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العنوان
Studies on antimicrobial activity of some bacterial isolates against extended spectrum beta-lactamases producing Gram-negative bacteria /
المؤلف
Hassouna,Nadia Abdel-Halim.
هيئة الاعداد
باحث / Yomna Nagy Hussein Elkholy
مشرف / Nadia Abdel-Halim Hassouna
مشرف / Mohammad Mabrouk Aboulwafa
مشرف / Walid Faisal Elkhatib
مشرف / Khaled Mohamed Anwar Abu Shanab
تاريخ النشر
2019
عدد الصفحات
272p.:
اللغة
الإنجليزية
الدرجة
الدكتوراه
التخصص
الصيدلة ، علم السموم والصيدلانيات (المتنوعة)
تاريخ الإجازة
1/1/2019
مكان الإجازة
جامعة عين شمس - كلية الصيدلة - ميكروبيولوجيا و مناعة
الفهرس
Only 14 pages are availabe for public view

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from 272

Abstract

One hundred and seventy-three bacterial isolates were recovered from soil samples from different Egyptian governorates with the characteristic morphology of Genus Streptomyces. The collected isolates were screened for their antimicrobial activity against an ESBL producer K. pneumoniae ATCC 700603 reference strain using agar plug method, and 30 isolates were found to have antimicrobial activity against the tested strain. The 30 positive Streptomyces isolates were further screened for their antimicrobial activity against the same K. pneumoniae ATCC 700603 strain and against different pathogenic bacteria harboring various ESBL resistance genes by agar well diffusion method using their CFCS.
The tested Streptomyces isolates exhibited various levels of antimicrobial activities with only 10 isolates showing activities against the entire tested ESBL producers as evidenced by the obtained inhibition zone diameters. Of these, two Streptomyces isolates coded W2 and H21 showed the largest inhibition zones against all tested ESBL producers. These isolates were selected for completing the present study and when needed the antimicrobial activity of these two selected isolates was tested using K. pneumoniae ATCC 700603 strain.
The highest two producers, were identified by 16S rRNA gene sequencing as Streptomyces fulvissimus isolate W2 and Streptomyces manipurensis isolate H21 and their assembled sequences were deposited in the NCBI GenBank database with the accession codes MH036743 and MH036744, respectively. Also,some physiological and biochemical characteristics of the two tested isolated were studied. Both could hydrolyze soluble starch and casein. Both showed growth at pH 5 to 9. Moreover, examination of their spore morphology and spore chain characteristics using a scanning electron microscope showed the features of Streptomyces bacterial species.
The antibiotic susceptibility pattern of Streptomyces fulvissimus isolate W2 showed its sensitivity towards amikacin, ciprofloxacin, doxycycline, erythromycin, ertapenem, gentamicin, levofloxacin, linezolid, meropenem and vancomycin and resistance towards amoxicillin, azithromycin, aztreonam, cefepime, cefotaxime, ceftazidime, ceftriaxone, cephoxitin, clindamycin, co-amoxiclav, cotrimoxazole and imipenem. However, Streptomyces manipurensis isolate H21 showed sensitivity towards amikacin, azithromycin, cephoxitin, ciprofloxacin, doxycycline, erythromycin, gentamicin, levofloxacin, linezolid and vancomycin and resistance towards amoxicillin, aztreonam, cefepime, cefotaxime, ceftazidime, ceftriaxone, clindamycin, co-amoxiclav, cotrimoxazole and imipenem.
Effect of different factors on growth and production of antimicrobial agent(s) by the selected Streptomyces isolates was done using one factor at a time (OFAT) method followed by response surface methodology (RSM) in submerged fermentation in shake flasks. Three variables were studied using OFAT; incubation period, incubation temperature and culture media. The optimal conditions obtained from the OFAT tests were used in the RSM model.
For both isolates, incubation period of 72 h was found to be optimum for antimicrobial agent(s) activity. Maximum activity was observed at incubation temperature of 28˚C. Soybean meal medium (M1) revealed to be the best cheapest culture media. These factors were used the RSM model at concentrations that supported maximum production. For Streptomyces fulvissimus isolate W2, the model consisted of 32 experiments and had an R2 of 0.85. Production using the optimized parameters of 72 h incubation at 28 ˚C using 250 rpm agitation rate and an inoculum size of 4% v/v added to initial pH 8.33 resulted in ~ 1.7 fold increase in the antimicrobial agent(s) production compared to production under unoptimized conditions. For Streptomyces manipurensis isolate H21, the model consisted of 23 experiments and had an R2 of 0.93. Production using the optimized parameters of 72 h incubation at 28˚C using 250 rpm agitation rate and an inoculum size of 2.75 % v/v added to initial pH 7 resulted in 2 fold increase in the antimicrobial agent(s) production (28 mm) compared to production under unoptimized conditions (26 mm). Neither Streptomyces fulvissimus isolate W2 nor do Streptomyces manipurensis isolate H21 could produce β-lactamase inhibitor as evidenced by the nitroefin colorimetric assay.The antimicrobial activity of Streptomyces manipurensis isolate H21 against the ESBL producer, K. pneumoniae ATCC 700603 reference strain was both intracellular and extracellular. However, Streptomyces fulvissimus isolate W2 exhibited only extracellular antimicrobial activity.
The antimicrobial activity spectra of CFCS of the two selected Streptomyces isolates against the tested organisms were different. Streptomyces fulvissimus isolate W2 exhibited strong antibacterial activity (IZ ≥ 30 mm) against 7 out of 10 and 4 out of 6 tested Gram negative and Gram positive organisms, respectively. It gave IZ diameter ranged between 20-30 mm against Candida parasilosis ATCC 22019, Candida krusei ATCC 14243 and Cryptococcus neoformans RCMB 0049001 and no activity against the rest tested fungi. However, Streptomyces manipurensis isolate H21 showed strong activity (IZ ≥ 30 mm) against Proteus vulgaris ATCC 33420 and Staphylococcus saprophyticus ATCC 49907 and moderate activity (IZ: 20-30 mm) against other tested Gram positive and Gram negative organisms. It showed antifungal activity against all tested fungi.
Antimicrobial activity of the selected Streptomyces isolates was relatively thermostable and temperature of 100˚C (30 min) or autoclaving (121˚C for 15 min) caused abolishment of activity as evidenced by the absence of inhibition zones in comparison to the control. The produced antimicrobial agent by the selected isolates could tolerate pH as high as 9 without loss of activity. No change in activity was found in case of proteinase K or trypsin treatment.For Streptomyces fulvissimus isolate W2, no effect on its antimicrobial activity against the ESBL producer, K. pneumoniae ATCC 700603 reference strain of its CFCS upon treatment with some divalent metals (Ca2+, Cu2+, Mg2+, Zn2+ , Fe2+), EDTA and SDS. In case of Streptomyces manipurensis isolate H21, Fe2+ (10 mM) and Zn2+ (1, 10 mM) caused 15 and 7% loss of the antimicrobial activity, respectively. The other tested divalent metals, EDTA and SDS had no effect. None of the CFCS of tested Streptomyces isolates contained surfactant and/or emulsifying agent(s).
Different methods of extraction were used as an attempt to purify the antimicrobial agent(s) from CFCS of the tested Streptomyces isolates. All extracts were tested for their antimicrobial activity against the ESBL producer, K. pneumoniae ATCC 700603 reference strain using agar well diffusion method. These extraction methods included; extraction of the liquid CFCS, sequential extraction of the liquid CFCS by multiple organic solvents, extraction of the dried CFCS and extraction of the lyophilized CFCS. The results revealed that extraction using methanol after lyophilization of CFCS could reasonably extract the antimicrobial agent(s) of both test isolates. Testing the antimicrobial activity of the methanolic extracts of the lyophilized CFCS of Streptomyces fulvissimus W2 and Streptomyces manipurensis H21 using agar well diffusion method showed IZ diameter of 28 ±0 and 20 ± 0.5 mm, respectively.The developed TLC chromatogram of Streptomyces fulvissimus isolate W2 showed separate bands using ethyl acetate: methanol at 30:70 ratios upon detection with UV and also upon detection with UV, vanillin sulphuric or ninhydrin. However, no complete separation (tailing without separate bands) was observed in case of ethyl acetate: methanol 40:60. This TLC chromatogram was subjected for bioautography. A bioautogram of the methanolic extract of lyophilized CFCS of Streptomyces fulvissimus W2 showed clear spots of inhibition on the surface inoculated plates; which were clearly defined at site of spotting and at the other end of the plate.
The chromatogram of the methanolic extract of lyophilized CFCS of Streptomyces manipurensis isolate H21 showed poor separation upon using ethyl acetate: methanol 40:60 and 30:70. A bioautogram with ethyl acetate: methanol 30:70 showed no spots of inhibition on surface of MHA inoculated plates.
The active methanolic extracts of lyophilized CFCS were loaded on sterile discs and tested against certain commercial antibiotic discs (MEM, AMC and A/S). Extract (50 mg/ml) of Streptomyces fulvissimus isolate W2 showed 15 mm inhibition zone, while extract of Streptomyces manipurensis isolate H21 gave 18 mm inhibition zone. Additionally the methanolic extracts of the lyophilized CFCS of both tested isolates were tested against some fungi using either agar well or disc diffusion method. Antifungal activity could be detected against only 2 Candida species (Candida parasilosis ATCC 22019 in case of Streptomyces fulvissimus W2 and Candida lusitaniae ATCC 3449 in case of Streptomyces manipurensis H21. The obtained inhibition zone diameters in both cases were 11±0 and 10±0 , respectively. No activity could be detected against the other tested fungi (Candida glabarta ATCC MyA-2950, Candida krusei ATCC 14243, Candida albicans ATCC 10231, Candida tropicalis ATCC 13803, Cryptococcus neoformans RCMB 0049001, Aspergillus niger RCMB 002005, Fusarium oxysporum RCMB 001018, Penicillium italicum RCMB 001004.
Minimum inhibitory concentrations (MICs) of the methanolic extracts of lyophilized CFCS of Streptomyces fulvissimus isolate W2 and Streptomyces manipurensis isolate H21 against K. pneumonia ATCC 700603 were found to be 3.125 and 5 mg/ml, respectively. Moreover, minimum bactericidal concentrations (MBCs) of the corresponding extracts were found to be 12.5 and 10 mg/ml, respectively.
Cytotoxicity of CFCS of Streptomyces fulvissimus isolate W2 revealed to be potent (percentage cytotoxicity was 95%); while moderate cytotoxicity (62%) was observed in case of CFCS of Streptomyces manipurensis isolate H21. Methanolic extract of lyophilized CFCS of Streptomyces fulvissimus isolate W2 showed potential cytotoxic activities against tested Caco-2 cell line (CD50= 5.04 μg/ml). However, methanolic extract of the lyophilized CFCS of Streptomyces manipurensis isolate H21 showed no in vitro cytotoxic activity (CD50= 1170 mg/ml).