Special Issue:

Veterinary Medicine between Sustainable Development and Public Health to Confront Global Changes

Antibacterial Effect of Zinc Oxide Nanoparticles on Drug Resistant E. coli Isolated from Chicken with a Zoonotic Perspective

Mohamed Ahmed Abaza1, Amany O. Selim2, Mona Abdallah3, Shimaa A.E. Atwa4, Hala El Daous5, Mona Abd-Allah Abd-Elrehim6, Mohamed M.S. Gaballa7, Reda R. Fathy1*

1Avian and Rabbit Diseases Department, Faculty of Veterinary Medicine, Benha University, 13736, Mushtuhur, Toukh, Qalioubia, Egypt: 2Bacteriological department, Animal Health Research Institute (AHRI), Banha branch, Agricultural Research Center (ARC), Doki, Giza, Egypt; 3Zoonoses department, Faculty of Veterinary Medicine, Benha University, 13736, Mushtuhur, Toukh, Qalioubia, Egypt; 4Biochemistry and Molecular Biology department, Faculty of Veterinary Medicine, Benha University, 13736, Mushtuhur, Toukh, Qalioubia, Egypt; 5Department of Hygiene and Veterinary Management, Faculty of Veterinary Medicine, Benha University, 13736, Mushtuhur, Toukh, Qalioubia, Egypt; 6Biochemistry department, Animal Health Research Institute (AHRI), Banha branch, Agricultural Research Center (ARC), Doki, Giza, Egypt; 7Pathology department, Faculty of Veterinary Medicine, Benha University, 13736, Mushtuhur, Toukh, Qalioubia, Egypt.

Received | June 02, 2024; Accepted | July 21 2024; Published | August 31, 2024

*Correspondence | Reda Refat Fathy, Avian and Rabbit Diseases Department, Faculty of Veterinary Medicine, Benha University, 13736, Mushtuhur, Toukh, Qalioubia, Egypt; Email: [email protected]

Citation | Abaza MA, Selim AO, Abdallah M, Atwa SAE, El Daous H, Abd-Elrehim MAA, Gaballa MMS, Fathy RR (2024). Antibacterial Effect of zinc oxide nanoparticles on drug resistant E. coli isolated from chicken with a zoonotic perspective. Adv. Anim. Vet. Sci. 12(s1): 75-89.

DOI | https://dx.doi.org/10.17582/journal.aavs/2024/12.s1.75.89

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

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

Recently, poultry producers has faced a great challenges in preserving the health and safety of their flocks due to the prevalence of various bacterial infections, particularly those caused by Escherichia coli (E. coli) (Swelum et al., 2021). Gram-negative pathogenic bacteria called E. coli are frequently found in the gastrointestinal tracts of chickens. These bacteria cause substantial economic losses, severely impair the growth performance of chickens, and can even be fatal, especially in young chicks who have just hatched (Jiddu Joseph et al., 2023). Because certain strains of E. coli are known to be zoonotic, these infections not only threaten the health of poultry but also have consequences for food safety and public health (Zachary et al., 2017). Several poultry species, specially, chickens, are particularly susceptible to E. coli spp. infection owing to several factors such as high population density, stress factors, and bad breeding conditions (Mughini-Gras et al., 2018).

Because E. coli infections in chickens can result in a range of clinical symptoms, such as respiratory, gastrointestinal, and reproductive problems, the poultry industry faces severe financial losses because of the infected birds’ higher mortality rates, decreased growth rates, and decreased egg production (Kromann and Jensen, 2022) . Furthermore, E. coli-contaminated the poultry products as meat and eggs and pose a significant transmission of pathogenic E. coli strains from poultry to human (Raheel et al., 2022). Zoonotic transmission of E. coli raises critical concerns, as it causes a wide range of symptoms among people, from minor gastrointestinal distress to severe and perhaps fatal infections (Mellata et al., 2018).

The extensive proliferation of antibiotic resistance has become more alarming in recent years and presents a significant worldwide threat to public health, resulting in millions of fatalities worldwide (Abdus Salam et al., 2023). This problem increased when bacteria form biofilms, which can greatly enhance bacterial resistance up to 1000 times and contribute to multidrug resistance (MDR) infections. The control measures implementation can lead to emergence of antibiotic- E. coli resistant strains, which increase the problem and reduces the available treatment choices for both poultry and human (Christodoulou, 2023). The lack of new powerful antimicrobial chemicals is connected to the growth in this resistance (Helal et al., 2023). Therefore, it is important to create new innovative alternative to prevent and control E. coli infections in poultry, in order to protect the poultry industry and ensure public health care (Serwecińska, 2020). To tackle this issue, this study was searching about novel techniques that have antibacterial properties and a high level of safety.

Nanotechnology has become a popular option because of its tiny particle size and enhances the absorption of materials. The use of nanoparticles show many optimistic results due to their unique physical and chemical properties, as well as their possible applications in disease prevention (Mubeen et al., 2021). Zinc is an essential micronutrient for health and biological procedures in the body. Inorganic forms of zinc, such as zinc oxide and zinc sulfate, have traditionally been used in poultry feed because it is inexpensive. However, its bioavailability is limited, which necessitates adding high levels to the feed which can lead to various problems (Hidayat et al., 2023). Zinc oxide nanoparticles (ZnO-NPs) have become an alternative for wider kinds of uses including veterinary field, however there are still concerns regarding their safety. ZnO-NPs have obtained considerable attention due to their antimicrobial properties and wide-ranging uses in industries such as agriculture and medicine (Xian-Qing et al., 2023). Contrasting to these findings, several additional research trials examining the positive impacts of zinc supplementation and didn’t record performance improvements. However, they generally noticed an improved bird’s immunological state (Yogesh et al., 2013). The central hypothesis for the this research was to assess the antimicrobial effectiveness of ZnO-NPs in vitro and in vivo against specific pathogenic bacterial strain of E. coli in broiler chicken as a potential alternative to antibiotics.

Therefore, this study aimed to investigate the zoonotic potential of E. coli infection, different serotypes prevalence, the development ability of biofilms, antibiotic resistance of E. coli among chicken and human populations. The results of this study could be utilized to advocate for the utilization of ZnO-NPs as a potent alternative antimicrobial supplement in chicken feed, given its safety, strong capacity to be absorbed by the body, and ability to kill bacteria.

Materials and Methods

Ethical Considerations

All procedures described in this study were ethically approved from the Ethical Approval Committee of the Faculty of Veterinary Medicine, Benha University, Egypt for the use of cell line, chicken, and human samples (No: BUFVTM 16-1-23). In this study we followed the ethical guidelines indicated by previously mentioned committee as the chicks were reared on caged system within a high level of sanitary conditions in segregated, cleaned, and disinfected rooms at the center of animal research at the faculty of Veterinary Medicine, Benha University, Egypt.

Samples Collection

Chicken samples: two hundred polled surveyed random samples (lung and liver 100 pooled samples each) were randomly collected from 100 diverse poultry farms in Qalioubia governorate, Egypt. Qalioubia governorate is recognized as one of the most prominent governorates for poultry production in Egypt. Liver and lung samples were hygienically collected under complete aseptic conditions. Each individual organ was carefully collected into a sterile labelled packet in an ice bag at 4°C without any unnecessary delay for bacteriological analysis.

Human samples: twenty samples (10 human urine samples and 10 nasal swab) were collected from persons who admitted to Benha Teaching Hospital at Qalioubia governorate, Egypt. All samples were transferred to the lab into sterile labelled packets in an ice box at 4°C, for bacteriological examination.

Bacteriological Isolation

For enrichment the collected samples were incubated in nutrient broth at 37°C for 24 h. The enriched samples were streaked on Eosin Methylene Blue agar (EMB) plates and incubated aerobically at 37°C overnight according to (Markey et al., 2013).

Biofilm Formation

Assessment of biofilm production was based on the distinctive appearance of colonies grown on Congo Red Agar (CRA) medium. Notably, colonies exhibiting a deep black hue, coupled with a dry and crystalline consistency, were indicative of robust biofilm production according to (Freeman et al., 1989).

Serological Identification of E. coli spp.

The serotyping process, pivotal for characterizing E. coli isolates that exhibited high biofilm production, was carried out using the slide agglutination technique. For serotyping, the “SEIKEN” antisera, a well-recognized reagent supplied by MAST ASSURE™, was employed. The serological reaction was executed by combining a 24 h-old colony of the tested E. coli strain, previously cultured on nutrient agar, with a drop of physiological saline on a slide. The resultant mixture was emulsified using a loop and thoroughly mixed with a drop of the designated “SEIKEN” antiserum according to (Markey et al., 2013)..

Antibiotic Sensitivity Assay

A panel of antibiotics commonly used for E. coli infections in both chickens and human was selected such as Norfloxacin, Gentamicin, DE Oxytetracycline, Chloramphenicol, Colistin, Trimethoprim + Sulfamethoxazole, Azithromycin, Amikacin, Neomycin and Ampicillin Clavulanic Acid. The Kirby-Bauer disk diffusion method was employed to determine the sensitivity profiles of the four serotypes of isolated E. coli strains O1, O119, O6, and O44 to antibiotics as described by Markey et al. (2013).

Zinc Oxide Nanoparticles (ZnO-NPs)

ZnO-NPs provided by the Nanoparticles Unit at the Animal Health Institute in Cairo, Egypt. The structure of the produced ZnO-NPs was analyzed using High-Resolution Transmission Electron Microscopy (HR-TEM). The ZnO-NPs’ physicochemical characteristics were examined via a UV-visible spectrophotometer (SHIMADZU-2600i, USA). The dimensions and electric charge of the produced ZnO-NPs were measured using a NANOTRAC-WAVE II Zeta sizer (MICROTRAC, USA). The surfaces plasmon resonance was measured by means of UV-Visual spectroscopy, whereas the ZnO-NPs average particle size was calculated via more than 300 particles with ImageJ software (National Institute of Health, Bethesda, MD, USA).

Cytotoxicity Assay

The cytotoxicity effect of ZnO-NPs was assessed applying the sulforhodamine B (SRB) assay, using Vero cell line obtained from Nawah Scientific Inc. Mokatam, Cairo, Egypt. Vero cells were cultivated in DMEM media with 100 mg/mL streptomycin, 100 units/mL penicillin, and 10% heat-inactivated fetal bovine serum. The cells were maintained at 37°C with 5% CO2. The cultured cells, consisting of 5 x 103 cells, were placed in 96-well plates and allowed to grow in a nutrient solution for a period of 24 h. After that, cells were exposed to different quantities of ZnO-NPs ranging from 0.01 µg/mL to 100 µg/mL that were suspended in the solution. After exposed for 72 h, the cells were treated with 150 µL of 10% trichloroacetic acid for fixation. Then, they were rinsed five times with distilled water. Then, 70 microliters of 0.4% solution of SRB dye were added to each well and kept in the dark condition at room temperature for 10 min. Following application of a 1% acetic acid wash and overnight air-drying, the protein-bound SRB stain was dissolved by the addition of 150µL of 10mM TRIS solution. The measurement of absorbance was conducted at a wavelength of 540 nm using BMG LABTECH®- FLUOstar Omega microplate reader (Allam et al., 2018).

In vitro Study

Preparation of bacterial cultures and biofilm formation: The isolated E. coli O6 strain was cultured overnight in nutrient media. The overnight cultures of bacterial isolates were properly diluted to 1:100 in Tryptic Soy Broth (TSB) which enriched by 1% glucose, conducive to biofilm development. Subsequently, aliquots of approximately 100μL from these diluted cultures were carefully dispensed into wells of a sterile microtiter plate. The plate incubated for 48 hours to promote the development of a strong biofilm. (Basumatari et al., 2021).

Evaluation of antimicrobial and antibiofilm efficacy of ZnO-NPs: The evaluation of biofilm elimination and antibacterial efficacy of ZnO-NPs performed using different concentrations of ZnO-NPs 100, 50, 25, 12.5, 6.25µg/mL into microtiter plate. To create acceptable benchmarks, negative control wells without bacterial growth and positive control wells without ZnO-NPs were maintained for each isolate. The microtiter plate was then incubated in a controlled environment at a temperature of 37°C for a duration of 24 h. The efficacy of ZnO-NPs in eliminating bacterial biofilms was assessed by inoculating samples onto nutritional agar and Congo Red Agar (CRA) media, using the method outlined by (Basumatari et al., 2021).

In vivo Study

Animal model: One hundred specified pathogen free (SPF) one-day-old chicks were acquired from Al-Fayemi hatchery in Egypt. These chicks were reared in a battery system under strict hygienic circumstances in sanitized rooms at the Animal Research Center at Benha University’s Faculty of Veterinary Medicine. They were used for field assessment of ZnO-NPs in starter feed ration against E coli infection challenge.

Experimental design: The chicks were divided into five distinct groups of twenty chicks per group. Group I served as the negative control which received no treatment, while group II represented the positive control for E. coli O6 strain infection without any intervention. Chicks in group III were receiving ZnO-NPs at one day old till the end of experiment with a dose of 50 mg/kg in feed. Group IV was given colistin as commercial antibiotic in DW which coincide with the time of challenge at 5 days of age, each challenged chick in group (II), (III) and (IV) received 0.1 ml of E. coli strain (5x1010) CFU for 3 successive days. Group V was designated as the ZnO-NPs toxicity group receiving ZnO-NPs in feed at concentration 50mg per kg. Daily activities included clinical observations, post-mortem examinations, and monitoring of both living and diseased chicks. At the 5th day post-infection, all birds from each group were euthanized in accordance with ethical standards.

Lesions in internal organs such as heart, lung, intestine, and spleen were visually scored as per established protocols (Peighambari et al., 1995). The identification and culture of bacteria were conducted utilizing tissue swabs that were obtained post-mortem from poultry in each group on the 5th day following infection. Bacterial re-isolation was executed employing the previously established isolation method. Histopathological examinations were conducted on samples from cecum, liver, lung, spleen, and heart. Additionally, comprehensive hematology and biochemical analysis were conducted, collected blood samples were determined by spectrophotometer in which sera were separated by centrifugation at 2500 RPM. for 15 min and kept in a deep freeze at -20°C till used for determination of the biochemical parameters: Serum L-MDA (Mesbah et al., 2004), liver function tests as serum AST, ALT (Murray, 1984) ALP (John 1982) and serum urea according to (Patton and Crouch, 1977) Serum uric acid according to (Young, 2001). Serum Creatinine (Henry, 1974), serum total protein (Tietz, 1994), Albumin (Doumas et al., 1971), Enzymatic Antioxidants as CAT (Luck, 1974), Non-Enzymatic Antioxidants as GSH (Moron et al., 1979).

Statistical Analysis

SPSS software (version 25.0; SPSS Inc., Chicago, IL, USA) was used for statistical analysis. The significance between the mean values was set at P < 0.05. We analyzed the statistical differences among treated and untreated groups by using Analysis of variance (ANOVA) according to (Booth et al., 1981).

RESULTS And DISCUSSION

Bacterial Isolation and Characterization

Twenty isolates tested positive for E. coli out of the 100-chicken lung pooled samples that were examined. On the other hand, the prevalence rate of E. coli in the chicken liver samples (44%) was significantly higher than other samples. Regarding the human urine samples had prevalence rate of E. coli 30% higher than that of urine samples (20%) (Table 1).

 

Table 1: Prevalence of E. coli among different tested chicken samples.

Number of tested samples/tested organ	Total samples No.	Positive No.	Prevalence %.
Lung	100	20	20%
Liver	100	44	44%
Urine	10	3	30%
Nasal swabs	10	2	20%

 

Biofilm Formation

Among twenty E. coli lung isolates, 5 were high-level biofilm producers, as indicated by extremely dark colonies; 9 were intermediate; and 6 were low-level biofilm producers. Eight of the liver isolates were very powerful, 25 were moderately strong, and 11 were weak. Two of the urine isolates were very productive, and the third was a partial producer. Finally, the two isolates from nasal swabs were both potent biofilm formers (Figure 1).

Serotyping Using E. coli Polyvalent and Monovalent O Antisera

The results revealed a predominance of strains belonging to serotypes O1, O6, O44, and O119. The isolates from lung samples, all 5 were typed as O6 and O119 serotypes. The 8 isolates from liver samples were typed as O6, O119, and O44 serotypes. The urine and nasal swab samples each had 2 isolates belonging to the O1 and O6 serotypes (Table 2).

 

Table 2: Serotyping of E. coli strains isolates that exhibited high biofilm production.

Sample	Number of high biofilm producers	Serotyping
Lung	5	2 (O6), 3(O119)
Liver	8	2 (O6), 3 (O119), 3 (O44)
Urine	2	1(O1), 1(O6)
Nasal swab	2	1(O1), 1(O6)

 

Table 3: Antibiotic sensitivity testing results for 4 different E. coli serotypes - O1, O6, O119, and O44.

Antibiotic disc/ E. coli	O1	O6	O119	O44
Norfloxacin	R	R	R	R
Gentamicin	S	R	S	S
De oxytetracycline	S	R	R	R
Chloramphenicol	R	R	S	S
Colistin	S	R	S	S
Trimethoprim + sulfamethoxazole	R	R	S	S
Azithromycin	S	S	S	S
Amikacin	R	R	R	R
Neomycin	R	R	R	R
Ampicillin clavulanic acid	R	R	R	R

 

R: Antibiotic-resistant, S: Antibiotic-susceptible

 

The Antibiotic Sensitivity Assay

The antibiotic sensitivity assay findings for four distinct serotypes of E. coli, namely O1, O6, O119, and O44, are presented in Table 3 and Figure 2. Across the serotypes, there was a high level of antibiotic resistance. Norfloxacin, amikacin, neomycin, and the ampicillin-clavulanic acid combination were all resistant to all four serotypes. O6 exhibited the most widespread multi-drug resistance, being resistant to 9 of the 10 antibiotics examined. O1 has intermediate levels of resistance, with 6 antibiotics resistant. O44 and O119 exhibited the least antibiotic resistance, being susceptible to 5 of the 10 antibiotics tested, including Gentamicin, Chloramphenicol, Colistin, Trimethoprim-sulfamethoxazole, and Azithromycin.

 

Characterization of ZnO-NPs

The results of Zetasizer showed that the average particle size of synthesized ZnO-NPs is 163.6 nm with a narrow size distribution (poly dispersity index (PDI) is 0.04). Surface charge of synthesized NPs is -25.0 mV (Figure 3A) confirmed by TEM results showed spherical shape nanoparticles well dispersed without agglomeration ranged in size from 19.5 to 23.8 nm (Figure 3B). ZnO-NPs showed strong absorption beak at 371 nm with optical density of 1.137 (Figure 3C).

Cytotoxicity Assay of ZnO-NPs

The cytotoxicity of synthesized ZnO-NPs was evaluated using Vero cell line through SRB assay at concentration ranged from 0.01- 100 µg/ml. In SRB assay, ZnO-NPs showed no observed toxicity till concentration of 100 µg/ml that cells have the same characteristic features compared to control ones and the cell viability rate ranged from 103.177 to 93.0227 % at concentration ranged from 0.01 to 100 µg/ml, IC50 concentration is >100 µg/ml (Figure 4).

In vitro Antibacterial Activity and Antibiofilm Effects of ZnO-NPs

At a concentration of 50 µg/ml, ZnO-NPs exhibited significant antibacterial effects against E. coli serotype O6 which was identified as a multi drug resistant isolate (Figure 5A). Additionally, ZnO-NPs demonstrated antibiofilm activity against E. coli serotype O6 at a concentration of 6.25 µg/ml (Figure 5B).

 

 

In vivo Study

Clinicopathological picture: In control E. coli serotype O6-positive group, chicks showed depression, pasty vent with severe profuse whitish and greenish diarrhea, respiratory manifestation as swelling of infraorbital sinus, conjunctivitis, and nasal discharge in some birds. While post-mortem lesions revealed petechial hemorrhage in the liver with impacted cecum along intestinal tract mainly at

 

 

duodenum and jejunum. Treatment with Colistin sulphate effectively mitigated the severity of clinical symptoms, although liver congestion persisted. In contrast, ZnO-NPs treated group resulted in no clinical signs and a near-normal appearance of internal organs, show casing its protective effects (Figure 6).

Quantitative analysis revealed that ZnO-NPs significantly reduced gross lesion scores in various organs compared to the E. coli-infected group, including the liver, cecum, intestine, spleen, heart, and lungs, suggesting their potential in mitigating E. coli serotype O6 infections in chicks. In the colistin treated group, revealed congested liver in comparison with ZnO-NPs group, there is no marked changes in the liver. In case of cecum, control positive group (E. coli serotype O6 infected) showed enteritis with distended cecum with gas in comparison the zinc oxide nanoparticle normal content of cecum as demonstrated in Table 4.

Histopathological picture: Histopathological examination across different organs revealed diverse outcomes, all examined organs from the negative control group (I) have typical histological alterations. While in E. coli-infected group (II), liver showed focal to diffuse areas of hepatocytic necrosis, eosinophilic fibrillar masses, infiltration of inflammatory cells and fragmented nuclei or complete nuclear absence indicative of severe tissue damage. ZnO-NPs treated group revealed preserved hepatic structure with minimal changes while, colistin treatment mitigated necrosis and inflammation. However, a toxic dose of ZnO-NPs induced mild to moderate hydropic vacuolization and portal area thickening. Lung tissue examination showed that chickens infected with E. coli displayed significant lung tissue alterations, including serofibrinous exudates,

 

Table 4: Gross lesion score in internal organs at 5th day post infection scorings.

Organs	Postmortem findings	Groups
		Group I	Group II	Group III	Group IV	Group V
Liver	Congestion Petechial haemorrhage	0d	3.6±0.33a	1.2 ±0.5c	1.67±0.3b	3.2±0.2 ab
Lung	Pneumonia	0d	3.4±0.33a	1.1±0.33c	2.33±1.15bc	2.6±0.33ab
Spleen	Congestion Mottled appearance	0d	2.9±0.5a	1.33±0.44c	1.4±0.4c	2.67±1.31b
Cecum	Impacted cecum Caecal core	0d	2.67±1.32a	1 ±0.37c	1.2±0.16c	2.1±0.3bc
Heart	Inflammation Haemorrhage	0d	3±0.49a	0.67±0.44c	1.4±0.8c	1.33±0.33b

 

Post-mortem lesion scores mean the following: (0) no, (1) mild, (2) moderate, and (3) severe lesion, was expressed as mean ± standard error. control negative (I), E. coli infection control positive (II), ZnO-NPs treatment (III), Colistin treatment (IV) and ZnO-NPs toxic dose (V).

 

cellular debris, neutrophil accumulation, and alveolar interval was widened with dilated and congested capillaries. ZnO-NPs maintained pulmonary structure while, colistin treatment alleviated symptoms, limiting infiltration. In the spleen, E. coli-infected groups displayed notable changes, included a reduction in certain regions, accompanied by lymphocyte apoptosis and the presence of nuclear debris ZnO-NPs caused mild expansion of blood sinusoids whereas, colistin treatment resulted in lesser lymphoid depletion. A toxic dose of ZnO-NPs significantly dilated blood sinusoids in the red pulp. Cecum tissue examination revealed that chickens infected with E. coli showed severe desquamation of the lining epithelium and necrotic debris mixed with inflammatory cells. ZnO-NPs treatment maintained a healthy cecum structure while, colistin treatment reduced changes and showing mild leukocytic infiltration, in toxic dose of ZnO-NPs group resulted in epithelial desquamation. In heart tissue examination, control group (II) showed that focal areas of disrupted and fragmentated cardiac muscle fibers with mononuclear inflammatory cells indicating an inflammatory response. On other hand, both ZnO-NPs and colistin treatments preserved overall heart muscle structure. Although a toxic dose of ZnO-NPs caused mild myocardial fibre changes as shown in Figure 7 and Table 5.

Biochemical Analysis

Table 6 showed the significant differences (P < 0.05) among the treatment groups in serum AST, ALT, ALP, TP, Albumin, Globulin, Urea, Creatinin and Uric acid levels. There was significant decrease in AST, ALP, ALT, Creatinin and Uric acid serum levels and significant increase in TP, Globulin, albumin and Urea levels in E. coli infected group and Zno-NPs treated group. On the other hand, there was no significant difference between E. coli infected group and colistin treated group except for ALT, AST, urea serum levels. Additionally, uninfected Zno-NPs toxic dose group showed significant decrease in ALT, TP, Albumin, globulin, Urea and uric acid levels and showed significant increase in AST, ALP and Creatinin levels when compared with control negative group. The results revealed that the lowest level of AST and A/G ratio showed in infected group treated with ZnO-NPs when compared to the other groups.

 

Table 5: Grading and scoring of histopathological lesions of examined sample.

Treatments	Degenerative and necrotic changes of hepatocytes	Inflammatory cells infiltration	Hepatic vasculatures/ sinusoidal dilatation
Group (I)	-	-	-
Group (II)	+++	+	-
Group (III)	-	-	-
Group (IV)	+	-
Group (V)	++	-

 

Table 6: Effect of different treatment on liver and kidney function tests in SPF chicks infected by E. coli.

Group/Parameter	ALT (U/L)	AST (U/L)	ALP (U/L)	TP (g/dl)	Albumin (g/dl)	Globulin (g/dl)	A/G ratio	Urea (mg/dL)	Creatinin (mg/dL)	Uric acid (mg/dL)
Control negative group	19.33 ±1.45b	84.33 ±1.45ab	1298.66 ±13.56c	4.93 ±0.04a	2.31 ±0.05bc	2.58 ±0.01a	0.89 ±0.02d	21.06 ±0.37b	0.45 ±0.01cd	8.16 ±0.31a
Control positive group	24.66 ±2.60a	93.00 ±6.35a	1391.00 ±8.08a	4.12 ±0.02b	2.40 ±0.02abc	2.04 ±0.03b	1.02 ±0.02abc	20.30 ±0.40b	0.47 ±0.01bcd	7.36 ±0.49ab
E. coli and ZnO-NPs group	16.66 ±3.75b	65.66 ±13.56b	1312.00 ±6.92b	4.61 ±0.16ab	2.08 ±0.01d	2.37 ±0.23ab	0.93 ±0.03cd	24.40 ±0.46a	0.45 ±0.02cd	6.03 ±0.81cd
Clostin treated group	19.00 ±1.15b	89.66 ±3.75ab	1376.66 ±20.49ab	4.84 ±0.35a	2.47 ±0.11ab	2.59 ±0.11a	1.05 ±0.05ab	20.00 ±0.46b	0.43 ±0.01d	5.76 ±0.08cd
ZnO-NPs toxicity	36.66 ±2.60a	90.66 ±2.02a	1371.00 ±7.23ab	4.99 ±0.13a	2.29 ±0.08bc	2.32 ±0.08ab	0.98 ±0.003bcd	20.16 ±0.49b	0.51 ±0.01ab	5.50 ±0.05cd

 

 

Regarding antioxidants, E. coli infection induced oxidative stress, as evidenced by elevated levels of MDA and catalase and decreased GSH. ZnO-NPs further increased these antioxidant markers, indicating enhanced antioxidant defences in response to infection. Colistin drug increased MDA and catalase levels but decreased GSH, suggesting a different antioxidant response. When ZnO-NPs were administered without infection, they increased MDA, catalase, and GSH levels compared to controls, indicating their influence on antioxidant parameters independently. These comprehensive results highlight the effectiveness of Colistin and ZnO-NPs in mitigating the impact of E. coli infection in chickens, offering valuable insights into potential therapeutic strategies and the associated biochemical responses (Table 7).

Infectious microorganisms provide a significant challenge to the poultry business since they negatively impact growth rate, efficiency, and result in substantial economic losses estimated by several millions $ due to enteric infections such as E. coli infection (Kromann and Jensen, 2022). The poultry industry frequently uses antibiotics as growth promoters and to control pathogenic microbes. Nevertheless, the extended utilization of antibiotics resulted in the development of antimicrobial resistance (Raquel et al., 2023), and the possibility for transmission to humans and the sharing of genes for resistance between multiple kinds of bacteria resulted in the emergence of MDR (multidrug-resistant) bacteria (Helal et al., 2023). Multidrug resistant bacteria are a major problem because they are resistant to multiple antibiotics which affect public health, veterinary medicine and agriculture. MDR bacteria affects a wide and diverse area as it is not limited to a specific region or group of people (Catalano et al., 2022). As a result, there have been global efforts to develop new and improved antimicrobial drugs, as well as creative and efficient methods for administering antibiotics. Combating MDR bacteria is possible only with the help of the complex use of traditional and non-traditional approaches. Thus, probiotics, bacteriophages, nanoparticles, and AMPs are different strategies with their benefits and limitations. Further studies and innovations in these areas and other novel approaches are believed to offer better and lasting strategies to tackle antibiotic resistance on an international level. (Zeinab and Rafik, 2023). 

 

Table 7: Effect of different treatments on antioxidants parameters in SPF chicks infected by E. coli

	MDA (n.mol\ul)	CAT(u\l)	GSH(u\l)
Control negative group	1.27±.08d	15.66±.18de	3.79±.02b
Control positive group	1.55±.08bcd	15.89±.65cd	3.78±.16b
E. coli and ZnO-NPs group	2.67±.03a	21.47±.37a	4.25±.06a
Clostin treated group	2.32±.10ab	16.95±.45c	2.11±.03d
ZnO-NPs toxicity	1.46±.03cd	18.33±.44b	4.14±.06a

 

Mean values with different superscript letters in the same row are significantly different at (P<0.05). Data are presented as (Mean ± Standard error). Antioxidants: Serum L-Malondialdehyde (L-MDA) (MDA (n.mol\ul), Catalase CAT(u\l) and Reduced Glutathione GSH(u\l)

 

A possible use in the poultry business is using trace minerals, particularly Zinc oxide in the form of nanoparticles ZnO-NPs, as a suitable alternative to bigger particles. The inclusion of ZnO-NPs in the diet of broiler chickens led to improved zinc uptake and bioavailability of Zn and shown potential antibacterial properties against the many pathogens (Hidayat et al., 2023). This study compared the efficiency of ZnO-NPs as antibacterial and antibiofilm against E. coli O6 serotype isolated from chickens and humans, in vitro with colistin. It also reviewed the side effects on the liver and kidney functions, antioxidants and toxicity when used as a dietary supplement.

The isolation results in this study showed that 44% of the samples were positive for E. coli by which the previous studies also reported that E. coli was highly prevalent in poultry, for example 66. 3% prevalence which was observed by Abdelkarim et al. (2020). This underlines the importance of poultry as the most common source of pathogenic and antibiotic-resistant E. coli strains that can be transmitted to humans through the food chain or physical contact (De Mesquita et al., 2022). The higher isolation rates from poultry samples imply that perhaps, eradicative hygienic measures and public health interventions are more effectively controlling the spread of E. coli in humans than in birds (Reed et al., 2023).

The prevalence of the highly capable biofilm producers E. coli. strains in different sample sources highlight the need of minimizing contamination, as biofilms have been linked to increased antibiotic resistance and virulence (Murugesan et al., 2022). The results revealed that the predominance of E. coli strains was present in serotypes O1, O6, O44, and O119. Lung samples which were positive for of 5 isolates, all of which were of serotypes O6 and O119. All the 8 liver isolates were further identified into the serotypes O6, O119, and O44. Urine samples and nasal swab had two isolates of serotype O1 and O6. The junction of these data suggests the potential for zoonotic transmission among diverse hosts. There is several evidence that support the fact that retail chicken meat can transmit pathogenic E. coli to humans (Dipak et al., 2021).

Antibiotic resistance exhibited a remarkable prevalence within different serotypes; however diverse resistance patterns were detected. Serotype O6 showed multidrug resistance against all tested antibiotics except Azithromycin. The occurrence of multidrug resistance in O6 is quite troublesome and suggests the probable idea for antibiotic selection for local poultry industry (Veloo et al., 2022). These findings highlight the need for tailored strategies to tackle antibiotic resistance in chickens recorded in various serotypes of E. coli.

The physicochemical profiles of synthesized ZnO-NPs were carefully investigated that would help to understand their possible antibacterial and antibiofilm activities. Further characterization as well as analysis using XRD test proved that the ZnO-NPs are crystalline and the atomic structure matches the previous literature works (Król-Górniak et al., 2023). The shape, nanostructure, size, and dispersion of the NPs were investigated using TEM where it was confirmed that the ZnO-NPs were successfully synthesized (El-Ghwas et al., 2022). Due to the smaller size of ZnO-NPs they have a large surface area to volume ratio which makes them more reactive and more capable to absorb more. This enhanced reactivity is essential for their antibacterial property since the destruction of bacterial cell membrane and interference of biofilm formation is directly proportional to the reactivity between the NPs and bacterial cells. Also, the large surface area provides a better opportunity to interact with microbial cells; this attribute increases the ZnO-NPs to penetrate and erode biofilms, making them suitable for use as antibiofilm agents (Hidayat et al., 2023). These property understandings can well account for the antibacterial and antibiofilm effects evidenced in ZnO-NPs.

Determining the biocompatibility of Vero cells requires first evaluating their cytotoxicity. In vitro tests on ZnO-NPs cytotoxicity against cell lines proved its cytotoxicity. From cytotoxic perspective, the ZnO-NPs are suitable for therapeutic applications due to their high cytocompatibility, as showed by their high cell viability IC` 54W32 value across the concentration range and lack of noticeable toxicity (De Berardis et al., 2010; Martinez et al., 2011).

The antibacterial and antibiofilm characteristics of ZnO-NPs were determined in vitro by testing them against E. coli serotype O6 isolates. The results indicated that 50 µg/ml had strong antibacterial effects and 6.25 µg/ml was effective in breaking up biofilms. Helal et al. (2023) investigated how quickly nano formulations can pass through biological barriers, such as biofilms, due to their small size, large surface area, and intense reactivity. These nano-formulations show promise for treating multidrug-resistant (MDR) pathogens and decreasing biofilm development. They also show a high preference against bacterial cell walls. Antibacterial mechanism of ZnO-NPs as shown by Krishnamoorthy et al. (2022), who involves making more malondialdehyde and reactive oxygen species (ROS) effects in bacterial cells. Which leads to membrane peroxidation, decreased permeability, denaturation of intracellular proteins, DNA damage, and membrane leakage. The information we’ve gathered supports the conclusions of (Mohd Yusof et al., 2020; Ashengroph et al., 2020), who also documented that ZnO-NPs had notable antibacterial effects against foodborne pathogens such S.aureus , E.coli, and Salmonella spp. that are important to poultry. According to these findings, ZnO-NPs may eventually take the place of traditional antibiotics in the raising of chickens. We investigate the impact of giving one-day-old SPF chicks 50 mg ZnO-NPs /kg of feed based on the earlier findings. Evaluating this supplementation to the commercial antibiotic colistin, we will examine whether it functions as an antibacterial agent against multidrug-resistant E. coli serotype O6.

At the time of the bacterial challenge, the results showed that ZnO-NPs successfully decreased clinical symptoms and post-mortem lesions, suggesting its potential for therapeutic use. These results are agreed with earlier research that demonstrated ZnO-NPs antibacterial properties in living organisms (Hameed et al., 2016). Our study’s results are consistent with those of Hidayat et al. (2023), who showed that ZnO-NPs had antibacterial properties against gastrointestinal infections in broiler chickens, including Enterococcus spp. and E. coli. ZnO-NPs is safe and does not upset the balance of commensal bacteria, hence they advised using it at a dosage of 100 mg/kg. Li et al. (2021) and Long et al. (2022) have also revealed equivalent outcomes, demonstrating that high bioavailability zinc supplements improve antioxidative activity and less liver damage in animals. Furthermore, our results are consistent with theirs because none of the treatment groups’ liver samples showed any symptoms of hepatotoxicity, such as hepatic bleeding or swelling.

The biochemical changes seen were a significant increase in the liver enzymes, aspartate aminotransferase (AST) and alanine aminotransferase (ALT); also seen was a significant decrease in the total serum protein, albumin and globulin. These changes suggest hepatocellular injury, and this could have a negative impact on the overall health and productivity of the poultry because the liver plays an important role in the metabolisms of protein. In comparison with the group III treated with ZnO-NPs, the level of which significantly decreased in globulin, albumin, AST, ALT, ALP and TP, the A/G ratio of the control positive group II sharply increased. This implies that there is a differential effect to ZnO-NPs, which calls for evaluation of dosage to prevent hepatotoxic effects. In the ZnO-NPs hazardous dosage group V, all the investigated biochemical parameters such as ALT, AST, ALP, TP, albumin, and globulin were found to be increased showing signs of liver stress not even when but by the toxic effect of ZnO-NPs alone. But there was relatively close similarity with the infected group IV treated with colistin, which indicates that both treatments are equally likely to have toll on the biochemical stress in the liver.

These results are in support with Sonwane et al. (2017) where they presented that hepatocellular damage could have caused hepatocyte membrane damage associated with hepatocyte content leakage during the detoxification of E. coli and bacterial toxins. This might partly explain why the liver enzymes are highly raised, which are very key indicators of liver dysfunction. AST and ALT enzymes as biomarkers in toxicological investigations are very sensitive to reveal that an enlarged liver is a sign of an underlying disease that suggests inflammation of the organ. In addition, the effects of the observed decrease in serum total protein, albumin and globulin levels would be to suggest that liver synthetic function is impaired in the face of the growing broiler industry and their overall health and productivity could be adversely affected. Kaneko et al. (1997) noted that renal affection and hepatocyte damage can cause failure in the plasma protein synthesis and therefore, protein losses due to low serum urea, uric acid, and creatinine.

Antioxidant activity and stress response are the two important factors that affect the health and production of poultry. In this study, the E. coli infection group treated with ZnO-NPs had the highest MDA, GSH in serum and significantly higher CAT level (P < 0. 05). The CAT, MDA, and GSH of Group V were significantly higher than those of the control group after the exposure to a more toxic number of ZnO-NPs. These enzymes include glutathione (GSH) that help in removal of free radicals and catalase (CAT) that help in the removal of hydrogen peroxide which are forms of oxidative stress. Similarly, Hafez et al. (2020) and Zhao et al. (2014) reported higher serum GSH and CAT activity in broiler chicks fed with ZnO-NPs. Equally, Abd-El Rhman et al. (2018) observed higher serum MDA concentrations in the E. coli infected chickens because of endotoxins resulting from E. coli infection. These endotoxins cause an increase in the production of ROS and RNS which in turn result in the damage of proteins and nucleic acids and increased lipid peroxidation. The applicability of these results in poultry farming is quite profound. Thus, improved antioxidant activity, which is supported by the increase in GSH and CAT, may help to decrease the level of oxidative stress resulting from bacterial infections and environmental stressors. This can result in better health status of the birds by lowering cellular damage, increasing immune function, and increasing general growth and production in poultry. In addition, the knowledge of the processes that occur within the framework of oxidative stress and its effect on health also paves the way for effective interventions to improve the birds’ resistance to infections and stress, for example using antioxidants or nanoparticles such as ZnO-NPs.

The following should be considered as future investigation and prospects: the chronic influence of ZnO-NPs on the health and performance of chickens, mechanism pathways of antibacterial activity of ZnO-NPs for efficient prevention and control of some poultry economic effective diseases, and large-scale field trials to evaluate the ZnO-NPs in the commercial environment.

CONCLUSIONS And RECOMMENDATIONS

The results of our study confirmed our main hypothesis, which aimed to assess the effectiveness of ZnO-NPs in combating pathogenic multi-drug-resistant E. coli bacteria in broiler chickens. We specifically focused on the antibacterial and antioxidant properties of ZnO-NPs, with the goal of finding a safe and potent alternative to antibiotics for the treatment and prevention of drug-resistant E. coli O6 infections. The in vivo study demonstrated that ZnO-NPs are capable of alleviating E. coli induced- signs, macroscopic and microscopic lesions, and possess a potent antioxidant agent. These findings suggest that ZnO-NPs could serve as an alternative to the antibiotic Colistin in the field to combat antibiotic resistance in both humans and chickens. This is one of the unintended consequences of the conventional wisdom that antibiotics are the most effective method of preventing and controlling bacterial infections.

ACKNOWLEDGEMENTS

The authors grateful thank Dr.Dalia EL-Masery at nanotechnology unit of Animal Health Research Institute (AHRI), Agricultural Research Center (ARC), Doki, Giza, Egypt for her valuable assistance during the practical part of research.

NOVELTY STATEMENT

Availability of ZnO-NPs as antibacterial agent for efficient prevention and control of some poultry economic diseases.

AUTHOR’S CONTRIBUTION

All authors participated in the experimental work design, analysis, observation, data collection, results, correction, and overlooking. Writing of the original outline and statistically analysis of results was done by Reda, Fathy, and all authors have revised and agreed on the manuscript

Conflicts of Interest

The authors declare no conflict of interest

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