Highlighting on the antimicrobial effect of silver nanoparticles on E. Coli Isolated from Newly Weaned Rabbits

Highlighting on the antimicrobial effect of silver nanoparticles on E. Coli Isolated from Newly Weaned Rabbits

El-Gedawy,A .A*, Yasser Hanna*, Mohamed A. Elkady**, Abeer F.I.Hassan*** , Amira.M.Ali***Marwa Ibrahem**,Mahmoud Abdelnaeem**,Asmaa E. Gamal Eldeen* and Rania,S.M.*

*Bacteriology Dept., Animal Health Research Institute, Dokki. **Bacteriology Dep., AHRI, Mansoura

***Veterinary hospital, faculty of vet. Med., Zagazig University.

Abstract:

Rabbits reared for meat in industrial farms exhibit the highest rates of antimicrobial usage compared to other food-producing animals. E. coli is principally responsible for neonatal and post-weaning colibacillosis in rabbits, which is frequently accompanied by enteritis and diarrhea.

A total of 300 rectal swabs  were obtained  from diarrheic  live  newly  weaned (30 - 40 days old) Newzeland white rabbits from 3 farms from different localities    in Shakira  province, which suffered from diarrhea, high morbidity and mortality rates. Bacteriological isolation of E.  coli from diarrheic weaned rabbits, Biochemical and Serological identification of isolated strains as well as detection of some virulence genes in isolated strains, antimicrobial Susceptibility Testing of isolated strains, assessment of the efficacy of silver nanoparticles on E. coli isolates, scanning electron microscopy (SEM) To determine the influence of the silver nanoparticles on the morphology of E. coli. Experimental modelling in E. coli free rabbits.

The   allover   incidence   of E. coli isolation from rabbits with diarrhea was 28.33% (85/300).

Serotypes of the isolated E. coli strain were O stereotype in order of frequency  O158,  O128, O125,  O18,  O119,  O148  and untypable  (25%,     15%,  20,10%, 10%, 5% and 15 %  isolates respectively). E. coli isolates were highly resistance to Ampicillin (AM) (83.3%; 70/85), Cefoxitine (CF) (60%; 51/85), Sulfamethoxazole (SXT) (56.5%; 3156/85), chloramphincol (CHI) (57.7% ;56/85), Kanamycin (KAN) and Streptomycin (S) (41/85; 48.8%).Hundred percent ( 6|6) of tested E. coli isolates carry eaeA  gene, while 66.7 %( 4/6) of the tested E. coli isolates were positive to tsh gene. The MIC50 of AgNPs-H2O2 was 0.625 μg/mL against E. coli. Electron microscopy scanning (SEM) was utilized to determine the differences in bacterial morphology after application of the of AgNPs-H2O2at 1 MIC and 2 MIC concentrations. Significant alterations (exhibited varying degrees of distortion) in the morphology of treated cells. Experimental infection results revealed that the nanotechnology-treated group exhibited a reduction in bacterial load and clinical signs, indicating potential treatment efficacy.

Key words: E.coli, Rabbits, Antimicrobial resistance, Silver nanoparticles.

INTRODUCTION:

Rabbits reared for meat in industrial farms exhibit the highest rates of antimicrobial usage compared to other food-producing animals, leading to alarming rates of antimicrobial resistance within the industry (Crovato et al. 2023).

Rabbits are raised for a variety of purposes,   including   their   use   in laboratory, and fur, which constitute a valuable   by-product, meat, besides being efficient converters of vegetable protein into high quality animal protein.  The  production  of rabbit  meat  on  an  industrial  scale has been very slow to develop due to    excessive    mortality    among growing rabbit which hinder mass production (Okerman, 1999) E coli   bacterium   is   a member  of     the  family Enterobacteriacae   facultative anaerobic, gram-negative short rods and  considered  a  common inhabitant of the gut of the worm- blooded   animals,   including   man (WHO, 1996).

1. coli  infection is the primary causative agent in most outbreaks of diarrhea in newly weaned rabbits. Several strains of varying virulence cause diarrhea in rabbits belong to different serotypes (Percy et al, 1993). E. coli is principally responsible for neonatal and post-weaning colibacillosis in rabbits, which is frequently accompanied by enteritis and diarrhea (Adriana Silva, 2024)

The enterotoxigenic E. coli (ETEC) is leading to infectious diarrhea worldwide (Wolf, 1997). The adherence of bacteria to  the  enterocytes  is mediated   by   intimin,   an   outer membrane protein encoded by eaeA that  mediates  close  attachment  of enteropathogenic bacteria to apical surfaces    of   epithelial    cells,    is required    for    formation    of   the attaching-effacing  lesions  and  for full  pathogenesis  of  the  bacteria  ( Nataro and Kaper, 1998).

Tsh gene, is another adhesion-related factor. The tsh gene encoding

 a temperature-sensitive hemagglutinin, was isolated   and characterized   by and may act as an adhesion, particularly in the initial stages of bacterial colonization. The Tsh autotransporter seems to be one of the factors associated with induce fluid accumulation in the rabbit gut (Maluta et al. 2014).

Antibiotic resistance in commensal bacteria from food animals is a global concern, with research focusing on the effects of antibiotic use on animals and the potential transmission of resistant bacteria to humans. The use of antibiotics in food-producing animals has led to the development of MDR food bacteria like E. coli. While studies have primarily focused on E. coli’s prevalence in other livestock animals, there is a significant lack of research on rabbits (Ramos et al. 2020).

Silver nanoparticles (AgNPs) exhibit biocidal effects against various bacteria. They can interact with the cell surface of Gram-negative bacteria, causing damage and structural changes that enhance bacterial permeability (Jones et al. 2008.

In Egypt there is a little literature on newly    weaned    rabbit     diarrhea causes by E. coli.

Therefore, this study aims to determine the occurrence and antimicrobial resistance patterns of E. coli in newly weaned rabbits. Evaluation of the antibactericidal efficacy of silver nanoparticles (AgNPs -H2O2) against E. coli this can be achieved by: Isolation of E. coli from diarrheic weaned rabbits,  Biochemical  and Serological identification of isolated strains as well as detection of  some virulence genes in isolated strains, antimicrobial Susceptibility Testing of isolated strains, assessment of the Efficacy of silver nanoparticles on E. coli isolates, Scanning electron microscopy (SEM) To determine the influence of the silver nanoparticles on the morphology of E. coli. Experimental modelling in E. coli free rabbits.

MATERIAL and Methods

Sample:

A  total  of  300  rectal  swabs  were obtained  from  live  newly  weaned (30 - 40 days old) Newzeland white rabbits from 3 farms from different localities (100 each)   in Shakira  province, which suffered from diarrhea, high morbidity and mortality rates during extended     periods     of     seasons (summer,  winter,  autumn and  spring). Samples were directly transferred to the   bacteriological   laboratory examination without delay.

Bacteriological examination:

 Isolation and biochemical identification of E. coli:

 Collected samples  were  enriched first  on buffered peptone  broth incubated aerobically at 37Cº for 24 hours,  then a loopful  from  each sample  was  inoculated  separately onto  MacConkey  agar  and  Eosin Methylene blue agar (EMB).  The inoculated plates were aerobically incubated at 37Cº for 24h. Suspected colonies were subjected to morphological and biochemical identification according to Cruickshank et al. (1982).

Serological identification:

Suspected E. Coli isolates were subjected to serological identification according to Quinn et al. (2002) for determination of (O) antigen using slide agglutination test.

Antimicrobial Susceptibility Testing:

Agar disc diffusion assay was used to test antimicrobial sensitivity according to the Clinical and Laboratory Standards Institute standards (CLSI, 2017). The antimicrobials used were ampicillin (AM; 10 μg), tetracycline (TE; 30 μg), gentamicin (GE; 10 μg), sulfamethoxazole (SXT;25 μg), amikacin (AK;30 μg), ceftriaxone (Cef;30 μg), Streptomycin (S 30 μg), Chloramphenicol (CHI 30 μg), Kanamycin (KAN 30 μg), Cefoxitin (Cf 30 μg), Nalidixic acid (Na 30 μg), Ofloxacine (Ofx 5 μg) and Ciprofloxacin (Cip 5 μg). These antimicrobials are commmon utilized in veterinary field.

 Molecular detection of virulence genes in E. coli isolates using PCR:

It was carried out on 6 isolates (two isolates from each farm).

- Extraction of DNA according to QIAamp   DNA mini kit instructions.

-Preparation of PCR master mix according to Emerald Amp GT PCR master mix (Takara).

- Cycling conditions of the primers during cPCR:

Table (1): Cycling conditions of the different primers   during cPCR .

- Evaluation of the Efficacy of silver nanoparticleson E. coli isolates:

Silver nanoparticles combined with Hydrogen peroxide (AgNPs-H2O2) were used to control some virulent organisms.  This study will make atrial to investigate the Efficacy of Silver nanoparticles combined with Hydrogen peroxide on E. coli isolates.

Antimicrobial effect of silver nanoparticles on E. coli isolates:

Silver nanoparticles (AgNPs-H2O2) was obtained as a commercial product from El-Delta Center for Nano silver Technology Company, Mansoura, Egypt. The stock solution of the product contained 45-nm silver nanoparticles (0.00004467 mL/liter), hydrogen peroxide (50% per liter) and natural herb mint (1 mL/liter) at a concentration of 5 mL/liter of water. The particles size was previously determined to be 30.17–67.92 nm with a zeta potential estimation of −0.192 mV (El-Gohary et al. 2020). The AgNPs-H2O2 mixture was prepared by diluting the stock solution in sterile dis- tilled water to achieve the desired commercial concentration. The minimum inhibitory concentrations (MIC50 and MIC90) of AgNPs-H2O2 were determined against E. coli isolates by the broth micro dilution method (CLSI; 2012). Briefly, microliter plate wells were supplemented with   various   concentrations of AgNPs-H2O2 ranging from 100, 50, 25, 10, 5, 2.5 1.25, 0.625, 0.312, 0.156 and 0.078 μg/ml. E. coli isolates colonies were added in Muller Hinton broth and adjusted to the density of a 0.5 McFarland standard  (1 × 108 cfu/ml). Each well received a final inoculum of 5 × 105 cfu/mL, and the plates were incubated for 24 h at 37 °C. A well E. coli isolates l alone and another well included E. coli isolates with AgNPs-H2O2 were used as reference control. The lowest agent con- centration that entirely prevents an organism’s observable growth is known as the MIC endpoint. The MIC50 and MIC90 were calculated using an orderly array method (Hamilton-Miller 1991) where the middle value was selected as MIC50. The MIC90 was determined in the same way by selecting the appropriate value from the orderly array.The inhibitory effect of nanoparticles product on E. coli isolates was performed using disc diffusion method according to (CLSI 2017).

Scanning electron microscopy (SEM):

To determine the influence of the silver nanoparticles combined with Hydrogen peroxide on the morphology of E. coli, SEM was used in conjunction with a few modifications to the technique reported by Bajpai et al. (2013). The Silver nanoparticles combined with Hydrogen peroxide were added at (MIC and 2MIC) concentrations to working cultures. After 1 hour at 37 °C, 1 ml sections of each tube were collected and centrifuged for 10 minutes at 4000 rpm. Fix with 2.5% buffered gluteraldehyde in 0.1 M PBS pH 7.4 at 4˚C for 2 hours. Following fixation in 1% osmic acid for 10 minutes, wash three times with PBS (10 minutes between each wash) (30min). Three PBS washes (10 minutes each), followed by 30 minutes of dehydration with an ascending sequence of ethyl alcohol concentrations (30, 50, 70, 90, and absolute alcohol) infiltrated with acetone. SEM samples were dried with liquid CO2, supplied by SPI supplies®, using a critical point drying device. In an SPI- Module TM Vac/Sputter, mounted on aluminum stubs and gold-coated. Scanning electron microscope JEOL JSM-5200LV (SEM, Hitachi, and Tokyo, Japan). Electron Microscope Unit at Tanta University.

- Experimental model:

Animals

 Thirty days old 120 rabbits were used in this study, divided into four groups. Rectal swabs were examined to assure pathogenic E. coli free rabbits. Good housing and feeding were supplied.

 They were infected orally with an infective dose of109mo. /ml. (Stas T.1999)

Strains:

1. coli, serotype O158, isolated from rectal swab of diarrheic rabbit (farm 1) in the current study.

Experimental design:

Animals were divided into 4groups (30 rabbits each)

G1: Control negative (non- infected)

G2: Control positive (infected and non-treated).

G3:  infected and treated with Silver nanoparticles combined with Hydrogen peroxide.

G4:  infected and treated with Norfloxacine.

- Treatments were added to drinking water from the first day of infection at a dose and duration according to the instruction of the producing companies.

- Clinical signs and mortality rates were recorded daily from 0 day of infection till slaughter day

Reisolation of experimental E.coli strain:

Reisolation rates of E. coli experimental strain were assessed from day 0 to slaughter day.  Two rectal swabs were taken from each rabbit from the day of infection, up to the day of slaughter. One swab was then streaked directly Onto EMB agar (Oxoid). The other was broken into a separate bijou bottle containing 2 ml of Mackonky broth (Oxoid) to determine the count of E. coli on each swab. For this, the broth was mixed on a vortex mixer (Stuart Scientific GB) for 60 sec, 0.1 ml was taken from the bijou bottle and 10-fold dilutions were prepared in sterile Mackonky broth at pH 7.2. Viable counts were then determined.

RESULTS:

Results of isolation and biochemical identification of E. coli:-

1. coli isolates were Gram negative, medium sized bacilli to coccobacilli, non sporulated and arranged single, in pairs or in short chains. Appeared as smooth, shinny,    strong lactose fermenting colon and on MacConkey's agar.

Characteristic greenish metallic sheen on EMB.   

Suspected E. coli isolates   were   lactose   fermenting colonies     and     positive     Indole, methyl red and catalase. Meanwhile, all isolates were negative oxidase, urea hydrolysis, citrate utilization, and voges-proskauer and not produced H2S.

The   allover   incidence of E .coli isolation from rabbits with diarrhea as shown in table (2) was 28.33% (85/300).  E. coli Was recovered with high  rate (46%)  from  farm  (3) ,   followed   by   Farm   (1):  (22%)  and  Farm  (2) :  (17%).  Seasonal prevalence    of   E. coli isolates from weaned rabbits revealed that the highest incidence occurred in summer season   (44%). Followed by spring (31%), then in autumn (22%), and finally in winter (13.33%).

Table (2): The prevalence of E. coli isolated from diarrheic Rabbits from different farm:

Table (3): Seasonal prevalence of E. coli recovered from weaned rabbits.

Results of serological identification of isolated E. coli:

Serotyping was applied to isolated E. coli (table 4), the isolated E. coli strain, from diarrheic rabbit were O stereotype in order  of  frequency  O158,  O128, O125,  O18,  O119,  O148  and untypable  (25%, 15%,  20%, 10%, 10%, 5%, and 15%  isolates respectively).

Table (4): E. coli serotypes recovered from diarrheic rabbits:

Results of antimicrobial Susceptibility Testing:

83. coli isolates were highly resistance to Ampicillin (AM) (83.3%; 71/85), Cefoxitine (CF) (60%; 51/85), Sulfamethoxazole (SXT) (56.5%; 3156/85), chloramphincol (CHI) (57.7%; 56/85), Kanamycin (KAN) and Streptomycin (S) (41/85; 48.8%) (Table 5). Resistance to other antimicrobial agents was always less than 30%.

Table (5): Antibacterial resistance of tested E. coli isolates

Results of cPCR FOR detection of E. coli virulence genes:

PCR assay was carried out on 6 E. coli isolates (two isolates from each farm) serotypes   (O158,   O128, O125, O119, O18 and O148) to detect two virulence genes (eaeA and tsh). It was found that 100 %( 6/6) of tested E. coli isolates carry eaeA gene, while 66.7 %( 4/6) of the tested E. coli isolates were positive to tsh gene

Table (6): Prevalence of virulence genes (eaeA, tsh,) using cPCR among Isolated E. coli isolates:

Fig (1): Agarose gel electrophoresis of conventional PCR for detection of eaeA showing amplification of 620 bp. fragment.

L (ladder): 100-600 bp.

(Pos): positive control.  

(Neg): negative control. 

Lanes: (1, 2, 3.4, 5, 6) positive samples.

Fig (2): Agarose gel electrophoresis of conventional PCR for detection of Tsh showing amplification of 620 bp. fragment.

L (Ladder): 100-600 bp.

(Pos): positive control.

(Neg): negative control.

Lanes (3, 4, 5and 6): positive samples.

Lane (1and 2): negative sample

Table (7): The distribution of minimum inhibition concentration (MIC) values of AgNPs concentrations against E. coli isolates:

Results of electron microscopy scanning:

SEM was utilized to determine the differences in bacterial morphology after application of the of AgNPs-H2O2 For 1 hour treatment, a sample suspension of E. coli was mixed with AgNPs-H2O2 at 1 MIC and 2 MIC concentrations, and the microstructure was examined (Fig. 3). Bacteria that had not been treated were used as a control. SEM was used to examine changes in bacterial morphology that had been treated and   had not been treated. Significant alterations (exhibited varying degrees of distortion) in the morphology of microbial cells treated with AgNPs-H2O2 (Fig 3B1, B2) were detected when compared to untreated cell which exhibited rod-like form with smooth undamaged surfaces (Fig 3A).

Figure (3): Scanning electron micrographs. (A): untreated E. coli, (B1):

1. coli treated with AgNPs-H2O2 at 1 MIC, (B2):E. coli treated with AgNPs-H2O2 at 2 MIC.

        Untreated E. coli               Treated E. coli with 1 MIC AgNPs-H2O2           Treated E. coli with 2 MIC AgNPs- H2O2

Table (8) Results of experimental infection with E. coli in 30 days old rabbits

Table (9) Reisolation rates of E. coli experimental isolate from experimentally infected rabbits:

DISCUSSION

Diarrhea is one of the major problems facing rabbitries in Egypt, causing high mortalities (Stakenborg et al.  2006).

Three hundred rectal  swabs from diarrheic rabbits were subjected for isolation and identification of E. coli The result indicated that 85 isolates were biochemically identified as E. coli, the allover incidence of E. coli isolation  from a rabbit with  diarrhea was 28.33% (85/300). This result is less than that isolated by Morsy et al.  (2002) as they recovered E. coli from newly weaned rabbit by 80% from examined samples (60/90). While Shahin et al. (2011) isolated E. coli by 65.7% from diarrheic rabbit.

Concerning, seasonal prevalence of E. coli infection   in weaned rabbits. The result showed  that,  summer  season  was found  to  be  the  most  important season   that   influenced   the   post weaning diarrhea  in rabbits. (44%) during summer compared to   22%,   13.33% and 31%   during autumn, winter and spring seasons respectively. This result higher than Habeeb  et al.  (1997)  who showed that, the mortality rate was found to be 18% in summer season, while no mortality    was    recorded    during winter season. While, Shehata et al. (1998) recorded 18.52% mortality rate during summer season compared to (3.70, 7.41and 7.41%) during spring, autumn and winter seasons, respectively.

Concerning, serological serotyping in the current study, 20 E coli isolates    recovered    from weaned   rabbits   were   distributed among   6   different   O    serotype groups besides untypable ones. The most    prevalent   serotypes were O158 (25%), O125 (20), O128 (15%)   followed by O18, O119 (10%), O148 (5%) and untypable strains were (15%).  Such results were similar to Percy et al.  (1993)  as they isolated 0128, and untyped from newly weaned diarrheic rabbits. Leroy et al. (1994) recovered 0128.  Saad  (1994) isolated E. coli O125 from weaned rabbits. Aisha and Youseif (1999) isolated   0128,   O125,   O158   and untyped strains.   Alshimaa  (2007) isolated     E.  coli serogroup  O125 from rabbits with enteritis.  Morsy et al.   (2002)   found   that,   Serotypes associated with diarrhoea in newly weaned  rabbits  in   Ismailia  were (O119,  O103,  O55,  O153,  O128) and  untypable  ones  with  variable percentages.  Blanco et al.  (1997) and Marches et al.  (2000) found that,   most    common    serotypes among E.  coli   strains associated with diarrhea in rabbit in order of frequency were  (0103,  049,  026, 0128, 092) on the other hand (Leroy et al 1994) recorded the isolation of  6  nonpathogenic  diarrheic  E. coli belonged to (0128) and (0132).

The majority of isolates had high resistance to Ampicillin (AM) (83.3%), Cefoxitin (CF) (60%), Sulfamethoxazole (SXT) (56.5%), chloramphenicol (CHI) (57.7%), Kanamycin (KAN), and Streptomycin (S) (48.8%). Antimicrobial resistance develops as a consequence of antibiotic consumption, along with transmission of resistant genes and bacteria across animals, animal products, humans and the surrounding area. Antibiotic-resistant bacteria may be spread directly between animals and people, as well as to soil, food, and underground water.  (Wichmann et al. 2014).

The highest drug resistance found could be due to the widespread use of antimicrobials as a therapeutic and preventative treatment in animal production farms (Mohammed et al. 2014) and (Taye et al. 2013). These findings show that rabbits may act as a reservoir for antibiotic-resistant E. coli, as well as a vector for their dissemination and a health danger. Many studies have indicated that isolated E. coli was resistant to erythromycin (100%), ampicillin (50%), tetracycline (75%), streptomycin (50%), but was highly susceptible to penicillin (100%), chloramphenicol (75%), gentamicin (75%) and amoxicillin (50%) recorded by (Kindu et al. 2019).

  Results   of PCR   analysis (table: 6) showed that (6/6) 100% of tested E. coli strains isolated from weaned rabbits with diarrhea carried (eaeA) virulence gene.  This result agreed with Blanco et al. (2005) who reported that, fecal culture examination of 20 rabbits yielded 48 E. coli isolates, 83% of which were eaeA positive. Alexis and James (2003) found that, (25%) of 28 rabbits were positive for eaeA gene.

Based on  the  obtained  molecular results, it explained the severity of clinical  signs  and  morbidity  and mortality of weaned rabbits where, The  intimin         (eaeA    gene) considered as indicator of attaching and  effacing  pathogenicity  factor. It was present in100% of tested isolates.  So, this  gene  is  clearly associated  with  diarrhoeagenic  E. coli  which  increased  the  severity and duration of diarrhea as well as mortality and rabbit inflammatory response (Mashood et al. 2009).

Concerning, tsh gene, is another adhesion factor.

The result of this study proved that   E.  coli    isolates    for    the presence  of temperature  sensitive hemagglutinin gene (tsh) revealed that (4/6)  66.7% of the examined E.coli strains from weaned rabbits with diarrhea bearing the virulence gene  (tsh).  Which go in parallel with Hanchun et al.  (2004)  who detected tsh gene in 93% of E. coli isolates from diseased animals with diarrhea  While,  this  result  not matched with Abhirrosh and Asit (2013) who did not detect tsh gene in the tested E. coli strains isolated from  diarrheic  rabbits.  Meanwhile, Maluta et a.l (2014) suggested that, EPEC might       induce       fluid accumulation in the rabbit gut. Hagedorn   et al. (2011) reported that, although tsh gene associated with the bird, it was also found in 46% of E. coli isolates.

Nanoparticles, such as silver nanoparticles, are considered alternatives to antibiotics for treating various infections.  Silver nanoparticles have large surface area to volume ratio, allowing for increased contact with bacteria and resulting in direct interaction with the bacterial cell wall to produce antibacterial activity (Castillo RR, et al. 2019). Results showed that the MIC50 of AgNPs-H2O2 was 0.625 μg/mL against E. coli. Ahmed et al. (2023) reported that AgNPs-H2O2 at concentrations of 0.625, 1.25, 2.5 and 5 μg/mL showed complete bacterial growth inhibition. Another study reported that the average MIC value of AgNPs against ESBL-producing E. coli was 27 μg/ml. Krishna et al (2018) reported that the in vitro antibacterial tests against C. jejuni showed a minimal inhibitory concentration of AgNP at the level of 50 ppm. Shafreen, et al. (2017) argued that silver nanoparticles suspensions prepared by biological methods and with concentrations higher than 100 μg/mL may lose their antibacterial effect on microorganisms.

SEM was used to examine the morphological alterations of both treated and untreated E.coli. Untreated E. coli cells (control) which exhibited usual rod-shaped characteristics, with normal and smooth cell surfaces (Fig. 3A). After 60 min of treatment with the AgNPs-H2O2 at 1 MIC, the majority of bacteria treated grew irregular with varying degrees and the membrane has burst in some cells (as shown in Fig.3B1). Greater effects to the E. coli cell membrane were seen after 60 min of treatment with AgNPs-H2O2 at 2 × MIC, nearly every cell exhibits membrane breakdown and rupture, (Fig.3B2).

According to Zhang et al. (2016), the unfavorable morphological changes of E. coli cells might be caused by the rupture of the E. coli membrane and the loss of intracellular components. Bacterial cells were likely destroyed as a result of the cytoplasmic membrane being broken or penetrated by an interfacial contacting inhibitory action on the surface (Kong et al., 2008). The targeted cells deformity induced by AgNPs-H₂O₂ depends on specific dose that was corroborated by other investigations (Diao et al, 2014). The recorded SEM pictures in this work are similar to prior studies on the effects of various antimicrobial drugs on E. coli cells, such as cinnamon oil (Ma et al. 2016 and Zhang et al. 2016). Other antibacterial agents include mustard EO (Turgis et al., 2009), cold nitrogen plasma, and clove oil (Cui et al. 2016), a thymus nanoemulsion (Moghimi et al. 2016). These SEM findings corroborate our antibacterial activity findings, which revealed that AgNPs-H₂O₂ was capable of killing E. coli at 1 MIC and were significantly more effective at 2 MIC.

Concerning experimental infection, the negative control group showed no signs of infection, validating the health status of the non-infected rabbits. In contrast, the positive control group exhibited clinical symptoms and significantly elevated bacterial load, indicating successful induction of E. coli infection and expressed high virulence as clinical manifestations and modalities occurred specially in control positive group which reached its peak at the 10^th day. The Norfloxacin-treated group demonstrated a notable reduction in bacterial load and clinical improvement compared to the positive control group. Similarly, the nanotechnology-treated group exhibited a reduction in bacterial load and clinical signs, indicating potential treatment efficacy. The findings highlight the potential of both Norfloxacin and nanotechnology in reducing E. coli infection. Norfloxacin, a widely used antibiotic, demonstrated its effectiveness in mitigating bacterial load and clinical symptoms. However, concerns about antibiotic resistance necessitate exploring alternative treatments. The use of nanotechnology in managing E. coli infection presents a promising avenue as it minimized bacterial shedding. Nanoparticles have shown antimicrobial properties and the ability to target specific pathogens, making them a potential alternative or adjunct to traditional antibiotics

CONCLUSION:

Silver nanoparticles products are considered a promising alternative medication for treatment E. coli infection and could reduce the  risk  of antimicrobial resistance.

Further intensive studies are required for implementation of the effective alternative medication to reduce the risk of antimicrobial resistance.

Ethics approval:

The study received approval from the Zagazig University Institutional Animal Care and Use Committee (ZU-IACUC) under approval number

ZU-IACUC/2/F/117/2023.