The Study Of The Detection For Bacterium With Drug-resistant Gene Bla_NDM-1 By Real-Time Quantitative PCR And The Survey For Bacterial Contamination Of Surface Water In A Northern City

In recent years, as the antibiotics largely and widely used in humans and livestock, drug-resistance bacteria to these antibiotics have also continued to increase. New mechanisms of drug-resistance are emerging. In2010, the researchers found a new type of drug-resistant bacteria which could produce New Delhi metallo-β-lactamase. This type of bacteria carries a new drug-resistant gene blaNDM-1and encodes a new metallo-p-lactamase NDM-1, which could hydrolyze almost all antibiotics that are brought into clinical application, and has caused great difficulties to clinical treatment. Infection of the bacteria carrying drug-resistant gene blaNDM-1will pose a serious threat to people’s health. Until now, the detection methods of blaNDM-1postive bacteria have not been very advanced. The current means include isolation, culture, susceptibility test screening of drug-resistant bacteria, and PCR test. These detection methods cost a longer period of time to culture and identification, so they are not conducive to the prevention and control of the drug-resistant bacteria. This subject investigated and detected the microbial contamination of surface water in a northern city, and made susceptibility test to the isolates so as to analysis the drug-resistant bacterial contamination of the surface water. In addition, this subject has established a real-time quantitative PCR test for rapid detection of bacteria carrying drug-resistant gene blaNDM-1and provided a new direction to the rapid screening tests of the polluted water with low concentration.Objective: 1. Because Real-time quantitative PCR has the superiority of rapid detection and accurate quantitative determination, we establish a real-time quantitative PCR test for rapid detection of bacteria carrying drug-resistant gene blaNDM-1.We design a pair of specific primers and optimize the detection conditions to detemiine the detection sensitivity, specificity, and precision.2. We select four rivers and lakes in a northern city, collect samples and detect the indicator bacteria respectively so as to analysis the situation of microbial contamination of the surface water. At the same time, we isolate the strains and then make drug susceptibility test, production of metallo-β-lactamase test, PCR test, in order to have a preliminary understanding of the situation of bacterial drug-resistance, enzyme production, and drug-resistant gene blaNDM-1in bacteria of the surface water.Methods:1. The study has established real-time fluorescent quantitative PCR test for rapid detection of bacteria carrying drug-resistant gene blaNDM-1.We design a pair of specific primers to the drug-resistant gene blaNDM-1.The synthesis of plasmid with drug-resistant gene blaNDM-1is transformed into recipient strains, so the transformants could be positive control of PCR test. At the same time, we optimize the detection conditions to determine the detection sensitivity, specificity, and precision. Because Real-time quantitative PCR has the superiority of rapid detection and accurate quantitative determination, we prepare DNA standards of various concentration gradients to establish a standard curve, which can achieve accurate quantification of the drug-resistant gene in water samples.2. We select four rivers and lakes of surface water in a northern city, collect samples respectively and detect the indicator bacteria like total bacterial count, total coliform, thermotolerant coliform bacteria, enterococcus. At the same time, we isolate the strains from the water samples of surface water and then make drug susceptibility test. To the strains of Minimum inhibitory concentration≥2μg/ml, we use E-test strip to detect metallo-β-lactamase. To the MBL-positive strains, PCR test is used to verify that whether the drug-resistant gene is Results:1. The method of Real time quantitative PCR:the study has successfully established a method of Real time fluorescent quantitative PCR to detect the bacteria carrying drug-resistant gene blaNDM-1, this technology can be completed within2h. we design a pair of specific primers and probes to the specific sequence of drug-resistant gene blaNDM-1.The amplified fragment length is106bp. The genomic DNA of the transformants with synthetic plasmid that carrying drug-resistant gene blaNDM-1, we prepare DNA standards of various concentration gradient to establish a standard curve:Y=-3.473X+18.537, R2=0.998, which can achieve accurate quantification of the drug-resistant gene in water samples. The sensitivity of this method detecting drug-resistant gene blaNDM-1is10copies/μl. To the artificially infected samples, the detection sensitivity is1.6×101cfu/ml. This method has excellent detection sensitivity.2. Microbial contamination of the Surface water:The numbers of total bacterial count, total coliform, thermotolerant coliform bacteria, enterococcus from the water samples of two rivers are much larger than it from lakes. The numbers of the thermotolerant coliform bacteria from four sampling points are23cfu/100ml-5.4×105cfu/100ml, among that, the numbers from the two rivers are1.3×103cfu/100ml-5.4×105cfu/100ml, the numbers from two lakes are23cfu/100ml-3.5×103cfu/100ml. The study has isolated and identified225strains from water samples. Among them,28strains are Gram-positive bacteria,197strains are Gram-positive bacteria. The antimicrobial susceptibility tests indicate that196strains are drug-resistant to one antibiotic at least,87.11%of the225strains. Among them,128strains are drug-resistant to three or more antibiotics,76strains are drug-resistant to five the antibiotics at least,20strains are drug-resistant to ten or more antibiotics. There are38strains with minimum inhibitory concentration to Imipenem≥2μ g/ml. We make metallo-P-lactamase testing to the38strains and find that13strains could produce metallo-β-lactamase. But bacteria carrying drug-resistant gene blaNDM-1is not found in all tested strains by PCR experiments.Conclusions: This project established real-time quantitative PCR method for a rapid detection of the bacteria carrying drug-resistant gene blaNDM-1successfully. This method can achieve accurate quantification of the drug-resistant gene blaNDM-1in water samples. Real-time quantitative PCR has the superiority of powerful specificity, high sensitivity and good precision, so it provides a new direction to the rapid screening tests of the polluted water with low concentrations.The study also made an investigation and detection of the microbial contamination of surface water in a city of the north, the results show that microbial contamination of the two rivers is more serious than the two lakes, The numbers of the thermotolerant coliform bacteria from the two lakes are within the scope of V grade of the surface water environmental quality standards, while The numbers of the thermotolerant coliform bacteria from the two rivers are far beyond the limit of the surface water environmental quality standards. We make experiments for microbiological detection on river water samples and find several pathogens like Streptococcus pneumoniae and Vibrio cholerae. These pathogens are likely pose a threat and injury to human health, Drug susceptibility tests are made to the isolated and identified strains, in order to have a preliminary understanding the contamination of drug-resistant bacteria. The bacteria in Surface water, especially in the rivers have high drug-resistant rate, broad drug-resistant spectrum, strong drug-resistant capability, therefore, we should strengthen the importance of microbial contamination in surface water, and conduct a wide range of investigation about microbial contamination so as to take effective measures to protect surface water environment.