Isolation And Identification Of Bovine Pathogenic Enterobacter Hormaechei And Establishment Of Experimental Animal Model

In recent years,Enterobacter hormaechei is considered to be more and more important pathogens,especially in hospitals.Enterobacter hormaechei is one of the most easily found drug-resistant bacteria.In the following up reports,Enterobacter hormaechei infection were also found in shrimp,fox and pig,mainly with clinics as uterine inflammation,fetal abortion,diarrhea and other symptoms.In this study,Enterobacter hormaechei were isolated from the samples of two calves with respiratory disease in Nanyang area;Then the experimental animal model of Enterobacter hormaechei infections in BALB/c mice was established to investigate the pathogenicity.The pathogenic bacteria were isolated and cultured from nasal swabs and lung tissue samples by conventional methods.The samples were respectively inoculated on LB solid medium,tryptone soybean agar(TSA)and eosin methylene blue medium.Each plate was repeated three times.Three passages of plate marking were carried out.Single colony was selected from each passage.Gram staining method and microscopic examination showed that the bacteria were dyed red,short rod-shaped,similar to the morphology of Enterobacter hormaechei.Secondly,15 routine biochemical test tubes were used to identify the biochemical characteristics.The results showed that Enterobacter hormaechei could decompose glucose,xylose,sorbitol,etc.the oxidase was negative,and acid and gas production were positive.O-F showed that the strain was fermentation type.The isolates could grow in anoxia and environment with aseptic paraffin oil sealed culture medium,which indicated that Enterobacter hormaechei belongs to anaerobic microorganism.The above physiological and biochemical identification results were consistent with the growth characteristics of Enterobacter hormaechei.PCR method was used with universal primers for 16S r RNA amplification.The amplicon was about 1500 bp,which was consistent with the size of the target fragment.Blast analysis of the sequencing results showed that the homology between the isolated strain and Enterobacter hormaechei on NCBI were more than 99%.The drug resistance tests of Enterobacter hormaechei to 26 kinds of drugs were detected by disk amplification method(K-B).According to the drug sensitivity tests,peptide and lincomycin antibiotics had no effect on Enterobacter hormaechei;Enterobacter hormaechei had strong resistance to tetracyclines and macrolides.It was also found that Enterobacter hormaechei was extremely sensitive to quinolones such as ciprofloxacin,enrofloxacin,norfloxacin and lomefloxacin,as well as to nitrofurantoin,compound sulfamethoxazole and trimethoprim.Therefore,these drugs should be considered as the clinical choice for the treatment of Enterobacter hormaechei infection.The isolated bacteria can cause the occurrence of respiratory diseases in cattle farms in this area,and because of the strong drug resistance of the isolated bacteria,the commonly used antibiotics is no effect,thus causing certain economic damage to cattle farms.So far,there is no report about the pathogenicity of this pathogen to ruminants,and because the target animal of the pathogen isolated in this study is cattle,which has high economic value,and it is difficult to obtain clean animals,this study intends to use BALB/c mice to establish animal pathogenic model,and establish three groups of experimental infection methods,namely nasal drip group,oral administration group and intraperitoneal injection group,the inoculum concentration was adopt to 2×10¹²CFU/m L,each mouse in the intraperitoneal injection group and the oral administration group was inoculated with 0.2 m L bacterial solution;mice in nasal drip group were inoculated with 20?L bacteria liquid.Through the three groups of experimental group mice infection model,the visible pathological changes of the experimental group lung were observed by naked eye:the volume became larger and the color became dark;The color of liver became dark;The spleen became dark red and larger;When bacteria were detected in various organs of mice in the infection group,it was found that the target band were detected in the lung,liver,spleen and kidney,but not in the blank control group.In the nasal drip group,congestion and edema of pulmonary alveolar septum vessels,cell proliferation,obvious thickening of alveolar wall,atrophy of some alveolar cavities and infiltration of inflammatory cells were observed;There were hemorrhage and inflammatory cell infiltration in lung alveoli in intraperitoneal injection group and perfusion group.There were inflammatory cells infiltration in hepatic sinusoids of mice in perfusion group and intraperitoneal injection group;In the nasal drip group,the hepatocytes became larger and irregular,and the hepatic lobular cells became larger.Acute splenitis,splenic congestion,bleeding,necrosis of small blood vessel wall,and the whole spleen tissue were almost occupied by red blood cells in the mice in the oral administration group;There was bleeding in spleen tissue in nasal drip group and intraperitoneal injection group.Therefore,it is concluded that the mouse model of Enterobacter hormaechei infection is preliminarily established.