Development Of High Throughput Assays For The Detection Of Bacterial Resistance Genes

Laboratory and clinical evaluation of DNA microarray for the detection of carbapenemase genesBackground:Bacteria resistant to first-line and second-line antibiotics have increased rapidly in recent years.The widely used extended-spectrum beta-lactam antibiotics ESBLs have also developed resistance to different degrees [1].Carbapenems are often used as the last line of defense against ESBLs infection because of their strong bactericidal ability and wide bactericidal range.However,with the widespread use of broad-spectrum antibiotics,carbapenem-resistant strains have become epidemic all over the world.The rapid global spread of carbapenem-resistant strains poses a serious challenge to medical treatment,and severely reduces the ability of human beings to treat bacterial infections.The spread of carbapenem-resistant bacteria is also closely related to high mortality and low cure rate in hospitals [2,3].It not only causes serious infection in hospitals,but also prevails in community environment.For example,healthy travelers from high epidemic areas may carry the corresponding drug-resistant bacteria and promote the international transmission of drug-resistant strains.This is because most of the carbapenem-resistant bacteria are transmitted through plasmids,which is easy to cause regional outbreaks.Therefore,it is an urgent task for many medical centers to detect and control the emergence and prevalence of carbapenem-resistant strains.The aim of this study is to develop a high-throughput,specific,sensitive and rapid DNA microarray-based diagnostic method,which can provide an effective means for the diagnosis,phenotypic confirmation and molecular epidemiological investigation of carbapenemase resistance genes.Methods: Eight common carbapenenenase genes were identified by literature research,including KPC,NDM-1,OXA-23,OXA-48,OXA-51,IMP,VIM and DIM.The primer and probes were designed by literature retrieval and software.Positive sample strains were collected to build the plasmid reference materials by artificial construction,including the sensitivity reference and the specific reference plasmids.The experimental conditions were optimized,and the multiple PCR amplification system and the PCR reaction cycle parameters were optimized.The ultra sensitive chemiluminescence image instrument(CL)was used to collect and process the chip signal,and the minimum detection limit(LOD)of the chip was detected by the sensitivity reference.Then the optimized method was used to detect clinical samples and Sanger sequencing verified the array results.Results: Eight carbapenemase genes could be detected with high sensitivity and specificity.The absolute LOD of this strategy to detect serially diluted plasmids of eight carbapenemase genes were 102-103copies/μL.Then,416 specimens collected from hospital were detected and the results showed 96.6% concordance between the phenotypic and microarray tests.Compared with Sanger sequencing,a specificity and sensitivity of 100% were recorded for bla NDM-1,bla IMP,bla VIM,bla DIM genes.The specificity for bla KPC,bla OXA-23,bla OXA-48,bla OXA-51 genes were 100% and the sensitivity were 98.5%,97.6%,95.7%,97.9% respectively.The overall consistency rate between the sequencing and microarray is 97.8%.Conclusions: Eight plasmids reference materials of KPC,NDM-1,OXA-23,OXA-48,OXA-51,IMP,VIM and DIM were constructed respectively.The primers and probes of the chip were screened,and 10 pairs of primers and 17 probes were determined.Meanwhile,the reaction system of chip was optimized,and two groups of five-weight PCR asymmetric system were established.The primer concentration and ratio were optimized to make each target gene get good amplification.The performance of the chip was evaluated,and the specificity evaluation results showed that the chip could effectively detect carbapenem negative bacteria and positive bacteria.Sensitivity evaluation showed that the minimum detection limit could reach 102~103copy / μL.Then,416 specimens collected from hospital were detected and the results showed 96.6% concordance between the phenotypic and microarray tests.Compared with Sanger sequencing,a specificity and sensitivity of 100% were recorded for bla NDM-1,bla IMP,bla VIM,bla DIM genes.The specificity for bla KPC,bla OXA-23,bla OXA-48,bla OXA-51 genes were 100% and the sensitivity were 98.5%,97.6%,95.7%,97.9% respectively.The overall consistency rate between the sequencing and microarray is 97.8%.Sequencing-based detection and clinical evaluation for Helicobacter pylori resistance to antibiotics and the genotype of CYP2C19 and IL-1βBackground: Helicobacter pylori(Hp)often colonize gastric mucosa,which can produce a variety of toxins,regulate signal pathway and secret virulence factors.The diseases including peptic ulcer,chronic gastritis,and duodenal ulcer have a certain relationship with Hp [11].Many kinds of digestion,occurrence and development of gastric lymphoma have correlation with Hp and it is listed as a class I carcinogen international cancer tissue.The drugs currently used for the treatment of Hp include clarithromycin,levofloxacin,furazolidone,tetracycline,metronidazole,amoxicillin and proton pump inhibitor PPI(omeprazole,pantoprazole,etc.),forming bismuth in protective layer and so on.With the increase of HP resistance,the eradication rate of triple therapy decreased significantly: two kinds of antibiotics had a drug resistance,the eradication rate was 50 to 60%,and the eradication rate of two antibiotics was only 10% [20].The ideal eradication rate of Hp was 95%,which could not be less than 80% for drug resistance.The resistance rate of Hp is different in regions.The metronidazole resistance rate in China is 40~70%,the resistance rate of levofloxacin and clarithromycin is 20~45%,amoxicillin and tetracycline are 0~5%,furazolidone is 0%~1%.But not bismuth and bismuth containing tetralogy of the scheme,the effect of resistance to the effect of the eradication rate also has a large difference.Meanwhile,the interleukin-1 beta(IL-1 beta)and the cytochrome P450(CYP2C19)involved in PPI metabolism also affect the eradication rate.The effect of fast metabolic therapy is lower than that of intermediate metabolism and slow metabolism [29].At present,drug sensitivity test is commonly used to determine antibiotic resistance,but the limitation of its long period,low operability,and accuracy are influenced by many factors,such as mixed infection of 10~15%,which limits its application and promotion.The purpose of this study was to establish a high-throughput sequencing platform that can simultaneously detect resistant genes of clarithromycin,levofloxacin,furazolidone,amoxicillin,tetracycline sensitivity and host genes of IL-1 beta,CYP2C19 polymorphism,which could provide accurate and efficient scientific basis for clinical treatment,clinical treatment of individual medication the,increase the eradication rate and reduce the overuse of antibiotics.Methods: The target genes were determined as well as the mutation sites of these genes and the polymorphism of the human host genes by literature review.Primers were designed in the conserved region after the corresponding gene sequence was downloaded.Hp positive samples were collected and positive reference materials were constructed after identified primers.Some positive reference plasmids were constructed by artificial synthesis.The multiplex PCR amplification system was optimized to enable each gene to be amplified effectively.Mixed samples were prepared with positive reference materials to detect the minimum detection limit that could be detected by sequencing technology.By using the optimized system and condition,we analyzed the antibiotic resistance and the polymorphism distribution of CYP2C19 and IL-1β in clinical samples by Sanger,PGM and Miseq sequencing detection.Results: We finally choosed ten resistance genes including 23 S r RNA,gyr A,PBP-1A,por D,oor D,16 S,r RNA,CYP2C19*2,CYP2C19*3,CYP2C19*17,IL-1β and their common mutations.Twelve pairs of primers for each locus were designed and the plasmids were established.A set of multiple amplification system for PGM sequencing were established.The results of the mixed sample sequencing showed that the mutation rate of Sanger sequencing could be 5%.Samples of gastric mucosa were collected from 307 Hospital and Huzhou central hospital.Gyr A of clarithromycin had the highest detect frequency.PBP-1A of amoxicillin,por D and oor D of furazolidone mutation site and mutation rate were detected,but the detection rate of known drug resistance sites was lower.23 S r RNA was the second,and 16 S r RNA of tetracycline detection rate was the lowest.The detection of host gene polymorphisms was 38.4% for homozygote fast metabolism,53% for heterozygote intermediate metabolism,8.96% for mutation slow metabolism,which was consistent with the proportion of host gene polymorphism reported in China.Conclusions: We established a method based on Sanger sequencing and next-generation sequencing that can detect resistance genes of H.pylori and host genes of human.The method has high specificity and specificity that could be applied to clinical therapy,which may help to improve the eradication rate of H.pylori.