التأثير المضاد للمايكروبات للبايوسيانين المنتج من بكتريا الزائفه الزنجاريه المعزولة سريريا

Antimicrobial effect of pyocyanin produced by clinical isolate of Pseudomonas aeruginosa

Abstract

Dermatophytose is caused by dermatophytes located in cutaneous layer of skin. It also infected nails and hair , it is consired a common fungal disease all of the world. This type of infection was determined among 18 patients (9 males and 9 females) involved in this study. Dermatophytes were isolated from 7(38.88%) of involved patients, while 11(61.11%) of them given a negative results. Distribution of fungal infection among genders showed the presence of two positive males and five positive females. Tinea capitis 2 (11.11%) was the only type of dermatophytoses that gave a positive culture for dermatophytes among males. Whereas, two of tinea capitis 2 (11.11%) and three of tinea corporis 3 (16.66%) were gave a positive culture of dermatophytes in females.
Pyocyanin (PCN), which is bule –green pigments mainly produced by Pseudomonas aeruginosa during the stationary phase of the growth curve bacteria and can easily solvent in water. PCN was successfully isolated from clinical isolate of Pseudomonas aeruginosa which is registered later in the data base of GenBank under the number MH382164. Chloroform method was used for purification of PCN from bacterial culture and its concentration was determined by spectrophotometer method.
Antimicrobial activity of pyocyanin (PCN) was determined against various species of bacteria and fungi. Antibacterial activity of PCN was tested against 14 strains of isolated bacteria by disk diffusion method. Diagnosis of isolates was performed depending on morphological characters, biochemical tests and by API-20 system. Isolated bacteria distributed between 8 strains of Gram negative and 6 strains of Gram positive. Antifungal action of PCN was
also tested against three strains of yeasts and seven isolates of dermatophytes which also registered in GenBank after molecular identification and sequences of Internal transcribed spacer (ITS) genes, including Trichophyton interdigitale (MH383047), Epidermophyton floccosum (MH383041), three strains of Microsporum canis (MH383044, MH383045, MH383046), and two strains of Microsporum ferrugineum (MH383042, MH383043). The MIC value of PCN was also determined against isolated organisms by CLSI methods.
The activity of PCN at 4 µg/ml on the isolated strains of bacteria was variable between Gram positive and negative. The most sensitive strains were Staphylococcus epidermidis, Salmonella spp., Streptococcus pneumonia, Citrobacter freundii and Enterobacter cloacae, which were showed a zone of inhibition 7, 5.6, 5.3, 5.1, and 5 mm, respectively. Other strains showed less sensitivity toward the 4 µg of PCN as with Streptococcus agalactiae, Streptococcus pyogenes, Acinetobacter baumannii, and E. coli with a zone of inhibition 2.2, 2.3, 2.3, and 2.4 mm, respectively. Streptococcus agalactiae needed less MIC value of PCN (3.5 µg/ml) to inhibit than other bacteria.
An antifungal effect of PCN at 4 µg/ml was tested against isolated fungi. Microsporum canis (MH383044), Epidermophyton floccosum(MH383041) and Candida albicans which revealed a zone of inhibition 6.9, 4.5, and 1.2 mm, respectively were the most sensitive fungi toward PCN with significant differences from most of fungi at p < 0.05. The isolates of Microsporum ferrugineum(MH383043), Microsporum canis (MH383046) and Cryptococcus terreus shown a variable sensitivity to PCN (2, 0.7, and 0.3 mm, respectively) with a significant differences from each other at p < 0.05. The MIC value of M. canis (MH383044) as the most sensitive fungal strain was determined at 3.8 µg/ml, while for C.albicans was 4 µg/ml.
Its effect on the antibacterial action of ampicillin and cefotaxime and on the action of griseofulvin as antifungal was also measured. Generally, the effect of PCN (4 µg/ml) on the antibacterial activity of ampicillin was variable on some isolated bacteria. It was decreased by the effects of PCN even at high concentration (8 µg/ml) as with E. coli and E. cloacae in comparison with ampicillin alone. Whereas, it enhanced ampicillin activity against S. pyogenes by decreasing MIC value from 6 to 4 µg/ml. The 2 µg/ml of PCN decreased the MIC action of ampicillin against all of bacterial strains to reach a concentration 2 µg/ml, except Acinetobacter baumannii which showed resistance to the largest MIC value (8 µg/ml) of ampicillin in compared with ampicillin alone.
At concentration 15 µg/ml of cefotaxime combined with PCN (4 µg/ml), all bacterial growth was also inhibited, except of Streptococcus pyogenes, Acinetobacter baumannii and Shigella spp. The lower concentration of cefotaxime (10 µg/ml) revealed no activity against all of bacteria strains, except of Staphylococcus aureus, Citrobacter freundii, E. coli and Klebsiella pneumoniae. The PCN didn’t show any enhancement effects on the action of cefotaxime at high concentration (20 µg/ml) against all of isolated bacteria. However, the most activity of PCN at 2 µg/ml on the cefotaxime was indicated by elevating its MIC value from 10 to 15 µg/ml against S. aureus, Citrobacter freundii, and E. coli or from 20 to more than 25 µg/ml against Acinetobacter baumannii. The MIC of cefotaxime against other strains was not changed in the presence of PCN.
Antifungal activity of griseofulvin was decreased after mixing with 2 µg/ml of PCN against M. canis and C. albicans through elevation of MIC value to 100 µg/ml.