Klebsiella pneumoniae yfiRNB operon affects biofilm formation, polysaccharide production and drug susceptibility

Abstract

Klebsiella pneumoniae is an opportunistic pathogen important in hospital-acquired infections, which are complicated by the rise of drug-resistant strains and the capacity of cells to adhere to surfaces and form biofilms. In this work, we carried out an analysis of the genes in the K. pneumoniae yfiRNB operon, previously implicated in biofilm formation. The results indicated that in addition to the previously reported effect on type 3 fimbriae expression, this operon also affected biofilm formation due to changes in cellulose as part of the extracellular matrix. Deletion of yfiR resulted in enhanced biofilm formation and an altered colony phenotype indicative of cellulose overproduction when grown on solid indicator media. Extraction of polysaccharides and treatment with cellulase were consistent with the presence of cellulose in biofilms. The enhanced cellulose production did not, however, correlate with virulence as assessed using a Caenorhabditis elegans assay. In addition, cells bearing mutations in genes of the yfiRNB operon varied with respect to the WT control in terms of susceptibility to the antibiotics amikacin, ciprofloxacin, imipenem and meropenem. These results indicated that the yfiRNB operon is implicated in the production of exopolysaccharides that alter cell surface characteristics and the capacity to form biofilms -a phenotype that does not necessarily correlate with properties related with survival, such as resistance to antibiotics.

Increased biofilm formation ability in Klebsiella pneumoniae after short‐term exposure to a simulated microgravity environment

“…; Huertas, Zarate et al. ). Therefore, the expressions of the cellulose synthase‐encoding gene bcsA and type 3 fimbriae‐encoding genes mrkABCDF and its regulatory gene mrkH were selected for further analysis through RNA‐seq and qRT‐PCR.…”

“…Two milliliters of 50 mmol/L citrate buffer (pH 4.6) with 0.1% cellulase (Sigma Chemical Co., St. Louis, Mo, USA) were added to the wells, which were then incubated at 45°C for 48 h (Huertas, Zarate et al. ). The biofilms were stained with crystal violet and quantified through spectrometry.…”

Structural insights into the regulatory mechanism of the Pseudomonas aeruginosa YfiBNR system

“…The YfiBNR system contains three protein members and modulates intracellular c-di-GMP levels in response to signals received in the periplasm (Malone et al, 2010 ). More recently, this system was also reported in other Gram-negative bacteria, such as Escherichia coli (Hufnagel et al, 2014 ; Raterman et al, 2013 ; Sanchez-Torres et al, 2011 ), Klebsiella pneumonia (Huertas et al, 2014 ) and Yersinia pestis (Ren et al, 2014 ). YfiN is an integral inner-membrane protein with two potential transmembrane helices, a periplasmic Per-Arnt-Sim (PAS) domain, and cytosolic domains containing a HAMP domain (mediate input-output signaling in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and a C-terminal GGDEF domain indicating a DGC’s function (Giardina et al, 2013 ; Malone et al, 2010 ).…”

“…The YfiBNR system provides a good example of a delicate homeostatic system that integrates multiple signals to regulate the c-di-GMP level. Homologs of the YfiBNR system are functionally conserved in P. aeruginosa (Malone et al, 2012 ; Malone et al, 2010 ), E. coli (Hufnagel et al, 2014 ; Raterman et al, 2013 ; Sanchez-Torres et al, 2011 ), K. pneumonia (Huertas et al, 2014 ) and Y. pestis (Ren et al, 2014 ), where they affect c-di-GMP production and biofilm formation. The mechanism by which activated YfiB relieves the repression of YfiN may be applicable to the YfiBNR system in other bacteria and to analogous outside-in signaling for c-di-GMP production, which in turn may be relevant to the development of drugs that can circumvent complicated antibiotic resistance.…”

The effect of aminoglycosides on horizontal gene transfer in Klebsiella pneumoniae

“…Kpn CH404 LM21 SHV-1: aadA7-gfpmut3; Spc R (Balestrino, et al, 2008) Kpn 16Kp1 Clinical isolate with bla NDM-1 encoding plasmid (Escobar-Pérez, et al, 2013) Kpn ATCC700603 Conjugation control (Rasheed, et al, 2000) E. coli J53 Az r (Fmet pro) (Jacoby, et al, 1996) Plasmids pUC19 ORI pMB1 derivative; Amp R (Yanisch -Perron, et al, 1985) pBAD18-yfiN ORI pMB1; Cam R (Huertas, et al, 2014) pMP2444 ORI pBBR1; egfp under control of the tac promoter; Gen R (Bloemberg, et al, 2000) pNDM-1 WT plasmid, 177Kb, IncA/C ORI; (bla NDM-1 and bla CARB-2 ); Resistant to β-lactams, aminoglycosides, sulphonamides, macrolides, lincosamide, streptogramin B, Cam (Escobar Pérez, et al, 2013) analyze the effect of antibiotics on exogenous DNA uptake, Kpn CH404 was grown in the presence of sub-lethal concentrations of the antibiotics Amk, Amp, Imp, Gen, Kan, Spc, Cip, and TS for 6 h at 37° C before precipitating cells by centrifugation and mixing with the DNA.…”