| Since the winter of 2010, there had a large range of pig diarrhea epidemic in many provinces in China, also included Anhui. Various days of old pigs can be infected. The clinical symptoms of suckling pig is the most serious, the characteristics included acute onset, high morbidity and mortality. According to the continuous monitoring and survey results of swine nationwide, the main pathogens that caused diarrhea were porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus, in addition with the presence of porcine rotavirus, Boka virus, porcine circovirus, Salmonella, Escherichia coli and other pathogens that related. Although the pathogenic bacteria were not caused by the main culprits for the current pig diarrhea severity, but bacterial pathogens can aggravate diarrhea, increase the mortality of diarrhea piglets. Salmonella and Escherichia coli were the important bacterial pathogens that caused diarrhea in piglets. With the irrational use of antibiotics in veterinary clinic, which caused the increasing number of drug-resistant strain of Salmonella and Escherichia coli, the effect of medication was not obvious in clinic. The formation of biofilm for bacteria is the result of bacterial enhanced the resistance to adverse environmental, such as the insensitivity to antibiotics, virulence change. In view of the pig diarrhea epidemic in Anhui, in order to providing scientific basis for effective prevention and control measures, two aspects including viral pathogens and bacterial pathogens were investigated in this study. Following works had been carried out with Salmonella and Escherichia coli:(1) Isolation and identification of Salmonella and pathogenic Escherichia coli were carried out in 601 samples (including feces and intestinal contents) by conventional method combined with PCR. The serotypes of isolated bacterial were identified by slide agglutination test.69 strains of Salmonella and 129 strains pathogenic Escherichia coli were isolated, the detection rate of Salmonella and pathogenic Escherichia coli were 11.5% and 21.5%. The detection rate of Salmonella in feces and intestinal contents were 13.1% and 8.8%, for pathogenic Escherichia coli were 7.5% and 44.7%, respectively. Salmonella derby was the dominant serotype of Salmonella, the dominant serotype of pathogenic Escherichia coli were O:55,O:26,O:125,O:128,O:6,O:111, O:15 and O:7,O:104.(2) Kirby-Bauer agar diffusion method was used to test antibiotic sensitivity (23 kinds of antibiotics) in isolated Salmonella and pathogenic Escherichia coli. The results showed that, tetracycline and doxycycline resistance rate of 59 strains Salmonella was high, which accounted for 71.1% and 69.6%, respectively. All of those Salmonella sensitive to piperacillin/tazobactam, aztreonam, cefepime, cefoperazone, ceftazidime and cefatriaxone.10.1% (7/69) of those isolated Salmonella can resistant to 7-10 kinds of antibiotics.4.3% (3/69) of those isolated Salmonella can resistant to 11 kinds of antibiotics. Tetracycline- doxycycline- amoxicillin- ampicillin- trimethoprim-sulfamethoxazole- florfenicol was the common multidrug- resistant spectrum. Tetracycline, ampicillin, amoxicillin, doxycycline, sulfamethoxazole and trimethoprim resistance rate of 129 strains isolated pathogenic Escherichia coli was 92.2%,92.2%, 93%,90.7%,89.1% and 87.6%, respectively. Those bacteria were sensitive to imipenem, piperacillin/tazobactam, nitrofurantoin and cefepime, the susceptibility rates were 98.4%,88.4%,88.4% and 86.8%, respectively.31.0%(40/129) of those isolated pathogenic Escherichia coli can resistant to 2-10 kinds of antibiotics.66.7%(86/129) of those isolated pathogenic Escherichia coli can resistant to 21 kinds of antibiotics. Amoxicillin- ampicillin- tetracycline- doxycycline -compound sulfamethoxazole-trimethoprim- gentamicin and ciprofloxacin was the common multidrug-resistant spectrum. The results indicate that, the extent of the drug resistance was gotten to be serious and the multidrug resistant spectrum was gotten to be width. There were 1 strain Salmonella and 38 strains pathogenic Escherichia coli belong to ESBLs producing strains, which were identificated by phenotypic confirmatory test. The results show that, the drug resistant of Salmonella and Escherichia coli were serious, and have a width multi drug resistant spectrum.(3) 96 well plates were used to detection the ability of biofilm formation in vitro for Salmonella and pathogenic Escherichia coli. The ability of biofilm formation were divided in to strong, middle, weak and negative. The results showed that, the rate of biofilm formation in vitro of Salmonella was 92.8%(64/69), bacteria that produce a large number of biofilm, a small amount of biofilm and none account for 14.5% (10/69),78.3%(54/69) and 7.2%(5/69), respectively. The rate of biofilm formation in vitro of pathogenic Escherichia coli was 96.1%(124/129), bacteria that produce a large number of biofilm, a moderate amount of biofilm, a small amount of biofilm and none account for 14.7%(19/129),20.9%(27/129),60.5%(78/129) and 3.9%(5/129), respectively. The results indicated that, both Salmonella and Escherichia coli have a common ability of formation biofilm, and a small amount of biofilm formation was the major.(4) 3 strains Salmonella and 2 strain pathogenic E. coli strains that have strong biofilm formation ability were selected. MIC of Salmonella and Escherichia coli under planktonic and biofilm state, to ciprofloxacin, florfenicol, doxycycline, Cefradine was detected by microplate assay. The ability of biofilm forming in different time and corresponding MIC were detected. The results showed that, compared with planktonic bacteria, the MIC of ciprofloxacin increase 2-16 times than that of biofilm state; and 8-16 times of florfenicol. Bacteria in different time points have different biofilm forming abilitiy. The MIC reached the maximum when bacteria owned the strongest ability of biofilm forming. The results showed that, after the biofilm formatted the MIC of the bacteria will be increased.(5) 8 strains Salmonella (including 3 strains of strong,3 strains of weak,2 strains of negative) and 8 strain pathogenic Escherichia coli (including 2 strains of strong,2 strains of middle,2 strains of weak,2 strains of negative) were selected.50% lethal dose (LD50) of bacteria, which own different ability of biofilm forming for Kunming mice was determined by Karber’s method. The result was analysised by SAS. The result showed that, the LD50 of strong biofilm formation Salmonella were 2.44 x 109, 8.75×106,2.80×107; weak biofilm formation Salmonella were 1.19×107,1.85× 107,4.77 × 107; negative biofilm formation Salmonella were 1.50×107,3.08 × 107; the LD50 of strong biofilm formation pathogenic Escherichia coli were 9.59 × 108, 6.87 × 108; in middle biofilm formation were 2.30 × 108,4.64 × 108; weak biofilm formation were 3.34 ×107,7.32 × 107; negative biofilm formation were 1.20 × 107, 2.75 × 107; the bacterial virulence distinguished between strong, weak and negative biofilm formation Salmonella were significant (P<0.05); the bacterial virulence distinguished between strong, weak and negative biofilm formation pathogenic Escherichia coli were significant (P<0.05). The results showed that, bacteria which have a better biofilm formation ability well owned a weaker the virulence. |
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