Introduction

Urinary tract infection (UTI) is one of the very common category of disease affecting around more than 15 million people annually in India ¹. The treatment includes the use of a wide range of antibiotics along with other medicines. Silk from immune‐challenged silkworm (Bombyxmori) on the other hand is having regenerative properties in injury treatment also can act as an antibacterial agent as reported by Choi et al., 2019 ². Being an excellent agent for anti- inflammatory and antibacterial properties, as per the studies of Manju Tiwari (2017) ⁸, it has been assumed that the muga silk already has antimicrobial and anti-inflammatory properties as studied by.  The recent development in the study of silk as a medicine has inspired the researchers to find out the usuability of the silk extracted from the cocoon of Antheraea assamensis as a potential medicine in the treatment of UTI. The primary interest is to investigate the applicability and efficiency of the extracted proteins from the cocoons of Antheraea assamensis and examine the anti-bacterial and anti-inflammatory activity of the proteins found in the organisms with respect to the bacteria found in Urinary tract infections. These methods will help to state that whether the silk proteins will be a potential medicinal agent against the UTI bacteria and also if any other benefiting properties with respect to the primary objective.

Methodology

Collection of Samples

Samples of patients suffering from Urinary Tract infections and admitted in SRL diagnostics, Ulubari, Guwahati were collected and the study was approved by Assam down town University Ethical Committee. The culturing procedure was done within 3 hours after the sample collection.

Selection of Bacterial Specie

For the experiment five bacterial species were selected- Escherichia coli, Staphylococcus  aureus, Klebsialla pneumonia, Enterococcus species and Enterobacter species on the basis of the occurrence in case of urinary tract infected patients. The isolation of the bacteria from urine is followed and explained in the following steps-

Isolation from Urine

The samples were evenly mixed in a peptone solution and was cultured on blood agar and chocolate agar plates respectively followed by quadrant streaking and incubation at 37⁰C for 24 hours.

Isolation of Bacteria

Microbial colonies were obtained using serial dilution method.

Bacterial Incubation

After the media was solidified, these plates was placed in an inverted position at 37°C ± 10°C for 24-72 hours in an incubator for incubation.

Bacterial List/Enumeration

Plate selection was conducted in which 30-300colonies in a colony counter.

Discrete bacterial colonies isolation

The selected bacterial colonies were transferred to nutrient agar slant aseptically for further purification.

VITEK® 2 Compact system (Version: 08.01) – For the confirmation of the UTI Bacteria VITEK was done to determine the specificity of the bacteria.

Procedure

After primary organism isolation, there is minimal handling with a simple standardized inoculum

The inoculum is placed  into the VITEK^® 2 Cassette at the Smart Carrier Station^™

The VITEK^® 2 Card and sample are linked via barcode

Once the Cassette is loaded, the instrument handles all subsequent steps for incubation and reading

Results are recorded at the end.

Storage conditions of the bacteria

Storage and culturing of the bacteria was performed with Brain Heart Infusion Agar for the Gram negative bacteria and TSA agar for Gram positive bacteria.

For the Silk Proteins

Fibroin Extraction

Pure Silk Fibroin was prepared following the Degumming, the Dissolution  and the Dialysis

Silk Sericin extraction

Small sections of autoclaved silk cocoons were filtrated with filter paper followed by centrifugation. Pelets of sericin were obtained and dried in oven at 100⁰C

Antimicrobial Sensitivity Test/Susceptibility Test

Antimicrobial Sensitivity Test was conducted following standard protocol.

Antimicrobial susceptibility tests /Disk Diffusion

Disk diffusion method was conducted to determine the antimicrobial substances against UTI causing bacteria.

Results and Discussion

Antimicrobial Results (Susceptibility Tests).

24 hours later the results are obtained; a zone of inhibition was seen in the samples, the. Silk fibroin and Sericin micro particles a without the drug was taken as negative control and had a good anti-bacterial results (susceptibility). Also with the positive control of two of the drugs a large zone of inhibition was observed. Olflaxin & ciproflaxin were used as Controls during the test.

Table 1: Bacterial Inhibition Zone Diameter with Crude Sericin and Fibroin.

It was seen during the results that the Silk proteins (namely fibroin and sericin) had antimicrobial capabilities when they were made to react against the pathogenic bacteria and had formidable results against the UTI.A special emphasis was given upon the anti-microbial properties found in the proteins and the anti-bacterial activities of the silk proteins found in the Antheraea assamensis. From the previous studies it was found that Bombyxmori cocoon Silk consists of two kinds of proteins i.e. Sericin and Fibroin which constitutes the overall protein content of the Silkworm cocoon.¹⁷ It was seen that the silk proteins namely fibroin and sericin showed antimicrobial properties against some bacteria and the same resultswere again obtained when protein was reacted against the pathogenic UTI bacteria.⁷ The core of the protein which constitutes around 70-80% of the total protein content is known as Fibroin and the remaining 20-30% constitutes of Sericin which is surrounded by other layers like inorganic components, colour pigments and wax.¹⁵ Overall there are three layers of sericin which covers up the core protein, i.e.   Fibroin and is composed of the inner, outer and middle layer which holds 4.5% , 10.5%  and 15% . In cold water Sericin is not soluble.²⁰ But because of  the long protein molecules due to its solubility in hot water as the  protein polymers break up as smaller fragments easily gets dispersed and hydrolyzed in the hot condition. Sericin possesses various biological properties like anti-bacterial, resistant  Ultraviolet light, antitumor, antioxidant,  and absorption of moisture; It has a potential to be utilized as an finishing agent for fibers (natural/man-made)in  textile industries, in cosmetics industry and forpolymeric biomedical products.¹⁶As per previous studies, Sericin has  biological properties like Anti-bacterial, antioxidant, antitumor activities, UV resistant and moisture absorbing properties; it can be used as a finishing agent for natural or man-made fibers of textile industries, in cosmetics industries as skincare, and used for biomedical polymeric products.³⁸

It was seen with the accordance with the Kirby disk Bauer test that Sericin had higher antimicrobial properties as compared to fibroin, with the highest inhibition found in Staphylococcus aureus with 30 mm and the lowest zone was found in fibroin with 18mm in Escherichia coli though it was still considered to have sensitive properties when reacted with E. Coli as per the standard Bacterial Inhibitor scale [Table 1].

Overall the results against the UTI with respect with Silk proteins of the Muga silk proved quite positive as both the sericin and fibroin was showing antimicrobial properties and can be considered as an antimicrobial agent for UTI infections.

Conclusion

It was observed that there was a pattern of Antibiotic sensitivity for the UTI bacteria when it was made to react with the silk based proteins in Muller Hinton agar along with the controls, it was observed that the Silk proteins namely fibroin and sericin has antibiotic properties in the crude form and has the potential to be used as an antibiotic in UTI Infections. Nearly all the bacteria shows susceptible ranges within the protocolsof Kirby-Bauer disk diffusion susceptibility test, the zone measuring sizes can be utilized as a potential microbial drug if further research is done.

This is the first report to illustrate the antimicrobial effects of the muga silkworm Antheraeaassama cocoon proteins and its effect as a potential treating agent against the Urinary tract infection bacteria (s). The sericin obtained from the cocoon of the silk was found to have more antimicrobial effects against the pathogenic bacteria compared to fibroin. However, extensive research is still needed to investigate the in vivo behavior and exploit the material withfurther modifications to make it a multi-functional agent against the UTI.

Acknowledgement

The research work was partially funded by a student project under Assam Science Technology and Environment council. The authors also acknowledges the infrastructure facility provided by the host institute, Assam down town University. We extend gratitude to BioAptagen Laboratories Pvt. Ltd., Research Park Guwahati, IIT and SRL diagnostics, Guwahati for technical help.

Conflict of interest

Authors report no conflict of interests.

Funding Sources

There are no funding source. 

References

1. Stamm, W. E., & Norrby, S. R. (2001). Urinary tract infections: disease panorama and challenges. The Journal of infectious diseases, 183 Suppl 1, S1–S4. https://doi.org/10.1086/318850
   CrossRef
2. Kweon, H. Y., & Cho, C. S. (2001). Biomedical applications of silk protein. International Journal of Industrial Entomology, 3(1), 1-6.
   CrossRef
3. Yamamoto, T., Miyajima, T., Mase, K., &Iizuka, T. (2003). Production of sericin a silk protein by the silkworm. BIO INDUSTRY, 20, 13-18.
4. Dandin, S. B., & Kumar, S. N. (2007). Bio-medical uses of silk and its derivatives. Indian Silk, 45(9), 5-8.
5. Kundu, B., Kurland, N. E., Yadavalli, V. K., &Kundu, S. C. (2014). Isolation and processing of silk proteins for biomedical applications. International journal of biological macromolecules, 70, 70-77.
   CrossRef
6. Wang, Z., Zhang, Y., Zhang, J., Huang, L., Liu, J., Li, Y. … & Wang, L. (2014). Exploring natural silk protein sericin for regenerative medicine: an injectable, photoluminescent, cell-adhesive 3D hydrogel. Scientific reports, 4(1), 1-11.
   CrossRef
7. Mondal, M., Trivedy, K., & NIRMAL, K. S. (2007). The silk proteins, sericin and fibroin in silkworm, BombyxmoriLinn.,-a review.
   CrossRef
8. Tiwari, M. (2017). The role of serratiopeptidase in the resolution of inflammation. Asian journal of pharmaceutical sciences, 12(3), 209-215.
   CrossRef
9. Kundu, S. C., Kundu, B., Talukdar, S., Bano, S., Nayak, S., Kundu, J. … & Ghosh, A. K. (2012). Nonmulberry silk biopolymers. Biopolymers, 97(6), 455-467.
   CrossRef
10. Gautam, R., Jain, A., & Kapoor, S. (2017). Silk Protein based Novel Matrix for Tissue Engineering Applications. Indian Journal of Science and Technology, 10, 31.
    CrossRef
11. Murphy, A. R., & Kaplan, D. L. (2009). Biomedical applications of chemically-modified silk fibroin. Journal of materials chemistry, 19(36), 6443-6450.
    CrossRef
12. Murphy, A. R., John, P. S., & Kaplan, D. L. (2008). Modification of silk fibroin using diazonium coupling chemistry and the effects on hMSC proliferation and differentiation. Biomaterials, 29(19), 2829-2838.
    CrossRef
13. Wenk, E., Murphy, A. R., Kaplan, D. L., Meinel, L., Merkle, H. P., &Uebersax, L. (2010). The use of sulfonated silk fibroin derivatives to control binding, delivery and potency of FGF-2 in tissue regeneration. Biomaterials, 31(6), 1403-1413.
    CrossRef
14. Vepari, C., Matheson, D., Drummy, L., Naik, R., & Kaplan, D. L. (2010). Surface modification of silk fibroin with poly (ethylene glycol) for antiadhesion and antithrombotic applications. Journal of Biomedical Materials Research Part A: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials, 93(2), 595-606.
15. Sofia, S., McCarthy, M. B., Gronowicz, G., & Kaplan, D. L. (2001). Functionalized silk‐based biomaterials for bone formation. Journal of Biomedical Materials Research: An Official Journal of The Society for Biomaterials and The Japanese Society for Biomaterials, 54(1), 139-148.
    CrossRef
16. Vepari, C., & Kaplan, D. L. (2007). Silk as a biomaterial. Progress in polymer science, 32(8-9), 991-1007.
    CrossRef
17. Craig, C. L., &Riekel, C. (2002). Comparative architecture of silks, fibrous proteins and their encoding genes in insects and spiders. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 133(4), 493-507.
    CrossRef
18. Omenetto, F. G., & Kaplan, D. L. (2010). New opportunities for an ancient material. Science, 329(5991), 528-531.
    CrossRef
19. Altman, G. H., Diaz, F., Jakuba, C., Calabro, T., Horan, R. L., Chen, J. … & Kaplan, D. L. (2003). Silk-based biomaterials. Biomaterials, 24(3), 401-416.
    CrossRef
20. Horan, R. L., Antle, K., Collette, A. L., Wang, Y., Huang, J., Moreau, J. E., … & Altman, G. H. (2005). In vitro degradation of silk fibroin. Biomaterials, 26(17), 3385-3393.
    CrossRef
21. Park, S. H., Gil, E. S., Shi, H., Kim, H. J., Lee, K., & Kaplan, D. L. (2010). Relationships between degradability of silk scaffolds and osteogenesis. Biomaterials, 31(24), 6162-6172.
    CrossRef
22. Meinel, L., Hofmann, S., Karageorgiou, V., Kirker-Head, C., McCool, J., Gronowicz, G. … & Kaplan, D. L. (2005). The inflammatory responses to silk films in vitro and in vivo. Biomaterials, 26(2), 147-155.
    CrossRef
23. Wray, L. S., Hu, X., Gallego, J., Georgakoudi, I., Omenetto, F. G., Schmidt, D., & Kaplan, D. L. (2011). Effect of processing on silk‐based biomaterials: Reproducibility and biocompatibility. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 99(1), 89-101.
    CrossRef
24. Etienne, O., Schneider, A., Kluge, J. A., Bellemin‐Laponnaz, C., Polidori, C., Leisk, G. G., …&Egles, C. (2009). Soft tissue augmentation using silk gels: an in vitro and in vivo study. Journal of periodontology, 80(11), 1852-1858.
    CrossRef
25. Etienne, O., Schneider, A., Kluge, J. A., Bellemin‐Laponnaz, C., Polidori, C., Leisk, G. G. … &Egles, C. (2009). Soft tissue augmentation using silk gels: an in vitro and in vivo study. Journal of periodontology, 80(11), 1852-1858.
    CrossRef
26. Wang, Y., Rudym, D. D., Walsh, A., Abrahamsen, L., Kim, H. J., Kim, H. S. … & Kaplan, D. L. (2008). In vivo degradation of three-dimensional silk fibroin scaffolds. Biomaterials, 29(24-25), 3415-3428.
    CrossRef
27. Meinel, L., Betz, O., Fajardo, R., Hofmann, S., Nazarian, A., Cory, E. … &Kirker-Head, C. (2006). Silk based biomaterials to heal critical sized femur defects. Bone, 39(4), 922-931.
    CrossRef
28. Hu, X., Shmelev, K., Sun, L., Gil, E. S., Park, S. H., Cebe, P., & Kaplan, D. L. (2011). Regulation of silk material structure by temperature-controlled water vapor annealing. Biomacromolecules, 12(5), 1686-1696.
    CrossRef
29. Hu, X., Lu, Q., Kaplan, D. L., &Cebe, P. (2009). Microphase separation controlled β-sheet crystallization kinetics in fibrous proteins. Macromolecules, 42(6), 2079-2087.
    CrossRef
30. Aghaz, F., Hajarian, H., Shabankareh, H. K., &Abdolmohammadi, A. (2015). Effect of sericin supplementation in maturation medium on cumulus cell expansion, oocyte nuclear maturation, and subsequent embryo development in Sanjabi ewes during the breeding season. Theriogenology, 84(9), 1631-1635.
    CrossRef
31. Arami, M., Rahimi, S., Mivehie, L., Mazaheri, F., &Mahmoodi, N. M. (2007). Degumming of Persian silk with mixed proteolytic enzymes. Journal of applied polymer science, 106(1), 267-275.
    CrossRef
32. Aramwit, P., Damrongsakkul, S., Kanokpanont, S., &Srichana, T. (2010). Properties and antityrosinase activity of sericin from various extraction methods. Biotechnology and Applied Biochemistry, 55(2), 91-98.
    CrossRef
33. Aramwit, P., Kanokpanont, S., Nakpheng, T., &Srichana, T. (2010). The effect of sericin from various extraction methods on cell viability and collagen production. International Journal of Molecular Sciences, 11(5), 2200-2211.
    CrossRef
34. Butkhup, L., Jeenphakdee, M., Jorjong, S., Samappito, S., Samappito, W., &Butimal, J. (2012). Phenolic composition and antioxidant activity of Thai and Eri silk sericins. Food Science and Biotechnology, 21(2), 389-398.
    CrossRef
35. Caleja, C., Barros, L., Antonio, A. L., Oliveira, M. B. P., & Ferreira, I. C. (2017). A comparative study between natural and synthetic antioxidants: Evaluation of their performance after incorporation into biscuits. Food chemistry, 216, 342-346.
    CrossRef
36. Cao, T. T., & Zhang, Y. Q. (2016). Processing and characterization of silk sericin from Bombyxmori and its application in biomaterials and biomedicines. Materials Science and Engineering: C, 61, 940-952.
    CrossRef
37. Capar, G., Aygun, S. S., &Gecit, M. R. (2009). Separation of sericin from fatty acids towards its recovery from silk degumming wastewaters. Journal of Membrane Science, 342(1-2), 179-189.
    CrossRef
38. Martínez, D. C. C., Zuluaga, C. L., Restrepo-Osorio, A., &Álvarez-López, C. (2017). Characterization of sericin obtained from cocoons and silk yarns. Procedia Engineering, 200, 377-383.
    CrossRef
39. Chirila, T. V., Suzuki, S., Bray, L. J., Barnett, N. L., & Harkin, D. G. (2013). Evaluation of silk sericin as a biomaterial: in vitro growth of human corneal limbal epithelial cells on Bombyxmorisericin membranes. Progress in biomaterials, 2(1), 1-10.
    CrossRef
40. Chlapanidas, T., Faragò, S., Lucconi, G., Perteghella, S., Galuzzi, M., Mantelli, M., …&Faustini, M. (2013). Sericins exhibit ROS-scavenging, anti-tyrosinase, anti-elastase, and in vitro immunomodulatory activities. International journal of biological macromolecules, 58, 47-56.
    CrossRef
41. Lee, J. M., & Johnson, J. A. (2004). An important role of Nrf2-ARE pathway in the cellular defense mechanism. BMB Reports, 37(2), 139-143.
    CrossRef
42. Deori, M., Devi, D., Kumari, S., Hazarika, A., Kalita, H., Sarma, R., & Devi, R. (2016). Antioxidant effect of sericin in brain and peripheral tissues of oxidative stress induced hypercholesterolemic rats. Frontiers in pharmacology, 7, 319.
    CrossRef