EVALUATION OF EFFICACY COMBINATION OF ZNO NANOPARTICLES AND RESISTED ANTIBIOTICS AGAINST AVIAN PATHOGENIC E.COLI

The antibiotic resistance of Avian Pathogenic E. coli is considered one of the biggest public health concerns worldwide and with the growing necessity to find unconventional approaches for formulating new forms of safe and price of effective antibiotics for monitoring the spread of resisted pathogens globally, zinc oxide (ZnO) has the prospective to influence many aspects because of their antimicrobial efficacy. Therefore, our study is intended to evaluate the in vivo antibacterial activity of the chemically synthesized PEG-6000 coated-ZnO nanoparticles in low (11.6mg/ml) and high (23mg/ml) doses alone and in combination with florfincol and streptomycin, experimentally. The obtained results showed that oral administration of a low dose of ZnO NPs (11.6 mg/ml) alone or with florfincol and streptomycin revealed mild efficacy against APEC infection, while a high dose of ZnO-NPs (23 mg/ml) whether alone or combined with florfincol and streptomycin for 3 days of treatment, gave good improvement, that was confirmed grossly, and by histopathological inspection of the liver and kidney. It could be concluded that not only do PEG-6000 Coated ZnO NPs have a powerful antibacterial effect against APEC in broilers, but they also can enhance the antibacterial activity of resisted antibiotics.


INTRODUCTION
Escherichia coli normally inhabitant in the poultry intestine, but under stress factors certain strains, became virulent, those called avian pathogenic E.coli (APEC), spread into the different internal organs, causing the systemic fatal disease colibacillosis (McPeake et al., 2005).APEC is the main reason for economic losses as a result of morbidity, mortality, costs attained in prevention and disease control, and the condemnation of poultry carcasses worldwide (Ronco et al., 2015).
Colibacillosis denotes two-thirds of the reported bacterial infections in poultry production (Souillard et al., 2011), and mainly affects birds 4-8 weeks of age, though adults can be affected either by primary or secondary infection (Rashid et al., 2013), causing several local and systemic infections like septicemia, omphalitis, swollen head syndrome, cellulitis, pericarditis, and perihepatitis (Paixao et al., 2016).The most predominant serogroups related to colibacillosis are O1, O2, and O78, and their incidence differs between farms and countries (Mehat et al., 2021).
Several approaches were performed for controlling APEC infections, such as hygiene improvement, competitive exclusion of probiotics usage, vaccination, and the introduction of immune-potentiates; however, each of the following had limited achievement (La Ragione et al., 2004).This has necessitated the use of antimicrobial chemotherapy to decrease the incidence and mortality of outbreaks of avian colibacillosis (Dheilly et al., 2012).However, E. coli, like many other bacteria, can create antibiotic resistance and modern reports have described increased resistance to those antimicrobial agents commonly used for treatment (Yang et al., 2004).With the running out of options to treat bacterial diseases, the appearance of nanoparticles has emerged as a novel antimicrobial.ZnO NPs have several advantages: high antibacterial effectiveness at low concentrations against E.coli (Zhang et al., 2010), minimal toxicity, and decreased side effects (Lara et al., 2010).
The antibacterial activity of the ZnO NPs is induced by the release of Zn2+, which has a significant influence on the active transport inhibition, amino acid metabolism, enzyme system distribution and the liberation of oxygen species from the surface of ZnO (OH-, H2O2, and O2-2), causing fatal damage to microorganisms, indicating that ROS formation is the key mechanism for antibacterial ZnO NPs activity (Sirelkhatim et al., 2015).
Water molecules' presence around ZnO NPs motivates ZnO-Zn bond formation between nanoparticles, leading to agglomerate formation (flocculation), that hinders the dispersibility and use of ZnO NPs (Yıldırım and Durucan, 2010).Thus the PEGylation process is used to mend the stability of ZnO NPs against precipitation through the prevention of sticking the particles together (Liufu et al., 2004).Our study was considered to investigate the in vivo efficacy of two different concentrations of PEG-6000 Coated ZnO NPs and the synergistic effect of resisted florfincol, and streptomycin with ZnO NPs.

PEG-6000 Coated ZnO Nanoparticles
In situ PEG-6000 Coated ZnO nanoparticles, with sizes of 20 and 50 nm were supplemented by the Avian and Rabbit Medicine Department, Assiut University, Assiut, Egypt (Aml et al., 2023).ZnO NPs physically were spherical shape by TEM; white powder; stable colloid in a mixture of water, ethanol, and PEG-6000, an absorption peak seen at ~ 432 cm -1 optical properties (Abs.) of λmax = 301 nm and 380 nm and average size (TEM) in the range of 19-67 nm.The intense absorption peaks at ~ 1006, 1362, 1473, and 2877 cm -1 were observed by IR spectra, approving the presence of polymer molecules covering ZnO nanoparticles.ZnO powder was used, ovened for 3 hours at 160 • C, then dissolved in distilled water, to avoid particle aggregation and deposition.The sample was vortexed (10 minutes) and then sonicated (90 minutes) and the obtained suspensions (100 mL with a 1M ZnO concentration), were considered as a stock solution and then to be diluted for chicken treatment usage.

E. coli strain
The E. coli strain serotype O2, resistant to florfenicol and streptomycin, was previously isolated from broiler chickens, suffering from high mortalities, serotyped, and used for challenges (Badry, 2018).The bacterial suspension was adjusted to contain 10 8 CFU/mL by the colony count technique.

Experiment Design
Chicks were divided into ten equal groups (10 chicks per each) in separated small floor pens, and at 15 days old, they were challenged with E. coli strain serotype O2, (0.1ml) of the inoculum containing 10 8 cfu/ml, by intra-tracheal route, except group (10) served as the negative control (not challenged, not treated).Chickens were observed daily for signs, mortalities, and postmortem lesions.After 27 hours from infection, chickens were treated with two different doses of PEG-6000 coated ZnO nanoparticles, by oral gavage, as shown in Table (1), in addition to the resisted antibacterial drugs florfincol and streptomycin (Sigma-Aldrich) in drinking water.All groups were examined clinically after the third-and fifth-days post-treatments (dpt).

Table 1: Experimental design
APEC recovery from treated groups was done by using an E. coli count on EMB agar.The livers of chicks were removed aseptically and homogenized.The homogenates were tenfold serially diluted before plating on the EMB, according to (Ahmed et al., 2020).The EMB agar plates were incubated overnight at 37 • C, and the E.coli colonies were counted.

Histopathological examination
Specimens from the liver and kidney from groups 1 and 2 after 5 days post-oral treatment were sampled, fixed in 10% neutral buffered formalin, dehydrated in graded alcohol series, cleared with xylene, and embedded in paraffin wax.Sections of 4 μm were cut and stained with hematoxylin and eosin (H&E) (Bancroft et al., 1996), inspected by a light microscope, and photographed by a digital camera.

Clinical signs
All infected treated groups suffered from mild clinical signs of colibacillosis compared with chicks in the positive control group (group 9), which exhibited typical signs of colibacillosis after 3 days from infection including symptoms of weakness, depression, loss of appetite, dyspnea, coughing, sneezing, gasping, and nasal discharge.Control negative (group 10), chicks remained active throughout the experimental period as they appeared healthy without any clinical signs.
The morbidity percentage was 100%, while there were no mortalities in all infected groups.

Post mortem lesions
In the control positive group (G 9), congestion in all organs with pericarditis, fibrinous perihepatitis (Fig. 1B), splenomegaly, air sacculitis, pneumonia, and enteritis were the most observed common lesions.The severity of the lesion increased and persisted until the end of the experiment.Group 7 and group 8 which were treated only with florfincol and streptomycin, respectively showed mild congested liver, mild air sacculitis, congested lung, and enteritis.Group 5 was treated with PEG-6000 coated ZnO NPs (low dose 11.6 mg/kg) and streptomycin showed mildly congested liver, nephritis, and highly congested lung, and there was a minor improvement in the p/m picture after the fifth day of treatment.
Group 3, which was treated with a low dose of PEG-6000 Coated ZnO NPs (11.6 mg/ kg) and florfincol showed a closely normal carcass appearance after 3 days posttreatment, but there was mild congestion in the liver after 5 days from treatment.Group 1 which was treated only with a low dose of PEG-6000 Coated ZnO-NPs (11.6 mg/ kg), showed normal carcass appearance with mild congested liver and pericarditis after 3-and 5days post-treatment.
Table 1: APEC recovery rate from slaughtered chicks after each treatment

Histopathology
The score of histopathological lesions in the liver and kidneys to detect the residual effect of PEG-6000 Coated ZnO NPs in low and high doses are summarized in Table (2).The lesions were as follows:

Liver:
The liver of control-negative chicks (G10) revealed normal hepatic architecture (Fig 3 . A). Examination of the liver from chicks infected with E. coli (control + ve G9) revealed a severe inflammatory reaction that was noticed mainly in the periportal area and consisted of severe congestion, severe periportal infiltration of heterophils, necrosis of the epithelial lining bile ductules, an interstitial inflammatory reaction, and multiple areas of mononuclear cellular infiltration.
Damage to the endothelial lining of blood vessels and thrombosis can also be seen (Fig. 3.B-E).The liver of chicks treated with PEG-6000 Coated ZnO NPs at dose of (11.6 mg/kg) G1 showed no infiltration of heterophils.Congestion and necrosis of the epithelial lining bile ductules were still present.There were sporadic areas of mononuclear cellular infiltration (Fig. 3.F-H).Administration of PEG-6000 Coated ZnO NPs at dose of (23 mg/kg) G2 for 3days showed great improvement, in which there was minor periportal mononuclear cellular infiltration and normal bile ductules, no necrosis of the bile ductular epithelium, and no heterophilic infiltration (Fig. 3 I-J).(control -ve) 0 0 ZnO NPs at a dose of 23 mg/kg G2 for 5 days showed congestion of the blood vessels and sinusoids, hemorrhage, and sporadic areas of mononuclear cellular infiltration (Fig. 3 K-L).
Table 2: Histopathological lesion score in liver and kidneys in PEG-6000 Coated ZnO-NPs low and high doses groups after 5 days post-treatment compared with control groups.

DISCUSSION
APEC strain is regarded as a sub-pathotype of extra-intestinal pathogenic E. coli (ExPEC) causing avian colibacillosis, which is considered a potential zoonotic agent and some of these strains cause severe human diseases such as hemorrhagic colitis and hemolytic uremic syndrome (Nolan et al., 2013).The pathogenicity of E. coli strain O2, in the control positive group by intratracheal inoculation revealed depression, closed eyes and respiratory symptoms (gasping, rales, and nasal discharge), these findings in agreement with (Barnes, 1994).All infected treated groups suffered from mild clinical signs of colibacillosis.The control negative group remained active throughout the experimental period.
At necroscopy, congestion was the most prominent picture in all organs combined with perihepatitis, pericarditis, air sacculitis, pneumonia, and splenomegaly which comes in line with that obtained by (Sharada and Ruban, 2010) (Radi et al., 2021).Also, the toxic effects of ZnO NPs may be attributed to their solubility, resulting in increased intracellular Zn2+, as nanoparticles were predicted to increase the occurrence of inflammatory reactions in different organs, especially the lymph nodes (Mokhtar et al., 2019).

CONCLUSION
Exploring the in vivo antibacterial potential of ZnO NPs by oral treatment against APEC exposed a very good clinical prognosis.The best treatment for APEC was achieved using PEG-6000 coated ZnO NPs (23mg/kg) for 3 days alone or synergistically with both resisted florfincol and streptomycin, enhancing their efficacy, which was confirmed by gross examination, reisolation, and histopathological inspection.Continuous usage of the same dose for 5 days led to internal binging of histopathological toxicity appearing in the liver and kidney, so it was important to pay attention to the dose of ZnO-NPs to avoid toxicity.Further effective combination regimens using ZnO NPs, and resistive antibiotics are recommended for treating multi-drug resistant APEC.

Fig. 1 :
Fig. 1: (A) 23-day-old chicks from the negative control group displaying normal post-mortem picture.(B) 23-day-old chicks from the positive control group showed cloudiness and thickness of the air sac accompanied by serous exudates, marked pericarditis, and fibrinous perihepatitis.

Fig. 4 :
Fig. 3: Photomicrograph of liver tissues (A) Control negative group, liver showing normal hepatic architecture.(B-F) Liver of bird infected with E. coli.(B) Severe periportal heterophilic infiltration and congestion.(C) Higher power showing infiltration heterophils.(D) Necrosis of the bile ductular epithelium.(E) Interstitial infiltration of inflammatory cells and focal mononuclear cellular infiltration.(F) Thrombosis of blood vessels.(G-H) Liver of birds treated with ZnO NPs low dose.(G) Portal area showing congestion of blood vessels and necrosis of bile ductular epithelium.(H) Interstitial infiltration of inflammatory cells and focal mononuclear cellular infiltration.(I-J)Liver of bird-treated ZnO NPs high dose for 3 days.(I) Portal area with mild mononuclear inflammatory reaction and normal bile ductules.(J) Normal hepatic architecture.(K-L) liver of the bird treated with ZnO NPs for 5 days.(K) Congestion and dilatation of sinusoids.(L) Mononuclear cellular infiltration.HE Brayner et al.,  2006), and may due to the PEGylation process, which has been proven to reduce the cytotoxicity of ZnO NPs effectively, while ZnO NPs at dose of 23mg/ml for 5 days showed congestion of blood vessels and sinusoids, hemorrhage and sporadic areas of mononuclear cellular infiltration.Similar lesions were observed in the liver of rats after oral administration of ZnO NPs (11 mg/kg) for five consecutive days(Ben-Slama et al.,  2015).It was detected that oral administration of ZnO NPs can cause degenerative changes, necrosis, focal leukocytic infiltration of the liver, minimal renal lesions, hyalinosis, and lymphocytic myocarditis in the heart.It has been claimed that ZnO NPs cause hepato-and nephrotoxicity through epigenetic changes in the gene expression of mtTFA, and PGC-1α that may subsequently cause mitochondrial dysfunction which activates the generation of ROS and oxidative stress