| Background and objectivePseudomonas aeruginosa(Pseudomonas aeruginosa, PA) is one of the main opportunistic pathogens in hospital causing variety of infections, especially in the intensive care unit, respiratory and burn department, under normal circumstances will not cause disease in humans, when human body immunity is low can enter, cause human infection. In recent years, the abuse of antibiotics is increasing seriouly, and because of its inherent and acquired resistance mechanisms and make it have serious drug resistance to variety of drugs, and multiple drug resistance(multi- drug resistant, MDR) even generic drug resistant(pan- drug resistant, PDR) pseudomonas aeruginosa appeared.Carbapenems antibiotics is one of the important means to cure multiple drug resistance pseudomonas aeruginosa. But as the abuse of antibiotics is more and more serious, Carbapenem antibiotics resistance is more and more serious,increased year by year, in the face of such super bacteria Humanity has been helpless, facing the plight of hopeless.The following several aspects has mainly caused pseudomonas aeruginosa Carbapenem antibiotics resistance: the production of Carbapenemases, excessiveexpression of active efflux pump, the change of the outer membrane permeability,integron mediated resistance change, etc., among them, the most reason is the generation of Carbapenemases and the change of the outer membrane permeability.According to Ambler classification method,Carbapenemases can be divided into three categories, pseudomonas aeruginosas mainly carry AmblerB metal enzymes, getting AmblerB metal enzymes is the main cause of Carbapenem antibiotic resistance.At present, the main six AmblerB metal enzymes are detected, respectively for the IMP,VIM, SPM, SIM, GIM and NDM.Pseudomonas aeruginosa carry mainly IMP and VIM.Early detection of OXA enzymes belong to narrow hydrolytic enzymes, such as OXA- 1, OXA-2,OXA-10, etc.In recent years, ultra-broad-spectrum OXA enzyme hydrolysising even carbapenem drugs have been reported increasly, these enzymes except 0XA-15 and OXA-32 extended-spectrum enzymes, Other broad- spectrum OXA enzymes mostly from mutations of OXA-10. The miss of outer-membrane protein D2(OprD2) protein can also lead to carbapenem-resistant in Pseudomonas aeruginosa, and lack of channel proteins are often accompanied by overexpression of efflux pump system system, both of which plays an important role.Integron is mobile genetic elements, Capable of carrying gene cassettes, and the spread of drug resistance among bacterial, At present, domestic and foreign researchers mainly found three types of integration,namely intⅠ, intⅡ, intⅢ, Pseudomonas aeruginosa mainly carry Ⅰ integron.Tt is Because of present situation of pseudomonas aeruginosa resistance at home and abroad, we design this topic, to statistics this region Carbapenem antibiotic resistance situation 、 explore its main Carbapenemases genotype, whether carry channel protein, integron, or the resistant genes box, Clear pseudomonas aeruginosa resistant, molecular mechanisms and popular situation in this region, provide laboratory basis for clinical anti-infection treatment.Methods:1. Strain collection:Collecting the Carbapenem antibiotics(imipenem or meropenem) resistancePseudomonas aeruginosa in the First Affiliated Hospital of Zhengzhou University between May 2015 and October 2015, VITEK automatic bacterial identification system identify to species,susceptibility card identify susceptibility,KB susceptibility disk diffusion test carbapenems-resistant strans again,Confirm the accuracy, susceptibility is determined by refer to U.S. clinical and laboratory Standards Institute(CLSI) 2012 edition, Place purpose strain in- 20 degrees.2. Analysis of drug resistanceCount distribution of department, specimen type, Resistance rate of commonly used antibiotics, and Compare the resistance situation between 86 strains carbapenems-resistant pseudomonas aeruginosa and 86 carbapenems-sensitive strain,statistical methods using chi-square test.3. phenotypic confirmatoryAfter the recovery of strains, single colonies were picked painted on blood agar,with EDTA double disk synergy test screening enzyme producing pseudomonas aeruginosa.4. The genetic testingWith specific primers sequence, using polymerase chain reaction, to detect the A and B, class D carbapenems, Oprd2 channel proteins and integron, after amplification electrophoresis, send positive results to sangon biological engineering company to sequencing,upload Sequencing results to NCBI、BLAST.Results:Department, type of specimen distribution: A total of 86 objective strains,including ICU 39, Interventional Radiology 12,Neurosurgery 12,other departments23.sputum 70,lavage 4,secretions 4,bile 2,drainage 2,urine 2, pus 2.Resistance rate statistics:As follows are some common drug resistance rates,amikacin40%,piperacillin74%, ceftazidime 53%, cefepime 47%,meropenem 89%, imipenem 90%, aztreonam74%, ciprofloxacin 51%,levofloxacin 56%.Statistical Analysis to compare 86 carbapenems antibiotic-resistant strains with 86 carbapenem resistant strains at the drug resistance rate of Common used antibiotics,using the chi-square test to analysis differences, each group P is less than 0.05, at α = 0.05 significance level, it is believed that carbapenem-resistant PA and carbapenems-sensitive PA was significant difference in the rateof resistance to other common types of antibiotics.Genetic Testing results:Class A, GES is not detected in objective strains, 86 strains of Pseudomonas aeruginosas were amplified 11 Class B metalloenzymes IMP, 2 SIM. 18 containing the OXA-10.86 strains of Pseudomonas aeruginosa, only 32 were amplified channel protein, 54 channel protein are missing, the missing rate is 63%.Integron detection:Of all the 86 Objective bacteria, 26 amplified class Ⅰ integrons, not amplified integron Ⅱ, Ⅲ, 4 amplified 1850 bp length gene cassette(dhfrXⅡ-orfF-aadA2),4have 2000 bp gene cassette(aadB-aaC6-Ⅱ-PSE1), 2 own 1270 bp gene cassette(aadA6-orfD), 2 possess 2000bp(imp26-qacG-aacA4), 13 carry 1000 bp gene cassette(aadA2), only 1 1600bp(aadA4-OXA142).Conclusion:1. carbapenems-resistant Pseudomonas aeruginosa are often resistant to many other antibiotics. Clinical monitoring should be strengthened to prevent the spread of drug-resistant2. The main mechanism of carbapenem-resistant in Henan Province is that carring carbapenemases and the missing of channel proteins3. In Henan Province Pseudomonas aeruginosa mainly carried class I integron,resistance gene cassette mainly cause resistance to aminoglycosides and carbapenems... |
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