Therapeutic Effects of Lycopene Alone or in Combination with Cephalexin on Chronic Prostatitis Caused by Staphylococcus aureus in a Rat Model

Abstract

Chronic bacterial prostatitis (CBP) is caused by bacterial infection, commonly treated with fluoroquinolones. Due to rising antibiotic resistance, alternative therapies such as phytotherapy are being explored. Lycopene, a potential antioxidant with anti-inflammatory properties, is a candidate for such therapy. This study aims to evaluate lycopene’s therapeutic effects alone or with cephalexin against chronic prostate infections induced by Staphylococcus aureus using the Wistar rat model. The CBP model was established by introducing S. aureus through the urethra into the prostatic duct in 25 rats, confirming infection via uriculture and spermoculture analysis. Infected rats (n = 21) were grouped randomly: G1 (control), G2 (lycopene), G3 (cephalexin), and G4 (lycopene/cephalexin), in addition to negative control (G5) with healthy rats. Treatments were administered intragastrically, two times per day for 2 weeks: lycopene (10 mg/kg), cephalexin (2.5 mg/kg), or both. Biological samples (blood, urine, and prostate specimens) were collected for microbiological and histological analysis. The results showed a significant reduction in bacterial counts in urine and prostate (p < 0.01), especially in the group treated with both lycopene and cephalexin. This group also exhibited notable anti-inflammatory effects compared to single-treatment and control groups. In conclusion, lycopene combined with cephalexin demonstrated a beneficial synergistic effect, indicating its potential as an effective treatment for CBP caused by S. aureus.

1. Introduction

Chronic prostatitis is a persistent inflammation that affects the prostate gland, typically resulting from infectious (bacterial or non-bacterial) causes. Chronic prostatitis syndrome is the most common urological problem encountered in young men and is also a significant urological problem in men above 50 years of age. Prostate cancer can rapidly develop as aresult of this poorly treated inflammation [1]. Chronic bacterial prostatitis (CBP) is the commonest cause of relapsing urinary tract infection (UTI) in men and is characterized by recurrent UTI and persistent pathogenic bacteria in the prostatic secretions [2], leading to painful symptoms, urinary and sexual problems, and a significant reduction in quality of life and psychological state [3]. Bacterial prostatitis is most commonly caused by infection with Gram-negative pathogenic microorganisms, principally members of the Enterobacteriaceae family, but organisms from other families can be responsible and are more likely in certain high-risk populations. Escherichia coli (E. coli) is the most common isolate from urine cultures and is the causative agent in the majority (approximately 50% to 90%) of cases. Other common isolates include the species Proteus, Klebsiella, Enterobacter, Serratia, Pseudomonas, and Gram-positive organisms include Enterococcus species [4]. One of the main bacterial problems facing public health today is Staphylococcus aureus (S. Aureus), which is most common in those suffering from urinary tract infections. While S. aureus has been linked to 0.5–6% of urinary tract infections, biofilm-forming S. aureus in urinary tract infection are thought to be responsible for up to 65% of infections and 80% of chronic illnesses. Urology is one of the key specialties where biofilm formation might pose a significant threat. Furthermore, the development of biofilms by strains responsible for acute prostatitis enhances their capacity to endure inside the prostatic secretory system, resulting in the recurring urinary tract infections that are typical of chronic bacterial prostatitis [5].

Nevertheless, long-term therapy has been recommended to eradicate the causative organism from the prostate and prevent relapses, especially if they are symptomatic. The long-term administration of antibiotics leads to potential adverse effects such as gastrointestinal, cardiovascular, and skin problems and the development of bacterial resistance. Although antibiotics such as fluoroquinolones and cefalexin are used, recurrence rates are high due to the formation of biofilms, poor penetration of the drugs into the prostate and the emergence of bacterial resistance [6]. Recently, due to the lack of proven effectiveness of conventional antibiotic therapy, the goal of treatment has shifted from complete healing to improving quality of life, and attention has been paid to herbal and other alternative treatments.

Many researchers have been interested in important phytochemicals such as lycopene, an antioxidant present throughout certain red fruits and vegetables including tomatoes and watermelon. Lycopene demonstrates a range of beneficial functions, including antioxidant [7], anti-inflammatory, and antiproliferative activities. These properties contribute to its potential in preventing and treating conditions such as heart failure and various types of cancer [8]. Notably, lycopene’s capacity to inhibit oxidative stress underlies its protective effects, helping to protect cells from damage and reduce the risk of chronic diseases [9]. The synergistic interaction of lycopene with other drugs and micronutrients is particularly emphasized because of its binding activity with dietary components crucial in cancer prevention [1].

Typical lycopene consumption in humans (intake: 0.6 to 10.9 mg/day) [10], and the adsorption of lycopene with a dose of 10 mg, is more ideal in comparison with other high doses (30 and 50 mg) [11]. Recent research has demonstrated the correlation among prostatitis, chronic oxidative stress, and lycopene. Other investigations show that the combination of the antibiotic ciprofloxacin and lycopene improves the effectiveness for treating E. coli-induced chronic prostate infections in the Wistar rat model [2]. The purpose of our research is to investigate the synergistic interaction between lycopene and cephalexin, a β-lactam antibiotic from the cephalosporin class, on chronic prostatitis induced by the Gram-positive bacterium S. aureus in Wistar rats.

The main objective of this experimental study is to thoroughly evaluate the therapeutic potential of lycopene, a carotenoid with well-documented antioxidant properties, administered alone or in combination with an antibiotic of the cephalosporin family, cephalexin. This evaluation is based on a rigorously established murine model of chronic bacterial prostatitis, experimentally induced through the inoculation of the pathogenic strain S. aureus. This study aims to determine the impact of these treatments on the histopathological, microbiological, and biochemical parameters associated with chronic prostatic inflammation, while exploring underlying mechanisms of action, including those related to oxidative stress, immune response, and bacterial load.

2. Materials and Methods

2.1. The Chemicals and Reagents Used in This Study

Lycopene (purity ≥ 90%), purchased from Sigma-Aldrich; Medetomidine (Domitor^®, Orion Pharma, Espoo, Finland); Cephalexin, obtained from Laboratory Hup Pharma; and Ketamine (Imalgene^® 1000, Merial, Lyon, France).

2.2. Animals

This experiment utilized 30 male adult Wistar rats, each weighing between 200 and 300 g: 5 healthy rats were used as negative control, and 25 rats were used to induce chronic bacterial prostatitis (CBP). The animals were maintained under regular environmental conditions at 22 °C, and 12 h light-dark cycle, with food and water provided ad libitum [12]. This study was conducted in accordance with the National Institutes of Health (NIH) guidelines. All experiments involving animals received approval from the local ethical council for animal care at the institution (University, Abdel Hamid Ibn Badis, Mostaganem, Algeria) (rat/mouse 20% upkeep, RN-01-20K12; (Pasteur institute, Algeria).

2.3. CBP Model

A developed CBP rat model was carried out according to a procedure noted in the literature [2]. The strain used in this experiment was S. aureus, a Gram-positive strain. This sample was acquired from a sperm culture of a patient suffering from chronic prostatitis from the private clinical laboratory “Bouziane”, located in Mostaganem, Algeria. The isolated strain was identified using the automated VITEK^® system, which provided a comprehensive antibiogram. The collected strain was then cultured for 18 h in Brain Heart Infusion Broth (BHIB) at 37 °C in an incubator. The S. aureus cells were wrung out, rinsed three times, and resuspended in physiological solution to have a concentration of 10⁸ CFU/mL. Animals were put under anesthesia with ketamine intramuscular injection (50 mg/kg), the vaginal region was then disinfected with 70% alcohol, and a pediatric catheter (0.9 mm outer diameter, 2.5 cm length) was subsequently placed into the urethra (Figure 1). A total of 0.2 mL of a S. aureus suspension was injected into the prostatic urethra using an insulin syringe. To avoid urine leakage due to rat movement, anesthesia was maintained for 1 h, allowing the bacteria to reach and invade the prostate. After 4 weeks, to confirm the establishment of CBP, urine and sperm sampling were performed as follows.

2.4. Collection of Urine

Urine was aseptically collected in a sterile environment near a Bunsen burner and placed in a sterile Petri dish. The urine sample was diluted in phosphate-buffered saline (PBS) and subsequently distributed over blood agar to assess the presence or absence of S. aureus.

2.5. Sperm Collection

Sperm collection in rats was conducted following the approach outlined in [13], the same technique employed in cats, where ejaculation is pharmacologically triggered via α-adeno-epididymal receptors that facilitate the contraction of the vas deferens, trigone, and urinary sphincters upon stimulation. Sampling was conducted in rats utilizing a pediatric urethral catheter (a sterilized polyethylene tube with an outside diameter of 0.9 mm and a length of 2.5 cm, truncated at its distal part) following the administration of medetomidine at a dosage of 140 μg/kg. The catheter was evacuated with a syringe under air pressure, and the sperm was collected in a sterile Eppendorf tube. Out of 25 samples collected in this investigation, 4 rats failed to ejaculate. The collected sperm was applied directly onto the slide and prepared for May-Grünwald Giemsa staining according to [2]. Approximately 10 μL of the remaining sperm was cultured on blood agar to determine the presence or absence of S. aureus and leukocytes.

2.6. Animal Distribution

Only rats with positive CBP were chosen for this experiment. Twenty-one male Wistar rats were divided into four groups of 06 rats in the control-positive group and 05 rats in the experimental groups, as well as 5 healthy rats as negative control.

Group 01: Control rats received 1 mL of physiological water orally.

Group 02: These rats were administered lycopene alone (10 mg/kg), from Sigma-Aldrich, dissolved in 2 mL of maize oil, via oral administration in two divided dosages daily for a duration of 2 weeks.

Group 03: These rats were treated with the antibiotic cephalexin, which was provided at a dose of 2.5 mg/kg, diluted in 2 mL of distilled water, via oral gavage in two divided doses daily for a duration of 2 weeks.

Group 04: These rats were treated with lycopene/cephalexin antibiotic (10 mg/kgbw of lycopene and 2.5 mg/kg of cephalexin), which was dissolved in 2 mL of distilled water and administered through an oral administration in two divided doses daily for 2 weeks.

Group 05: This is the negative control group, where healthy rats received 1 mL of physiological water orally.

2.7. Animal Sacrifice and Specimen Collection

After 2 weeks of treatment, animals were weighed and anesthetized by intramuscular injection of ketamine at a dose of 150 mg/kg body weight (bw)., urine samples were recuperated by cystocentesis for bacterial culture, and blood samples were collected from the retroorbital region using a hematocrit capillary for Prostate-Specific Antigen analysis (PSA), while the prostate was dissected rapidly. From each rat, a portion of prostate tissue was cut and sonicated in 10 mL of PBS for 10 min (under aseptic conditions) for quantitative microbiological study under a series of dilutions in tissue homogenate. The last portion was fixed in 10% buffered formol to evaluate the histological study.

2.8. Prostate-Specific Antigen

PSA (Prostate-Specific Antigen) analysis is a test used to measure the levels of Prostate-Specific Antigen (PSA) in plasma. It is primarily employed in the assessment of prostate health, particularly for the diagnosis and monitoring of prostate cancer. Since PSA is a protease, its enzymatic activity can be measured using various laboratory techniques, such as ELISA or immunohistochemical analysis [14]. In this study, the analysis was carried out using the ELISA technique.

2.9. Histological Study

Prostates were preserved in a 10% (v/v) formalin solution. The preserved tissues were dehydrated in four acetone baths, cleaned in toluene, and then embedded in paraffin using a Leica TP1020. The specimens were sliced at 5 µm with a Leica RM 2135 rotating microtome. The sections were mounted on slides and stained with hematoxylin and eosin (H&E) [15].

2.10. Statistical Study

Data are presented as mean ± SD, with a p value of <0.05 being statistically significant. Statistical analysis was conducted using one-way ANOVA, followed by Tukey’s t-test for multiple comparisons. All analyses were conducted using the Past3.exe program (version 2018).

3. Results

3.1. CBP Detection

According to the microbiological results, out of 25 rats, 21 (84%) exhibited chronic bacterial prostatitis. The presence of S. aureus with beta hemolysis around each colony on blood agar as well as GRAM staining confirms the presence of this bacterium.

Sperm staining using May-Grünwald Giemsa revealed the presence of a significant number of leukocytes in all 21 rats (Figure 2). The four rats that did not ejaculate were excluded from the experiment.

3.2. Body Weight Evolution

Body and prostate weight, which were enregistered at the end of the experiment, revealed a different change (

Table 1. Body and prostate weight in different groups presented in mean ± standard deviation.

Groups	Body Weight (g)	Prostate Weight (g)
Negative control group	276.24 ± 11.47 b	0.54 ± 0.05
Positive control group	171.4 ± 38.66	0.34 ± 0.01 a
Lycopene group	218.94 ± 25.95 a	0.47 ± 0.12 a
Cephalexin group	222.5 ± 11.46 a	0.53 ± 0.05
Lycopene/cephalexin	275.34 ± 11.47 b	0.55 ± 0.11

a: significant difference at p < 0.05; b: highly significant difference at p < 0.001.

). It was observed that lycopene and cephalexin treatment alone, 218.94 ± 25.95 g and 218.94 ± 25.95 g, respectively, induced a significant increase in body weight when compared to the CBP control one (171.4 ± 38.66 g). In addition, the lycopene and cephalexin combination treatment (275.34 ± 11.47 g) was found to exhibit a high significant increase in body weight compared the control group.

Prostate weight in control and lycopene groups (0.34 ± 0.01 and 0.47 ± 0.12 g, respectively) reduced significantly compared to other groups. This decrease may be explained by atrophy in prostatic tissues.

3.3. Prostate-Specific Antigen Analysis

No significant difference (p ˃ 0.05) was observed in the Prostate-Specific Antigen concentration in either treated or untreated animals; PSA values ranged from 2.84 ± 0.008 × 10⁻² ng/mL to 2.62 ± 0.003 × 10⁻² ng/mL (the results are shown in

Table 2. Prostate-Specific Antigen parameters in different groups presented as mean ± standard deviation.

Groups	PSA(×10−2 ng/mL)
Negative control group	2.83 ± 0.002
Positive control group	2.64 ±0.008
Lycopene group	2.66 ± 0.003
Cephalexin group	2.62± 0.004
Lycopene/cephalexin	2.84 ± 0.001

).

3.4. Preostate and Urine Cultures

The quantitative microbiological result of both the prostate tissues and urine cultures of rats showed that there was a highly significant decrease in colonies number (CFU) in the cefalexin and lycopene/cefalexin groups compared to the control one (p < 0.05) (

Table 3. Quantitative microbiological results of urine and prostate tissue culture presented as mean ± standard deviation.

Groups	log10 CFU/g of Prostate Tissue	log10 CFU/100 µL of Urine
Negative control group	00.00	00.00
Positive control group	6.43 ± 0.40	5.21 ± 0.16
Lycopene group	5.48 ± 0.36	4.66 ± 0.75
Cephalexin group	3.48 ± 0.37 a	2.92 ± 0.89 a
Lycopene/cephalexin	1.19 ± 0.39 a	1.08 ± 0.05 a

CFU, colony-forming units. a: p < 0.05 compared with control group.

). A non-significant decrease (p ˃ 0.05) in CFU in the lycopene-treated group was recorded, and also compared to the positive control.

3.5. Prostate Histological Results

After two weeks of treatment, several structural changes were observed in the histological sections on CBP tissues compared to in the treated groups (Figure 3). Interestingly, an intense infiltration of monocytes and macrophages, interstitial fibrosis, and glandular edema were observed in the acini and interstitial tissues in the CBP group (Figure 3A). In contrast, microscope observation in the lycopene-treated group revealed a decrease in inflammatory cell infiltration in the glandular and peri-glandular tissues, as well as mild and focal cell infiltration (Figure 3B). Moreover, the group treated with cephalexin showed a decrease in cellular infiltration but with the presence of edema (Figure 3C). In addition, in the group treated with lycopene/cephalexin, the acini recovered their size, and their lumen contained some lymphocytes, a normal aspect of fibrous tissue, and no edema (Figure 3D).

4. Discussion

Chronic prostatitis of infectious origin constitutes the highest prevalent urological disease that affects men under 50 years of age [16]. Several germs can be encountered. Gram-negative bacilli are the most common germs. It has been identified that S. aureus, a Gram-positive microorganism, also plays a role in chronic bacterial prostatitis etiology [17].

In the present work, a reproducible rat model of chronic bacterial prostatitis (CBP) was developed by introducing S. aureus bacteria, which traveled through the urethra and penetrated the prostatic tract, inducing prostatic infection. After four weeks of inoculation 21 rats (84%) out of 25 rats developed chronic bacterial prostatitis; urine and sperm samples were taken to confirm the infection. Semen collection was performed to avoid complications of prostate biopsy surgery, especially to avoid rat death, and to improve on the protocol of [2], where there were six rat deaths due to complications following surgery. Since 80% of the sperm fluid from prostate exhibited imbalance, the high leukocyte count established by May-Grünwald Giemsa staining as well as the presence of S. aureus in the sperm culture are sufficient to confirm prostate infection. During the two weeks of treatment, the significant decrease in body weight in the untreated group compared to the treated groups was caused by the loss of appetite in the rats with prostatitis. Prostate antigen (PSA) is a serine protease, elaborated by the prostatic ductal epithelium. Generally, the concentration of PSA is elevated in patients with adenocarcinoma, and even in many of the non-malignant conditions of the prostate, especially benign prostatic hyperplasia (BPH) [18]. Also, it was evaluated in bacterial prostatitis according to [16], which noted that the concentration of PSA can increase only in the first week after infection in acute prostatitis, and then decrease and remain stable in the following two weeks (chronic prostatitis) [19]. This observation in PSA analysis is in agreement with our results. On the other hand, the quantitative microbiological study of urine and prostate tissue indicated that treatment with cephalexin only or in combination with lycopene significantly decreases the colony count (CFU). It is noted that cephalexin, an antibiotic used in chronic prostatitis treatment due to S. aureus [20,21], belongs to the family of β-lactam, which inhibits cell wall production and peptidoglycan synthesis. This disorder of cell structure leads to membrane permeability, so lycopene can easily cross the membrane, which reinforces its antimicrobial effect. This suggests that there is a possibility of synergy between lycopene and the tested antibiotic.

Lycopene is well known as an antioxidant that contributes to the protection of white blood cells and immunostimulants and involves anti-inflammatory effects [22]

Chronic prostatitis, particularly when caused by S. aureus, can lead to significant histological changes in prostate tissue, including marked inflammatory cell infiltration, interstitial fibrosis, vascular congestion, and massive interstitial edema. These changes are indicative of the body’s response to persistent bacterial infection and inflammation. The treatment of such conditions remains challenging due to increasing antibiotic resistance and the complex nature of the disease. This infection is characterized by significant inflammatory cell infiltration, which can lead to fibrosis in the prostate tissue. This fibrosis is associated with lower urinary tract symptoms (LUTSs) and impaired urethral function, as well as persistent inflammation and infection inducing vascular congestion and interstitial edema [23].

This opportunistic Gram-positive pathogen (S. aureus) is able to colonize prostatic tissues by activating Toll receptors, particularly TLR2 and TLR4, expressed on immune and epithelial cells. This activation triggers the NF-κ B signaling pathway, which plays a central role in initiating and maintaining inflammation by stimulating the expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6. In addition, S. aureus is able to form biofilms on prostatic tissues, which promotes the persistence of the infection by making it more resistant to antibiotic treatments and the immune response of the host, thus contributing to the chronicity of the disease [24,25].

Lycopene is also suggested in many studies to reduce the production and expression of inflammatory factors [23,26], further explaining that the possible mechanism of lycopene in reducing inflammatory factors could be that lycopene is a lipophilic compound closely associated with the cell membrane, where carotenoids regulate the activities of redox-sensitive transcription factors and kinases. It works by neutralizing reactive oxygen species (ROS), thereby reducing tissue oxidative stress and positively regulating the activity of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Lycopene also inhibits the activation of NF-κ B, which reduces the production of pro-inflammatory mediators (IL-6, TNF-α, COX-2) and moderates leukocyte infiltration in tissues [27]. Other studies show that lycopene exerted an anti-inflammatory effect by downregulating the levels of inflammatory factors, and also exerted an antioxidant effect [28]. Our results and their interpretations are supported by these studies. The association of lycopene with an antibiotic treatment such as cefalexin is based on pharmacological synergy. Cefalexin is a β-lactamine antibiotic that works by inhibiting bacterial wall synthesis, causing the lysis of bacterial cells. However, in the context of chronic prostatitis, its effectiveness may be reduced by the formation of bacterial biofilms, intense tissue inflammation, and impaired permeability of prostate tissues [29]. This combined action allows for better bacterial eradication and an overall improvement of the prostate condition. Several studies have examined the efficacy of lycopene in chronic prostatitis and compared it to other natural anti-inflammatory drugs. An animal model of chronic prostatitis/pelvic pain syndrome (CP/CPPS) [30,31,32] showed that lycopene significantly reduced pro-inflammatory cytokines such as TNF-α, IL-1β, IL-2, and IL-6. Another comparative study analyzed the effects of lycopene alone and in combination with other natural compounds such as quercetin and curcumin, a pharmacological synergy where the combination resulted in a more pronounced reduction in inflammatory mediators and oxidative stress than each compound administered alone. It has been shown in rats that the addition of lycopene to ciprofloxacin treatment increased the antibacterial and anti-inflammatory efficacy of the treatment, compared with the antibiotic alone. This synergistic combination not only improves treatment outcomes but may also allow for lower antibiotic doses, thus reducing the risk of side effects and antibiotic resistance.

5. Conclusions

Based on the findings of this study, it became clear that S. aureus installation in prostate tissues alters, endocrine homeostasis, sperm parameters, and prostate histology, which causes a deterioration in male reproductive function. Nonetheless, the combination of lycopene and cephalexin as a treatment for these alterations can detoxify the prostate damage. This study highlights the importance of lycopene as a potential therapeutic natural agent to reinforce cephalexin treatment in chronic infection and improve male function. Further studies are necessary to identify the plausible mechanisms of action involved and the synergistic effect between these molecules. However, these preliminary results justify further research on a larger animal model, including in-depth analyses to assess the therapeutic potential of this approach.