Research Article| Volume 57, ISSUE 2, P309-315, March 2018

Appraisal of Biofilm Formation in Diabetic Foot Infections by Comparing Phenotypic Methods With the Ultrastructural Analysis


      Diabetic patients are more prone to the development of foot ulcers, because their underlying tissues are exposed to colonization by various pathogenic organisms. Hence, biofilm formation plays a vital role in disease progression by antibiotic resistance to the pathogen found in foot infections. The present study has demonstrated the correlation of biofilm assay with the clinical characteristics of diabetic foot infection. The clinical characteristics such as the ulcer duration, size, nature, and grade were associated with biofilm production. Our results suggest that as the size of the ulcer with poor glycemic control increased, the organism was more likely to be positive for biofilm formation. A high-degree of antibiotic resistance was exhibited by the biofilm-producing gram-positive isolates for erythromycin and gram-negative isolates for cefpodoxime. Comparisons of biofilm production using 3 different conventional methods were performed. The strong producers with the tube adherence method were able to produce biofilm using the cover slip assay method, and the weak producers in tube adherence method had difficulty in producing biofilm using the other 2 methods, indicating that the tube adherence method is the best method for assessing biofilm formation. The strong production of biofilm with the conventional method was further confirmed by scanning electron microscopy analysis, because bacteria attached as a distinct layer of biofilm. Thus, the high degree of antibiotic resistance was exhibited by biofilm producers compared with nonbiofilm producers. The tube adherence and cover slip assay were found to be the better method for biofilm evaluation.

      Level of Clinical Evidence


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to The Journal of Foot and Ankle Surgery
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Hefni A.A.
        • Ibrahim A.M.R.
        • Attia K.M.
        • Moawad M.M.
        • El-ramah A.F.
        • Shahin M.M.
        • Al-Molla M.
        • Al-Satar L.A.
        Bacteriological study of diabetic foot infection in Egypt.
        J Arab Soc Med Res. 2013; 8: 26-32
        • Smith K.
        • Collier A.
        • Townsend E.M.
        • O'Donnell L.E.
        • Abhijit B.M.
        • Butcher J.
        • Mackay W.G.
        • Ramage G.
        • Williams C.
        One step closer to understanding the role of bacteria in diabetic foot ulcers: characterizing the microbiome of ulcers.
        BMC Microbiol. 2016; 16: 54
        • Clokie M.
        • Greenway A.L.
        • Harding K.
        • Jones N.J.
        • Vedhara K.
        • Game F.
        • Dhatariya K.K.
        New horizons in the understanding of the causes and management of diabetic foot disease: report from the 2017 Diabetes UK Annual Professional Conference Symposium.
        Diabet Med. 2017; 34: 305-315
        • Babapour E.
        • Haddadi A.
        • Mirnejad R.
        • Angaji S.-A.
        • Amirmozafari N.
        Biofilm formation in clinical isolates of nosocomial Acinetobacter baumannii and its relationship with multidrug resistance.
        Asian Pac J Trop Biomed. 2016; 6: 528-533
        • Omar A.
        • Wright B.J.
        • Schultz G.
        • Burrell R.
        • Nadworny P.
        Microbial biofilms and chronic wounds.
        Microorganisms. 2017; 5: 1-15
        • Phillips P.L.
        • Wolcott R.D.
        • Fletcher J.
        • Schultz G.S.
        Biofilms made easy.
        Wounds Int. 2010; 1: 1-6
        • Tsuneda S.
        • Aikawa H.
        • Hayashi H.
        • Yuasa A.
        • Hirata A.
        Extracellular polymeric substances responsible for bacterial adhesion onto solid surface.
        FEMS Microbiol Lett. 2003; 223: 287-292
        • Kim L.
        Riddle of biofilm resistance.
        Antimicrob Agents Chemother. 2001; 45: 999-1007
        • Wilson M.
        • Devine D.A.
        Medical Implications of Biofilms.
        Cambridge University Press, Cambridge, UK2003
        • Nasr R.A.
        • AbuShady H.M.
        • Hussein S.H.
        Biofilm formation and presence of icaAD gene in clinical isolates of staphylococci.
        Egypt J Med Hum Genet. 2012; 13: 269-274
        • Oliveira A.
        • de Lourdes RS Cunha M.
        Comparison of methods for the detection of biofilm production in coagulase-negative staphylococci.
        BMC Res Notes. 2010; 3: 260
        • Hoiby N.
        • Bjarnsholt T.
        • Givskov M.
        • Molin S.
        • Ciofu O.
        Antibiotic resistance of bacterial biofilms.
        Int J Antimicrob Agents. 2010; 35: 322-332
        • Han A.
        • Zenilman J.M.
        • Melendez J.H.
        • Shirtliff M.E.
        • Agostinho J.G.
        • Stewart P.S.
        • Mongodin E.F.
        • Rao D.
        • Rickard A.H.
        • Lazarus G.S.
        The importance of a multifaceted approach to characterizing the microbial flora of chronic wounds.
        Wound Repair Regen. 2011; 19: 532-541
        • Saginur R.
        • St Denis M.
        • Ferris W.
        • Aaron S.D.
        • Chan F.
        • Lee C.
        • Ramotar K.
        Multiple combination bactericidal testing of Staphylococcal biofilms from implant-associated infections.
        Antimicrob Agents Chemother. 2006; 50: 55-61
        • Curtis Jones E.
        • John Kennedy P.
        Treatment options to manage wound biofilm.
        Adv Wound Care (New Rochelle). 2012; 1: 120-126
        • Rhoads D.
        • Wolcott R.D.
        • Sun Y.
        • Scot Dowd E.
        Comparison of culture and molecular identification of bacteria in chronic wounds.
        Int J Mol Sci. 2012; 13: 2535-2550
        • Swarna S.R.
        • Radha Madhavan S.
        • Devaraj G.
        • Thamaraiselvi S.
        A study of biofilm on diabetic foot ulcer.
        Int J Res Pharma Biomed Sci. 2012; 3: 1809-1814
        • Wagner F.W.
        The vascular foot, a system of diagnosis and treatment.
        Foot Ankle. 1981; 2: 64-122
        • Holt J.G.
        Bergey's Manual of Determinative Bacteriology.
        ed 9. Lippincott, Williams & Wilkins, Philadelphia, PA2000
        • Clinical Laboratory Standards Institute
        ed 11. Performance Standards for Antimicrobial Susceptibility Testing. Vol. 32, No. 3. Clinical Laboratory Standards Institute, Wayne, PA2012
        • Christensen G.D.
        • Simpson W.A.
        • Bisno A.L.
        • Beachy E.H.
        Adherence of biofilm producing strains of Staphylococci epidermidis to smooth surfaces.
        Infect Immun. 1982; 37: 318-326
        • Freeman D.J.
        • Falkiner F.R.
        • Keane C.T.
        New method of detecting slime production by coagulase negative Staphylococci.
        J Clin Pathol. 1989; 42: 84-87
        • Mathur T.
        • Singhal S.
        • Khan S.
        • Upadhyay D.J.
        • Fatma T.
        • Rattan A.
        Detection of biofilm formation among the clinical isolates of Staphylococci: an evaluation of three different screening methods.
        Indian J Med Microbiol. 2006; 24: 25-29
        • James G.A.
        • Swogger E.
        • Wolcott R.
        Biofilms in chronic wounds.
        Wound Repair Regen. 2008; 16: 37-44
        • Wolcott R.D.
        • Gontcharova V.
        • Sun Y.
        • Zischkau A.M.
        • Dowd S.E.
        Bacterial diversity in surgical site infections: not just aerobic cocci anymore.
        J Wound Care. 2009; 18: 317-323
        • Abdulrazak A.
        • Bitar Z.I.
        • Al Shamali A.A.
        • Ahmed Mobasher L.
        Bacteriological study of diabetic foot infections.
        J Diabetes Complications. 2005; 19: 138-141
        • Viswanathan V.
        • Jasmine J.J.
        • Snehalatha C.
        • Ramachandran A.
        Prevalence of pathogens in diabetic foot infection in South India type 2 diabetic patients.
        J Assoc Physicians India. 2002; 50: 1013-1016
        • Sharma V.K.
        • Khakda P.B.
        • Joshi A.
        • Sharma R.
        The common pathogens which were isolated from diabetic foot infections in Bir hospital.
        Kathmandu Univ Med J. 2006; 4: 295-301
        • Sivanmaliappan T.S.
        • Sevanan M.
        Antimicrobial susceptibility patterns of Pseudomonas aeruginosa from diabetes patients with foot ulcers.
        Int J Microbiol. 2011; : 1-4
        • Mottola C.
        • Matias C.S.
        • Mendes J.J.
        • Melo-Cristino J.
        • Tavares L.
        • Cavaco-Silva P.
        • Oliveira M.
        Susceptibility patterns of Staphylococcus aureus biofilms in diabetic foot infections.
        BMC Microbiol. 2016; 16: 119
        • Zubair M.
        • Malik A.
        • Ahmad J.
        Clinico-microbiological study and antimicrobial drug resistance profile of diabetic foot infections in North India.
        Foot (Edinb). 2011; 21: 6-14
        • Zubair M.
        • Malik A.
        • Ahmad J.
        The impact of creatinine clearance on the outcome of diabetic foot ulcers in north Indian tertiary care hospital.
        Diabetes Metab Syndr. 2011; 5: 120-125
        • Stepanovic S.
        • Vukovic D.
        • Dakic I.
        • Savic B.
        • Svabic Vlahovic M.
        A modified microtitre-plate test for quantification of staphylococcal biofilm formation.
        J Microbiol Methods. 2000; 40: 175-179
        • Taj Aldeen S.J.
        • Gene J.
        • Al Bozom I.
        • Buzina W.
        • Cano J.F.
        • Guarro J.
        Gangrenous necrosis of the diabetic foot caused by Fusarium acutatum.
        Med Mycol. 2006; 44: 547-552
        • Croes S.
        • Deurenberg R.H.
        • Boumans M.L.
        • Beisser P.S.
        • Neef C.
        • Stobberingh E.E.
        Staphylococcus aureus biofilm formation at the physiologic glucose concentration depends on the S. aureus lineage.
        BMC Microbiol. 2009; 9: 229
        • Sugandhi P.
        • Prasanth D.A.
        Spectrum of microbial infections in diabetic foot ulcers.
        Indo Am J Pharm Res. 2014; 4: 3428-3434
        • Maclean L.C.W.
        • Tyliszczak T.
        • Gilbert P.U.
        A high-resolution chemical and structural study of framboidal pyrite formed within a low temperature bacterial biofilm.
        Geobiology. 2008; 6: 471-480
        • Wallace P.K.
        • Arey B.
        • Mahaffee W.F.
        Subsurface examination of a foliar biofilm using scanning electron and focused-ion-beam microscopy.
        Micron. 2011; 42: 579-585
        • Smith K.
        • Perez A.
        • Ramage G.
        • Lappin D.
        • Gemmell C.G.
        • Lang S.
        Biofilm formation by Scottish clinical isolates of Staphylococcus aureus.
        J Med Microbiol. 2008; 57: 1018-1023
        • Wu H.
        • Moser C.
        • Wang H.Z.
        • Hoiby N.
        • Song Z.-J.
        Strategies for combating bacterial biofilm infections.
        Int J Oral Sci. 2015; 7: 1-7
        • Sun F.
        • Qu F.
        • Ling Y.
        • Mao P.
        • Xia P.
        • Chen H.
        • Zhou D.
        Biofilm-associated infections: antibiotic resistance and novel therapeutic strategies.
        Future Microbiol. 2013; 8: 877-886