Evaluation of Biofilm Formation and Frequency of Multidrug-resistant and Extended Drug-resistant Strain in Pseudomonas aeruginosa Isolated from Burn Patients in Isfahan

Document Type : Original Article


1 Department of Microbiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

2 Department of Infectious Disease Research, Imammosa Kazem Hospital, Isfahan University of Medical Sciences, Isfahan, Iran


Background: Pseudomonas aeruginosa is a biofilm-forming bacterium which can result in serious health problems, particularly in burn patients. Biofilm has been assumed to protect the bacteria from environmental fluctuations such as antimicrobial agent. Mucoid strains generate extensive levels of the alginate exopolysaccharide, which is an important factor of its biofilm. Materials and Methods: Totally, 100 isolates of P. aeruginosa has been gathered from wound infections of burn patients. Polymerase chain reaction of exoA gene has been carried out to confirm the bacteriologic identification of isolates. The biofilm-forming capacity has been specified by capsule staining and microtiter plate test as qualitative and quantitative determination, respectively. Antimicrobial susceptibility of the isolates has been specified by disk diffusion method. Results: All the isolates carried the exoA gene. The antibiotic resistance was imipenem (90%); levofloxacin (93%); aztreonam (87%); piperacillin-tazobactam (85%); tobramycin (92%); polymyxin b (PB) (2%); and ceftazidime (CAZ) (32%). Totally, multidrug-resistant (MDR) and extended drug-resistant (XDR) isolates were 19% and 75%, respectively. Fortunately, pan drug-resistant (PDR) strain has not been observed. The assessment of biofilm formation has shown that 7% of the isolates were nonbiofilm (N), weak (W) 67%, moderate (M) 22%, and strong (S) 4%. Conclusions: As a result, the findings of this survey indicated that PB and CAZ were the most effective antibiotics against P. aeruginosa, which of course indicate a serious problem about the emergence of the PDR strains. There was no relationship between the patterns of biofilm production and antibiotic susceptibility, but high frequency of MDR/XDR and biofilm producer strains has been detected.


Rezaee MA, Behzadiyan-Nejad Q, Owlia P, Pirayeh SN. In vitro activity of imipenem and ceftazidim against mucoid and non-mucoid strains of Pseudomonas aeruginosa isolated from patients in Iran. Arch Iran Med. 2002;4:251-4.  Back to cited text no. 1
Ugargol AR, Srikanth N, Shilpa K, Patil S. Characterisation and detection of virulence factors, alginate and phospholipase 'C' in Pseudomonas aeruginosa in a Tertiary Care Hospital. Int J Health Sci Res 2014;4:82-7.  Back to cited text no. 2
Rezaei E, Safari H, Naderinasab M, Aliakbarian H. Common pathogens in burn wound and changes in their drug sensitivity. Burns 2011;37:805-7.  Back to cited text no. 3
Japoni A, Alborzi A, Kalani M, Nasiri J, Hayati M, Farshad S. Susceptibility patterns and cross-resistance of antibiotics against Pseudomonas aeruginosa isolated from burn patients in the South of Iran. Burns 2006;32:343-7.  Back to cited text no. 4
Moghoofei M, Fazeli H, Poursina F, Nasr Esfahani B, Moghim S, Vaez H, et al. Morphological and bactericidal effects of amikacin, meropenem and imipenem on Pseudomonas aeruginosa. Jundishapur J Microbiol 2015;8:e25250.  Back to cited text no. 5
Aloush V, Navon-Venezia S, Seigman-Igra Y, Cabili S, Carmeli Y. Multidrug-resistant Pseudomonas aeruginosa: Risk factors and clinical impact. Antimicrob Agents Chemother 2006;50:43-8.  Back to cited text no. 6
Vaez H, Faghri J, Isfahani BN, Moghim S, Yadegari S, Fazeli H, et al. Efflux pump regulatory genes mutations in multidrug resistance Pseudomonas aeruginosa isolated from wound infections in Isfahan hospitals. Adv Biomed Res 2014;3:117.  Back to cited text no. 7
Shu JC, Chia JH, Siu LK, Kuo AJ, Huang SH, Su LH, et al. Interplay between mutational and horizontally acquired resistance mechanisms and its association with carbapenem resistance amongst extensively drug-resistant Pseudomonas aeruginosa (XDR-PA). Int J Antimicrob Agents 2012;39:217-22.  Back to cited text no. 8
Dobbin C, Maley M, Harkness J, Benn R, Malouf M, Glanville A, et al. The impact of pan-resistant bacterial pathogens on survival after lung transplantation in cystic fibrosis: Results from a single large referral centre. J Hosp Infect 2004;56:277-82.  Back to cited text no. 9
Hammond AA, Miller KG, Kruczek CJ, Dertien J, Colmer-Hamood JA, Griswold JA, et al. An in vitro biofilm model to examine the effect of antibiotic ointments on biofilms produced by burn wound bacterial isolates. Burns 2011;37:312-21.  Back to cited text no. 10
Byrd MS, Pang B, Hong W, Waligora EA, Juneau RA, Armbruster CE, et al. Direct evaluation of Pseudomonas aeruginosa biofilm mediators in a chronic infection model. Infect Immun 2011;79:3087-95.  Back to cited text no. 11
Kennedy P, Brammah S, Wills E. Burns, biofilm and a new appraisal of burn wound sepsis. Burns 2010;36:49-56.  Back to cited text no. 12
Charlesworth CJ, Saran VV, Volpiana LK, Woods HL. The role of biofilm structure in the mechanism of gentamicin and ciprofloxacin antibiotic resistance in P. aeruginosa PAO1 biofilms. J Exp Microbiol Immunol 2008;12:27-33.  Back to cited text no. 13
Cotton LA, Graham RJ, Lee RJ. The role of alginate in P. aeruginosa PAO1 biofilm structural resistance to gentamicin and ciprofloxacin. J Exp Microbiol Immunol 2009;13:58-62.  Back to cited text no. 14
Lawley R, Curtis L, Davis J. The food safety hazard guidebook: Royal Society of Chemistry; 2012.  Back to cited text no. 15
Hengzhuang W, Song Z, Ciofu O, Onsøyen E, Rye PD, Høiby N. OligoG CF-5/20 Disruption of mucoid Pseudomonas aeruginosa biofilm in a murine lung infection model. Antimicrob Agents Chemother 2016;60:2620-6.  Back to cited text no. 16
Wood LF, Leech AJ, Ohman DE. Cell wall-inhibitory antibiotics activate the alginate biosynthesis operon in Pseudomonas aeruginosa: Roles of sigma (AlgT) and the AlgW and Prc proteases. Mol Microbiol 2006;62:412-26.  Back to cited text no. 17
Ahangarzadeh RM, Behzadiannezhad Q, NAJJAR PS, Oulia P. Higher aminoglycoside resistance in mucoid Pseudomonas aeruginosa than in non-mucoid strains. Arch Iranian Med. 2002;5:108-10.  Back to cited text no. 18
Slack MP, Nichols WW. Antibiotic penetration through bacterial capsules and exopolysaccharides. J Antimicrob Chemother 1982;10:368-72.  Back to cited text no. 19
Costerton JW, Irvin RT, Cheng KJ. The bacterial glycocalyx in nature and disease. Annu Rev Microbiol 1981;35:299-324.  Back to cited text no. 20
Diaz E, Haaf H, Lai A, Yadana J. Role of alginate in gentamicin antibiotic susceptibility during the early stages of Pseudomonas aeruginosa PAO1 biofilm establishment. J Exp Microbiol Immunol 2011;15:71-8.  Back to cited text no. 21
DeVries CA, Ohman DE. Mucoid-to-nonmucoid conversion in alginate-producing Pseudomonas aeruginosa often results from spontaneous mutations in ALGT, encoding a putative alternate sigma factor, and shows evidence for autoregulation. J Bacteriol 1994;176:6677-87.  Back to cited text no. 22
Khan AA, Cerniglia CE. Detection of Pseudomonas aeruginosa from clinical and environmental samples by amplification of the exotoxin A gene using PCR. Appl Environ Microbiol 1994;60:3739-45.  Back to cited text no. 23
Prescott LM, Harley JP, Klein D. Microbiology. Dubuque, Iowa: Wm. C. C Brown; 1996.  Back to cited text no. 24
O'Toole GA. Microtiter dish biofilm formation assay. J Vis Exp 2011. pii: 2437.  Back to cited text no. 25
Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 2012;18:268-81.  Back to cited text no. 26
Choy MH, Stapleton F, Willcox MD, Zhu H. Comparison of virulence factors in Pseudomonas aeruginosa strains isolated from contact lens- and non-contact lens-related keratitis. J Med Microbiol 2008;57(Pt 12):1539-46.  Back to cited text no. 27
Ghanbarzadeh Corehtash Z, Khorshidi A, Firoozeh F, Akbari H, Mahmoudi Aznaveh A. Biofilm formation and virulence factors among Pseudomonas aeruginosa isolated from burn patients. Jundishapur J Microbiol 2015;8:e22345.  Back to cited text no. 28
Jabalameli F, Mirsalehian A, Khoramian B, Aligholi M, Khoramrooz SS, Asadollahi P, et al. Evaluation of biofilm production and characterization of genes encoding type III secretion system among Pseudomonas aeruginosa isolated from burn patients. Burns 2012;38:1192-7.  Back to cited text no. 29
Kádár B, Szász M, Kristóf K, Pesti N, Krizsán G, Szentandrássy J, et al. In vitro activity of clarithromycin in combination with other antimicrobial agents against biofilm-forming Pseudomonas aeruginosa strains. Acta Microbiol Immunol Hung 2010;57:235-45.  Back to cited text no. 30
Ghadaksaz A, Fooladi AA, Hosseini HM, Amin M. The prevalence of some Pseudomonas virulence genes related to biofilm formation and alginate production among clinical isolates. J Appl Biomed 2015;13:61-8.  Back to cited text no. 31
Prasad SV, Ballal M, Shivananda PG. Slime production a virulence marker in Pseudomonas aeruginosa strains isolated from clinical and environmental specimens: A comparative study of two methods. Indian J Pathol Microbiol 2009;52:191-3.  Back to cited text no. 32
[PUBMED]  [Full text]  
Prince AA, Steiger JD, Khalid AN, Dogrhamji L, Reger C, Eau Claire S, et al. Prevalence of biofilm-forming bacteria in chronic rhinosinusitis. Am J Rhinol 2008;22:239-45.  Back to cited text no. 33
Coban AY, Ciftci A, Onuk EE, Erturan Z, Tanriverdi Cayci Y, Durupinar B. Investigation of biofilm formation and relationship with genotype and antibiotic susceptibility of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis. Mikrobiyol Bul 2009;43:563-73.  Back to cited text no. 34
Hou W, Sun X, Wang Z, Zhang Y. Biofilm-forming capacity of Staphylococcus epidermidis, Staphylococcus aureus, and Pseudomonas aeruginosa from ocular infections biofilm-forming capacity of human flora bacteria. Invest Ophthalmol Vis Sci 2012;53:5624-31.  Back to cited text no. 35
Srifuengfung S, Tiensasitorn C, Yungyuen T, Dhiraputra C. Prevalence and antimicrobial susceptibility of Pseudomonas aeruginosa mucoid and non-mucoid type. Southeast Asian J Trop Med Public Health 2004;35:893-6.  Back to cited text no. 36
Shawar RM, MacLeod DL, Garber RL, Burns JL, Stapp JR, Clausen CR, et al. Activities of tobramycin and six other antibiotics against Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Antimicrob Agents Chemother 1999;43:2877-80.  Back to cited text no. 37
Nichols WW, Dorrington SM, Slack MP, Walmsley HL. Inhibition of tobramycin diffusion by binding to alginate. Antimicrob Agents Chemother 1988;32:518-23.  Back to cited text no. 38
Abidi SH, Sherwani SK, Siddiqui TR, Bashir A, Kazmi SU. Drug resistance profile and biofilm forming potential of Pseudomonas aeruginosa isolated from contact lenses in Karachi-Pakistan. BMC Ophthalmol 2013;13:57.  Back to cited text no. 39
Owlia P, Nosrati R, Alaghehbandan R, Lari AR. Antimicrobial susceptibility differences among mucoid and non-mucoid Pseudomonas aeruginosa isolates. GMS hygiene and infection control. 2014;9(2).  Back to cited text no. 40
Burns JL, Saiman L, Whittier S, Larone D, Krzewinski J, Liu Z, et al. Comparison of agar diffusion methodologies for antimicrobial susceptibility testing of Pseudomonas aeruginosa isolates from cystic fibrosis patients. J Clin Microbiol 2000;38:1818-22.  Back to cited text no. 41
Church D, Elsayed S, Reid O, Winston B, Lindsay R. Burn wound infections. Clin Microbiol Rev 2006;19:403-34.  Back to cited text no. 42
Shahcheraghi F, Feizabadi MM, Yamin V, Abiri R, Abedian Z. Serovar determination, drug resistance patterns and plasmid profiles of Pseudomonas aeruginosa isolated from burn patients at two hospitals of Tehran (IRAN). Burns 2003;29:547-51.  Back to cited text no. 43
Nikbin VS, Abdi-Ali A, Feizabadi MM, Gharavi S. Pulsed field gel electrophoresis & plasmid profile of Pseudomonas aeruginosa at two hospitals in Tehran, Iran. Indian J Med Res 2007;126:146-51.  Back to cited text no. 44
[PUBMED]  [Full text]  
Ghazi M, Khanbabaee G, Fallah F, Kazemi B, Mahmoudi S, Navidnia M, et al. Emergence of Pseudomonas aeruginosa cross-infection in children with cystic fibrosis attending an Iranian referral pediatric center. Iran J Microbiol 2012;4:124-9.  Back to cited text no. 45
Nikokar I, Tishayar A, Flakiyan Z, Alijani K, Rehana-Banisaeed S, Hossinpour M, et al. Antibiotic resistance and frequency of class 1 integrons among Pseudomonas aeruginosa, isolated from burn patients in Guilan, Iran. Iran J Microbiol 2013;5:36-41.  Back to cited text no. 46
Yousefi S, Nahaei M, Farajnia S, Ghojazadeh M, Akhi M, Sharifi Y, et al. Class 1 integron and imipenem resistance in clinical isolates of Pseudomonas aeruginosa: Prevalence and antibiotic susceptibility. Iran J Microbiol 2010;2:115-21.  Back to cited text no. 47
Shahcheraghi F, Badmasti F, Feizabadi MM. Molecular characterization of class 1 integrons in MDR Pseudomonas aeruginosa isolated from clinical settings in Iran, Tehran. FEMS Immunol Med Microbiol 2010;58:421-5.  Back to cited text no. 48