1887

Abstract

Multidrug-resistant has emerged as one of the deadliest opportunistic nosocomial pathogens that forms biofilm for the establishment of chronic infections. Herein, we made an attempt to identify the genes involved in biofilm formation in the strain ATCC13883. To achieve this, we constructed mini-Tn5 transposon insertion mutants and screened them for biofilm production. We observed that the biofilm formation was enhanced in the mutant where the gene was disrupted. WcaJ is the initiating enzyme of colanic acid synthesis and loads the first sugar (glucose-1-P) on the lipid carrier undecaprenyl phosphate. The absence of this glycosyltransferase results in the absence of colanic acid, which renders a non-mucoid phenotype to the mutant. Further, to determine the effect of mucoidy on antibiotic susceptibility, we tested the sensitivity of the strains towards different groups of antibiotics. Unlike the mucoid strains, the resistance of the non-mucoid cells was greater for polymyxins, but less for quinolones. Capsular polysaccharides are known to have a protective effect against phagocytosis, therefore we assessed the role of colanic acid in virulence by conducting infection studies on murine macrophages. Surprisingly, the Δ strain was less efficient in macrophage activation and was not readily phagocytosed. Thus, the presence of colanic acid appeared to increase the immunogenicity of . Overall, the results indicate that the presence of colanic acid increases the vulnerability of towards both polymyxins and macrophages, implying that the mucoid strains are less threatening as compared to their high biofilm forming non-mucoid counterparts.

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/content/journal/micro/10.1099/mic.0.000827
2019-08-01
2019-08-20
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References

  1. Rice LB. Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE. J Infect Dis 2008;197:1079–1081 [CrossRef]
    [Google Scholar]
  2. Flemming HC, Wingender J. The biofilm matrix. Nat Rev Microbiol 2010;8:623–633 [CrossRef]
    [Google Scholar]
  3. Balestrino D, Ghigo JM, Charbonnel N, Haagensen JAJ, Forestier C. The characterization of functions involved in the establishment and maturation of Klebsiella pneumoniae in vitro biofilm reveals dual roles for surface exopolysaccharides. Environ Microbiol 2008;10:685–701 [CrossRef]
    [Google Scholar]
  4. Stevenson G, Andrianopoulos K, Hobbs M, Reeves PR. Organization of the Escherichia coli K-12 gene cluster responsible for production of the extracellular polysaccharide colanic acid. J Bacteriol 1996;178:4885–4893 [CrossRef]
    [Google Scholar]
  5. Podschun R, Fischer A, Ullman U. Expression of putative virulence factors by clinical isolates of Klebsiella planticola. J Med Microbiol 2000;49:115–119 [CrossRef]
    [Google Scholar]
  6. Ren G, Wang Z, Li Y, Hu X, Wang X. Effects of lipopolysaccharide core sugar deficiency on colanic acid biosynthesis in Escherichia coli. J Bacteriol 2016;198:1576–1584 [CrossRef]
    [Google Scholar]
  7. Patel KB, Toh E, Fernandez XB, Hanuszkiewicz A, Hardy GG et al. Functional characterization of UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferases of Escherichia coli and Caulobacter crescentus. J Bacteriol 2012;194:2646–2657 [CrossRef]
    [Google Scholar]
  8. Chaput C, Spindler E, Gill RT, Zychlinsky A. O-antigen protects gram-negative bacteria from histone killing. PLoS One 2013;8:e71097 [CrossRef]
    [Google Scholar]
  9. Zhang X-S, García-Contreras R, Wood TK. Escherichia coli transcription factor YncC (McbR) regulates colanic acid and biofilm formation by repressing expression of periplasmic protein YbiM (McbA). ISME J 2008;2:615–631 [CrossRef]
    [Google Scholar]
  10. Manat G, Roure S, Auger R, Bouhss A, Barreteau H et al. Deciphering the metabolism of undecaprenyl-phosphate: the bacterial cell-wall unit carrier at the membrane frontier. Microb Drug Resist 2014;20:199–214 [CrossRef]
    [Google Scholar]
  11. Matatov R, Goldhar J, Skutelsky E, Sechter I, Perry R et al. Inability of encapsulated Klebsiella pneumoniae to assemble functional type 1 fimbriae on their surface. FEMS Microbiol Lett 1999;179:123–130 [CrossRef]
    [Google Scholar]
  12. Ranjit DK, Young KD. Colanic acid intermediates prevent De Novo shape recovery of Escherichia coli spheroplasts, calling into question biological roles previously attributed to colanic acid. J Bacteriol 2016;198:1230–1240 [CrossRef]
    [Google Scholar]
  13. Danese PN, Pratt LA, Kolter R. Exopolysaccharide production is required for development of Escherichia coli K-12 biofilm architecture. J Bacteriol 2000;182:3593–3596 [CrossRef]
    [Google Scholar]
  14. Yu M, Xu Y, Xu T, Wang B, Sheng A et al. WcaJ, the initiating enzyme for colanic acid synthesis, is required for lipopolysaccharide production, biofilm formation and virulence in Edwardsiella tarda. Aquaculture 2015;437:287–291 [CrossRef]
    [Google Scholar]
  15. Huang TW, Lam I, Chang HY, Tsai SF, Palsson BO et al. Capsule deletion via a λ-Red knockout system perturbs biofilm formation and fimbriae expression in Klebsiella pneumoniae MGH 78578. BMC Res Notes 2014;7:13 [CrossRef]
    [Google Scholar]
  16. Davey ME, Duncan MJ. Enhanced biofilm formation and loss of capsule synthesis: deletion of a putative glycosyltransferase in Porphyromonas gingivalis. J Bacteriol 2006;188:5510–5523 [CrossRef]
    [Google Scholar]
  17. Ledeboer NA, Jones BD. Exopolysaccharide sugars contribute to biofilm formation by Salmonella enterica serovar typhimurium on HEp-2 cells and chicken intestinal epithelium. J Bacteriol 2005;187:3214–3226 [CrossRef]
    [Google Scholar]
  18. Lopez-Torres AJ, Stout V. Role of colanic acid polysaccharide in serum resistance in vivo and in adherence. Curr Microbiol 1996;33:383–389 [CrossRef]
    [Google Scholar]
  19. Biswas S, Brunel JM, Dubus JC, Reynaud-Gaubert M, Rolain JM. Colistin: an update on the antibiotic of the 21st century. Expert Rev Anti Infect Ther 2012;10:917–934 [CrossRef]
    [Google Scholar]
  20. Beiter K, Wartha F, Hurwitz R, Normark S, Zychlinsky A et al. The capsule sensitizes Streptococcus pneumoniae to alpha-defensins human neutrophil proteins 1 to 3. Infect Immun 2008;76:3710–3716 [CrossRef]
    [Google Scholar]
  21. Spinosa MR, Progida C, Talà A, Cogli L, Alifano P et al. The Neisseria meningitidis capsule is important for intracellular survival in human cells. Infect Immun 2007;75:3594–3603 [CrossRef]
    [Google Scholar]
  22. Meredith TC, Mamat U, Kaczynski Z, Lindner B, Holst O et al. Modification of lipopolysaccharide with colanic acid (M-antigen) repeats in Escherichia coli. J Biol Chem 2007;282:7790–7798 [CrossRef]
    [Google Scholar]
  23. Fàbrega A, Madurga S, Giralt E, Vila J. Mechanism of action of and resistance to quinolones. Microb Biotechnol 2009;2:40–61 [CrossRef]
    [Google Scholar]
  24. Allen PM, Fisher D, Saunders JR, Hart CA. The role of capsular polysaccharide K21b of Klebsiella and of the structurally related colanic-acid polysaccharide of Escherichia coli in resistance to phagocytosis and serum killing. J Med Microbiol 1987;24:363–370 [CrossRef]
    [Google Scholar]
  25. Wang S, Shi H, Li Y, Shi Z, Zhang X et al. A colanic acid operon deletion mutation enhances induction of early antibody responses by live attenuated Salmonella vaccine strains. Infect Immun 2013;81:3148–3162 [CrossRef]
    [Google Scholar]
  26. Hancock RE, Diamond G. The role of cationic antimicrobial peptides in innate host defences. Trends Microbiol 2000;8:402–410 [CrossRef]
    [Google Scholar]
  27. Wyres KL, Wick RR, Gorrie C, Jenney A, Follador R et al. Identification of Klebsiella capsule synthesis loci from whole genome data. Microb Genom 2016;2:e000102 [CrossRef]
    [Google Scholar]
  28. Römling U, Sierralta WD, Eriksson K, Normark S. Multicellular and aggregative behaviour of Salmonella typhimurium strains is controlled by mutations in the agfD promoter. Mol Microbiol 1998;28:249–264 [CrossRef]
    [Google Scholar]
  29. Ferrières L, Hémery G, Nham T, Guérout AM, Mazel D et al. Silent mischief: bacteriophage Mu insertions contaminate products of Escherichia coli random mutagenesis performed using suicidal transposon delivery plasmids mobilized by broad-host-range RP4 conjugative machinery. J Bacteriol 2010;192:6418–6427 [CrossRef]
    [Google Scholar]
  30. Larsen R, Wilson M, Guss A, Metcalf W. Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria. Arch Microbiol 2002;178:193–201 [CrossRef]
    [Google Scholar]
  31. Kleiner D, Paul W, Merrick MJ. Construction of multicopy expression vectors for regulated over-production of proteins in Klebsiella pneumoniae and other enteric bacteria. Microbiology 1988;134:1779–1784 [CrossRef]
    [Google Scholar]
  32. Obadia B, Lacour S, Doublet P, Baubichon-Cortay H, Cozzone AJ et al. Influence of tyrosine-kinase Wzc activity on colanic acid production in Escherichia coli K12 cells. J Mol Biol 2007;367:42–53 [CrossRef]
    [Google Scholar]
  33. Nelson DE, Young KD. Penicillin binding protein 5 affects cell diameter, contour, and morphology of Escherichia coli. J Bacteriol 2000;182:1714–1721 [CrossRef]
    [Google Scholar]
  34. Kumar A, Mallik D, Pal S, Mallick S, Sarkar S et al. Escherichia coli O8-antigen enhances biofilm formation under agitated conditions. FEMS Microbiol Lett 2015;362:fnv112 [CrossRef]
    [Google Scholar]
  35. Naves P, del Prado G, Huelves L, Gracia M, Ruiz V et al. Measurement of biofilm formation by clinical isolates of Escherichia coli is method-dependent. J Appl Microbiol 2008;105:585–590 [CrossRef]
    [Google Scholar]
  36. Hammar M, Arnqvist A, Bian Z, Olsén A, Normark S. Expression of two csg operons is required for production of fibronectin- and Congo red-binding curli polymers in Escherichia coli K-12. Mol Microbiol 1995;18:661–670 [CrossRef]
    [Google Scholar]
  37. CLSI Twenty-Second Informational Supplement Performance standards for antimicrobial susceptibility testing, CLSI document M100-S22. 2012
    [Google Scholar]
  38. March C, Cano V, Moranta D, Llobet E, Pérez-Gutiérrez C et al. Role of bacterial surface structures on the interaction of Klebsiella pneumoniae with phagocytes. PLoS One 2013;8:e56847-ee56847 [CrossRef]
    [Google Scholar]
  39. Devi KSP, Behera B, Mishra D, Maiti TK. Immune augmentation and Dalton's Lymphoma tumor inhibition by glucans/glycans isolated from the mycelia and fruit body of Pleurotus ostreatus. Int Immunopharmacol 2015;25:207–217 [CrossRef]
    [Google Scholar]
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