| Proteus mirabilis(PM) is an opportunistic pathogen that can infect humans and animals, which has wide host ranges, such as goat, pig, chicken, calf, fox, macaque, mink, pigeon and fish. PM can cause human food poisoning and urethral infection, however, it cause different diseases when infected other animals, such as diarrhea, wound infection, otitis media, peritonitis and meningitis. Proteus mirabilis is facultative anaerobic bacteria, and can also be breeding under the condition of low temperature. The bacterium is extremely easy to cause pollution of animal food and lead to human food poisoning. Therefore, it was very important for PM to public health. But until the end of the 1970 s, researchers were gradually begun to concern about the pathogenicity of PM and had different levels of study from aspects of etiology, pathogenesis, etc. In recent years of China, the reports of the human and animals infected by PM showed an ascendant trend which caused great harm to the human health and safety of the livestock and poultry breeding industry.Goat diarrhea is a common disease and frequently occurring disease, which is caused by many factors, mainly reasons of the bacterial, viral and parasitic infection. In our laboratory, we previously investigated the etiology detection of diarrheal goat in Chengdu, and found the PM has a close relationship with the goat diarrhea. However, there is less date of etiology of PM at present. To further understand the biological characteristics, therefore, this study was to isolate, indentify the PM from goats and to detect the some virulence genes. The results obtained are as follows. 1 Isolation and identification of PM from goats 1.1Cultivate characteristic dyeing and morphologyWe collected 140 diarrhea and healthy goat fecal swabs respectively from seven goat yards in 5 regions of Sichuan Province(Qingbaijiang, Jianyang and Xichang, Panzhihua, Beichuan). The samples were inoculated into BPW, cultured 12 h for enrichment in advance under 37?, then switched it into SC enrichment medium cultured 12 h under 37?. Finally, the samples were switched it into SS agar medium and the LB solid medium cultured 12 h under 37?. On the SS agar medium, it can be seen that smooth, flat, transparent or translucent circular colonies with black center. In LB plate, the typical "migration" growth phenomenon was observed, and the bacteria are spread to growth. The suspected colonies were selected to Gram staining under a microscope, and were observed to be gram-negative bacilli with medium-sized size, no capsule and no spore forming. 1.2 Identified by PCRWe selected the suspected colonies to inoculated into LB medium for 12 h under 37?. By the phenol chloroform method, the total DNA of PM was extracted. The specific fragment of 225 bp was amplified by using the primers of the target strain of the strain of the strain of the exotic strain of the urease regulator(ure R). The 5 positive amplification products were detected by random selection and then were connected the T vector. The sequencing results confirmed as the specific fragment of PM. There were a total of 41 strains of PM detected from goat feces, 31 isolations(31/140) of which were from the goat diarrheal fecal samples and 10 strains(10/140) from healthy fecal samples. The detection rate of PM in diarrhea samples was significantly higher than that in healthy fecal samples(p=0.02) by SPPSS software, which indicated that PM was closely related to the diarrhea of goats. 2 Detection of drug resistance of PM from goatsUsing K-B method, we detected the resistance of 20 strains PM of goat to 6 major categories( penicillins, sulfonamides, fluoroquinolones, aminoglycosides, cephalosporins, beta lactam etc) of a total of 15 kinds commonly used antimicrobial drugs(ceftazidime, ceftriaxone, amikacin, streptomycin, new debbi Ming, norfloxacin, nalidixic acid, cefepime, cephalothin, amoxicillin, ampicillin, gentamicin, card that mildew prime, levofloxacin, ciprofloxacin and other 15 kinds of antibacterial drug). The results showed that drug resistance rates were 30%?35%?15%?100%?100%?85%?100%?40%?90%?100%?100%?100%?95%?100%?100% to the above drugs, respectively. Resistance spectrums were 9-13 kinds of antimicrobial drugs, which suggested that the goat PM resistance were very serious. In addition, the significant differences in PM resistance spectrum and drug resistance rate existed in the different regions, for example, the generally resistance to pencillins appeared to the PM from the Panzhihua region, which the generally resistance to the beta lactam presented in the PM from the Jianyang region. The phenomenon was possibly due to relation to the drug habit of goat in different regions. Therefore, the perdic-drug sensitivity test could provide reliable guidance for prevention and treatment of PM infection in goats. 3 Pathogenicity of PM from goatsWe randomly selected 5 PM strains(numbered PM1~PM5) from goats diarrheal feces. The five strains were inoculated in the LB broth, respectively. The bacterial concentration were adjusted to 5×108CFU/m L by OD600 value. The 2-month-old SPF female kunming mice was intraperitoneal inject with 1×108CFU/m L(0.2ml each), and the sterile saline water was as control. The each strain was inoculated with 5 mice. The results showed that the controls group exhibited a healthy status in the whole assay, however, the infection group was involved in depression, unresponsive, closed eyes and curled up, get together and trembling and other symptoms at about 8h. The mice begun to die after 10 h infection and all died within 24 h. In pathological anatomy, the death mice could be seen obvious filling and expansion and full of yellow atherosclerosis dilute liquid contents in intestinal tract. There appeared different degrees of swelling in spleen. No significant pathological change was observed in the heart, liver and other organs. Therefore, the above results indicated that the PM has a strong pathogenecity to mice. 4 Detection of main virulence factors of PM from goatIt was important to study and identify the virulence factors during bacterial infection and pathogenesis. In this study, we selected 8 virulence factors, including the urec gene encoded by the functional urease subunit, the zap A gene encoded by the functional metalloproteinase subunit, the mrp A gene encoded by main structure of MPR fimbrial, the uca A gene encoded by the functional subunit of urethral epithelial adhesion, the rsb A gene encoded by the functional subunit to regulation of "fog tendril" migration, the pmf A gene encoded by the functional subunit of main structure of Pmf fimbrial and the atf A gene encoded by functional subunit of optimum temperature of pilus. According to the reported method, we synthesized the 8 couples of primers of the above virulence genes and detected the prevalence rate of 8 virulence genes in 10 PM isolates from healthy fecal samples and 20 isolates from diarrhea fecal samples. The results exhibited that the carrying rate of ure C gene in healthy and diarrheal samples was 60%(6/10) and 80%(16/20), respectively; the Zap A gene was 50%(5/10) and 85%(17/20), respectively. The significant difference of carrying rates was observed in both genes between healthy and diarrheal goats by statistical analysis of SPSS software(p=0.04 and 0.02). however, there was no significant difference in detection rate of mrp A?uca A?rsb A?pmf A?atf A?atf C genes between healthy and diarrheal goats. Therefore, the PM carrying the urec and Zap A genes might be associated with the cause of diarrhea in goats. |
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