The influence of biofilm formation on carbapenem resistance in clinicalKlebsiella pneumoniaeinfections: phenotype vs genome-wide analysis
Authors: Ragupathi Naveen Kumar Devanga , Sethuvel Dhiviya Prabaa Muthuirulandi , Dwarakanathan Hariharan Triplicane , Murugan Dhivya , Umashankar Yamini , Monk Peter N. , Karunakaran Esther , Veeraraghavan Balaji 
Published: July 2020

Klebsiella pneumoniaeis one of the leading causes of nosocomial infections. Carbapenem-resistant (CR)K. pneumoniaeare on the rise in India. The biofilm forming ability ofK. pneumoniae further complicates patient management. There is still a knowledge gap on the association of biofilm formation with patient outcome and carbapenem susceptibility, which is investigated in the present study.

K. pneumoniaeisolates from patients admitted in critical care units with catheters and ventilators were included.K. pneumoniae(n= 72) were tested for antimicrobial susceptibility as recommended by CLSI 2019 and subjected to 96-well microtitre plate biofilm formation assay. Based on optical density at 570 nm isolates were graded as strong, moderate and weak biofilm formers. Subset of strong biofilm formers were subjected to whole genome sequencing and a core genome phylogenetic analysis in comparison with global isolates were performed. Biofilm formation was compared for an association with the carbapenem susceptibility and with patient outcome. Statistical significance, correlations and graphical representation were performed using SPSS v23.0.

Phenotypic analyses showed a positive correlation between biofilm formation and carbapenem resistance. Planktonic cells observed to be susceptiblein vitroexhibited higher MICs in biofilm structure. The biofilm forming ability had a significant association with the morbidity/mortality. Infections by stronger biofilm forming pathogens significantly (P<0.05) resulted in fewer ‘average days alive’ for the patient (3.33) in comparison to those negative for biofilms (11.33). Phylogenetic analysis including global isolates revealed the clear association of sequence types with genes for biofilm mechanism and carbapenem resistance. Carbapenemase genes were found specific to each clade. The known hypervirulent clone-ST23 withwcaG,magA,rmpA,rmpA2andwzcwith a lack of mutation for hyper-capsulation might be poor biofilm formers. Interestingly, ST15, ST16, ST307 and ST258 – reported global high-risk clones werewcaJnegative indicating the high potential of biofilm forming capacity. GeneswabGandtreCfor CPS,bcsAandpgaCfor adhesins,luxSfor quorum sensing were common in all clades in addition to genes for aerobactin (iutA), allantoin (allS), type I and III fimbriae (fimA,fimH,mrkD) and pili (pilQ,ecpA).

This study is the first of its kind to compare genetic features of antimicrobial resistance with a spectrum covering most of the genetic factors forK. pneumoniaebiofilm. These results highlight the importance of biofilm screening to effectively manage nosocomial infections byK. pneumoniae. Further, data obtained on epidemiology and associations of biofilm and antimicrobial resistance genetic factors will serve to enhance our understanding on biofilm mechanisms inK. pneumoniae.