,and a large number of progeny phages can be released when a small dose of phage infects bacteria.However,the activity is easily affected under high temperature and extremely acidic and alkaline conditions.（3）Within 20 h of monitoring,the bacteriophage was completely inhibited when combined with 1/2MIC ceftiofurr,and the synergistic effect was the most significant.However,when combined with doxycycline and ampicillin,the synergistic effect was only within 1/2MIC,8h.In conclusion,in this experiment,the isolation rate and drug resistance of Salmonella and carbapenem-resistant Escherichia coli in Jiaodong area showed certain differences among different poultry,and multiple drug resistance was widespread in the three types of poultry,especially showing universal resistance to commonly used antibiotics.Salmonella ST type has strong correlation with serotype and is mainly transmitted by cloning.The ST type of CREC showed the characteristics of"small aggregation and large dispersion",and the transmission among poultry was mainly non-clonal.Strains with the same ST type are more closely related.In terms of the mitigation of drug resistance,it was found that the bactericidal effect of the combination of some phages and biogens was better than that of antibiotics and phages alone,and the combination effect was related to the type and concentration of antibiotics,which provided a new idea for the treatment of bacterial diseases. |
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