K. PNEUMONIAE VE P. MIRABILIS TESPİTİ İÇİN TOZ ANTOSİYANİN İÇERİKLİ-ÜREAZ TESTİNİN RASYONEL HAZIRLANMASI

Abstract

Bu çalışmada, üreaz-pozitif bakteriler olan Klebsiella pneumoniae ATCC 54475 ve Proteus mirabilis ATCC 25933'ün hızlı, seçici, hassas tespiti için Brassica oleracea var. Capitata (kırmızılahana) lliyofilize ekstresi ilize ekstresi (RCE) içeren kolorimetrik ortam geliştirilmiştir. Ana bileşen kırmızı lahana, pH'a bağlı renk değişiminden sorumlu antosiyanin gruplarıdır. Üre testi, 0.01 M sodium fosfat tampon ve sodium azit içinde hazırlanmış belirli antosiyanin, üre konsantrasyonu içerir. Testteki üre, K. pneumoniae ve P. mirabilis'ten salınan üreaz enzimi ile hidrolize edilir ve sırasıyla test ortamını alkalin yapan, antosiyaninin proton kaybetmesine neden olan ve pembeden yeşile renk değişimini indükleyen amonyum (NH3) molekülleri üretir. Ayrıca Escherichia coli ATCC 25922, üreaz negatif kontrol bakterisi olarak kullanıldı. K. pneumoniae ve P. mirabilis'in tespiti, antosiyanin konsantrasyonu ve inkübasyon süresinin bir fonksiyonu olarak incelenmiştir. Antosiyanin, hidroklorikasit (HCl) ilavesi nedeniyle sıvı formdaki serbest protonun girişimini atmak için üreaz testinde liyofilizetoz formunda kullanıldı. Görsel sonuçların kantitatif teyiti için dijital görüntü analizi olan Delta E (∆E) ölçümleri ile sağlanmıştır. Sonuçlar, K. pneumoniae ve P. mirabilis için sırasıyla 150 ve 210 dakika inkübasyonda pembeden yeşile renk değişiminin gözlendiğini göstermektedir. Burada, antosiyanin içeren üreaz testini başarıyla hazırlayarak sonuçların çıplak göz ve dijital görüntü analizi ile K. pneumoniae ve P. mirabilis'in kolorimetrik tespitini gerçekleştirdik.

Keywords

• antosiyanin
• dijital görüntü analizi
• hızlı üreaz testi
• üreaz pozitif bakteri

RATIONAL PREPARATION OF POWDER ANTHOCYANIN INCORPORATED UREASE TEST FOR DETECTION OF K. PNEUMONIAE AND P. MIRABILIS

Abstract

In this present study, we developed Brassica oleracea var. Capitata (red cabbage) extract (RCE) powder- incorporated colorimetric media for rapid, selective, sensitive detection of urease-postive bacteria, Klebsiella pneumoniae ATCC 54475 and Proteus mirabilis ATCC 25933. The major component of RCE is anthocyanin groups which are in charge of pH dependent color change. The ureas test contains certain concetration of anthocyanin, urea prepated in 0.01 M sodium phosphate buffer and sodium azide. The urea in the test was hydrolyzed by urease enzyme released from K. pneumoniae and P.mirabilis and produce ammonium (NH3) molecules which sequentially made the test media alkaline, caused deprotonation of anthocyanin and induced the color change from pink to green. Also, Escherichia coli ATCC 25922 was used a urease-negative control bacteria. The detection of K. pneumoniae and P. mirabilis was examined as a function of anthocyanin concentration and incubation time. The anthocyanin was used lyophilized powder form in the urease test to discard the in interference of free proton in liquid form owing to addition of hydrochloric acid (HCl). We also performed the digital image analysis, Delta E (∆E) for quantitative confirmation of the visual results. It was observed that the results showed that the color change from pink to green in 150 and 210 minutes of incubaiton for K. pneumoniae and P. mirabilis, respectively. Here in, we successfully prepared anthocyanin-incorporated urease test and perform colorimetric detection of K. pneumoniae and P. mirabilis with naked eyes and digital image analysis.

Keywords

• rapid urease test
• Anthocyanin
• digital image analysis
• urease-postive bacteria

References

1. Referans1. Cohen N, Fedewa S, Chen AY. Epidemiology and Demographics of the Head and Neck Cancer Population. Oral Maxillofac Surg Clin North Am 2018;30:381-95
2. Referans2. Chandra H, Singh C, Kumari P, et al. Promising Roles of Alternative Medicine and Plant-Based Nanotechnology as Remedies for Urinary Tract Infections. Molecules 2020;25:5593.
3. Referans3. Zaha DC, Bungau S, Aleya S,et al. What antibiotics for what pathogens? The sensitivity spectrum of isolated strains in an intensive care unit. Sci Total Environ 2019;687:118-127.
4. Referans4. Bjerrum L, Grinsted P, Søgaard P. Detection of bacteriuria by microscopy and dipslide culture in general practice. Eur J Gen Pr 2001; 7: 55-58.
5. Referans5. Collee JG, Miles RS, Watt B. Mackie and McCartney Practical Medical Microbiology, 14th ed.; Churchill Livingstone: New York, NY, USA, 1996.
6. Referans6. Lenke RR, Van Dorsten J. The efficacy of the nitrite test and microscopic urinalysis in predicting urine culture results. Am J Obstet Gynecol 1981;140:427-429.
7. Referans7. Pourjavaher S, Almasi H, Meshkini S, et al. Development of a colorimetricpHindicatorbased on bacterial cellulose nanofiber sand redcabbage (Brassica oleraceae) extract. Carbohydr Polym 2017;156:193-201.
8. Referans8. Pereira Jr VA, de Arruda INQ, Stefani R. Active chitosan/PVA films with anthocyanins from Brassica oleraceae (Red Cabbage) as Time–Temperature Indicators for application in intelligent food packaging. Food Hydrocoll 2015;43: 180-188.

9. Referans9. Haddar W, Ben Ticha M, Meksi N, et al. Application of anthocyanins as natural dye extracted from Brassica oleracea L. var. capitata f. rubra: dyeing studies of wooland silk fibres. Nat Prod Res 2018; 32: 141-148.
10. Referans10. Sharma G, Bala R. Digital Color Imaging Handbook, CRC Press, 2017; 816.
11. Referans11. Gupta K, Hooton TM, Naber KG, et al. International clinical practice guidelines for the treatment of acute uncomplicated cystitis and pyelonephritis in women: A 2010 update by the Infectious Diseases Society of America and the European Society for Microbiology and Infectious Diseases. Clin Infect Dis 2011; 52: 103-120.
12. Referans12. Wiedemann B, Heisig A, Heisig P. Uncomplicated urinary tract infections and antibiotic resistance-epidemiological and mechanistic aspects. Antibiotics 2014;3:341-352.
13. Referans13. Hooton TM, Bradley SF, Cardenas DD, et al. Diagnosis, Prevention, and Treatment of Catheter-Associated Urinary Tract Infection in Adults: 2009 International Clinical Practice Guidelines from the Infectious Diseases Society of America. Clin Infect Dis 2010;50:625-663.
14. Referans14. Stefaniuk E, Suchocka U, Bosacka K,et al. Etiology and antibiotic susceptibility of bacterial pathogens responsible for community-acquired urinary tract infections in Poland. Eur J Clin Microbiol Infect Dis 2016;35:1-7.
15. Referans15. Abbo LM, Hooton TM. Antimicrobial Stewardship and Urinary Tract Infections. Antibiotics (Basel) 2014;3:174-192.
16. Referans16. Adeolu M, Alnajar S, Naushad S, et al. Genome-based phylogeny and taxonomy of the “Enterobacteriales”: proposal for Enterobacterales ord. nov. divided into the families Enterobacteriaceae, Erwiniaceae fam. nov., Pectobacteriaceae fam. nov., Yersiniaceae fam. nov., Hafniaceae fam. nov., Morganellaceae fam. nov., and Budviciaceae fam. nov. Int J Syst Evol Microbiol 2016;66:5575-5599.
17. Referans17. Calzi A, Grignolo S, Caviglia I, et al. Resistance to oral antibiotics in 4569 Gram-negative rods isolated from urinary tract infection in children. Eur J Pediatr 2016;175:1219-1225.
18. Referans18. Behzadi P,Behzadi E,Ranjbar R. Urinary tract infections and Candida albicans. Cent Eur J Urol. 2015;68:96-101.
19. Referans19. Gatermann S, John J, Marre R. Staphylococcus saprophyticus urease: characterization and contribution to uropathogenicity in unobstructed urinary tract infection in rats. Infect Immun 1989; 57:110-116
20. Referans20. Johnson DE, Russell RG, Lockatell CV, et al. Contribution of Proteus mirabilis urease to persistence, urolithiasis, and acute pyelonephritis in a mouse model of ascending urinary tract infection. Infect Immun 1993;61:2748-2754.
21. Referans21. Mobley HLT, Hausinger RP. Microbial ureases: significance, regulation, and molecular characterization. Microbiol Rev 1989;53:85-108.
22. Referans22. Clegg S, Murphy CN. Epidemiology and virulence of Klebsiella pneumoniae. Urinary Tract Infections: Molecular Pathogenesis and Clinical Management 2017;435-457.
23. Referans23. Aldridge KE, Hodges RL. Correlation studies of entero-set 20, API 20E and conventional media systems for Enterobacteriaceae identification. J Clin Microbiol 1981;13:120-125.
24. Referans24. Tominaga T. Rapid detection of Klebsiella pneumoniae, Klebsiella oxytoca, Raoultella ornithinolytica and other related bacteria in food by lateral-flow test strip immunoassays. J Microbiol Methods 2018;147:43-49.