Study On The Preparation And Application Of CTX-M-38 Type ESBLs Polyclonal Antibody

With the usage of broad spectrum antibiotics, especially the third generation cephalosporins application, more infectious diseases were controlled and obtained better therapeutic efficacy. On the other hand, the overuse or abuse of antibiotics had made the clinic face a severe test gradually. Because selection pressure was formed, more and more multiple antibiotic resistant strains emerged recently. Many genetic transfer elements which contained resistant genes can transfer among the strains isolated from the clinic frequently, as result, the antibiotics resistance was spreaded at the same time. Without effective antibiotics, The clinic had no available project to treat the infectious disease sometimes. According to the results of latest research, we found that the antibiotic resistant mechanisms of the multiple antibiotic resistant strains involved in producing inactive and modification enzyme,changing membrane permeability and forming efflux pump. However, the most important things, especially for the Gram-negative bacterial, is producing extended-spectrumβ-lactamases (ESBLs).ESBL-producing strain was first reported in 1983. And nowdays, it has become an increasing problem in daily clinical life worldwide. The ESBLs-producing strains include Enterobacteriaceae, non-fermentative bacilli and so on; the ESBLs-types located in the single strain reveal diversity according to districts; different ESBLs type has different antibiotics resistance. So, it is very important for us to confirm the prevailing ESBLs types and its antibiotics resistance. And we must detect the ESBLs-producing strains early if we want to provide a correct treatment theory for the clinic.when all of these works are finished successfully, we can decrease the ESBLs-producing strains emerging rate and intercept the ESBLs-producing strains spreading indeed.Due to different antibacterials are used to treat infectious disease in different district, the prevalence of ESBL-types differs from country to country and from laboratory to laboratory.In most parts of china, the isolating rate of CTX-M type ESBLs was at a high level. The CTX-M-1 type ESBLs was first confirmed by Bauernfeind in German. Now, there were more subtypes were reported all over the world. The CTX-M type ESBLs may locate in more kinds of strains separated from different laboratory. The hydrolytic substrates of CTX-M type ESBLs include penicillins,the first and second generation cephalosporins. Some subtypes can hydrolyze cefecime. Because of the difference of stereospecific blockade between Cefotaxime and ceftazidime, the hydrolytic activity of enzyme to Cefotaxime is more stronger than to ceftazidime. And some strains producing CTX-M type ESBLs are noticeable sensitive in vitro. According to whole genome sequence nucleotide homology, the CTX-M type ESBLs can be divided into 5 clusters. The nucleotide homology is less than 90%among subtypes which belong to the different cluster, and more than 94%among subtypes which belong to in same group. The mutational sites among groups may locate at anywhere in the DNA sequences. The further researchs show that all of the DNA mutational sites are away from the enzyme active center, as result, all the CTX-M genotypes possess some common properties. Confirming the common properties is useful to inhibit the resistent activity of enzyme of CTX-M type ESBLs.Separating and identifying the ESBLs-producing strains in time is very important for the clinic to treat the infectious disease early and inhibit the resistent isolates spread effectively. Nowdays, there are three kinds of methods to identify the enzyme-producing strains in the clinical laboratory. The ESBLs-postive isolates are selected by double-disc synergy test and are confirmed by enzymes inhibitor enhancing test or E-test. Both of these methods can be performed base on the isolates separating and would consume a long time. The most available measure is the molecular biology technique which can detect the ESBLs-postive strains specificly and quickly. However, due to the high cost and the sophisticated laboratory requirement, molecular biology technique is inapplicable to be performed in clinical laboratory.In order to solve the problem discussed above,46 nonduplicate ESBLs-positive E.coli isolates were collected from the first affiliated hospital. ESBLs-Producing isolates were confirmed by double-disc synergy test and enzymes inhibitor enhancing test; The enzyme encoding genes were amplified by PCR; The construction and expression of the vecter of pET-28-CTX-M were preformed by gene recombination technique; The antibiotic resistence was detected by liquid dilution; we prepared and purified the polyclonal antibody through classical immunology methods; we established a quickly detective method of double antibody sandwich ELISA to confirm CTX-M type ESBLs. At last, we inhibited the enzyme abilities by Kirby-Bauer disc agar diffusion test to explore the application of the polyclonal antibody in seroimmunity.The experiment is divided into three parts. Part I:The analysis of expression and antibiotic susceptibilities of CTX-M-38 type extended-spectrum-lactamaseMethods:1.1.46 Multiple antibiotic resistant E.coli isolates were detected corresponding to the phenotype of producing ESBL detected by double-disc synergy screening test, and confirmation for producing ESBL was carried out by enzymes inhibitor enhancing test.2. Acorrding to nucleotide homology diversity of CTX-M-38 type ESBLs, specific primers were designed by DNAstar analysing software.3. CTX-M-38 type ESBLs encoding gene was amplified by PCR.4. The expressing vecter of pET-28-CTX-M-38 was constructed by gene recombination technique.5. Using CaCl2 transforming method to introduce the expressing vector of pET-28a-CTX-M-38 into E.coli BL21.6. Selecting the positive strains by kanamycin resistance and PCR.7. The insert element of pET-28-CTX-M-38 was detected by DNA sequencing to identified the mutation of CTX-M-38 type encoding gene.8. The antibiotic susceptibilities of the transformant of BL21-CTX-M-38 was carried out by liquid dilution test.9. The enzyme activities of culture supernatant and bacteria sonicate were tested by Kirby-Bauer disc agar diffusion to reflect its distribution.Results:1. The isolates was resistant to Amoxicillin and sensitive to Amoxicillin/clavulanic acid. There was synergy phenomenon in the juncture. All of these indicated that the screening test was positive. 2. The diameter of Ceftazidime was 8mm, and the diameter of Ceftazidime/ clavulanic acid was 26mm, the difference between them was larger than 5mm. All of these indicated that the confirmation test was positive.3. According to the agarose gel electrophoresis, the molecular weight of amplifying products corresponded to the design.4. Using PCR to detect the plasmids derived from transformants, the products located at 900bp in the agarose gel electrophoresis. The result indicated that the expressing vectors were constructed successfully.5. the transformant of BL21-CTX-M-38 could grow in the LB liquid medium (100 u g/ml kanamycin), the result indicated that the transformant of BL21-CTX-M-38 was constructed successfully.6. The mutation point in DNA sequence between insert element and Bla CTX-M-38 located at 663, the thymine was replaced by adenine. According to the analysis of amino acid sequence, however, the mutation was samesense mutation, the codon 221 of them all was alanine.7. According to the Kirby-Bauer disc agar diffusion test, the enzyme activities from bacteria sonicate was stronger than culture supernatant. The result showed that the product mainly lied inside the bacteria.8. The CTX-M-38 transformant was obviously resisitant to ampicillin and piperacillin, and their MICs were more than 32μg/ml. The transformant was noticeable resistant to the first,second and third generation cephalosporins tested in the study too. The MICs of cephazoline,cefuroxime,cefepime and cefaclor were all more than 16μg/ml, and the MIC of ceftriaxone was 32μg/ml. At the same time, the hydrolytic activity of enzyme to Cefotaxime was 4 times as much as it to the ceftazidime, the former was resistant and its MIC was more than 32 u g/ml, and the latter was sensitive in vitro and its MIC was 8μg/ml. On the other hand, The CTX-M-38 transformant was stably sensitive to imipenemwas and its MIC was less than 4μg/ml. Different from other CTX-M type ESBLs, the MIC of aztreonam was less than 8μg/ml, the result indicated that the transformant was sensitive to aztreonam. The transformant was sensitive to piperacillin/ tazobactam and its MIC was less than 16/4μg/ml. To the other antibiotics including beta-lactamase inhibitors including Amoxicillin/clavulanic and ampicillin/sulbactam, however, the transformant was resistance and their MIC were more than 32/16μg/ml. The MIC of cefoperazone/sulbactam was equal to 32/16μg/ml, which indicated that the transformant was intermediate to the antibiotic. The transformant was also resistance to gentamicin, minocycline, ciprofloxacin and levofloxacin.Part II:the polyclonal antibody preparation of CTX-M-38 type ESBLsMethods:1. The CX-M-38 transformant expressed the extended-spectrum beta-lactamases in BL21 E.coli with IPTG derivation.2. The bacterial protein was extracted by water boiling extraction technique.3. The inclusion body protein was extracted by sonication method.4. The expressing products of CTX-M-38 transformant was detected by SDS-PAGE electrophoresis technique.5. The CTX-M-38 type ESBLs was reclaimed and purified by elution diffusion method.6. The polyclonal antibody of CTX-M-38 type ESBLs was preparation by classical immunology methods. 7. The antibody titer of polyclonal antibody was detected by indirect enzyme-linked immunosorbent assay.8. The polyclonal antibody prepared in research was purified by Octanoic acid precipitation method.9. The specificity of the polyclonal antibody was confirmed by Western blot.Results:1. The SDS-PAGE electrophoresis results showed that there was a noticeable protein band in the supernatant, whole bacterial proteins and inclusion body protein lane respectively, and the molecular weight was about 30kDa, which correspond to the design.All of these indicated that the transformants could express enzyme protein significantly with IPTG induction.2. The SDS-PAGE electrophoresis results showed that the expressing products quantity was supernatant, whole bacterial proteins and inclusion body protein in turn. All these results indicated that the expressing products mainly lie in the inclusion body.3. According to the results of indirect enzyme-linked immunosorbent assay, we found that the experimental result was positive when the polyclonal antibody was diluted from 1:1000 to 1:16000, and the experimental result was negative when the antibody was diluted more than 1:32000. we could conclude that the antibody titer was 1:16000 and the polyclonal antibody was prepared successfully.4. The Western blot results showed that different dilutions of antibodies reacted to the same protein band (about 30kDa), which molecular weight was correspond to the CTX-M-38 type ESBLs. All of these results suggested that the specificity of polyclonal antibodies was better. PartⅢ:Study on the application of polyclonal antibody of CTX-M-38 type ESBLsMethods:1. The reaction optimal ratio between CTX-M-38 type ESBLs and polyclonal antibody was confirmed by double agar diffusion test. Selectinig 4h culture medium as the experimental antigen concentration,Series of antibody titers were prepared by fold dilution method.2. The Kirby-Bauer disc agar diffusion was carried out to explore the Inhibitory effect of enzyme activity by polyclonal antibody.3. The enzyme labelled antibody was prepared by modified heptaiodic acid sodium method.4. The enzyme labelled antibody was purified by 50%Saturated ammonium sulfate precipitation method.5. Based on double-antibody sandwich principle, we designed a quick detection method of enzyme-linked immunosorbent assay for CTX-M-38 type ESBLs.6. The parallel testing was carried out to detect the products of CTX-M-38 transformant. According the result, we obtained the information of experimental repeatability and intraasay variation.7.146 ESBLs-producing E.coli strains were detected by PCR and double-antibody sandwich ELISA respectively.Comparing with the results of the two groups, we evaluated the experimental specificity.8. Using double-antibody sandwich ELISA to detect 27 isolates of ESBL-producing E.coli strains(not carrying CTX-M-38 type ESBLs). According to the result, we evaluated the experimental cross-reactivity.9. The culture medium of CTX-M-38 type ESBLs-producing E.coli was detected at various time, Aaccording to the result, we confirmed the optimal detective time of ESBLs.Result:1. According to the double agar diffusion test result, we found that the Precipitation line located at the middle of two reactions when the antibody titer was 1:64. all of the results indicated that the optimal experimental ratio was 1:64.2. The antibiotic diameter of antibody group was different from the control group(p<0.01) in K-B disc agar diffusion, the result indicated that the polyclonal antibody could inhibit the enzyme activity to some degrees.3. The inhibition effect of polyclonal antibody to amoxicillin, ceftazidime, cefepime and aztreonam was obvious. The diameter of these four antibiotics of antibody group turned larger than that of control group, which indicated that the CTX-M-38 transformant turn sensitive or the MIC of them decreased to some extent. It looked like that the anbibody had no effect on the cefotaxime and antibiotics including beta-lactamase inhibitors, the diameters of them were similar between the antibody group and control group.4. According the results of the parallel testing, the CV was 1.55%, which indicated that the intraasay variation was small and the experimental repeatability was better.5. There were 6 positive isolates detected by PCR among 146 strains, and 11 isolates of double-antibody sandwich ELISA. According to the statistics,p>0.05, we could conclude that there was no difference between the two groups and double-antibody sandwich ELISA had better Specificity.6. There was 1 positive strains detected by double-antibody sandwich ELISA among 27 strains, which indicated that the antibody possessed cross-reactivity and the method may have the phenomenon of false-positive. 7. The enzyme protein could be confirmed after the host bacteria was cultured 2h, and the result became significant after 4h. The amount of enzyme protein exceed the linear range of detection when cultured 8h,18h and 24h.As result, there was no change in absorbance. Combination of test procedures, this study was to determine the optimal detection time for ESBLs was culture 4h.Conclusion:1. The expressing products mainly lies inside the inclusion body, The transformant is obviously resistant to most of antibiotics.And there are some differences between the CTX-M-38 and other ESBLs types.2. The polyclonal antibody of CTX-M-38 type ESBLs is prepared, purified and identified. The polyclonal titer is 1:16000, the reaction between the antibody and enzyme protein possess higher specificity.3. Polyclonal antibody can inhibit the enzyme activity to some extent. Double-antibody sandwich enzyme-linked immunosorbent assay establish in research possesse better repeatability and specificity. The optimal detection time for ESBLs is culture for 4h.