Study On One Mutant Of Pseudomonas Aeruginosa Which Show Increased Antibiotics Resistance To Carbapenems

Pseudomonas aeruginosa is one of the three common nosocomial opportunistic pathogens in hospitals. According to the survey of CHINET bacterial resistance, P. aeruginosa is the second largest Gram negative pathogenic bacteria which could infect immunocompromised patients in China. P. aeruginosa show strong resistance to a variety of antimicrobials due to its intrinsic and acquired resistance machnisms. It brings great challenge to the clinic treatment.BK3 is a transposon mutant of PAO1 which showed an increased resistance to carbapenems. The transporson has been inserted in the intergenic region between PA4639 and PA4638 according to the results of random PCR and sequencing analysis. PA4639 and PA4638 are in the same operon.Mutants of PAO1(A4639-4638), PAO1(A4639) and PAO1(A4638) have been constructed together with the overexpression strains of PAO1(pAK4639-4638) PAO1(pAK4639) and PAO1(pAK4638). The sensitivity of the strains to three kinds of carbapenems were determined by the plate diffusion method. The results showed that increased antibiotics resistance of BK3 is caused by overexpression of PA4638-46339) and PA4639 plays more important role.The protein of PA4639 coded contains a Ⅱ type of signal peptide and lipobox through the analysis of YRCPDR, TMHMM and NCBI-CDD. The results show that it is a hypothesis lipoprotein which belongs to lipoprotein-16-super family. The family includes YajG. Furthermore, this hypothesis lipoprotein had been found in OMVs of PAO1 by microarray technology in other reported study. So PA4639 could be associated with OMVs.The activity of the β-Lactamase in the extracellular space has been increased in PAO1(pAK4639-4638) in which PA4639-4638 had been overexpressed. Disk synergy test shows that the increased β-Lactamases are neither ESBLs nor MBLs. However, the activity of the β-Lactamase ΔampC(pAK4639-4638) is not increased, this result revealed that the overexpression of PA4639-4638 increase the activity of AmpC. On the other hand, PAO1ΔampC(pAK4639) still showed increased resistance to carbapenems compared with PAO1ΔampC (pAK1900). The result indicated that overexpression of PA4639 also induce other resistance mechanisms which needs further investigation.The virulence of PAO1 has been decreased when PA4639 was overexpressed in plant infection model. According to the previous report, PA4639 could be associated with OMVs which transports virulence factors from intracellular to extracellular. Lipoproteins could decrease the production of outer membrane vesicle (OMVs) according to the previous report. Therefore the decreased virulence of PAOl(pAK4639-4638) could probably be caused by the decreased production of OMVs from the overexpression of PA4639 which codes a potential lipoproteins. Those need further research.