EFFECT OF PRODIGIOSIN ON BIOFILM FORMATION IN CLINICAL ISOLATES OF PSEUDOMONAS AERUGINOSA

Abstract

From the duration between August to December 2019, a total of 120 burn and wound specimens were collected from Baghdad hospitals. The obtained specimens were streaked directly on macConkey and cetramide agar plates, followed by further biochemical testing; 46 isolates were identified as Pseudomonas aeruginosa. Furthermore, the identification was confirmed using Vitek-2 compact system. Moreover; from the 120 obtained specimens, four isolates were identified as Serratia marcescens (prodigiosin producers) using biochemical tests and Vitek-2 compact system. Mineral salt broth with pepton (0.5%) was used, followed by prodigiosin pigment extraction, purification and determination the concentration of the prodigiosin pigment, During prigment extraction, the four isolates of S. marcescens (S1, S2, S3, S4) were cultured singly and also cultured by mixing them randomly. The results showed that mixing Serratia isolates led to further results, the production of pigment was better by mixing two isolates (S1+S2). Antibiotic susceptibility testing of all (46) P. aeruginosa isolates to Ciprofloxacin, Amikacin, Tobramycin, Cefotaxime, Azteronam and Meropenem was assessed using Kirby-Bauer disk diffusion assay. The result revealed that 46 (100%) isolates were resistant to Amikacin; additionally, 50%, 48%, 43% and 28% isolates were resistant to Tobramycin, Cefotaxime, Ciprofloxacin and Azteronam respectively. On the other hand, Meropenem revealed to be most effective drug since it recorded the highest sensitivity percentage of 98%. Moreover, the bacterial ability for the formation of biofilm was assessed for 30 selected P. aeruginosa isolates (multi drug resistant isolates) using microtiter plate assay, the result indicated that only 5 strong isolates were biofilm producer whereas 18 and 7 isolates were moderate and weak biofilm producers respectively. DNA of P. aeruginosa were successfully extracted from overnight cultures, and PCR was conducted to amplify constitutional genes 16srRNA, AlgD, the bands were confirmed with gel electrophoresis, the results showed that genes 16srRNA (956 bp) and AlgD (313bp) were detected. The presence of AlgD gene was tested in 17 selected P. aeruginosa isolates (strong and moderate biofilm producers) using polymerase chain reaction technique; however, only 2 isolates (P9 and P21) harbored AlgD gene. The minimal inhibitory concentrations of prodigiosin and Ciprofloxacin were assessed for P. aeruginosa isolates (P9 and P21) using broth microdilution method; even more, the synergistic effect of prodigiosin combined with the Ciprofloxacin was also assessed for the same tested isolates. The result revealed that Ciprofloxacin minimum inhibitory concentration values were 31.2 μg/ml and 15.6 μg/ml for P. aeruginosa(P9 and P21) isolates respectively, Additionally, the minimum inhibitory concentration value for prodigiosin pigment were 87.5 μg/ml and 175 μg/ml for P. aeruginosa (P9 and P21) isolates respectively; regarding the synergistic effect the Ciprofloxacin combined with the prodigiosin, the result revealed that their minimum inhibitory concentration values of Ciprofloxacin were 15.6 μg/ml and 7.8 μg/ml for P. aeruginosa (P9 and P21) isolates respectively while the values of prodigiosin were 2.7 μg/ml and 1.36 μg/ml for P. aeruginosa (P9 and P21) isolates respectively. To perform the reduction biofilm assay, P.aeruginosa (P9 and P21) isolates were treated with two sub MICs of (prodigiosin, ciprofloxacin and synergisim between them). The results showed that the biofilm inhibition of P9 isolates was ( 84%, 82% and 85% under the effect of sub MIC 1, and were 81%, 80% and 83% under the effect of sub MIC 2 ) and for P21 isolates was ( 81%, 79% and 82% under the effect of sub MIC1, and were 80%, 77% and 81% under the effect of sub MIC2)