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| Pseudomonas cinsi bakterilerin bazı metabolik aktivite, antimikrobiyal etki ve protein profillerinin incelenmesi / Investigation of some metabolic activity, antimicrobial effect and protein profiles of Pseudomonas bacteria Yazar:GÖKÇEN YUVALI ÇELİK Danışman: PROF.DR. YAVUZ BEYATLI Yer Bilgisi: Gazi Üniversitesi / Fen Bilimleri Enstitüsü / Biyoloji Ana Bilim Dalı Konu:Biyoloji = Biology Dizin: | Onaylandı Doktora Türkçe 2005 169 s. | ||
| PSEUDOMONAS CİNSİ BAKTERİLERİN BAZI METABOLİK AKTİVİTE, ANTİMİKROBİYAL ETKİ VE PROTEİN PROFİLLERİNİN İNCELENMESİ (Doktora Tezi) Gökçen YUVALI ÇELİK GAZİ ÜNİVERSİTESİ FEN BİLİMLERİ ENSTİTÜSÜ Ocak 2005 ÖZET Araştırmada Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas stutzeri, Pseudomonas cepacia, Pseudomonas putida ve Pseudomonas luteola türlerine ait toplam 30 adet suş kullanılmıştır. Suşlann 15 adeti bilimsel kuruluşlardan temin edilirken, diğer 15 adeti ise atık su ve topraklardan izole edilmiştir ve suşlar, Analitik Profil İndeksin kullanımı (API 20NE) ile tanımlanmışlardır. Bakterilerin toplam hücre protein profillerinin sodyum dodesil sülfat poliakrilamid jel elektroforezi (SDS-PAGE) ile analizi yapılmış ve proteinlerin molekül ağırlıkları 4,5-250 kD arasında bulunmuştur. SDS-PAGE analizi, aynı türe ait suşların protein profillerinin birbirine benzer ve diğer türlerden farklı olduğunu göstermiştir. Pseudomonas’ların Poli-B- hidroksibütirat (PHB) üretimleri spektrofotometrik olarak belirlenmiş ve PHB verimlerinin hücre kuru ağırlığına göre %l,58-6737 arasında değiştiği bulunmuştur. En yüksek PHB verimi gösteren Pseudomonas cepacia G13 (%67,37) susunun farklı sürelerde, farklı karbon ve azot kaynaklarında PHB üretimi araştırılmıştır. Susun farklı sürelerde maksimum PHB verimine 48. saatte vardığı tespit edilmiştir. Susun kontrol (%67,37) grubuna göre farklı karbon ve azot kaynaklarında PHB verimlerinin daha düşük olduğu belirlenmiştir. Çalışmada, şeker pancarı melası ve peynir altı suyu kullanılarak PHB’ın ekonomik eldesi araştırılmıştır. Farklı melas konsantrasyonlarının PHBüretimine etkisi incelenmiştir. Bu amaçla, en yüksek PHB verimine sahip Pseudomonas cepacia G13 susu seçilerek, %l-%6 melas konsantrasyonlarında geliştirilmiş ve 48.saatte PHB üretimi incelenmiştir. G13 susunun %3 melas konsantrasyonunda en yüksek PHB verimi olan %70,00 değerine vardığı tespit edilmiştir. P. cepacia G13 susu, peynir altı suyu besiortamında geliştirilmiş ve 48. saatteki PHB veriminin kontrol grubuna göre çok dûşfik olduğu belirlenmiştir. Araştırmada, Pseudomonas spp. suşlarının bazı patojen ve kontaminant test bakterileri (Yersinia enterocolitica ATCC1501, Staphylococcus (litrem ATCC 25923, Bacillus thuringiensis RSKK 380, Micrococcus luteus NRLL-B 4375, Bacillus subitlis ATCC 6633, B adil us megaterium RSKK 578, Escherichia cott ATCC 11230, Bacillus cereus F2, Micrococcus flavus) üzerindeki genel inhibisyon etkileri ağar difuzyon metodu ile incelenmiştir. Staphylococcus aureus ve Bacittus thuringiensis bakterilerinin Pseudomonas suşlarının antimikrobiyal aktivitesinden daha fazla etkilendikten tespit edilmiştir. Pseudomonas spp. suşlarının birbirlerine karşı olan inhibisyon etkilerinin incelenmesinde de yine ağar diffizyon metodu kullanılmış ancak, suşların hiçbirinin birbirleri üzerinde inhibisyon etkisi göstermediği belirlenmiştir. Çalışmada, Pseudomonas suşların bazı laktik asit bakterileri (Lactobacillus casei C-20, Lactobacülus plantarum ATCC 20246, Lactobacittus bulgaricus ATCC 11843, Lactobacülus brevis B-40, Lactococcus dmcetylactis P- 90, Lactococcus cremoris TO (2-2), Leuconostoc cremoris BlLeu, Leuconostoc mesenteroides B2Leu, Streptococcus thermophillus ATCC 14425) üzerinde ağar difuzyon metodu kullanılarak antimikrobiyal etkileri incelenmiştir. Pseudomonas suskunun hiçbirinin laktik asit bakterileri üzerinde inhibitörik etki göstermediği tespit edilmiştir. Buna karşın laktik asit bakterilerinin Pseudomonas suşları üzerinde önemli ölçüde inhibisyon etki gösterdiği belirlenmiştir. Pseudomonas spp. suşlarının arnikasın (30 ug), tobramisin (10 ug), aztreonam (30 ug), piperarasilin (100 ug), siprofloksasin (5 ug), gentamisin (10 pg), meropenem (10 ug)» piperasibn/tazobaktam (100 ug/10 ug), oflaksasin (5 ug), seftazidim (30 ug),tetrasiktin (30 ug), sefoperazon (75 ug), hnipenem (10 ug), sefepim (30 ug) ve tikarsihn/klavulanik asit (75 ug/100 ug) antibiyotiklerine karşı duyarlılık testleri disk difuzyon yöntemine göre yapılmış, sonuçlarPseudomonas cepacia G13, which had produced the highest percentage yield of PHB in nutrient broth medium were selected and cultivated in l%-5% molasses concentrations at appropriate periods (48 h). It was found that strain was produced higher PHB (%70.00) in 3% molasses concentration. It was also observed that PHB productivity by this strain was decreased by the increasing molasses concentration. G13 strain cultivated in whey and was determined that the yield of PHB production was lower than to control medium. The general inhibition of Pseudomonas isolates on pathogenic and contaminated test bacteria {Yersinia enterocolitica ATCC 1501, Staphylococcus aureus ATCC 25923, Bacillus thuringiensis RSSK 380, Micrococcus luteus NRLL-B 4375, Bacillus subtilis ATCC 6633, Bacillus megaterium RSKK 578, Escherichia coli ATCC 11230, Bacillus cereus F2, Micrococcus flavus) were investigated by using agar diffusion method. From the results, It was determined that Staphylococcus aureus and Bacillus thuringiensis were significantly inhibited by Pseudomonas strains. Also, on the studing of inhibition effects among Pseudomonas strains, none of the strains were inhibited each others. Antimicrobial effects of Pseudomonas strains on some lactic acid bacteria (Lactobacillus casei C-20, Lactobacillus plantarum ATCC 20246, Lactobacillus bulgaricus ATCC 11843, Lactobacillus brevis B-40, Lactococcus diacetylacus P-90, Lactococcus cremoris TO (2-2), Leuconostoc cremoris BILeu, Leuconostoc mesenteroides B2Leu, Streptococcus thermophilus ATCC 14425) were investigated by using agar diffusion method. None of the Pseudomonas strains had showed inhibition effect on lactic acid bacteria. It was determined that lactic acid bacteria had significantly high inhibition effect on Pseudomonas strains. Antibiotic susceptibility tests of Pseudomonas spp. strains to arnikasın (30 ug), tobramisin (10 ug), aztreonam (30 ug), piperarasilin (100 ug), siprofloksasin (5 ug), gentamisin (10 ug), meropenem (10 ug), piperasilin/tazobaktam (100 ug/10 ug), oflaksasin (5 ug), seftazidim (30 ug), tetrasifclin (30 ug), sefoperazon (75 ug), imipenem (10 ug), sefepim (30 ug) and tikarsilin/klavulanik asit (75 ug/100 ug) were determined by using the disk diffusion method. The results were described according to NCCLS standards. It was determined that 59% of Pseudomonas strains were susceptibility, 28% were resistant and 13% were intermediate | |||
| IV INVESTIGATION OF SOME METABOLIC ACTIVITY, ANTIMICROBIAL EFFECT AND PROTEIN PROFILES OF PSEUDOMONAS BACTERIA (Ph. D. Thesis) Gökçen YUVALI ÇELİK GAZI UNIVERSITY INSTITUTE OF SCIENCE AND TECHNOLOGY January 2005 ABSTRACT In the study, a total of thirty strains belonging to Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas stutzeri, Pseudomonas cepacia, Pseudomonas putida and Pseudomonas luteola were analysed. Fifteen of thirty strains were obtained from Scientific Researc Centers and the other fifteen strains were isolated from the waste water and soil. Strains were identified by using Analytical Profile indeks (API 20 NE). Total cell protein profiles of bacteries were analysed by sodium dodecyl sulphate poryacrylamide gel electrophoresis (SDS-PAGE) and the molecular weights of proteins were found ranging between 4.5 and 250 kD. SDS-PAGE analysis showed that those strains belong to the same category had similar protein profiles and that they were different from the others. Poly-B-hydroxybutyrate (PHB) productions of Pseudomonas bacteria were determined by spectrophotometric method and they were found that PHB yields ranged from %1.58-6737 according dry cell weight. Pseudomonas cepacia G13 strain which has the highest PHB yields (%6737) was analysed in terms of its PHB production in different incubation times and in different carbon and nitrogen sources. It was determined that strain was produced maximum PHB (%6737) at 48 th hours of incubation. The yields of PHB of this strain in different carbon and nutrient sources were lower than PHB production in control medium. Economical production of PHB was investigated by using sugar beet molasses and whey. The effects of different molasses concentrations on PHB production were investigated. For this reason,Pseudomonas cepacia G13, which had produced the highest percentage yield of PHB in nutrient broth medium were selected and cultivated in l%-5% molasses concentrations at appropriate periods (48 h). It was found that strain was produced higher PHB (%70.00) in 3% molasses concentration. It was also observed that PHB productivity by this strain was decreased by the increasing molasses concentration. G13 strain cultivated in whey and was determined that the yield of PHB production was lower than to control medium. The general inhibition of Pseudomonas isolates on pathogenic and contaminated test bacteria {Yersinia enterocolitica ATCC 1501, Staphylococcus aureus ATCC 25923, Bacillus thuringiensis RSSK 380, Micrococcus luteus NRLL-B 4375, Bacillus subtilis ATCC 6633, Bacillus megaterium RSKK 578, Escherichia coli ATCC 11230, Bacillus cereus F2, Micrococcus flavus) were investigated by using agar diffusion method. From the results, It was determined that Staphylococcus aureus and Bacillus thuringiensis were significantly inhibited by Pseudomonas strains. Also, on the studing of inhibition effects among Pseudomonas strains, none of the strains were inhibited each others. Antimicrobial effects of Pseudomonas strains on some lactic acid bacteria (Lactobacillus casei C-20, Lactobacillus plantarum ATCC 20246, Lactobacillus bulgaricus ATCC 11843, Lactobacillus brevis B-40, Lactococcus diacetylacus P-90, Lactococcus cremoris TO (2-2), Leuconostoc cremoris BILeu, Leuconostoc mesenteroides B2Leu, Streptococcus thermophilus ATCC 14425) were investigated by using agar diffusion method. None of the Pseudomonas strains had showed inhibition effect on lactic acid bacteria. It was determined that lactic acid bacteria had significantly high inhibition effect on Pseudomonas strains. Antibiotic susceptibility tests of Pseudomonas spp. strains to arnikasın (30 ug), tobramisin (10 ug), aztreonam (30 ug), piperarasilin (100 ug), siprofloksasin (5 ug), gentamisin (10 ug), meropenem (10 ug), piperasilin/tazobaktam (100 ug/10 ug), oflaksasin (5 ug), seftazidim (30 ug), tetrasifclin (30 ug), sefoperazon (75 ug), imipenem (10 ug), sefepim (30 ug) and tikarsilin/klavulanik asit (75 ug/100 ug) were determined by using the disk diffusion method. The results were described according to NCCLS standards. It was determined that 59% of Pseudomonas strains were susceptibility, 28% were resistant and 13% were intermediateVI susceptibility against fifteen antibiotics tested. Proteolytic activities of Pseudomonas isolates were tested in Skim Milk Agar (SMA) medium. A total of eighteen strains belonging to Pseudomonas aeruginosa, Pseudomonas fluorescens and Pseudomonas cepacia were determined that had a proteolytic activities. Proteolytic activities of these strains were found between 14.1-26.5 mm with an average 20.9 mm zone diameter. The highest proteolysis 26.5 mm was found in Pseudomonas aeruginosa No:15 strain. In this study, lipolytic activities of Pseudomonas isolates were tested in Tribfitirin Agar (TA) medium. Seventeen strains had showed lipolytic activities ranging between 14.8-32.8 with an average 23.2 mm zone diameter. From the results the strains which had showed proteolytic activities had also showed lipolytic activities. The highest zone diameter of lipoh/sis 32.8 mm was found in Pseudomonas aeruginosa No:20 strain. Science Code Key Words Page Number Advisor :216..Pseudomonas, isolation, protein profile, bioplastic (PHB) production, antimicrobial activity, antibiotic susceptibilty, proteolytic activity, lipolytic activity. :152 :Prof. Dr. Yavuz BEYATLI | |||