| Objective: To explore the resistance and resistance rate of endoscopic Pseudomonas aeruginosa biofilms to three high-level disinfectants,o-phthalaldehyde,glutaraldehyde,and peracetic acid,and the correlation with the disinfectant resistance gene qac E△1-sul1.Method: 1.Establishment of Pseudomonas aeruginosa biofilmTeflon material pipeline is connected with silica gel return pipe,liquid storage bottle and peristaltic pump to form a closed system for circulating perfusion.2.Count and observation of living bacteria in biofilmThe suspension of standard strain ATCC 27853 was continuously poured into Teflon pipeline.The biofilm formed on Teflon pipeline was separated by ultrasonic oscillation and vortex oscillation on the 2nd,4th and 6th day after perfusion.Take 1 m L diluted bacterial suspension and inoculate it into the nutrition plate after gradient dilution.Using Scanning electron microscopy to observe the biofilm under different culture days,determine the optimal growth days of biofilm.3.The resistance and resistance rate of the tested strain biofilm to three disinfectantsThe minimum inhibitory concentration test and the minimum bactericidal concentration test were used to detect the resistance and resistance rate of the standard strain ATCC 27853 biofilm and the 22 test strain biofilms to the three disinfectants.4.PCR detection of disinfectant gene qacE△1-sul1PCR(polymerase chain reaction)was used to detect the qac E△1-sul1 gene of 22 test strains,and the correlation between qac E△1-sul1 gene and the resistance of the three disinfectants was analyzed.5.Statistical methodsSPSS 20.0 statistical software was used to analyze the data.The measurement data is expressed in the form of mean value ± standard deviation.The data were analyzed by one way ANOVA,rank sum test and chi square test.The statistical test level α=0.05,P<0.05 was statistically significant.Results:1.The growth model of Pseudomonas aeruginosa biofilm was established,and 6 days was the best biofilm growth time.2.The MIC value of o-phthalaldehyde,glutaraldehyde and peracetic acid to ATCC 27853 biofilm was 5300 mg/L,4000 mg/L and 600 mg/L,respectively.MBC value is the same as MIC value.3.The MIC value range and MBC value range of the resistance of o-phthalaldehyde to the 22 strains biofilms of experimental bacteria was 4700 mg/L ~ 5500 mg/L and 5100 mg/L ~ 5500 mg/L,respectively.The MIC value range and MBC value range of the resistance of glutaraldehyde to the biofilm of 22 strains of experimental bacteria was 2000 mg/L ~ 8000 mg/L and 4000 mg/L ~ 10000 mg/L,respectively.The MIC value range and MBC value range of the resistance of peracetic acid to the biofilm of 22 strains of experimental bacteria was 400 mg/L ~ 800 mg/L and 400 mg/L ~ 1200 mg/L,respectively.4.The MIC value of standard strain ATCC 27853 biofilm was used as the standard to determine the resistance.The resistance rate of o-phthalaldehyde,glutaraldehyde and peracetic acid were 63.64%,36.36% and 9.10%,respectively.The MBC value of standard strain ATCC 27853 was used as the standard to determine the resistance.The resistance rate of o-phthalaldehyde,glutaraldehyde and peracetic acid were 86.36%,59.10% and 36.36%,respectively.5.By using the paralleling test,the resistance rates of o-phthalaldehyde,glutaraldehyde and peracetic acid to test strains were 86.36%,59.10% and 36.36%,respectively;By using the series test,the resistance of o-phthalaldehyde,glutaraldehyde and peracetic acid to test strains were 63.64%,36.36% and 9.10%,respectively.6.The positive rate of disinfectant resistance gene qac EΔ1-sul1 was 18.00%.There was no significant difference of MIC values and MBC values of three disinfectants between gene positive strains biofilm and gene negative strains biofilm.7.When the MIC value and MBC value of the standard strain ATCC 27853 biofilm were used as the criteria for disinfectant resistance,both the parallel test and the series test showed that there was no significant difference in the positive rate of qac E△1-sul1 between the resistant group and the non-resistant group.Conclusions: 1.Pseudomonas aeruginosa endoscope was cultured in biofilm,and 6 days was the best biofilm growth time.2.The test strain biofilm has a certain degree of resistance to clinical use of high-level disinfectants,and the resistance to o-phthalaldehyde is the highest,the resistance to glutaraldehyde is the second,and the resistance to peroxyacetic acid is the lowest.3.The results of polymerase chain reaction showed that 4 out of 22 test strains were positive for qac E △ 1-sul1 gene,and the positive rate was 18.18%.4.There is no obvious relationship between the MIC and MBC values of the test strains biofilms with positive or negative qac EΔ1-sul1 gene between three disinfectants.5.The parallel test and the series test showed that there was no obvious relationship between the resistance group and the non-resistance group test strains biofilms with the positive rate of qac E△1-sul1. |
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