The Determination And Correlation Of Various Virulence Genes,ESBL,Serum Bactericidal Effect And Biofilm Formation Of Clinically Isolated CKP And HvKP And Evaluate The Effect Of NAC And SAL On CPS Biosynthesis

1.The determination and correlation of various virulence genes,ESBL,serum bactericidal effect and biofilm formation of clinically isolated c KP and hv KP from respiratory tract infected patientsBack ground:The Klebsiella pneumoniae strains that are commonly recognized by clinicians and microbiologists are termed as classic K.pneumoniae(c KP).A capsule-associated mucopolysaccharide web is known as hypervirulent Klebsiella pneumoniae(hv KP)as it enhances the serum resistant and biofilm production.Aim was to determine and correlate of various virulence genes,ESBL,serum bactericidal effect and biofilm formation of clinical isolated c KP and hv KP from respiratory tract infected patients.Materials and Methods:A total of 96 K.pneumoniae strains were isolated from sputum of Respiratory tract infected patients.The isolates were performed string test,antimicrobial susceptibility test and ESBLs.ESBL and virulence gene of K.pneumoniae were amplified by PCR.Serum bactericidal and biofilm assays were performed by NHS and Microtiter plate method respectively.Results: Out of 96 isolated,39 isolates(40.6%)were identified with HV phenotypes on the basis of modified string test.The number of c KP strains exhibiting resistance to the tested antimicrobials was significantly higher than that of the hv KP strains.ESBL were identified in more number of c KP strains(28/57 [49.1%])than in hv KP strains(11/39 [28.2%])(P <.040).Moreover,single and multiple types of ESBL gene were present in c KP strains,whereas,only single gene was present in hv KP strains.The prevalence of virulence associated gene of K.pneumoniae as K1,K2,Rmp A,Uge,Kfu and Aerobactin were strongly associated with hv KP strains than c KP strains.However,no significant difference was found in FIM-1 and Mr KD3 genes.ESBL producingc KP and hv KP were significantly associated with strong biofilm formation(both P < 0.05)and highly associated with bactericidal effect of serum(both P < 0.05)than c KP strains.However,neither biofilm formation nor bactericidal effect of serum was found with significant difference in between ESBL producing c KP and ESBL producing hv KP strains(both P > 0.05).Conclusion: Although the hv KP possess more virulence gene,but there didn't show any significant difference between biofilm formation and bactericidal effect of serum compared with ESBL producing c KP strains.2.The determination of effect of sodium salicylate and N-acetylcystine on Capsular polysaccharide biosynthesis,biofilm formation and preformed biofilm of K1 and K2 gene of hypervirulent Klebsiella pneumoniae(hv KP)from Respiratory tract infected patientsBack ground:The strains of K.pneumoniae carry tremendous CPS loads that could protect the bacteria from phagocytosis,kill by serum factors and enhances the biofilm production.There is a high need of a chemotherapeutic agent that reduces the production of CPS and thus can be effective as an adjunct therapy for the infections that are caused by HV strains of K.pneumoniae.The main aim of the experiment is to assess the effect of(SAL)and N-acetylcystine(NAC)on biosynthesis of capsular polysaccharide,biofilm formation and preformed biofilm of hv KP.Materials and methods:Clinical isolate of K1 and K2 serotypes of hv KP strains were used in the following experiments: the effect of SAL and NAC on hypermucoviscosity phenotype,CPS production,K1 and K2 gene expression and biofilm formation and on preformed biofilm.Results:SAL significantly reduced the bacterial hypermucoviscosity and the CPS levels on concentration dependent manner.Furthermore,RT-PCR analysis showed that SAL downregulated the m RNA levels of cps biosynthesis genes.NAC neither affected the mucoviscosity and CPS production and nor the gene expression.However,both SAL and NAC inhibited the biofilm formation and eradicated the pre-formed biofilm.SAL inhibited biofilm formation by 48% at concentration(5 ?g/ml)and 84% at concentration(100 ?g/ ml)and it disrupted the pre-formed biofilm by 12% at concentration(5 ?g/ml)and 51% at concentration(100?g/ml).NAC inhibited biofilm formation by 9% and 26% at concentration(0.5 and 1 mg/ml)and disrupted the pre-formed biofilm by 38% and 49% at concentration(0.5 and 1 mg/ml)respectively.Thus,the NAC is more active to eradicated the mature pre-formed biofilm than inhibited biofilm formation.The combination of SAL and NAC showed that the percent of reduction in biofilm formation or eradication was significantly increased with all combinations.Conclusion:SAL decreases the formation of biofilm but has less effect on eradication of mature pre-formed biofilm.NAC disrupts the mature biofilm but has less effect on the formation of biofilm of hv KP.Therefore,the combination of both NAC and SAL has shown the best results for the reduction in biofilm formation and eradication of preformed biofilm than alone NAC or SAL.