Molecular Identification Of Escherichia Coli And Its Aminoglycoside Modifying Enzyme Genes

Escherichia coli belongs to gram-negative bacteria.Most of them are considered to be the human intestinal symbiotic bacteria,which will cause intestinal infection under certain conditions.Some special serotypes have pathogenicity,which can cause serious septicemia.E.coli widely exists in the natural ecological environment,and hospitals,communities and other social environments.Some pathogenic E.coli often cause severe epidemic diarrhea and pleurisy of people with low immunity.Since the discovery of antibiotic,it has been widely used in clinical treatment.However,the lack of timely and accurate pathogen detection methods lead to the abuse and misuse of antibiotics,cause the E.coli generally appears to drug resistance.Many antibiotics including aminoglycoside antibiotics cannot achieve the purpose of effective treatment,the treatment of multidrug-resistant E.coli infection has become very difficult.At present,the gold standard method of manual culture combined with drug sensitivity test has been gradually unable to meet the clinical needs,which is time-consuming and laborious.The automatic detection instruments are very expensive,which is difficult to apply to the basic medical units.In recent years,molecular detection methods have been gradually popular because of their advantages such as accurate detection,time saving,etc.Based on the previous experience in detection of pathogenic bacteria such as Klebsiella pneumoniae and Pseudomonas aeruginosa,this study established two rapid detection methods for E.coli and several typical aminosaccharide antibiotic resistance genes.Compared with the traditional culture method,these methods greatly shortens the time needed for the detection,and plays a key role in rapid selection of appropriate antibiotics for clinical treatment.The main molecular methods used in this study were PCR,LAMP,multiplex PCR,and multiplex qPCR.Firstly,the specific gene of E.coli was selected by bioinformatics,which named O3K₁9870 hypothetical protein gene?GenBank ID:13702648?.PCR and LAMP identification systems were established for this specific gene.240 E.coli and 170 non-E.coli?Pseudomonas aeruginosa,Klebsiella pneumoniae,Staphylococcus aureus,Staphylococcus epidermidis,Enterococcus faecalis,Shiga bacillus?were used for the specificity evaluation.It was proved that E.coli could be detected specifically by the two methods.In addition,in order to detect the clinical practicability,it is necessary to evaluate the sensitivity of the established detection system with two different templates including positive plasmid and bacteria lysate.The results show that the detection methods of PCR and LAMP with very high sensitivity,and the detection limit can reach 4 copies/reaction and 5 copies/reaction respectively.Secondly,the aminoglycoside modifying enzyme resistance genes?ant,aac,aph?were found from the Antibiotic Resistance Genes Database?ARDB?,and all the subtypes of the three kinds of drug resistance genes were downloaded from the NCBI database.Muti-sequences alignment of three classes ARG were performed to find respective conserved sequence,and common sequences were selected to design the primers.Four PCR methods for aac?6'?-I,aac?3?-II,ant?3'?,and aph?3'?drug resistance genes detection were constructed,and these detection systems were optimized and the sensitivity were evaluated.Finally,these detection methods were applied to 240 clinical strains of E.coli.The results showed that 99 strains with aac?6'?-I resistance gene were detected,and the positive rate was 41.08%.155 strains with aac?3?-II resistance gene,and the positive rate was 64.32%.53 strains with ant?3'?resistance gene,and the positive rate was 22.0%.40 strains with aph?3'?resistance gene,and the positive rate was 16.6%.The compliance rate of experimental results compared with the known drug resistance phenotype of E.coli was 93.75%,which indicated that the drug resistance genes detection systems established in this study are very accurate and completely show the advantages of molecular detection method.In addition,the detection limit of the four drug resistance genes reached 3 copies/reaction,4 copies/reaction and 5 copies/reaction which proved that these methods have very high sensitivity.Finally,the four aminoglycoside resistance genes aac?6'?-I,aac?3?-II,ant?3'?,and aph?3'?were used to construct quadruple PCR and quadruple qPCR reaction systems.The results showed that the four aminoglycoside modifying enzyme resistance genes could be detected simultaneously by quadruple PCR and quadruple qPCR resistance gene detection system,and the detection method of quadruple qPCR was especially time-saving,with only two hours.In this study,a new specific gene which can be used for the specific identification of E.coli was successfully excavated,and two molecular detection methods PCR and LAMP were constructed.Both the two methods can detect E.coli pathogens quickly and sensitively.In this study,a multiplex PCR and multiplex qPCR method for the detection of aminoglycoside modified enzyme resistance genes were also established.These methods can be used not only to detect the drug resistance genes of E.coli,but also with the potential to detect the aminoglycoside modifying enzyme resistance genes in other pathogenic bacteria.