Enhanced Neomycin Antibiotic Properties Through Proteolytic Enzyme in Escherichia coli Induced Infection in Broiler Chicks

Muhammad Shahkar Uzair1, Muhammad Mushtaq1, Muhammad Shuaib1*, Saqib Nawaz2, Mehboob Ali2, Faiz ur Rehman2, Abubakar Sufyan3,

Usman Zeb4, Muhammad Ayaz5 and Muhammad Aamir Khan5

1Department of Poultry Science, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar, Pakistan.

2College of Veterinary Science, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar, Pakistan.

3Department of Livestock and Poultry Production, Bahauddin Zakariya University, Multan, Pakistan.

4Institute of Biotechnology and Genetic Engineering, The University of Agriculture, Peshawar, Pakistan.

5Directorate General (Research) Livestock and Dairy Development Department, Khyber Pakhtunkhwa, Peshawar.

ABSTRACT

The study aimed to explore the beneficial aspect of serratiopeptidase (proteolytic enzyme) combined with low profile antibiotic (Neomycin) in chickens against the E. coli infection at the finisher stage. Both in-vitro and in-vivo studies were carried out to evaluate the antimicrobial activities and potency of this combination. A total of one hundred and eighty day-old chicks were randomly allotted to 6 groups i.e. G1 (negative control), G2 (positive control), G3 (standard antibiotic neomycin only), SN-1 (Serratiopeptidase @40g/L+Neomycin@10mg/L), SN-2 (Serratiopeptidase @50mg/L+ Neomycin @20mg/L), and SN-3 (Serratiopeptidase @ 60mg/L+ Neomycin @30mg/L). Each group consisted of thirty birds with 5 replicates (6 birds/ replicate). On day 21, fresh inocula of E. coli (1x109cfu) were orally administered to all the groups except negative control. The results revealed that birds in SN-3 group showed significantly larger zone of inhibition, increased feed intake, weight gain and improved feed conversion ratio compared to standard and control groups. Moreover, reduced (p<0.05) mortality and morbidity index was found in SN-3 group among all the treated groups. It was concluded, that use of serratiopeptidase combination with neomycin has better impact on broiler chickens to enhance the antimicrobial effect of Neomycin against E. coli infection and to enhance production parameters.

Article Information

Received 14 November 2022

Revised 25 April 2023

Accepted 21 May 2023

Available online 30 June 2023

(early access)

Published 16 July 2024

Authors’ Contribution

MSU conducted research trials, data collection. MM, MS data evaluation, data analysis, manuscript writing and review. SN, MA, FUR lab analysis, proteolytic enzymes and antibiotics dose preparation. AS, UZ, MA, MAK data evaluation, manuscript writing and review.

Key words

Serratiopeptidase, Histopathology, Mortality, Neomycin, In-vitro

DOI: https://dx.doi.org/10.17582/journal.pjz/20221114101149

* Corresponding author: shoaibwzr@gmail.com

0030-9923/2024/0005-2127 $ 9.00/0

Copyright 2024 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Serratiopeptidase (Serratia E-15 protease) is a proteolytic enzyme prepared from plants materials through Enterobacterium serratia sp. E-15 found in the intestine of Bombyx mori (silkworm). It has a higher anti-inflammatory effect as compared to other proteolytic enzymes and can easily and safely digest non-living tissues which are obtained as a side-product of healing process. For instance, it dissolves scar tissue, arterial plaque, cysts, blood clots and fibrosis. In sinusitis it also acts as mucolytic agent (Robert, 2009). Serratiopeptidase is more active in lungs infections as it removes dead scar tissues and mucous that leads to natural healing of the body tissue through replacement of dead tissues with healthy ones that make possible lung functions normal. It has anti carcinogenic effect as well as it dissolve the outer surfaces like fibroblast breasts which help the body immune system to work against these cancerous substances in the body (Robert, 2009).

Escherichia coli are the naturally occurring microbial flora in poultry and humans gut that cause gastrointestinal diseases. Most of E. coli strains are not pathogenic while some pathogenic strains are responsible for causing lesions in immune-compromised hosts (Akond et al., 2009). E. coli are associated through more severe ailments and occasionally by life threatening infections such as meningitis, endocarditis, urinary tract infection (UTI), septicemia, and epidemic diarrhea in adults and children (Akond et al., 2009). Moreover, in poultry it causes yolk sac infection, omphalitis, cellulitis, swollen head syndrome, coligranuloma, and colibacillosis (Gross, 1994).

Neomycin is an aminoglycoside, which is comprised of two or more amino sugars linked by glycosidic bond (Fischer et al., 2006). It is naturally produced by the bacterium Streptomyces fradiae (Nobel Foundation, 2008). Its Production needs particular nutrient environments in either stationary or submerged aerobic conditions. The compound is then secluded and filtered from the bacterium. Individual effects of these antibiotic and seratopeptidase have been studied in animal model. It is hypothesized in this study that neomycin and seratopeptidase may have synergestic effect against E. coli infection. Based on this hypothesis, the present study was planned to assess the efficacy of Seratiopeptidase with Neomycin against E. coli in-vitro. It was also the objective of this study to evaluate the efficacy of seratiopeptidases with neomycin on overall performance of broiler chicks.

MATERIALS AND METHODS

Birds husbandry

This study was conducted in the Department of Poultry Science, the University of Agriculture Peshawar, Pakistan. A total of 180 days old chicks were purchased from the local market. They were randomly allocated to six groups including G1 (negative control) means no infection and no treatment, G2 (positive control) means only infection and no treatment, G3 (standard) means infection treated with standard antibiotic Neomycin @ 30mg/L, SN-1 (Serratiopeptidase @40g/L+ Neomycin@10mg/L), SN-2 (Serratiopeptidase @50mg/L+ Neomycin @20mg/L), and SN-3 (Serratiopeptidase @ 60mg/L+ Neomycin @30mg/L). Each group was comprised of 5 replicate (6 birds per replicate). The birds were reared under standard managmental condition for 35 days. Feed was presented ad libitum and birds had free access to water. The birds were vaccinated against Newcastle disease, infectious bursal disease and infectious bronchitis.

In-vitro antimicrobial effect of serratiopeptidase with neomycin

Liver tissue was collected from E. coli infected bird obtained from Poultry Research Unit of the Department of Poultry Science, University of Agriculture, Peshawar, enriched in nutrient broth and incubated at 37°C for 24 h. After incubation the broth was checked for growth and compared with negative control. The inoculum from optimized growth of E. coli was taken from nutrient broth and streaked over nutrient agar plates, incubated at 37°C for 24 h for colony morphology. After incubation, the plate was observed for typical E. coli colonies. A single colony was picked and subcultured in fresh media followed by confirmation through gram staining. Each of neomycin (30 μg/Disc), serratiopiptidase and neomycin combination at the rate of 1 µl +10 µg, 2 µl +20 µg and 3 µl +30 µg were placed on ager plate at recommended distance (24mm from each other and 12mm from plate edges). All the plates were properly labelled, sealed with parafilm, and incubated at 37 °C for 48 h. Zones of inhibition against the E. coli were recorded after 48 h. DMSO (dimethyl sulfoxide) 10% was poured on discs taken as negative control (Buwa and Staden, 2006).

In-vivo antimicrobial activities of serratiopeptidase with neomycin

Chicks were placed in metabolic cages 2 weeks before inoculation with E. coli. Concentrated inocula (1.0 x 109cfu/ml) were prepared in order to induce E. coli infection in treated birds (Griffin, 1995). Briefly, E. coli on nutrient agar plates were shifted to 10 ml sterilized peptone water test tube and incubated at 37oC for 18-24 h to activate the culture. The tested organism were transferred twice in nutrient broth, and then incubated again for 18-24 h at 37oC. The cell concentration of 1.0 x 107cfu /ml was achieved by diluting the active culture serially in nutrient broth (Eman and Hoda, 2008).

Inoculation of E. coli

At day 21 of the experimental trail, fresh and enriched E. coli inocula (1.0 x 109 CFU/ml) was administered orally through drinking water to all treatment groups according to standard protocol developed by Eman and Hoda (2008). Simultaneously, serratiopeptidase and neomycin were given at the dose rate of 40mg/L+10mg/L, 50mg/L+20mg/L, and 60mg/L+30mg/L through drinking water to groups SN-1, SN-2 and SN-3, respectively. During experiment chicks were assessed weekly for their feed intake, weight gain, morbidity, and mortality.

Statistical analysis

The data were analyzed through statistical packages scientific analysis system (SAS) by using complete randomized design (CRD) according to standard procedure developed by Steel and Torrie (1981). Level of significance P<0.05 was considered for variables and clinical signs were categorized as mild (listlessness, loss of appetite) labeled as +, moderate (loss of appetite, listlessness, cough, diarrhea) labeled as ++, and severe (listlessness, loss of appetite, cough, diarrhea, labored breathing, yellow droppings, soiled vent) labeled as +++. For Pairwise comparison we performed least significant difference test (LSD).

RESULTS

In the current experiment, significant effects of antimicrobial activity of neomycin and serratiopeptidase against E. coli was observed in in-vitro test. Highest (22.33 mm) zone of inhibition was recorded at SN-3 treatment (60mg+30mg) as compared to G1 and other treatments while lowest zone of inhibition was observed in G3 group as shown in Figure I. Results regarding the feed intake have been presented in Table I. Post-infection feed intake decreased (p<0.05) in G2 group compared to the other treatments groups while higher (p<0.05) feed intake was recorded in G1 and SN-3 groups as compared to other groups. Similarly, post infection weight gain was reduced (p<0.05) in G2 group of the birds that was not treated with serratiopeptidase and neomycin, followed by the standard group. While the highest (p<0.05) weight gain was recorded in the SN-3 group among all groups (Table II). Infected birds of G2 and G3 showed poor FCR compared to G1 group while improved (p<0.05) FCR was observed in all treated group compared to G2 and G3. However, numerically best FCR was noted in SN-3 groups among treated groups as shown in the Table III. Results regarding mortality of broiler chicks during the induced pathogenic infection have been shown in the Table IV. The highest mortality was recorded in G2 group, G3 and SN-I groups as compared to other treatment groups. Decreased (p<0.05) mortality was observed in SN-3 group among G2, G3, SN-1, SN-2 groups. Morbidity index indicated that the birds in groups of positive control, standard and SN-I showed the highest disease signs while, birds in G1, SN-2 and SN-3 groups showed improved health status.

 

Table I. Effect of serratiopeptidase and neomycin on feed intake (g) in broiler chicks (Mean±SE).

Groups	Week-1	Week-2	Week-3	Week-4	Week-5	Overall
G1	118.3±2.90	299.6±9.06	480.3±8.37	781.6±16.4a	1002±11.0a	2682±47.1a
G2	118±1.52	303.3±5.20	475±12.5	621.6±24.5c	844.6±29.2dc	2362.6±68.6d
G3	116±2.30	292±6.42	466.6±6.76	705±6.80b	893.3±11.6bc	2473±19.0bc
SN-1	118±1.52	297±4.72	472±6.80	701±3.21b	813.6±40.9d	2401.6±38.3cd
SN-2	117.6±2.02	302±3.78	476.3±4.66	735±8.18b	933±19.3ba	2564±29.4b
SN-3	119.6±2.60	298.6±6.74	481±4.93	774.6±15.1a	1009.3±7.62a	2683.3±11.2a
P-value	0.784	0.378	0.788	<0.010	<0.010	<0.010

 

Means having different superscripts in columns are significantly different at p<0.05. G1, negative control; G2, positive control; G3, standard antibiotic; SN-1, Serratiopeptidase @40g/L+Neomycin@10mg/L; SN-2, Serratiopeptidase @50mg/L+ Neomycin @20mg/L; and SN-3, Serratiopeptidase @ 60mg/L+ Neomycin @30mg/L.

 

Table II. Effect of serratiopeptidase and neomycin on weekly weight gain (g) in broiler chicks (Mean±SE).

Groups	Week-1	Week-2	Week-3	Week-4	Week-5	Overall
G1	108.3±1.20	215.3±2.02	303±5.85	385.6±6.00a	457.6± 11.3b	1470±25.8a
G2	108.3±1.85	219±0.57	303±4.72	241± 5.56d	307.6± 6.69e	1179±13.00e
G3	108.3±1.20	218.6±1.45	302±6.24	248.6±14.49d	330.3±5.33d	1208±21.0d
SN-1	109.6±2.90	216.6±3.84	303±3.60	330± 5.50c	399.3± 2.84c	1358.6±12.1c
SN-2	109.3±2.02	215.3±2.18	302±1.52	354.3±2.60b	409.6± 5.45c	1390.6±6.11b
SN-3	108±0.57	217.6±2.72	303.6±3.92	375±5.77a	475.3±6.83a	1479.6±12.4a
P-value	0.980	0.785	0.998	<0.010	<0.010	<0.010

 

Means having different superscripts in columns are significantly different at p<0.05. See Table I for details of group.

 

Table III. Effect of serratiopeptidase and neomycin on weekly feed conversion ratio (FCR) in broiler chicks (Mean±SE).

Groups	Week-1	Week-2	Week-3	Week-4	Week-5	Overall
G1	1.08±0.01	1.38±0.02	1.58±0.00	2.02±0.01c	2.18±0.03cb	1.81±0.00b
G2	1.08±0.02	1.38±0.02	1.56±0.02	2.57±0.06b	2.74±0.09a	2.00±0.03a
G3	1.06±0.02	1.33±0.02	1.54±0.05	2.85±0.14a	2.70±0.06a	2.04±0.02a
SN-1	1.07± 0.02	1.36±0.01	1.55±0.00	2.12±0.03c	2.03±0.10c	1.96±0.03b
SN-2	1.07±0.03	1.39±0.03	1.57±0.02	2.07±0.02c	2.27±0.02b	1.84±0.02b
SN-3	1.10±0.02	1.36±0.01	1.58±0.03	2.06±0.02c	2.11±0.02cb	1.81±0.01b
P-value	0.742	0.246	0.923	<0.010	<0.010	<0.010

 

Means having different superscripts in columns are significantly different at p<0.05. See Tables I and II for details of groups.

 

Table IV. Effect of serratiopeptidase and neomycin on mortality (%) and morbidity index in broiler chicks (Means±SE).

Groups	Mortality (%)	Morbidity index†
G1	00±00b	+
G2	1.00±0.57a	+++
G3	1.00±00a	++
SN-1	1.00±00a	++
SN-2	0.66± 0.33ba	+
SN-3	00±00a	+
P- value	0.053	*****

 

† Morbidity index: Mild (+), Moderate (++), Severe (+++). For statistical details and details of groups, see Tables I, II and III.

 

DISCUSSION

In the present study single or combined effect of neomycin and serratiopeptidase was assessed against E. coli infection in broiler chicken in vitro as well as in vivo. The present results revealed that using neomycin with serratiopeptidase in broiler chicken synergistically improved the antibacterial effect of Neomycin against E. coli infection. Treatment @ 60mg+30mg in SN-3 group significantly increased the zone of inhibition than control and other treatment groups. The best results in the aforementioned group of treatment might be due to the activity of serratiopeptidase helping in reducing the bio-film layer of bacteria, also reducing the fluid in the tissues. These results were in similar with the results documented by Sabin et al. (2014), and Selan et al. (2015). They reported that combination of enzyme with antibiotics showed maximum zone of inhibition against different culture of bacteria as compared to individual effect of antibiotics. The feed intake in experimental birds showed significant variation after E. coli induction. Feed intake post infection showed significant decrease in G2 group (positive control) as compared to other treatments groups. However, significantly higher feed intake was recorded in control and SN-3 groups than all other groups. In previous studies, Balevi et al. (2000) reported that addition of enzyme does not result in any improvement in feed intake of broiler chicks which is contrary to our study findings. While, Fathi et al. (2016) depicted that addition of enzyme had significant effect on feed intake of broiler chicks due to enhancement of enzymatic activity and growth performance. Post infection weight gain was significantly reduced in G2 group followed by the G3 group. Highest weight gain was recorded in SN-3 group among other treated groups that was similar to G1 group. Previous findings of Islam et al. (2004) and Youssef et al. (2014) also supported our findings regarding body weight gain of broiler chicken. The improved weight gain might be linked to mitigation of infection, enhancing the metabolism and feed intake that resulted positive impact. Pre-infection of weekly FCR showed non-significant alteration while post-infection at week 4 and onwards showed significant variation. Positive control (G2 group) and standard (G3 group) showed significantly poor FCR post infection than rest of the groups. Control group and other treatment groups (SN-1, SN-2 and SN-3) showed significantly best FCR. These results were consistent with results documented by Youssef et al. (2014) and Mahmoud et al. (2017). They reported that better FCR in the treated groups might be due to improved digestive enzyme efficiency. Similarly, Swain et al. (1996) also reported similar findings which were in close agreement with current study results. The highest mortality percentage was recorded in G2, G3, and SN-1 group as compared to other treatment groups. While, the morbidity index indicated that the birds in groups of G2, G3, SN-1 showed more disease signs as compared to birds in G1, SN-2 and SN-3 that showed improved health status at post infection phase. This might be due to synergistic effect of seratopeptidase enzyme with neomycin in combating infection. Similar results were also documented by Griggs and Jacob (2005), Jalaludeen et al. (2005), and Shawkat et al. (2015) as they found decreased morbidity and mortality in chicks after enzyme addition. This effect might be ascribed to improvement in digestive enzyme activity and also producing inhibiting compounds after immune response.

CONCLUSIONS

It is concluded from the results of the current study, that seratiopeptidase combination with neomycin @ 60mg/L and 30mg/L in broilers is highly effective in obtaining production parameters, lowers mortality rate and improving morbidity index and a positive impact on antibacterial activity of neomycin in-vitro.

ACKNOWLEDGMENTS

I would like to express my gratitude to my primary supervisor, Dr. Muhammad Mushtaq, who guided me throughout this project. I wish to acknowledge the help provided by the other teaching staff in the faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture Peshawar, Pakistan. I would also like to show my deep appreciation to my supervisory committee who helped me to finalize my project. The research work is part of M.Sc (Hons) degree program and as self-supported.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

IRB approval

The study was approved by the Board of Study (No.245/PS/AUP, dated July 27, 2020), conducted at the Department of Poultry Science, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture Peshawar, KP, Pakistan.

Ethical statement

The experimental procedures used in the study were according to the guidelines of the Ethical Review Committee of the Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture Peshawar. Proper approval was taken by the aforementioned authority before start of experimental trial

Statement of conflict of interest

The authors have declared no conflict of interest.

REFERENCES

Akond, M.S., Alam, S.M., Hassan, R. and Momena, S., 2009. Antibiotic resistance of Escherichia coli isolated from poultry and poultry environment of Bangladesh. Int. J. Fd. Saf., 11: 19-23.

Balevi, T., Ucan, U.S., Coskun, B., Kurtoglu, V. and Cetingul, S., 2000. Effect of a commercial probiotic in the diet on performance and humoral immune system in layers. Hayvancilik Arastirma Dergisi, 10: 25-30.

Buwa, L.V. and Staden, J.V., 2006. Antibacterial and antifungal activity of traditional medicinal plants used against venereal diseases in South Africa. J. Ethnopharmacol., 103: 139-142. https://doi.org/10.1016/j.jep.2005.09.020

Eman, M.A. and Hoda, M.Z., 2008. Studies on the effect of garlic preparation on Escherichia coli O157:H7 causing enteritis in lambs. Egypt. J. clin. Pathol., 21: 102-129.

Fathi, M., Haydari, M. and Tanha, T., 2016. Effects of zinc oxide nanoparticles on antioxidant status, serum enzymes activities, biochemical parameters and performance in broiler chickens. J. Livest. Sci. Technol., 4: 07-13.

Fischer, J., Ganellin, C. and Robin, C., 2006. Analogue-based drug discovery. John Wiley and Sons. 507. ISBN 9783527607495. https://doi.org/10.1002/3527608001

Griffin, P.M., 1995. Escherichia coli O157:H7 and other enterohaemorrhagic Escherichia coli. (ed. M.J. Blazer). pp. 10-18.

Griggs, J.P. and Jacob, J.P., 2005. Alternatives to antibiotics for organic poultry production. J. appl. Poult. Res., 14: 150-156. https://doi.org/10.1093/japr/14.4.750

Gross, W.G., 1994. Diseases due to Escherichia coli in poultry. In: Escherichia coli in domestic animals and humans (ed. C.L. Gyles). CAB International, UK., pp. 237-259. ISBN 0-85198-921-7

Islam, M.W., Rahman, M.M., Kabir, S.M.L., Kamruzzaman, S.M. and Islam, M.N., 2004. Effects of probiotics supplementation on growth performance and certain haemato-biochemical parameters in broiler chickens. Bangladesh J. Vet. Med., 2: 39-43. https://doi.org/10.3329/bjvm.v2i1.1933

Jalaludeen, A., Sabiha, M.K.A. and Elizabeth, V.K., 2005. Effect of supplementation of probiotic on the growth performance of broiler chicken. Ind. J. Poult. Sci., 40: 73-75.

Mahmoud, A., Adel, I., Attia, A., Zenat, I., Reda, A., Mahmoud, S. and El-Sayed, A., 2017. The effectiveness of dietary sunflower meal and exogenous enzyme on growth, digestive enzymes, carcass traits, and blood chemistry of broilers. Environ. Sci. Poult. Res., 24: 12319-12327. https://doi.org/10.1007/s11356-017-8934-4

Nobel Foundation, 2008. The Nobel Prize in Physiology or Medicine 1952. Retrieved., 2008-10-29. https://www.nobelprize.org/prizes/medicine/2008/summary/

Robert, R., 2009. The Miracle Enzyme™ is Serrapeptase, the 2nd gift from Silkworms. Rayleigh Avenue, Davenham, Cheshire, CW9 8LE, UK., pp. 11-108. ISBN: 1910521000, 9781910521007

Sabine, N.T., Frasch, M., Schaller, E.F., Stange, L.T. and Zabel, 2014. Synergistic effects of antimicrobial peptides and antibiotics against Clostridium difficile. Antimicrob. Agents Chemother., 58: 5719-5725. https://doi.org/10.1128/AAC.02542-14

Selan, L., Papa, R. and Tilotta, M., 2015. Serratiopeptidase: A well-known metalloprotease with a new non-proteolytic activity against S. aureus biofilm. BMC Microbiol., 15: 207. https://doi.org/10.1186/s12866-015-0548-8

Shawkat, A., Sadeq, M., Shubiao, W., Robert, A. and Mingan, C.S., 2015. Towards the control of necrotic enteritis in broiler chickens with in-feed antibiotics phasing-out worldwide. J. Anim. Nutr., 1: 1-11. https://doi.org/10.1016/j.aninu.2015.02.004

Steel, R.G.D. and Torrie, J.H., 1981. Principle and procedures of statistics. McGraw Hill Book Co. New York

Swain, B.K., Johri, T.S., Shrivastav, A.K. and Majumdar, S., 1996. Performance of broilers fed on high or low fibre diets supplemented with digestive enzymes. J. appl. Anim. Res., 10: 95-102. https://doi.org/10.1080/09712119.1996.9706134

Youssef, A., Walid, S., El-Tahawy, El-Hamid, A.E., El-Hamid, A., Nizza, A., Bovera, F., Al-Harthi, M.A. and El-Kelway, M.I., 2014. Effect of feed form, pellet diameter and enzymes supplementation on growth performance and nutrient digestibility of broiler during days 21-37 of age. Arch. Tierzucht., 57: 1-11. https://doi.org/10.7482/0003-9438-57-034