| Background Pseudomonas aeruginosais is a most important opportunistic pathogen in the human body, and it can lead to infection of multiple organ or multiple sites. It is difficult to treat thepseudomonas aeruginosa because the long cycle of antibiotic medication, complicated with Pseudomona infection, In recent years, with the various instruments and invasive treatment method and antibacterial drug abuse, infection rate and drug resistance rate of pseudomonas aeruginosa in patients have been increasing obviously. It is one of the problems to be solvedhow to treat effectively resistant pseudomonas aeruginosa. The study found that pseudomonas aeruginosa was divided into mucinous and non-mucinous according to the morphological characteristics of the bacteria. The mucinous of bacteria was associated with biofilm formation. The studies showed that the colonization of non-mucoid pseudomonas aeruginosa was more durable, and the infection was more difficult to remove. In addition, mucoid pseudomonas aeruginosa was isolated from patients with chronic and persistent infection, and theisolated rate of mucoid pseudomonas aeruginosa was much lower than the non-mucoid pseudomonas aeruginosa. Different types of pseudomonas aeruginosa often changed with each other, and the mucus type sometimes changed to non-mucus type, especially for the new changes of the mucus type. With infection continues to develop, it is difficult to restore the non-mucinous type. When long-term infection of bacteria in the body, itcould causethe drug resistance. Previous study found that material secreted by mucoid pseudomonas aeruginosaand adjacent to the bacteria, platelet and fibrin were aggregated to form bacterial biofilm, which led to the antibacterial drugs. In addition, Pseudomonas aeruginosa biofilm inhibited immune cells or antibodies and bacterial contact, causing bacteria not be cleaned. Pseudomonas aeruginosa is a kind of special strains, and it easy to produce resistance.The resistance mechanism is very complexincluding active efflux system disorders, produce inactivated enzyme activity or modifying enzymes and membrane permeability reduction. Efflux system usually by intracellular mexb, mexd and Mex E protein identification of various antibiotics and antibiotic and intracellular MEXA, mexb and Opr M formation membrane transport channel, and formed a channel to discharge the antibiotics with the aid of outer membrane Opr M, oprj and Oprn.The widespread use of antibacterial drug stimulated bacteria drug-resistant gene change or generated new resistance genes.Outer membrane protein Opr D2 porin channels is carbapenem quickly into the green channel of the Pseudomonas aeruginosa, oprd2 deletion or lower expression is often lead to an important reason for of carbapenem resistant Pseudomonas aeruginosa. Carbon is one of the most effective drugs for the treatment of resistant gram negative bacilli infection. It is also the last line of defense to protect the body from injury. At present, carbapenem antibiotics is effective drug for treating resistant pseudomonas aeruginosa in clinical treatment. However, with the carbapenem antibiotics are widely used for treatment of pseudomonas aeruginosa, its drug resistance is becoming more and more severe, and the resistant rateto imipenemreached 46.9%. The resistance of pseudomonas aeruginosa including the production of carbon, the enzyme, the metal enzyme, the outer membrane protein and the active efflux pump. So far, the relationship and mechanism between different genotypes of mucus type pseudomonas aeruginosa and drug resistance is not clearObjective: To investigate the correlation of different genotypes of mucoid pseudomonas aeruginosa with antibiotic resistance, and to propose the theory reference for clinical rational use of antibiotics for treating different genotypes of mucoid Pseudomonas.Methods: Strains source 110 strains of pseudomonas aeruginosa were isolated from the patients from January 2012 to December2015 in 202 Hospital of the Chinese people’s Liberation Army. 100 strains from sputum samples, 8 strains from pus and secretions, 2 strains from other samples. Strain identification The strain identification was performed by routine identification method. Specimens were cultured and separated strictly accordance with the "national clinical laboratory procedures". The colony morphology diversity of Mucus pseudomonas aeruginosa including colorless, visible viscous, dew drop shaped, jelly like colony and colony margin irregular. The inoculation loop was used to carry out the metallic luster, no special odor of the colonies, and all the strains were identified as pseudomonas aeruginosa by Compact Vitek2 automatic microbial analyzer. Drug sensitivity test Paper method was used to measure the sensitivity of 10 kinds of antibiotics. The quality control strains waspseudomonas aeruginosa ATCC27853. The first drug sensitivity results were recorded under constant temperature 35 C at 24 h, and second drug sensitivity results were recorded at 48 h, and then the third drug sensitivity results were recorded at 72 h. The results of the sensitivity tests were interpreted in accordance with the standards of the Clinical Laboratory Standards Committee of the United states in 2011. PCR method Designing specific primers for the different genes of the pseudomonas aeruginosa and the primer specificity was performed by using Blast in the NCBI database in order to avoid the complementary sequence and the hairpin structure at the 3 ’end. Polymerase chain reaction(PCR) was performed to detect the Amp C, IMP-1,SPM-1,Opr D,KPC,VIM-1and VIM-2 gene. 20 ul product and 2ul bromophenol blue were mixed in 2% agarose gel electrophoresis by ethidium bromide staining. After 5min, DNA bands were observed by UV Gel imaging system.Statistical analysis The data was analsed using chi square test or s’ Fisher exact test in Spss18.0 software, and all statistical tests were two-sided probability test. P<0.05 was considered as statistically significant.Results: The results of antibiotic resistance of mucoid pseudomonas aeruginosa The results showed that the resistance ratesof mucoid pseudomonas aeruginosa to amikacin, tobramycin, gentamicin, piperacillin/tazobactam, levofloxacin, ceftazidime were 0~4.5%, and the resistance rates to imipenem, meropenem, cefepime and ciprofloxacin were 7.3~9.1% at 24 h. Compared with the results at 24 h, the resistance rate of mucoid pseudomonas aeruginosa to imipenem and meropenem was 13.6%, and the resistance rates to amikacin, tobramycin, gentamicin, cefepime, piperacillin/tazobactam, levofloxacin and cefotaxime, ceftazidime were 6.4~11.8 at 48 h, and the difference was statistically significant(P<0.05). Compared with the results at 48 h, the resistance rates mucus pseudomonas aeruginosa to all bacteria were not significantly different at 72h(P>0.05). The relationship between different genotypes of mucoid Pseudomonas aeruginosa and drug resistance The results demonstrated that strains harboring Amp C positive gene were 12 strains(80.00%), IMP-1 positive gene were 2 strains(13.33%), VIM-1 positive gene were 5 strains(33.33%), VIM-2 positivegene were 9 strains(60.00%), oprd2 gene positive 4 strains(26.67%), Opr D2 deletion were 11 strains(73.33%), SPM-1 positive gene 4 strains(26.67%), and KPCgene positive were 5 strains(33.33%).Conclusion: The sensitive rate and drug resistance rate of mucoid pseudomonas aeruginosa on the different antibiotics were significantly different, and there were significantly different for resistance rate to imipenem and meropenem; The resistant mechanism of mucoid pseudomonas aeruginosa to carbapenem are closely related to Amp C, VIM-2 and Opr D2 genes. |
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