Identification Of Brucella Isolates And The Molecular Resistance Mechanism From JiLin Regions

Brucellosis is a zoonotic disease caused by Brucella spp, which is regarded as one of the B communicable diseases according to the People's Republic of Communicable Diseases Prevention Law. Brucellosis has a wide popularity around the world, especially in developing countries. The persistent worldwide prevalence of brucellosis has been a serious threat to human health and has caused serious public health concerns in the global health problem. There are 500⁶00 million patients around the world. New cases came up to about 50 million annually and the economic losses were up to 30 billion dollars in U.S. Early diagnosis is important means to control brucellosis outbreak. Currently, the detection of this zoonosis is based on microbiological and serological laboratory tests. The pathogen can be detected in blood cultures, however, the sensitivity of this technique is low, and a long incubation period is necessary and represents a great risk of infection for laboratory technicians. The serological laboratory tests are unsatisfactory in early disease due to low sensitivity. Because of high safety, high sensitivity and specificity, molecular biology diagnosis of brucellosis has been a research focus, but it has not been a real clinical application due to high cost and techniques. It is imperative to find a diagnostic technique which has strong sensitivity, high specificity, simple and low cost to substitute or complement diagnostic methods which were being used.This study wanted to isolate Brucella from clinical blood specimens and analyzed the major epidemic strains of JiLin regions. Fluorescence quantitative PCR were established to detect the Brucella spp and to differentiate B. melitensis, B. abortus and B. suis by means of amplification specific gene fragment. The methods were expected to become a new means of detection of Brucella because of high sensitivity and specificity which were compared with the isolation and conventional PCR. This study also analyzed the susceptibility of Brucella which were isolated from clinical specimens and designed induce experiment to explore the resistance mechanism of quinolone.1. Isolation and Culture of Brucella isolates from JiLin regionsLaboratory tests were required to diagnose brucellosis since the symptoms were nonspecific and often atypical. Cultures were considered the gold standard in the laboratory diagnosis of brucellosis. In this study, first blood culture was used two-phase blood culture bottles. Pure culture of Brucella was used L-B agar medium, Brandt medium and liver infusion medium. 14 strains Brucella were isolated from 106 collected blood samples of suspected patients. The culture positive rate was 13.21%. Incubation time were from 5d¹7d. Brucella were differentiated by biochemical reactions, dye sensitivity test, serum agglutination test, CO₂ requirement test and H₂S production test. 7 stains were B. melitensis 3 biovar, 3 stains were B. melitensis 1 biovar, 2 stains were B. abortus 3 biovar, 2 stains were B. suis 1 biovar. B. melitensis 3 biovar were dominant isolates from clinical blood samples. This was consistent with isolates which were isolated historically in JiLin regions.2. Identification of Brucella spp. with fluorescence quantitative PCRIn this study, Quantitative PCR of SYBR Greenâ… were established to detect Brucella spp. Quantitative methods were absolute quantification which were based on Ct value. The amount of template was based on standard curve. Primers were designed according to IS711 gene. The concentration of primers and templates were optimized by matrix. The optimal amount of primer was 0.4μl, template volume was 2.0μl. The slope of standard curve was -3.615, the correlation coefficient was -0.99, amplification efficiency was 90%. Brucella spp showed positive result while the other strains and the negative control showed negative result. Melting curve had demonstrated only a single peak. The reaction system did not form primer dimers and had no nonspecific amplification. CV% were less than 5%. This indicated that the detection system was stable in addition to good repeatability.The sensitivity of fluorescence quantitative PCR was evaluated by counting the number of bacteria. The copy numbers of target gene were 3.826×10¹ copies /μl, Ct value were 33.21; in simulated blood samples, the copy number were 3.523×10¹ copies /μl, Ct values were 35.87. 36 clinical blood specimens were amplified positive. The positive rate was 33.96%.Established quantitative PCR methods could quickly identify the Brucella spp. The reaction process was only 25 minutes. The methods had achieved the expected results with high specificity, sensitivity and reproducible in detection of the simulated samples and clinical specimens. In the future, we should optimize the method of extraction of bacterial DNA in blood samples to make more effective in clinical application.3. Identification of Brucella species with fluorescence quantitative PCRB. melitensis, B. abortus and B. suis are the most important Brucella of public health. Three independent quantitative PCR were established to differentiate Brucella species. The slope of standard curve of B. melitensis, B. abortus and B.suis were -3.241, -3.447 and -4.608 respectively. Amplification efficiency of B. melitensis, B. abortus came up to 102% and 94%, while amplification efficiency of B. suis were low with 66%. The relationship between the Ct and the DNA copy number was linear (R² = 1).The methods could amplify the corresponding Brucella species, while the other species and the negative control showed negative. Melting curve had demonstrated only a single peak, the reaction system did not form primer dimers and had no nonspecific amplification. CV% were less than 5% . This indicated that the detection system were stable in addition to good repeatability.The sensitivity of B. melitensis and B. abortus were 10CFU/ml³0CFU/ml, minimum detectable fragments were 10¹ copies/μl, but B. suis were 100CFU/ml, minimum detectable fragments were 10² copies/μl. In simulated blood samples, the sensitivity of B. melitensis and B. abortus were 10¹ copies/μl, the sensitivity of B. suis were 10² copies/μl. 28 clinical blood specimens were amplified positive with identification system of B. melitensis. 6 clinical blood specimens were amplified positive with identification system of B. abortus. 2 clinical blood specimens were amplified positive with identification system of B. suis. B. melitensis were dominant, which was consistent with isolation.In summary, the methods could quickly differentiate Brucella species with high specificity. Identification system of B. melitensis, B. abortus were high efficient and sensitive. Identification system of B. suis was less efficient, but did not affect the specificity. In the future, we should further optimized system for B. suis to improve efficiency and sensitivity of amplification.4. Mechanism resistance to quinolone in BrucellaIn this study, Clinical isolates of quinolone-sensitive were carried out drug-induced experiments in vitro. MICs were determined before and after induction. MICs of the sensitive strains before induction were only 0.25μg/ml⁰.5μg/ml. This showed that quinolones had good potential to treat brucellosis. Two resistant strains were chosed after induction with Levofloxacin several times. MICs increased from 0.25μg/ml 0.5μg/ml to 64μg/ml¹28μg/ml which were up to 128 and 256 times. Resistant strains had cross-resistance with other quinolones. This revealed that the drug had therapy limitations to Brucella infection.GyrA gene were amplified and sequenced. Typical gryA gene mutation occurred in drug-resistant strains and the amino acid sequences were deduced. GyrA amino acid shifted from Alanine (GCU) to Valine (GUU) in 87-bit. Aspartic acid (GAU) replaced with Glycine (GGG) and Valine (GUG) in 91-bit. The results showed that the GyrA mediated quinolone resistance in Brucella spp.To sum up, the results showed that major pathogen of Brucellosis were B. melitensis, B. abortus and B. suis. The majority is B. melitensis biover 3 in JiLin regions. Quantitative PCR that we established could detect Brucella spp with high sensitivity, specificity and stability. Quantitative PCR of Brucella species could differentiate B. melitensis, B. abortus and B. suis. The methods were sensitive, rapid, specific compared with traditional methods. Two induced resistant strains were obtained and had cross-resistance with other quinolones. There were mutations in QRDR gyrA gene.