Study On Biological Characteristics Of Serratia Marcescens From Cattle And Establishment Of PCR Method

Serratia marcescens is an important conditioned pathogen of humans and animals.In addition to causing lung,urethra and blood infections,it can cause infection of insects,reptiles,fish,poultry and mammals.In this study,the biological characteristics,drug resistance and pathogenicity of 5 S.marcescens from blood samples of beef cattle with respiratory diseases(ZZCCN01)and milk samples of dairy cows with mastitis(ZZCNJ01,ZZCNJ02,ZZCNJ03,ZZCNJ04)were studied,and constructed a PCR detection method for S.marcescens,aiming to provide a reference for the clinical detection and prevention.1.The characteristics and 16S r DNA and gyr B sequence analysis of S.marcescens from cattleColony morphology analysis found that ZZCCN01 does not produce pigments,and white colonies on ordinary nutrient agar,blood agar,Mac Conkey and SS medium,lilac colonies on Eosin Methylene blue medium;ZZCNJ01 does not produce pigment,the colony morphology was the same as ZZCCN01;ZZCNJ02,ZZCNJ03 and ZZCNJ04 can produce pigment,the colony on ordinary nutrient agar was light red in the center and milky white at the edge at 37?;when cultured at 28?,there were orange-yellow colonies on ordinary nutrient agar,golden yellow colonies on blood agar,bright red colonies on Mac Conkey medium,violet black colonies with slight metallic luster on Eosin Methylene blue medium,and pink colonies on SS medium.Biochemical test results showed that ZZCCN01 galactose and sorbitol were negative,ZZCNJ01 galactose and sorbitol were positive;ZZCNJ02,ZZCNJ03 and ZZCNJ04 galactose were negative,sorbitol was positive.The phylogenetic tree of 16S r DNA and gyr B sequences showed100%homology among ZZCNJ02,ZZCNJ03 and ZZCNJ04,and they were respectively in three different branches with ZZCCN01 and ZZCNJ01.2.Drug resistance of S.marcescens and the detection of drug resistance-associated genesThe resistance of 5 strains of S.marcescens to 27 common antibiotics was studied by disc agar diffusion method.According to the drug sensitivity results of imipenem,doxycycline,streptomycin,ZZCNJ02,ZZCNJ03 and ZZCNJ04 were consistent,and were different from ZZCCN01 and ZZCNJ01.Simultaneously,5 S.marcescens from cattle showed resistance or decreased susceptibility to?-lactam drugs such as cefazolin,amoxicillin,ampicillin,oxacillin,meropenem,imipenem.Among the?-lactam resistance gene detection,only the result of OXA-58 gene was positive,and the remaining genes such as KPC,SME,VIM,IMI,GES,CTX-M,NDM-1,OXA-23,OXA-48,acr A,acr B,Mex B and Mex R were all negative.3.Study on pathogenicity of S.marcescens from cattle on miceBy artificially infecting mice,the LD₅₀of 5 S.marcescens on mice was determined,and the pathogenicity was explored from four aspects:clinical symptoms,pathological changes,bacteriological examination and histopathology.The mice infected by intraperitoneal injection showed depression,shortness of breath and other clinical symptoms.Dissection of dead mice revealed different degrees of congestion in the heart,liver,spleen,and lung,and the corresponding strains were isolated from the brain,heart,liver,spleen,lung,kidney,and blood,indicating that the death of mice was related to bacteremia.Histopathological analysis showed that the spleen was slightly congested,the splenic nodules were dilated,and the lesions of other organs were slight.The LD₅₀of ZZCCN01,ZZCNJ01,ZZCNJ02,ZZCNJ03 and ZZCNJ04 were respectively 2.5×10⁸CFU/ml,1.25×10⁸CFU/ml,2.483×10⁷CFU/ml,4.416×10⁷CFU/ml and 8.831×10⁷CFU/ml.4.The establishment of PCR detection method for S.marcescensSelected a pair of specific primers based on the highly conserved Lux S gene(Lux S-F3:5'-AAGGACGCGAGACCACTC-3',Lux S-R3:5'-TCGCCGTCCAACACT TCC-3').The total volume of the reaction system is 25?L,2×Taq DNA Master Mix12.5?L,upstream and downstream primers 1?L(10?mol/L),DNA 2?L,dd H2O 8.5?L.The specific bands of 640bp can be obtained at 95?5min;95?40s,63?30s,72?40s,35 cycles,and 72?for 10min.The DNA detection limit is 1.13×10^-3?g/m L.The above PCR detection method was used to detect 80 oral and nasal swabs of beef cattle with respiratory diseases and 48 milk samples of cows with mastitis.Four milk samples were positive,and the coincidence rate with the results of traditional bacteria isolation and identification was 100%.Conclusion:1)During the evolution of S.marcescens,the characteristics of culture,biochemical and drug resistance,and pathogenicity were also changing adaptively.Whether the occurrence of dairy cow mastitis and beef cattle bacteremia is related to this,and whether S.marcescens from beef cattle will become a potential pathogens for various of animals(including humans),which is to be studied further.2)The specific PCR detection method of S.marcescens was successfully established,which can be applied to the rapid detection in clinical samples.