Synergistic action of naringenin and amikacin in the treatment of avian colibacillosis

Lankun Yi; Xu Chen; Zhen Zhu; Qing Liu; Yang Huang; Jianing Pang; Zeyu Wang; Ding Guan; Tenghe Ma; Mingze Cao; Yongbiao Wan

doi:10.1186/s12917-026-05745-9

. 2026 Jul 25;22:632. doi: 10.1186/s12917-026-05745-9

Synergistic action of naringenin and amikacin in the treatment of avian colibacillosis

Lankun Yi ^1,^#, Xu Chen ^1,^#, Zhen Zhu ¹, Qing Liu ¹, Yang Huang ¹, Jianing Pang ¹, Zeyu Wang ¹, Ding Guan ¹, Tenghe Ma ¹, Mingze Cao ^1,^✉,^#, Yongbiao Wan ^1,^✉

PMCID: PMC13711978 PMID: 42934705

Abstract

Background

Avian colibacillosis is an infectious disease caused by pathogenic Escherichia coli (E. coli) and is one of the significant causes of economic losses in the global poultry industry. The overuse of antibiotics has led to the emergence of resistant strains, and the presence of antibiotic residues in chickens and eggs poses a threat to human health. Therefore, exploring safe adjuvants to enhance the efficacy of existing antibiotics is one of the critical issues in poultry farming and public health research. Naringenin (NG), a naturally occurring flavonoid, has been extensively studied as a therapeutic agent for various diseases, but its efficacy in combination with antibiotics for treating avian colibacillosis has not yet been explored.

Results

In this study, we established a model of avian colibacillosis and treated it with NG and amikacin (AMK) alone or in combination. Compared with monotherapy, the combination of NG and AMK significantly decreased bacterial loads in the heart, liver, spleen, lung, and kidney by 5.0–6.0 log₁₀ CFU/g (p < 0.01). Histopathological analysis revealed that the combination treatment markedly alleviated pathological damage in the lung and duodenum compared to the AMK alone group. Furthermore, the combination therapy significantly exerted anti-inflammatory and antioxidant effects, as evidenced by reduced serum levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and malondialdehyde (MDA), alongside increased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities (p < 0.05). 16 S rRNA sequencing demonstrated that the combination treatment significantly modulated the gut microbiota composition, decreasing the relative abundance of Proteobacteria from 44% in the infection group to 1.2% (p < 0.01), while increasing Firmicutes and Bacteroidetes to levels comparable to healthy controls.

Conclusions

The research results provides evidence for the therapeutic potential of NG as an effective antibiotic synergist when combined with AMK against avian colibacillosis.

Keywords: Avian colibacillosis, Naringenin, Amikacin, Synergistic antibacterial effect, In vivo antibacterial

Background

Avian pathogenic Escherichia coli (APEC) is one of the most important bacterial pathogens affecting poultry [1]. Most APEC strains are opportunistic pathogens. Their infection is closely associated with improper feeding management and secondary bacterial infections under immunosuppressive conditions. Avian colibacillosis causes massive economic losses to the poultry industry, reaching approximately 300 million yuan annually [2]. In livestock and poultry production, antibiotics serve a vital function in the prevention, treatment, and control of infectious diseases. However, the inappropriate overuse of antibiotics for growth promotion, as well as long-term application at sub-therapeutic dosages, accelerates the development of bacterial drug resistance, including Escherichia coli (E. coli), and the emergence of multidrug-resistant (MDR) bacteria, which brings great challenges to clinical treatment [3, 4]These MDR strains not only reduce the therapeutic efficacy of clinical drugs in poultry farming but also transfer resistance genes to human pathogens, thereby threatening public health safety [5].

Natural products have been widely used in poultry production in recent years to advance the performance, hold poultry productiveness, prevent and control the enteric pathogens and reduce the application frequency of antibacterial agents [6]. Compared with conventional antibiotics, most natural products exhibit relatively low toxicity and a lower risk of inducing bacterial resistance under conventional application conditions [7].

Naringenin (NG) is a naturally occurring flavonoid with multiple pharmacological effects, low toxicity, and wide availability. Wang [8] et al. have shown that NG, a flavonoid with strong anti-inflammatory activity, can alleviate non-alcoholic fatty liver disease in mice caused by dietary methionine-choline deficiency. In recent years, the antibacterial effect of NG has also been widely recognized. Studies have shown that naringenin is a powerful inhibitor of cell signaling and inhibits biofilm formation in Vibrio harveyi and E. coli O157:H7 [9]. Currently, the use of antibiotic-plant extract combinations has been proven to be an outstanding achievement in reducing antibiotic consumption and can be used for combined treatment of various diseases, which will delay the development of antibiotic resistance [10]. However, to date, few studies have been conducted on the therapeutic effect of NG and antibiotic on avian E. coli infection.

The present study investigated the therapeutic efficacy of NG in combination with amikacin (AMK) against poultry E. coli infection using in vivo experiments, building on our group’s previous in vitro research demonstrating that NG synergizes with AMK to exert antibacterial effects by disrupting the bacterial cell wall and cell membrane [11]. To the best of our knowledge, this is the first study to demonstrate the synergistic therapeutic effect of NG and AMK in an avian colibacillosis model, providing a novel antibiotic-sparing strategy and a potential lead for developing safe natural-product-based adjuvants against drug-resistant E. coli infections in poultry.

Results

Blood biochemical analysis

We hypothesized that combined treatment with naringenin (NG) and amikacin (AMK) would reduce the elevated levels of liver function enzymes including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT), and restore the decreased levels of total protein (TP) and albumin (ALB) induced by APEC strain C7F3 infection, thereby alleviating hepatic and renal injury in chicks. All blood samples for serum biochemical analysis were collected on day 8 post-challenge. The results of blood biochemical tests are presented in Table 1. In the PC group, serum levels of ALT, AST, ALP, and GGT were significantly elevated, exceeding the upper reference limits by 2.3-, 2.9-, 2.5-, and 3.6-fold, respectively (p < 0.01). In contrast, TP and ALB levels in the PC group were significantly decreased, falling below the normal range (reduced by 55% and 79%, respectively, p < 0.01). Following monotherapy with NG or AMK, these biochemical parameters improved, approaching normal reference values. Notably, in the combination therapy group (NG + AMK), all measured parameters (ALT, AST, ALP, GGT, TP, and ALB) returned to normal ranges, and showed no significant differences compared with the NC group (p > 0.05 for all).

Table 1.

Blood biochemical index results

Parameter	PC	NG	AMK	NG + AMK	NC
ALP (U/L)	496 ± 0.08^a	313 ± 0.05^b	301 ± 0.02^b	232 ± 0.05^c	127 ± 0.03^d
GGT (U/L)	97.5 ± 1.2^a	74.8 ± 0.5^b	70.2 ± 0.8^b	37.5 ± 0.26^cd	27.7 ± 0.56^d
ALB (g/l)	8 ± 0.02^c	23.2 ± 0.05^ab	25.7 ± 0.02^ab	35.3 ± 0.06^a	38 ± 0.03^a
TP (g/l)	28.3 ± 0.41^c	36.7 ± 0.53^b	38. 1 ± 0.23^b	57.8 ± 0.27^a	63.4 ± 0.34^a
ALT (U/L)	89. 1 ± 0.02^a	59. 1 ± 0.06^b	65.5 ± 0.03^b	34 ± 0.02^cd	27 ± 0.05^d
AST (U/L)	176.2 ± 1.32^a	105.9 ± 1.18^b	127 ± 0.54^b	32 ± 0.98^d	37.8 ± 2.81^cd

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Samples were collected at 8 days post-challenge with APEC strain C7F3. Values are mean ± SD; one-way ANOVA with LSD test. Different letters in the figure indicate significant differences (p<0.05); the same letters indicate no significant differences (p>0.05)

NC negative control, PC positive control, NG naringenin, AMK amikacin

Expression of inflammatory factors in serum

We hypothesized that combined treatment with NG and AMK would downregulate the elevated levels of pro-inflammatory cytokines including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) caused by APEC strain C7F3 infection, thereby mitigating the inflammatory response in chicks. All tissue samples for inflammatory cytokine detection were collected on day 8 post-challenge. The results, as shown in Fig. 1, illustrate the effects of the PC group, the NG treatment group, the AMK treatment group, and the NG + AMK treatment group on the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in the serum. Compared to the NC group, the serum levels of IL-1β, IL-6, and TNF-α in the positive control group were significantly elevated (p < 0.05). After treatment with NG or AMK, the serum levels of pro-inflammatory cytokines decreased compared to the PC group, with the NG treatment group demonstrating significantly superior anti-inflammatory effects compared to AMK (p < 0.05), consistent with previous research confirming the excellent anti-inflammatory properties of NG. Following treatment with NG + AMK, the serum levels of inflammatory cytokines IL-1β, IL-6, and TNF-α were significantly reduced (p < 0.01), showing no significant difference compared to the NC group (p > 0.05).

Fig. 1 — Effect of NG + AMK on chicken serum pro-inflammatory cytokines. Note: NC, negative control; PC, positive control; NG, naringenin; AMK, amikacin. Samples were collected at 8 days post-challenge with APEC strain C7F3. Values are mean ± SD; one-way ANOVA with LSD test. Different letters in the figure indicate significant differences (p < 0.05); the same letters indicate no significant differences (p > 0.05)

Changes in antioxidant indicators in serum

We hypothesized that combined treatment with NG and AMK would increase the reduced levels of antioxidant indicators including glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), and decrease the elevated content of malondialdehyde (MDA) caused by APEC strain C7F3 infection, thereby enhancing the antioxidant capacity and reducing oxidative damage in chicks. All samples for antioxidant index detection were collected on day 8 post-challenge. The effects of different drug group treatments on the peroxidation-related factors GSH-Px, SOD, and MDA in the serum of E. coli-infected chicks are shown in Fig. 2. Compared with the NC group, the activities of GSH-Px and SOD in the serum of E. coli-infected chicks were significantly decreased (p < 0.05), while the MDA content was significantly increased (p < 0.05). After NG treatment, the activities of GSH-Px and SOD in the serum of chicks were significantly higher than those in the infected group (p < 0.05), and the MDA content was significantly reduced (p < 0.05). There was no significant difference in the antioxidant effect between the AMK treatment group and the NG treatment group (p > 0.05). The levels of oxidative stress-related factors GSH-Px, SOD, and MDA in the NG + AMK treatment group were regulated to a level that showed no significant difference compared with the NC group (p > 0.05). The above results indicate that NG + AMK treatment ameliorated the E. coli-induced oxidative stress state in the body.

Fig. 2 — Effect of NG + AMK on antioxidant indicators in chicken serum. Note: NC, negative control; PC, positive control; NG, naringenin; AMK, amikacin. Samples were collected at 8 days post-challenge with APEC strain C7F3. Values are mean ± SD; one-way ANOVA with LSD test. Different letters in the figure indicate significant differences (p < 0.05); the same letters indicate no significant differences (p > 0.05)

Organ index

We hypothesized that combined treatment with NG and AMK would alleviate the abnormal changes in organ indices including heart index, liver index, spleen index, lung index and kidney index caused by APEC strain C7F3 infection, thereby protecting organ development and reducing organ injury in chicks. As shown in Table 2, compared with the NC group, the organ indices of chicks significantly increased after E. coli infection (p < 0.05). Compared with the PC group, the organ indices decreased after oral administration of NG or AMK, but there was no significant difference in the impact on organ indices between the two groups (p > 0.05). Notably, with the exception of the heart index, the organ indices in the NG and AMK groups were equivalent to those in the NC group. The NG + AMK group had the greatest impact on the organ indices, with a significant reduction observed after treatment (p < 0.05), and there was no significant difference compared with the negative control group (p > 0.05).

Table 2.

Chicken organ index in each group

Group	Heart	Liver	Spleen	Lung	Kidney
PC	10.02 ± 1.08^a	46.88 ± 0.77^a	1.74 ± 0.06^a	9.6 ± 0.29^a	16.87 ± 1.53^a
NG	8.61 ± 0.3^b	37.06 ± 0.64^b	1.45 ± 0.03^b	7.36 ± 0.24^b	12.3 ± 0.27^b
AMK	7.74 ± 0.05^b	38. 12 ± 0.41^b	1.51 ± 0.28^b	7. 13 ± 0.03^b	12. 15 ± 0.16^b
NG + AMK	6.33 ± 1.86^bc	32.23 ± 1.26^c	1.2 ± 0.34^b	5.54 ± 0.23^bc	10.96 ± 0.18^bc
NC	6.22 ± 0.41^c	30.41 ± 3.98^bc	0.97 ± 0. 11^bc	5.46 ± 0.21^bc	10.78 ± 1.7^bc

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NC negative control, PC positive control, NG naringenin, AMK amikacin

Samples were collected at 8 days post-challenge with APEC strain C7F3. Values are mean ± SD; one-way ANOVA with LSD test. Different letters in the figure indicate significant differences (p < 0.05); the same letters indicate no significant differences (p > 0.05)

Changes in bacterial load of organs

We hypothesized that combined treatment with NG and AMK would significantly reduce the bacterial load in the liver, spleen, and lung induced by APEC strain C7F3 infection, thereby inhibiting bacterial colonization and dissemination in chicks. The effects of NG + AMK treatment on the bacterial load in the heart, liver, spleen, lungs, and kidneys of chicks infected with E. coli are shown in Fig. 3. Compared to the NC group, there was no significant difference in the bacterial load in any of the organs in the combined treatment group (p > 0.05). In the AMK treatment group, the bacterial load in the liver, spleen, and lungs was significantly lower than that in the NG treatment group (p < 0.05), while there was no significant difference in the bacterial load in the heart and kidneys (p > 0.05). After treatment with NG and AMK alone, the bacterial load in all organs was significantly reduced compared to the PC group (p < 0.05), but it was significantly higher than that in the NG + AMK treatment group (p < 0.05).

Fig. 3 — Changes in bacterial load in chick tissue. Note: A negative control group; B aringenin+amikacin group; C naringenin group; D amikacin group; E positive control group. Samples were collected at 8 days post-challenge with APEC strain C7F3. Values are mean ± SD; one-way ANOVA with LSD test, p < 0.05

Histopathological analysis

We hypothesized that combined treatment with NG and AMK would alleviate histopathological damage in the duodenum and lung induced by APEC strain C7F3, thereby improving intestinal mucosal integrity and reducing lung injury in chicks. Tissues were collected at 8 days post-challenge.

E. coli infection caused duodenal intestinal damage and pulmonary hemorrhage in chicks. Figure 4 showed the histological images of the lungs and duodenum of chicks in different treatment groups. In the NC group, the duodenum exhibited intact morphology, with no rupture of intestinal villi, orderly arrangement, and no hemorrhage. In the positive control group, the duodenal villi were ruptured and deformed, with shedding of the intestinal mucosa. After treatment with NG or AMK, the morphological structure of the duodenal villi showed some recovery, and the condition improved. Following NG + AMK treatment, the duodenal intestinal structure of the chicks tended towards a healthy state, with intact structure, no shedding of the intestinal mucosa, and the intestinal villi maintaining their complete morphology.

Fig. 4 — Effects of NG + AMK on chickens lung and duodenum pathological result. Note: A, F: negative control group; B, G: positive control group; C,H: naringenin+amikacin group; D, I: naringenin group; E, J: amikacin group; A, B, C,D, E are lung sections; F, G, H, I, J are duodenal sections; scale bar: 100 μm; magnification: 100×

After E. coli infection, severe hemorrhage occurs in the lungs, accompanied by congestion and blood stasis, cell necrosis, structural loss, and severe pathological damage. The lung changes in the NG treatment group and the AMK treatment group are similar, with reduced lung hemorrhage. In the NG + AMK group, there is almost no hemorrhage or blood stasis in the lungs, the lung structure is normal, and there are no significant pathological changes.

16S rRNA sequencing of cecum microbiota

α-diversity analysis

As shown in Fig. 5, the Chao1, ACE, and Richness indices were significantly higher in the NC group than in the PC group (p < 0.05). However, no significant differences in these α-diversity indices were observed among the NG, AMK, and NG+AMK groups (p > 0.05).

β-diversity analysis

As illustrated in Fig. 6, principal coordinate analysis (PCoA) revealed distinct clustering between the NC and PC groups. Statistical analysis confirmed a significant difference in overall gut microbial community structure between the NC and PC groups (p < 0.05), while no significant separation was observed among the treatment groups.

Analysis of OTU in chicken intestinal microbiota

Cecal contents were collected on day 8 post-infection for 16 S rRNA gene sequencing of the cecal microbiota. Venn analysis was performed among the three sample groups, as shown in Fig. 7. A total of 320 OTUs were shared by the NC group, PC group, and NG + AMK treatment group. After excluding shared OTUs, the NC group and NG + AMK treatment group exhibited 318 and 305 unique OTUs, respectively, both of which were numerically higher than those in the PC group. The number of OTUs shared between the NC group and NG + AMK group was 184, which was greater than the 37 OTUs shared between the PC group and NG + AMK group. These results indicate that NG + AMK treatment tended to increase the similarity of cecal microbial composition between treated chickens and the NC group.

Fig. 7 — Venn diagram of species composition. Note: NC, negative control; PC, positive control; NG, naringenin; AMK, amikacin. Samples were collected at 8 days post-challenge with APEC strain C7F3

Microbial diversity of chicken intestine

Based on 16S rRNA gene sequencing, microbiota composition was analyzed at the genus level (Fig. 8). The dominant genera were affiliated with the phyla Firmicutes, Bacteroidetes, and Proteobacteria.

As shown in Fig. 8, E. coli infection markedly increased the relative abundance of Proteobacteria from 0.6% in the NC group to 44%, while this value decreased to 1.2% following combined NG + AMK treatment.

The abundance of Firmicutes was 82.6% in the NC group, 45.2% in the PC group, and 76.5% in the NG + AMK group, respectively. These results indicated that E. coli infection decreased the abundance of Firmicutes, whereas NG + AMK treatment effectively elevated its abundance toward a normal level.

Bacteroidetes displayed a similar changing pattern: its abundance was decreased in the PC group compared with the NC group, and was restored by NG + AMK treatment.

In summary, NG + AMK treatment effectively modulated gut microbial composition at the genus level, increasing the relative abundance of phyla Firmicutes and Bacteroidetes while reducing that of Proteobacteria, thereby contributing to the restoration of gut microbial structure.

LEfSe analysis

The dominant microbiota in the NC group, PC group, and NG + AMK group were compared through LEfSe analysis, as shown in Figs. 9 and 10. Figure 9 depicts the evolutionary cladogram of differential species, and Fig. 10 presents the histogram of significantly enriched biomarker microorganisms under LEfSe analysis, with the LDA threshold set at 4. The results showed that the six biomarker microorganisms enriched in NC group belonged to the Bacteroidetes and Firmicutes phyla, all of which are beneficial bacteria, such as Oscillospiraceae, Alistipes, Rikenellaceae, etc. Among the six biomarker microbial communities enriched in the PC group, they originated from the Proteobacteria phylum, including Enterobacterales, Enterobacteriaceae, Escherichia, Shigella, etc. In the NG + AMK group, a total of 10 biomarker species were co-enriched, with a composition similar to the NC group, predominantly belonging to the Bacteroidetes and Firmicutes phyla, such as Barnesiella, Bacteroides, Oscillibacter, etc., all of which are beneficial intestinal bacteria. In summary, E. coli infection reduced the abundance of beneficial bacteria, Bacteroidetes and Firmicutes, in the chicken gut microbiota, while increasing the abundance of Proteobacteria. NG + AMK treatment reversed this change by increasing the abundance of beneficial Bacteroidetes and decreasing the abundance of Proteobacteria, thereby improving the structure of the intestinal microbiota.

Fig. 9 — Group cladogram from LEfSe analysis. Note: NC, negative control; PC, positive control; NG, naringenin; AMK, amikacin. Samples were collected at 8 days post-challenge with APEC strain C7F3. Note: The cladogram shows taxonomy levels of phylum, class, order, family and genus from inside out. Each small circle in each layer represents a species at the taxonomic level of that layer. Red, green and blue represent significantly different species enriched in NC group, PC group and NG + AMK group, respectively, while yellow circles represent species without significant differences. The greater the relative abundance of each species, the larger the circles in the layer

Fig. 10 — Biomarker species score diagram from LEfSe analysis. Note: NC, negative control; PC, positive control; NG, naringenin; AMK, amikacin. Samples were collected at 8 days post-challenge with APEC strain C7F3

Discussion

Bacterial diseases in animals are prevalent in the farming process, with high incidence rates, and are typically controlled through vaccination and antibiotic treatment to manage their morbidity and mortality [12]. However, the extensive use of antibiotics has led to the continuous selection and enrichment of pre-existing resistant bacteria, severely affecting both animal and human health. It is estimated that approximately 1.27 million of the 7.7 million deaths resulting from bacterial infections are associated with drug-resistant pathogens, and the global health crisis driven by antimicrobial resistance continues to intensify [13–17]. Antimicrobial resistance also undermines global health by exacerbating food insecurity, given that it compromises the effective treatment of infectious diseases in livestock [18]. Overuse of antibiotics in animal agriculture and the release of bioactive antimicrobial compounds into the environment further drive the selection and proliferation of resistant bacterial strains. These processes heighten the risk of untreatable bacterial infections in both humans and animals, potentially precipitating severe future public health crises.

Therefore, identifying effective antibiotic alternatives has become a current research focus. This study investigated the synergistic effect of naringenin (NG) combined with amikacin (AMK) against avian colibacillosis, and further explored its potential mechanisms in terms of anti-inflammatory, antioxidant, and gut microbiota modulation.

In this study, NG combined with AMK significantly reduced serum levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and suppressed oxidative stress markers (MDA), while increasing antioxidant enzyme activities (SOD, GSH-Px). Furthermore, NG possesses notable antioxidant and anti-inflammatory properties, which may help alleviate intestinal oxidative stress and inflammatory damage caused by E. coli infection. These beneficial effects may act synergistically with its antimicrobial activity to enhance the therapeutic outcome of amikacin treatment. Consistent with our findings, previous studies have reported that NG possesses potent antioxidant and anti-inflammatory properties [9, 19]. However, the present study further demonstrates that when combined with AMK, NG not only retains but also enhances these effects in the context of E. coli infection. This synergistic effect may be attributed to the complementary mechanisms of the two agents: AMK exerts rapid bactericidal activity, while NG simultaneously mitigates excessive inflammatory responses and oxidative damage induced by bacterial infection. This combined effect likely contributes to the accelerated clinical recovery observed in the combination group compared to monotherapy groups.

NG has also been reported to protect against liver and kidney damage caused by anti-tuberculosis drugs [20]. In the present study, NG combined with AMK significantly reduced liver indices, restored serum biochemical parameters (ALT, AST), and alleviated hepatic pathological damage. These findings suggest that NG exerts a protective effect against E. coli-induced liver injury. The hepatoprotective effect may be mediated through its antioxidant activity, as oxidative stress is a key contributor to infection-induced tissue damage. Furthermore, the combination therapy significantly reduced bacterial loads in the liver, spleen, and lungs, and alleviated pathological damage in the lungs and duodenum. This indicates that the addition of NG enhances bacterial clearance, potentially by reducing tissue damage and thereby preserving host defense mechanisms.

Gut microbiota dysbiosis is commonly associated with enteric infections and antibiotic treatment. In this study, 16 S rRNA sequencing revealed that E. coli infection significantly increased the relative abundance of Proteobacteria (a phylum containing many pathogenic species), while decreasing Firmicutes and Bacteroidetes. Treatment with NG combined with AMK restored the gut microbiota structure to a profile similar to that of healthy controls. Notably, NG alone also partially reversed these changes, suggesting that NG contributes to microbiota modulation independently of its antibacterial activity. These results are consistent with previous studies showing that flavonoids such as chlorogenic acid, curcumin, and NG can modulate gut microbiota composition, alleviate inflammation, and enhance intestinal barrier integrity [21]. The combined therapy in this study appears to exert dual benefits: directly reducing pathogen load through AMK, while NG promotes recovery of commensal microbiota and reduces inflammation.

Conclusion

In this study, a synergistic combination of naringenin (NG) and amikacin (AMK) was identified and shown to significantly enhance therapeutic efficacy against avian colibacillosis compared with monotherapy. Mechanistically, the combination exerted anti-inflammatory and antioxidant effects, alleviated liver damage, and modulated gut microbiota structure by increasing beneficial bacteria (Firmicutes and Bacteroidetes) while reducing potentially pathogenic Proteobacteria. These findings demonstrate that NG combined with AMK achieves synergistic therapeutic effects through multiple mechanisms, providing a scientific basis for the application of NG as an adjuvant to AMK in treating avian colibacillosis.

Methods

Ethical approval

All animal experiments followed the guidelines for the care and use of laboratory animals issued by the U.S and has gained ethical approval for this study [22]. Our study was conducted according to the Animal Administration and Ethics Committee of Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS (Permit No. 2024-023). According to the AVMA Guidelines for the Euthanasia of Animals, chicks were euthanized by cervical dislocation following anesthesia induced via intravenous injection of an overdose of sodium pentobarbital [23].

Materials and bacterial strains

The clinical isolate E. coli C7F3 was isolated from chicken manure collected at three large-scale commercial layer farms in Handan, Hebei Province, with their serotype identified as O119. A single colony was inoculated in 15 mL Luria–Bertani (LB) broth (Guangdong Huankai Microbial Sci. & Tech. Co., Ltd., Guangzhou, China) and cultured at 220 rpm for 12 h at 37 °C. After centrifugation at 3500 rpm, the bacterial pellet was collected and resuspended in an equal volume of physiological saline (Chenxin Pharmaceutical Co., Ltd., Jining, China). Finally, physiological saline was used to adjust the strain concentration to 1.5 × 108 colony forming units (CFU)/mL for further analyses.

In this study, blood samples were collected once from each chicken via the wing vein at the time of euthanasia. All animal procedures were conducted at the Laboratory Animal Center of the Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences.

Animals and treatment

A total of 60 one-day-old male huangma broiler chickens (weigh of 40 ± 5 g) were purchased from Yuzhong Ji’an Broiler Farm, Gansu, China. The chickens were fed until they were 7 days old and were then randomly divided into 5 groups: the negative control (NC) group (500 µL of normal saline), the positive control (PC) group (500 µL of E. coli C7F3 bacterial suspension at 1.5 × 108 CFU/mL), the naringenin (NG, Aladdin Biochemical Technology Co., Ltd., Shanghai, China) treatment group (500 µL of E. coli C7F3 bacterial suspension at 1.5 × 108 CFU/mL, 50 mg/kg NG), the amikacin (AMK, Solarbio Life Sciences, Beijing, China) treatment group (500 µL of E. coli C7F3 bacterial suspension at 1.5 × 108 CFU/mL, 15 mg/kg AMK), and the combination of naringenin and amikacin hydrochloride treatment (NG + AMK) group (500 µL of E. coli C7F3 bacterial suspension at 1.5 × 108 CFU/mL, 50 mg/kg NG + 15 mg/kg AMK). A schematic timeline illustrating the key procedures of the animal experiment is presented in Fig. 11.

Fig. 11 — Schematic timeline of the animal experimental design. Created with BioRender (https://BioRender.com/pe3nntn)

Each group consisted of 12 chicks, with the infected group and the control group housed separately. Until the end of the experiment, all groups were fed a basal diet without growth promoters and antibiotics. All diets were fed for ad libitum intake with free access to water. Except for the NC group, the chicks were injected with 0.5 mL of 1.5×108 CFU/mL E. coli C7F3 bacterial suspension into the abdominal air sac, while the NC group chicks were injected with an equal volume of physiological saline [24]. After the onset of clinical symptoms, chicks in the NG, AMK, and NG+AMK groups were administered NG, AMK, and their combination, respectively, via gavage. The chicks were administered continuous treatment for 5 days, once daily, and samples were collected 3 days after drug withdrawal.