Study On Extended-spectrum Beta-lactamases Among Escherichia Coli Isolates Collected In Henan Province

Escherichia coli (E.coli) is the most important pathogen in community-acquired and nosocomial infections. The isolated rate of E. coli keeps at a high level in the laboratory of clinical microbiology and it is frequently reported all over the world. The therapeutic efficacy to E. coli has been regarded as a common factor affected the patients' prognosis and pathogenesis. In order to solve this problem and obtain better anti-infection efficacy, the research on the E. coli has become a hot topic in the anti-infective territory. The study focusing on the mechanism of resistance is the key point.With the overuse or abuse of broad spectrum antibiotics, especially the third generation cephalosporins application, multiple antibiotic resistant E.coli possess a high rate of occurrence. The strains separated from clinical laboratory not only are capable of hydrolyzing mostβ-lactams, but also are highly resistant to fluoroquinolones and aminoglycosides. In phenotype, the mechanism of resistance includes producing inactive and modification enzyme,changing membrane permeability and forming efflux pump. On the other hand, vertical transmission and plasmid-mediated transmission are the main mechanism of resistant gene spreading among strains in molecular level. According to the results of latest research, producing extended-spectrumβ-lactamases (ESBLs) play an important role in the multiple antibiotic resistant of wild-type E.coli. More than one ESBLs type lie in the same strain provides the resisrant ability obviously.ESBLs belongs toβ-lactamases. Due to the DNA sequence diversities, ESBLs can be divided into different types, and most types are plasmid-mediated. This kind ofβ-lactamases can hydrolyze oximino-cephalosporins obviously.that is to say, the ESBL-produing strains is highly resistant to the first,second and third generation cephalosporins, penicillins and monobactam. On the other hand, it is susceptible to cephamycin,carbapenem, andβ-lactamase inhibitors can inhibit its hydrolyzing antivity. ESBL-producing strain was first reported in 1983, and nowdays, it has become an increasing problem in daily clinical life worldwide. Prevalence characterization of extended-spectrum beta-lactamases among Escherichia coli Isolates divided into five parts. (1) The ESBLs from wild-type strains turns on diverse types, and new subtypes are frequently reported. (2) the whole genome sequence nucleotide homology of TEM or SHV subtypes is very higher, but the homology of other ESBLs subtypes is very lower. (3) most ESBLs derived from TEM-1,TEM-2 or SHV-1 enzymes. (4) the prevalence of ESBL-types differs from country to country and from laboratory to laboratory. (5) the antimicrobial-resistant phenotype among all of ESBL types and subtypes is different.In HeNan province, the first ESBL-producing strain was confirmed by liu xinzheng in 2000. And researchs most focus on The antibiotics resistant profile of wild-type ESBL-producing E.coli nowdays. Only a few studies regarded the topic that reveals the prevalence and the diversity of ESBLs among Escherichia coli isolates have been reported. Without effective information of the prevalence characterization and difference among all kinds of ESBLs types, the clinic is difficult to treat infections. As the result, patients have to afford much more. Henan has a large number population, and its ecnomics is at low level. To determine the prevalence and the diversity of ESBL among Escherichia coli isolates, and confirm the antibiotics resistant profile of different subtypes is very important for the clinic to treat infection reasonably and reduce the cost of medicine.In order to explore the prevalent characteration and diversity of ESBLs among Escherichia coli isolates, to find the role of ESBLs encoded-gene localization, and to explore the antibiotics resistant profile of ESBL enzymes dominated in ESBL-positive E.coli isolates in henan,138 nonduplicate ESBL-positive isolates were collected from the first affiliated hospital, the third affiliated hospital of zhengzhou university and the people's hospital of henan province. ESBL-Producing isolates were confirmed by double-disc synergy test and enzymes inhibitor enhancing test. ESBL epidemiology was described by PCR and DNA sequencing. Detecting the antibiotics sensitivity of all strains containing different plasmid profiles and ESBL-types by E-test. The statistics were analyzed by chi-square criterion. The ESBLs expressing vectors was constructed by gene recombination. The antibiotics sensitivities of transformant were determined by microdilution.Through above tests, we try to describe the a complex ESBLs epidemiology in henan, and to reveal the relationship in plasmid profile and ESBL-type, plasmid profile and antibiotics sensitivity, ESBL-type and antibiotics sensitivity, and to identify the antibiotics resistant of ESBL dominated. At last, we provide a new theory for the treatment of patients infected with E.coli.The experiment is divided into three parts. Part I:The study on the epidemiology of E.coli producing ESBLsMehtods:1. Multiple antibiotic resistant E.coli isolates were detected corresponding to the phenotype of producing ESBLs detected by double-disc synergy screening test, and confirmation for producing ESBLs was carried out by enzymes inhibitor enhancing test.2. Acorrding to nucleotide homology diversity of all kinds of ESBLs types, universal primers and specific primers were designed by DNAstar analysing software.3. ESBL-producing isolates confirmated by enzymes inhibitor enhangcing test were analysed by PCR to detect ESBLs type.4. The ESBLs types among E.coli dominated in henan were identified by DNA sequeemng.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 products of PCR and difference among all the ESBL types corresponded to the design.4. The DNA sequeening result revealed thay these were TEM-1,SHV-12,CTX-M-1,CTX-M-14,CTX-M-38,OXA-1 and OXA-20 ESBLs types among E.coli in henan province.5. In 138 E.coli isolates, TEM-1 typeβ-lactamases was encoded in 80.4%and donimated in ESBL-positive E.coli isolates in henan. Respectively, SHV-12,CTX-M-1,CTX-M-14,CTX-M-38,OXA-1 and OXA-20 ESBL type were encoded in 60.9%,32.6%,58.7%,6.5%,4.3%and 2.0%.6. In 138 ESBL-producing strains,1-5 ESBLs types could exist in single isolate. There were 32.6%,26.1%,21.7%,17.4%and 2.2%strains which contained two types,three types, one type, four types and five types respectively.7. TEM-1 typeβ-lactamases donimated in ESBL-positive isolates which only contained one ESBL type. And then, CTX-M-14. SHV-12 and CTX-M-1 were encoded in 4.3%.2.2%and 2.2%respectively.8. SHV-12+CTX-M-14 donimated in ESBL-positive isolates which contained two ESBL types (10.9%). And then, TEM-1+CTX-M-14. SHV-12+TEM-1. TEM-1+CTX-M-1. TEM-1+CTX-M-38 and OXA-1+CTX-M-1 were encoded in 8.7%.6.5%.2.2%.2.2%and 2.2%respectively.9. SHV-12+TEM-1+CTX-M-1 donimated in ESBL-positive isolates which contained three ESBL types (10.9%). And then, SHV-12+TEM-1+CTX-M-14. TEM-1+CTX-M-14+CTX-M-38. SHV-12+OXA-1+CTX-M-14 and SHV-12+ OXA-20+CTX-M-14 were encoded in 8.7%.2.2%.2.2%and 2.5%respectively.10. SHV-12+TEM-1+CTX-M-1+CTX-M-14 donimated in ESBL-positive isolates which contained four ESBL types (8.7%).and then, SHV-12+TEM-l+CTX-M-14+OXA-1,SHV-12+TEM-1+CTX-M-14+CTX-M-25 and SHV-12+TEM-1+ CTX-M-1+CTX-M-25 were encoded in 4.3%.2.2%and 2.2%respectively.11. SHV-12+TEM-1+CTX-M-1+CTX-M-25+CTX-M-14 was the only one pattern in the isolates which contained five ESBL types. PartⅡ:the relationships among the plasmid maps,ESBLs type patterns and antibiotics resistance of ESBL-producing E.coliMethods:1. Using molecular biological technique to reveal the plasmid map of 138 ESBL-producing strains of E.coli.2. The ESBL-type pattern among strains was confirmed by PCR and DNA sequencing.3. The common antibacterials minimum inhibition concentrations of ESBL-producers which contained different plasmid map was detected by E-test.4. The common antibacterials minimum inhibition concentration of ESBL-producers which contained different ESBL-type patterns was detected by E-test.5. All of the experimental data were analyzed by SPSS 10.0 statistical package program.the relationships among the plasmid maps,ESBLs type patterns and antibiotics resistance of ESBL-producing strains of E.coli were analyzed by chi-square criterion,and the level of significant difference wasα=0.05.Results:1. The ESBL-producing strains of E.coli had 1-5 plasmids, which molecule was about 21kb,9.0kb,7.0kb,3.0kb and 2.5kb.2. There were five kinds of plasmid maps in the 138 strains. The plasmid of 21kb existed in every isolate. The strain which contained two kinds of plasmid (21kb+2.5kb) was encoded in 50, single plasmid (21kb),five kinds,four kinds of plasmid (without 9.0kb) and three kinds of plasmid(21kb,3.0kb, 2.5kb) were encoded in 27,22,21 and 18 respectively.3. The strains which contained different plasmid map were resistant to penicillins, the first and second generation cephalosporins, cefotaxime and Ceftriaxone (MIC≥256μg/ml). All of them were sensitive to imipenem and amikacin (MIC≦12μg/ml and 0.25μg/ml).and every strain was sensitive to Ceftazidime in vitro (MIC≦4μg/ml). Different strain had a different result to cefepime (MICs was32,48,48,16,64μg/ml)4. There had no significant difference between the drug resistence and plasmid map of ESBL-producing strains of E.coli (p>0.5/0.9)5. There had no significant difference between the ESBL-type patterns and plasmid map of ESBL-producing strains of E.coli (p>0.05)6. There had no significant difference between the drug resistence and the ESBL-type patterns of ESBL-producing strains of E.coli (p>0.5/0.9)PartⅢ:Study on the antibiotics resistance of dominated ESBLs among E.coli isolatesMethods:1. Using gene recombination technical to construct the expressing vector of pET-28a-TEM-1,pET-28a-SHV-12 and pET-28a-CTX-M-14.and the vector was identified by PCR.2. Using CaCl2 transforming method to introduce the expressing vector of pET-28a-TEM-1 and pET-28a-CTX-M-14 into E.coli BL21, and introduce the expressing vector of pET-28a-SHV-12 into E.coli JM109. Selecting the positive strains by kanamycin resistant and PCR.3. Using microdilution to detected the antibiotics resistence of the E.coli BL21, E.coli JM109 and the transformants of BL21-TEM-1,BL21-CTX-M-14 and JM109-SHV-12. the results were judged according to the standard of NCCLs.Results:1. Using PCR to detect the plasmids derived from transformants, the products located at 900bp in the agarose gel electrophoresis. All of these results indicated that the expressing vectors were constructed successfully.2. The E.coli BL21 and E.coli JM109 were sensitive to all kinds of antibiotics in vitro.3. TEM-1 transformant was obviously resisitant to penicillins. The MICs of ampicillin and piperacillin were more than 32μg/ml respectively. Parts of the first generation cephalosporins could be hydrolyzed by TEM-1 transformant. The MIC of cephazoline was more than 16μg/ml. The third,quaternary generation cephalosporins tested in the study could efficiently inhibit the TEM-1 transformant. The MICs of cefotaxime and Ceftazidime were less than 1μg/ml. Theβ-lactamase inhibitors including clavulanic acid,sulbactam and tazobactam could inhibit the activity of the enzyme and reduce the resistance of TEM-1 producing strains. The MICs of the Amoxicillin/clavulanic,ampicillin/sulbactam and piperacillin/tazobactam were all less than 8/4μg/ml. the transformant was stably sensitive to carbapenems and monobactam tested in the study. The MICs of imipenem and aztreonam was less than 4μg/ml and 16μg/ml respectively.4. The SHV-12 transformant could effectively hydrolyze all the penicillins tested in the study. The MICs of ampicillin and piperacillin were more than 16μg/ml respectively. And the trasnformant was resistant to the first,third generation cephalosporins, the MICs of cephazoline and cefotaxime were more than 16μg/ml respectively,and the MIC of Ceftazidime was 32μg/ml.β-lactamase inhibitors including clavulanic acid,sulbactam and tazobactam could inhibit the activity of the enzyme and reduce the resistance of SHV-12 producing strains, the antibiotics containing the inhibitor could inhibit the strains producing enzyme. the transformant was stably sensitive to carbapenems tested in the study, the MIC of imipenemwas less than 4μg/ml. The SHV-12 transformant was intermediate to the aztreonam and its MIC was 16μg/ml, indicated that the transformant expressed a few enzyme or the activity of the enzyme had changed.5. The CTX-M-14 transformant was obviously resisitant to ampicillin and piperacillin, and their MICs were more than 32μg/ml,64μg/ml respectively. 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 32 times as much as it to the ceftazidime, the former was resistant and its MIC was more than 32μg/ml, and the latter was sensitive and its MIC was 1μg/ml. On the other hand, The CTX-M-14 transformant was stably resitant to aztreonam and its MIC was more than 16μg/ml. the MIC of imipenemwas less than 4μg/ml indicated than the transformant was stably sensitive to it. The transformant was also sensitive to Amoxicillin/clavulanic,ampicillin/sulbactam and piperacillin/tazobactam, and their MICs were all less than 8/4μg/ml. Quinolones and tetracyclines antibiotics tested in the study could inhibit the transformant effectively, the MIC of ciprofloxacin and tetracycline was less than 0.06μg/ml,less than 4μg/ml respectively.Conclusion:1. There are TEM-1,SHV-12,CTX-M-1,CTX-M-14,CTX-M-38,OXA-1 and OXA-20 ESBL types among E.coli in henan province, and TEM-1,SHV-12 and CTX-M-14 are the main ESBLs type which donimate in the ESBL-positive E.coli strains.1-5 ESBLs types can exist in single isolate. The strains which contain two ESBL types at the same time are the main prevalent isolates in henan.2. There are no significant difference between the plasmid map and ESBL types,ESBL types and drug resistance,drug resistance and plasmid map.3. The expressing products of BL21-TEM-1 belonged toβ-lactamase, and the transformant is obviously resistant to penicillins. The inhibitor can inhibit the activity of TEM-1 type enzyme. The expressing products of BL21-CTX-M-14 and JM109-SHV-12 belong to ESBLs, and the transformants are obviously resistant to penicillins and cephalosporins, The inhibitor can inhibit the activity of. CTX-M-14 and SHV-12 type ESBL.