
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00798-3
10.1016/j.psj.2024.104219
104219
IMMUNOLOGY, HEALTH AND DISEASE
Therapeutic efficacy of compound organic acids administration on methicillin-resistant Staphylococcus aureus-induced arthritis in broilers
Liu Gengsong *1
Gui Yanyao *1
Shi Wen *1
Yang Hongchun *
Feng Shufeng *
Liang Si *
Zhou Congcong *
Zhou Qiaoyan *
Li Haizhu *
Li Gonghe *
Si Hongbin *†‡
Ou Changbo ouchangbo2004@163.com
⁎†‡2
⁎ College of Animal Science and Technology, Guangxi University, Nanning, 530004, China
† Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning, 530004, China
‡ Guangxi Key Laboratory of Animal Reproduction, Breeding and Disease Control, Nanning, 530004, China
2 Corresponding author: ouchangbo2004@163.com
1 These authors contributed equally to this work and share first authorship.

01 9 2024
12 2024
01 9 2024
103 12 1042195 5 2024
9 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Avian arthritis is a common disease in the poultry industry, and the etiology is complex. Bacterial arthritis is usually caused by Staphylococcus aureus (S. aureus) infection. This study explored the minimum inhibitory concentration (MIC) of different organic acids against S. aureus MRSA85 and found that vanillic acid, suberic acid, itaconic acid, salicylic acid, and other organic acids had significant inhibitory effects on this strain, especially cinnamic acid, which exhibited the best inhibitory effect. The Fractional Inhibitory Concentration Index (FICI) test further revealed the synergistic effect among some compound organic acids, which can significantly enhance the antibacterial efficiency against MRSA85 while reducing the risk of bacterial resistance. Under the low concentrations (1/2 or 1/4 MIC) conditions, the MIC of the compound organic acids against S. aureus remains unchanged, and it can even enhance the sensitivity of antibiotic-resistant S. aureus to Oxacillin. Furthermore, the compound organic acids could effectively promote the recovery of S. aureus-induced arthritis in broiler models, reduce inflammatory responses, and lower down bacterial loads and inflammatory cytokine levels in joints, which indicated that the effects of the Compound 2 is comparable to that of the trimethoprim-sulfamethoxazole group. These results support the potential and application value of organic acids and their compounds, including Compound 1 to 3, as novel antibacterial agents in the treatment of S. aureus infections.

Key words

organic acid
Staphylococcus aureus
arthritis
MRSA
broiler
==== Body
pmcINTRODUCTION

Staphylococcosis is a zoonotic infectious disease mainly caused by Staphylococcus aureus (S. aureus), commonly found in livestock and poultry. It is a common acute or chronic infectious disease in animals. S. aureus can infect humans, cattle, poultry, pigs, rabbits, and other animals. Particularly in poultry, various clinical symptoms often occur after infection, such as arthritis, tenosynovitis, foot pad swelling, omphalitis, and staphylococcal septicemia, causing significant losses to the poultry industry. The disease is developing rapidly, and antibiotic treatment is prone to drug resistance, which is very detrimental to prognosis recovery and may even lead to permanent joint damage (Mohammad et al., 2020), causing significant economic losses to poultry farming (Pal et al., 2020). S. aureus often induces bacterial chondronecrosis with osteomyelitis, a primary cause of lameness in poultry. Clinical symptoms of this disease mainly include lameness, reduced mobility, and poor health status (Mcnamee and Smyth, 2000), particularly prevalent in broiler chickens around 35 d of age (Wideman Jr et al., 2015; Mohammad et al., 2020). Therefore, finding effective treatment strategies targeting S. aureus is crucial for the sustainable economic development of broiler chickens.

Due to the rapid development of staphylococcal disease and the possible emergence of multi-drug resistant strains, which lead to the ineffectiveness of antibacterial therapy, the growing demand for antibiotic-free agriculture has promoted the use of antibiotic alternatives. Therefore, we need to find other more effective means. In poultry, lameness and joint swelling caused by S. aureus typically occur when the pathogen infiltrates deeper tissues after penetrating damaged skin or mucous membranes. The disease becomes more severe and the incidence increases when the natural immune defense system of the host is compromised (Szafraniec et al., 2022). In clinical practice, animal models infected with S. aureus are commonly used for related research. Various methods are employed to induce staphylococcal arthritis, with the primary approach being the direct injection of the bacteria into avian joints to induce purulent arthritis (Mosleh et al., 2016). In chicken arthritis induced by S. aureus, rapid aggregation of immune cells and expression of pro-inflammatory cytokines are observed. In severe inflammatory reactions, activated macrophages produce a large number of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), leading to cartilage and joint capsule damage (Nematollahi et al., 2022; Jiang et al., 2023). Inflammation also causes histopathological changes in tissues, resulting in severe joint destruction and even irreversible damage. Additionally, S. aureus can colonize in the joint cavity of turkeys, causing local necrotizing lesions, heterophil infiltration, and fibroblast proliferation (Miner et al., 1968). S. aureus easily develops resistance to antibiotics, and prolonged antibiotic therapy is detrimental to poultry health and growth (Lin et al., 2016).

Organic acids have shown promising antibacterial effects. When organic acids reach the intestines, they change the intestinal environment by lowering the pH value and can directly kill pathogens. Previous studies indicated that boswellic acid had many positive effects on human health, especially in the treatment of inflammation, arthritis, and asthma (Jaroš et al., 2022). Flufenamic acid, a nonsteroidal anti-inflammatory drug, is approved clinically for the relief of arthritis inflammation and pain and the effective amelioration of local infections caused by methicillin-resistant S. aureus (MRSA) in mice (Zhang et al., 2020). The combination of gentamicin and ascorbic acid can effectively alleviate arthritis induced by S. aureus, reduce the bacterial load in blood, spleen, and synovial tissues, alleviate the clinical symptoms of arthritis, and reduce the serum levels of TNF-α and interferon-γ (IFN-γ) (Mal et al., 2012). Additionally, the combination of salicylate and chloroquine can treat S. aureus-induced arthritis and prevent and treat cartilage damage.

Organic acids have shown positive effects on S. aureus-induced arthritis, but their single use still has limitations. Moreover, it remains uncertain whether organic acids can sustain their antimicrobial activity in treating S. aureus infections in poultry. We found that the crude organic acids extracted from Portulaca oleracea had a good inhibitory effect on S. aureus infection in vitro (Liu et al., 2022b). Therefore, the chessboard experiment was used to identify combinations of organic acids that work synergistically in inhibiting bacterial growth. Subsequently, animal experiments were carried out to assess the therapeutic effects of these combined organic acids. Most of the acids in these organic acid combinations come from the previously studied crude organic acid extract of purslane, while a small portion of acids are used in combination with the crude organic acids to enhance their antibacterial effects (Liu et al., 2022b). Clinical scores, as well as levels of IL-6, IL-10, TNF-α in serum, and histopathological changes in tissues, were evaluated at 2 different time points for both the infected and treated groups, aiming to assess the efficacy of the combined organic acids.

MATERIALS AND METHODS

Strains and Reagents

Three strains of S. aureus were used in this study: 2 field strains, MRSA85 and MRSA29, and 1 standard strain (ATCC 29213). The strain ATCC29213 was obtained from the First People's Hospital of Nanning, China. Both MRSA85 and MRSA29 strains were isolated from clinical samples and stored at Guangxi University. Organic acids and Oxacillin sodium salt were purchased from Macklin Biochemical Technology Co., Ltd (Shanghai, China). Trimethoprim-sulfamethoxazole (TMP-SMX) was purchased from TEYI Pharmaceutical Group in Guangdong, China.

Preparation of Compound Organic Acids Solution

Based on the synergistic effects among organic acids and their cost, 3 compound organic acid formulations were prepared for subsequent experiments.

Compound 1 is a mixture of organic acids consisting of salicylic acid, gallic acid, itaconic acid, lactobionic acid, protocatechuic acid, vanillic acid, and coumaric acid in the ratio of 10:5:5:1:1.5:1.5:1.

Compound 2 is a mixture of organic acids consisting of salicylic acid, gallic acid, protocatechuic acid, and coumaric acid in the ratio of 15:7:2:1.

Compound 3 is a mixture of organic acids consisting of salicylic acid, gallic acid, protocatechuic acid, and vanillic acid in the ratio of 15:6:2:2.

These compound organic acid solutions were prepared using sterilized water at the above proportions, with a final concentration of 0.25‰.

Preparation of S. aureus Bacterial Suspension

The strain stored in the -80 °C refrigerator was removed, inoculated into fresh autoclaved BHI medium, and incubated overnight at 37 °C and 220 round per minute to activate it. The activated bacterial liquid was then inoculated onto BHI agar solid medium in a clean bench and cultured at 37 °C for 16-18 h in a thermostatic incubator. Afterwards, individual colonies were selected and inoculated into a sterile broth medium and cultured with shaking until they reached the logarithmic phase. The colonies were diluted with fresh sterile broth, and the final concentration was adjusted to 106 colony forming unit (CFU)/mL through plate counting.

Determination of Minimum Inhibitory Concentration

The minimum inhibitory concentration (MIC) was determined using a trace broth dilution method as recommended by CLSI (Clinical and Laboratory Standards Institute) (Li et al., 2022). Different organic acid solutions were diluted in multiple ratio on a 96-well plate, then an equal volume of bacterial suspension with the concentration of 106 CFU/mL was added, and negative and positive control groups were set. Then, the samples were cultured in a 37 °C incubator for 16-18 h. The results were only effective when the bacterial growth of the negative and positive control groups was consistent with the actual situation, and the minimum drug concentration corresponding to the complete inhibition of bacterial growth was considered as the MIC.

Determination of Fractional Inhibitory Concentration Index

In a 96-well microplate, solutions of organic acids A and B at different concentrations were added according to a predetermined protocol, with 3 replicates per concentration. S. aureus in the logarithmic phase was adjusted to a concentration of approximately 1 × 106 CFU/mL and added to each well containing the organic acid to give a final volume of 200 μL. The results were read after incubation at 37 °C for 18-24 h. According to the results of the MIC, the fractional inhibitory concentration index (FICI) of the 2 organic acids was calculated to evaluate the synergistic antibacterial effect.FICI=MICABMICA+MICABMICB

Explanation of results: The results of the FICI are generally used to explain the type of interaction between the 2 drugs. FICI ≤ 0.5 indicates Synergism effect between the 2 drugs; 0.5 < FICI ≤ 1 indicates cumulative effect between the 2 drugs; 1< FICI ≤ 4 indicates no interaction effect between the 2 drugs; FICI > 4 indicates Antagonism effect between the 2 drugs (Wang et al., 2023).

Study on the Resistance of Drug-Resistant S. aureus to Compound Organic Acids

10 μL of MRSA85 bacterial suspension (the concentration is 1.2 × 1010CFU/mL) in logarithmic growth phase were inoculated into 4 mL of compound organic acids broth at concentrations of 1/2 or 1/4 of the MIC. The inoculated broth was cultured in a constant temperature shaking incubator at 37 °C, denoted as the first generation. Subsequently, 10 μL of the first generation bacterial suspension was transferred into 4 mL of compound organic acids broth and cultured under the same conditions to generate the second generation. This iterative process was repeated to generate 25 consecutive generations of Staphylococcus aureus passage, with each generation timed according to the growth cycle of the bacteria. Every 5 generations, the sensitivity of the organic acids was assessed through MIC testing. MIC values for the compound organic acids were recorded every 5 generations. The tests were conducted in triplicate, and drug resistance development curves were plotted.

Study on the Reversal Effect of Compound Organic Acids on the Antibiotic Resistance of Drug-Resistant S. aureus

Methicillin-resistant S. aureus was continuously subcultured for 25 generations at the final concentration of 1/2 or 1/4 MIC compound organic acids. The detailed steps are as described above. The susceptibility to Oxacillin for Methicillin-resistant S. aureus was tested simultaneously after every 5 generation of culture.

Experimental Animal Group and Treatment

Sixty 8-wk-old 3-yellow chickens with a body weight of about 600 grams were purchased from Guangxi Fufeng Co., Ltd (Nanning, China), and all chickens were randomly divided into 6 groups, with ten chickens in each group. The room temperature was 22 ± 2 °C, the air humidity was 55 ± 20%, and the light time was 12 h per day. The chickens were allowed to eat and drink freely. The experiments were performed in strict accordance with the standards of the Animal Management and Ethics Committee of Guangxi University (No. GXU-2022-082).

The blank control group (BC) were inoculated with sterile PBS without medication. The infected control group (NC) were inoculated with S. aureus without medication. The positive control group was inoculated with S. aureus and administrated with TMP-SMX at the dose of 25 mg/kg body weight. The other 3 compound organic acids groups (Compound 1, 2 and 3) were inoculated and administrated with organic acids. Methods for inoculation of S. aureus are as follows. The right joint cavity of each chicken in the infection groups was injected with 0.2 mL (1.2 × 1010 CFU/mL) of S. aureus suspension (none of animals died, but there were obvious arthritis symptoms at this injection dosage), while the right joint cavity of each chicken in the blank control group was injected with 0.2 mL sterilized PBS. After inoculation, the chickens were allowed to freely drink water which was added with 0.25 ‰ compound organic acid for at least 8 h once a day. They were allowed to drink sterile water at a fixed time every day for the rest time until the end of the experiment. The chickens of the healthy control group and the infected control group drank sterile water throughout the trial period. The clinical symptoms of chickens in each group were observed throughout the experimental period. On the 7th and 14th day after inoculation with S. aureus, 5 chickens randomly selected from each group were sacrificed and observed for necropsy changes. Then the liver, kidney, and spleen were weighed, and the articular cartilage was fixed and prepared into paraffin sections for HE staining and histopathological observation. The articular synovial fluid was taken and stored in a sterile tube. The clinical score and severity score of arthritis were monitored on d 7 and 14 after inoculation and the assessment criteria are referenced from previous literature (Nematollahi et al., 2022) (please see the supplemental files).

Determination of Bacterial Load in Synovial Fluid of Joint Cavity

The obtained joint cavity synovial fluid was weighed, and then diluted to an appropriate gradient. 100 μL of synovial fluid was absorbed onto the brain-heart infusion agar culture medium, spread it evenly, and then incubated statically in a constant temperature incubator at 37°C. Colony counting was performed after 24 h.

Determination of Inflammatory Factors in Serum

Blood samples from chicken were collected into sterile tubes, and allowed to stand at room temperature for 30 min. Then, centrifuged the samples at 4,000 rpm at 4°C for 5 min. Collected the supernatant and stored it in a -80 °C freezer for future use. Detected the expression levels of inflammatory factors IL-1β, IL-6, and TNF-α in serum from chickens with S. aureus arthritis using the ELISA kit according to the manufacturer's instructions.

Data Statistics and Analysis

The MIC results were expressed in modal values because no variation among replicated results was observed. All data are presented as mean ± SD. Statistical analysis was performed using SPSS Statistics 26, and graphs were drawn using GraphPad Prism 8 software (USA). One-way ANOVA was used to compare differences between groups, followed by Dunnett's multiple comparison procedure. A P-value of < 0.05 was considered statistically significant. To assess chicken body weight, clinical scores, arthritis severity scores, organ indices, bacterial load in synovial fluid, and inflammatory cytokines. The results were expressed as "mean ± standard deviation." P< 0.05 indicated a significant difference, while P> 0.05 indicated no significant difference. P< 0.01 indicated a highly significant difference.

RESULTS

MIC of Different Organic Acids on S. aureus

According to the drug sensitivity test results of organic acids on S. aureus, most organic acids have certain inhibitory effects on S. aureus, and the MICs of various organic acids on S. aureus are shown in Supplementary Table 1. Especially notable are the relatively significant inhibitory effects of vanillic acid, adipic acid, oxalic acid, and salicylic acid on S. aureus MRSA85. Among these organic acids, cinnamic acid exhibits the best inhibitory effect on S. aureus MRSA85, and the MIC value is 0.781 mg/mL. Therefore, this study will focus on investigating the inhibitory effects of the above organic acids on S. aureus MRSA85.

FICI of Complex Organic Acids Against S. aureus

The chessboard experiment was further employed to investigate the potential of organic acids as novel antimicrobial agents, and the FICI was used to measure the synergistic effects between the combined organic acids. As shown in Table 1, protocatechuic acid significantly increased the sensitivity of S. aureus to vanillic acid, adipic acid, salicylic acid, ferulic acid, vanillic acid, and cinnamic acid. Vanillic acid significantly increased the sensitivity of S. aureus to adipic acid, gallic acid, salicylic acid, ferulic acid, cinnamic acid, and L-malic acid. Adipic acid significantly increased the sensitivity of S. aureus to gallic acid, succinic acid, salicylic acid, ferulic acid, and L-malic acid. Gallic acid significantly increased the sensitivity of S. aureus to salicylic acid. Lactic acid significantly increased the sensitivity of S. aureus to ferulic acid and vanillic acid. Succinic acid significantly increased the sensitivity of S. aureus to salicylic acid. Salicylic acid significantly increased the sensitivity of S. aureus to ferulic acid, vanillic acid, cinnamic acid, and citric acid. These results indicate that the combined use of organic acids can significantly reduce the dosage, resulting in a noticeable decrease in costs during clinical application.Table 1 FICI and action of Complex organic acids against S. aureus.

Table 1Organic acid A	Organic acid B	FICI	Action	
Protocatechuic acid	Vanillic acid	0.38	Synergism	
Suberic acid	0.50	Synergism	
Gallic acid	1.50	\	
Lactobionic acid	1.00	\	
Itaconic acid	2.50	\	
Succinic acid	0.63	\	
Salicylic acid	0.38	Synergism	
Ferulic acid	0.38	Synergism	
Caffeic acid	0.62	\	
Coumaric acid	0.25	Synergism	
Cinnamic acid	0.25	Synergism	
L- malic acid	0.75	\	
Citric acid	0.62	\	
Vanillic acid	Suberic acid	0.25	Synergism	
Gallic acid	0.38	Synergism	
Lactobionic acid	3.00	\	
Itaconic acid	2.50	\	
Succinic acid	0.63	\	
Salicylic acid	0.38	Synergism	
Ferulic acid	0.56	\	
Caffeic acid	0.62	\	
Coumaric acid	0.31	Synergism	
Cinnamic acid	0.19	Synergism	
L- malic acid	0.25	Synergism	
Citric acid	0.62	\	
Suberic acid	Gallic acid	0.38	Synergism	
Lactobionic acid	0.75	\	
Itaconic acid	2.50	\	
Succinic acid	0.50	Synergism	
Salicylic acid	0.25	Synergism	
Ferulic acid	0.62	\	
Caffeic acid	0.75	\	
Coumaric acid	0.38	Synergism	
Cinnamic acid	0.75	\	
L- malic acid	0.38	Synergism	
Citric acid	0.62	\	
Gallic acid	Lactobionic acid	1.25	\	
Itaconic acid	3.00	\	
Succinic acid	1.00	\	
Salicylic acid	0.31	Synergism	
Ferulic acid	0.75	\	
Caffeic acid	0.63	\	
Coumaric acid	0.63	\	
Cinnamic acid	0.75	\	
L- malic acid	1.25	\	
Citric acid	0.62	\	
Lactobionic acid	Itaconic acid	2.50	\	
Succinic acid	1.00	\	
Salicylic acid	1.12	\	
Ferulic acid	0.25	Synergism	
Caffeic acid	1.12	\	
Coumaric acid	0.19	Synergism	
Cinnamic acid	0.75	\	
L- malic acid	1.00	\	
Citric acid	0.75	\	
Itaconic acid	Succinic acid	0.63	\	
Salicylic acid	1.25	\	
Ferulic acid	0.75	\	
Caffeic acid	1.50	\	
Coumaric acid	1.13	\	
Cinnamic acid	0.75	\	
L- malic acid	2.50	\	
Citric acid	1.25	\	
Succinic acid	Salicylic acid	0.19	Synergism	
Ferulic acid	0.56	\	
Caffeic acid	0.75	\	
Coumaric acid	0.75	\	
Cinnamic acid	0.75	\	
L- malic acid	0.75	\	
Citric acid	0.75	\	
Salicylic acid	Ferulic acid	0.25	Synergism	
Caffeic acid	0.75	\	
Coumaric acid	0.25	Synergism	
Cinnamic acid	0.38	Synergism	
L- malic acid	0.75	\	
Citric acid	0.38	Synergism	
Ferulic acid	Caffeic acid	2.00	\	
Coumaric acid	1.25	\	
Cinnamic acid	1.50	\	
L- malic acid	1.25	\	
Citric acid	1.00	\	
Caffeic acid	Coumaric acid	1.25	\	
Cinnamic acid	0.75	\	
L- malic acid	0.75	\	
Citric acid	0.75	\	
Coumaric acid	Cinnamic acid	0.75	\	
L- malic acid	0.75	\	
Citric acid	0.62	\	
L- malic acid	0.75	\	
Citric acid	0.62	\	
L- malic acid	Citric acid	0.75	\	
Note: \ indicates that these 2 organic acids are nonsynergistic.

Compound Organic Acids Are Not Easy to Induce Drug Resistance in S. aureus

Under the pressure of the antibiotic environment, different bacteria will develop resistance to antibiotics through different mechanisms to protect themselves from damage. In this study, we investigate the development of drug resistance in S. aureus (MRSA85) to compound organic acids. As shown in Figure 1A, the results showed that the MIC values of compound organic acids against MRSA85 kept the same even if it continuously passes through 25 generations under the pressure of 1/4 MIC or 1/2 MIC compound organic acid. These results had proved that the compound organic acids are not easily induce bacteria to produce drug resistance.Figure 1 MIC determination of compound organic acids and oxacillin against S. aureus MRSA85 after continuous passage for 25 generations under the induction of 1/2 and 1/4 MIC compound organic acids. (A) MIC of 3 compound organic acids against S. aureus MRSA85. (B) MIC of oxacillin against S. aureus MRSA85 after compound organic acids treatment.

Figure 1

The Compound Organic Acids Reduce the Antibiotic Resistance of Drug-Resistant S. aureus

As shown in Figure 1B, when using compound organic acids, the MIC of Oxacillin to MRSA85 changed under continuous passage for 25 generations under the pressure of 1/4 MIC Compound 2. Specifically, from the 15th generation, the MIC of Oxacillin to MRSA85 reduced from 0.8 mg/mL to 0.4 mg/mL. Similarly, when MRSA85 were continuously passaged for 25 generations under the pressure of 1/2 MIC Compound 1, the MIC of Oxacillin to MRSA85 also changed, with a 1-fold decrease starting from the 15th generation. These results demonstrated that compound organic acids enhance the sensitivity of drug-resistant S. aureus to antibiotics.

Compound Organic Acid Promotes Recovery of Staphylococcal Arthritis

The inclusion of organic acids in various combinations in the daily drinking water of chickens significantly enhanced their growth performance to varying degrees. The weight of the NC group was significantly lower than that of the BC group (P < 0.01), indicating that the NC group ate less food after the bacterial attack (Figure 2). There was no significant difference between the TMP-SMX group and the Compound 2 group, indicating that the effect of improving the growth performance of the Compound 2 group was similar to that of TMP-SMX. The scores at the 2 sampling times were significantly different between the BC and NC groups in the clinical and arthritis severity scores (Figure 3, Figure 4). There were significant differences between the Compound groups and the NC group. There was no significant difference between the TMP-SMX group and the Compound groups, indicating that the improvement effect of chicken clinical scores in the Compound group was similar to that of TMP-SMX.Figure 2 Body weight of chickens from different groups on d 7 (A) or 14 (B) postinoculation with S. aureus MRSA85. *Indicates P < 0.05, **indicates P < 0.01 when compared with the NC group.

Figure 2

Figure 3 Effects of compound organic acid 1, 2 and 3 on limp score on d 7 (A) or 14 (B) postinoculation with S. aureus MRSA85. *Indicates P < 0.05, **indicates P < 0.01 when compared with the NC group.

Figure 3

Figure 4 Effects of compound organic acid 1, 2 and 3 on arthritis severity score on d 7 (A) or 14 (B) postinoculation with S. aureus MRSA85. *Indicates P < 0.05, **indicates P < 0.01 when compared with the NC group.

Figure 4

Histological changes were assessed by HE staining of the tissues on d 7 and 14 postinfection (Figure 5). In both samples, the BC group exhibited normal histological features of synovial, articular cartilage, and skeletal structures. Severe degeneration and necrosis of synovial cells were observed in both the NC group and the TMP-SMX and Compound groups, with congestion, edema, and fibrinoid xenophilic exudate, and bacterial colonies observed in the lesions. However, the degeneration and necrosis of synovial cells in the TMP-SMX and Compound groups were moderate, and the presence of xenophilic and mononuclear cells was mainly limited to the fixed peripheral area. In the infected control group, higher inflammatory cell infiltration was existed.Figure 5 Effects of compound organic acid 1, 2 and 3 on histopathology changes on d 14 postinoculation with S. aureus MRSA85. Arrows indicate lesion site including inflammatory cell infiltration.

Figure 5

The Compound Organic Acid Reduces Bacterial Load and Inflammatory Cytokine in the Joint

The bacterial burden in the tissues following S. aureus infection was assessed on d 7 postinfection (Figure 6). In the BC and NC groups, the bacterial count was 0 and 7 × 108 CFU/g. respectively. The bacterial loads in the combination and TMP-SMX treatment groups were 7.77 × 106 CFU/g and 3.11 × 106 CFU/g, which were significantly lower than those in the NC group. By measuring the inflammatory cytokines in chicken serum, we assessed the occurrence of inflammation (Figure 7). In the NC group, the levels of IL-1β, IL-6, and TNF-α were significantly increased (P < 0.05). However, in the Compound 2 and 3 groups, the level of IL-1β, IL-6, and TNF-α levels were significantly decreased on d 14 (P < 0.05). Additionally, TMP-SMX treatment significantly reduced the levels of IL-1β, IL-6, and TNF-α on the 7th and 14th day (P < 0.05). In contrast, the Compound groups significantly reduced the levels of IL-1β and IL-6 (P < 0.01), while TNF-α levels did not differ significantly between the Compound 2 and 3 groups (P > 0.05).Figure 6 Effects of compound organic acid 1, 2 and 3 on tissue bacterial load on d 7 (A) or 14 (B) postinoculation with S. aureus MRSA85. *Indicates P  < 0.05, **indicates P  < 0.01 when compared with the NC group.

Figure 6

Figure 7 Determination of IL-6, IL-1β, and TNF-α in chicken serum by ELISA method on d 7 (A–C) or 14 (D–F). *Indicates P < 0.05, **indicates P < 0.01 when compared with the NC group.

Figure 7

DISCUSSION

Previous data indicates that by scientifically selecting and combining different organic acids, compound organic acids can achieve stronger antibacterial effects than the use of a single acid (Konuk and Ergüden, 2017; Fouilloux et al., 2023). This synergistic effect not only enhances antibacterial efficiency but also reduces the required concentration of each organic acid, thereby minimizing potential toxicity and side effects. The strategy of compound usage allows us to achieve the desired antibacterial effect with lower dosages, which is significant in reducing treatment costs (Kragh and Truelstrup Hansen, 2019). Different organic acids exert their effects on bacteria through various mechanisms, such as disrupting cell walls, interfering with intracellular metabolic pathways, and inhibiting protein or nucleic acid synthesis (Long et al., 2017; Tian et al., 2017; Shu, 2022). For example, previous studies have shown that salicylic acid affects the virulence of pathogenic bacteria by reducing their ability to form biofilms and increasing the population of persistent cells. Salicylic acid can also significantly reduce the binding of fibronectin and fibrinogen, leading to an overall reduction in virulence. Protocatechuic acid and gallic acid exhibit good bactericidal activity through mechanisms such as disruption of cell membranes and disruption of metabolic processes. Studies have shown that gallic acid not only has antibacterial effects against a variety of bacteria, but also enhances the effectiveness of antibacterial compounds such as ciprofloxacin, ampicillin, and gentamicin through synergistic effects(Cai et al., 2019; Cao et al., 2019; Zhang et al., 2019; Scicutella et al., 2021; Szafraniec et al., 2022; Keyvani-Ghamsari et al., 2023; Sykes et al., 2024). When these organic acids are combined in appropriate proportions, they can attack bacteria from multiple angles simultaneously, which not only increases the breadth of their antibacterial action but also reduces the chance of bacteria developing resistance (Ya-Wei et al., 2017; Xuan et al., 2023). The current experimental results emphasize the importance of organic acid ratio design and optimization in the development of novel antibacterial formulations. By conducting thorough research on the interactions between different organic acids and their combined effects on bacterial growth, it is possible to design more effective composite antibacterial formulations with broad application potential. Furthermore, given the biological differences among different bacterial strains, developing customized composite formulations specifically targeted at particular types of bacteria or pathogens represents an important direction for future research.

This study found that the compounded organic acids were not easy to induce bacteria to develop drug resistance, and the MIC values did not change after continuous passage for 25 generations. This finding is of great significance for the current crisis of antibiotic resistance. Different from traditional antibiotics, the use of compound organic acids reduces the possibility of bacteria developing drug resistance and provides a new strategy for long-term antibacterial treatment. Furthermore, the experimental results also showed that the compound organic acids had the potential to reduce bacterial resistance to Oxacillin, which was particularly significant under the pressure of using low concentrations of compound organic acids. This is because the compound organic acids affect the expression of bacterial resistance genes or interfere with the development path of resistance through other mechanisms (Liu et al., 2022a; Wang et al., 2022). This provides a new idea for reversing the drug resistance of the existing drug-resistant bacteria.

Studies have shown that complex organic acids slow the development of drug resistance by affecting the expression of drug-resistance genes of bacteria, interfering with the drug-resistance transmission mechanism, or directly destroying the physiological function of drug-resistant bacteria (Jenic et al., 2021). Experimental studies have shown that compound organic acids have the potential to prevent the development of antibiotic resistance. However, there are still multiple challenges to overcome before their clinical application, including determining the most effective composite ratios, evaluating the safety of long-term use, and understanding their effects in different types of bacterial infections (Ma et al., 2022). Future research should also explore the possibility of combining compound organic acids with existing antibiotics and their potential applications in various scenarios. Compound organic acids provide a new perspective for considering drug resistance management and antibacterial treatment.

S. aureus-induced arthritis in chickens is usually caused by blood-borne transmission or infection from trauma. Skin damage caused by improper feeding and management, cracked and ulcerated footpads, vaccination procedures, and mosquito bites are common causes of staphylococcus arthritis (Nematollahi et al., 2022). Currently, the prevention and control of Staphylococcus aureus arthritis in chickens mainly relies on the administration of antibiotics. However, due to the transfer of drug-resistant gene plasmids, the drug resistance has been continuously strengthened, resulting in a gradual decrease in the sensitivity of Staphylococcus aureus to various antibiotics (Lee et al., 2022). Furthermore, due to the scarcity of blood vessels in the joint area, as well as the formation of invalid cavities and soft tissue scarring in the lesion site, it is difficult for therapeutic drugs to enter the joint cavity. This makes it challenging for antibiotics to effectively treat S. aureus-induced arthritis in chickens. Therefore, finding effective prevention methods is of significant importance for the control of S. aureus-induced arthritis in chickens (Nematollahi et al., 2021).

This study demonstrates that, in the context of S. aureus-induced arthritis, compound organic acids can significantly promote the growth of chickens. Notably, there was no significant difference between the TMP-SMX treatment group and Compound 2, indicating that the Compound 2 had a similar effect on promoting chicken growth performance as TMP-SMX. This finding suggests that compound organic acids may serve as a safer and more environmentally friendly alternative to traditional antimicrobial agents with similar effects (Khan et al., 2022). Additionally, compound organic acids have a certain effect in reducing S. aureus-induced joint inflammation and swelling, thereby indirectly promoting the overall health and growth performance recovery of animals (Sultana and Bishayi, 2020).

To investigate the inflammatory response of S. aureus-induced arthritis, inflammatory cytokines in chicken serum were measured, particularly the 3 key inflammatory markers IL-1β, IL-6, and TNF-α. These cytokines play crucial roles in the inflammatory response, regulating immune responses and promoting the aggregation and activation of inflammatory cells (Nematollahi et al., 2021; Nematollahi et al., 2022). The results of the study indicate that in the Compound groups, the levels of IL-1β, IL-6, and TNF-α were significantly reduced in the Compound 2 group on d 14, suggesting that this treatment method effectively suppresses inflammation caused by S. aureus infection. Additionally, TMP-SMX significantly decreased the levels of IL-1β, IL-6, and TNF-α on both d 7 and d 14, further demonstrating its effectiveness in inhibiting inflammation in S. aureus-induced arthritis. However, the Compound 2 and 3 groups showed significant reduction in IL-1β levels, while TNF-α levels did not change much on d 7. The Compound 2 and 3 groups had the same therapeutic effect as TMP-SMX on d 14. This may indicate that different treatment strategies have varying effects on these cytokines, or that TNF-α may not be as sensitive to treatment as IL-1β and IL-6.

CONCLUSION

Different organic acids have an inhibitory effect on S. aureus and cinnamic acid in particular shows the best antibacterial activity. The use of the compounded organic acids enhanced the antibacterial effect and showed a synergistic effect in effectively reducing the MIC of the bacteria. This finding points to the importance of complex organic acids in reducing drug use and in reducing the development of resistance. The current research has further confirmed that the compound organic acids are not easy to induce bacterial resistance, and can significantly improve the clinical symptoms of S. aureus arthritis, reduce the bacterial load in the joints and the inflammatory reaction. Therefore, this study demonstrates the potential of organic acids and their complexes as novel antibacterial agents, providing a new strategy and direction for the treatment of S. aureus infection.

Appendix Supplementary materials

Image, application 1

ACKNOWLEDGMENTS

The project is supported by National Key Research and Development Program (2023YFD1800100 ), Guangxi Science and Technology Program (GUIKE AD23049005 ), and scientific research startup funds of Guangxi University.

DISCLOSURES

The authors declare that they have no conflict of interest. The funders had no role in study design, data collection, and interpretation or in the decision to submit the work for publication.

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104219.
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