In vivo antibacterial activity of Streptomyces tuirus against MRSA-infected model

Abstract

Antibiotic resistance poses a global health risk, prompting researchers to focus on discovering new effective therapeutics from microbial sources. Hence, this study assessed the antimicrobial activity of the ethyl acetate crude extract of Streptomyces tuirus against MRSA in mice, providing the first in vivo validation of its previously reported in vitro activity. BALB/c mice (23 ± 1.2 g) were divided into 6 groups (6 mice/group). Groups I and II served as negative controls for saline and DMSO, respectively, while Group III received only the crude extract (10 mg/kg (post safety assessment). The crude extract was obtained using ethyl acetate extraction following submerged fermentation. Groups IV, V, and VI were intraperitoneally infected with 10⁸ CFU/ml MRSA. Subsequently, Group IV received no treatment, Group V was administered 10 mg/kg of the crude extract, and Group VI received 150 mg/kg of vancomycin. The treatment was given intraperitoneally for 7 days. At the end of the experiment, blood, liver, and kidney samples were collected. A hematological analysis was performed, followed by biochemical assessment for aspartate aminotransferase (AST), alanine aminotransferase (ALT), C-reactive protein (C-RP), and creatinine, along with histopathological examinations. MRSA infection induced significant systemic alterations. Although vancomycin was effective, it remained associated with elevated AST, ALT, and creatinine. However, the crude extract improved the hematological and biochemical parameters. It decreased AST, ALT, and creatinine by 42.94% (P-value 0.001), 43.33% (P-value 0.006), and 32.79% (P-value 0.0003), respectively, compared to the untreated infected Group. The crude extract of S. tuirus demonstrated in vivo antibacterial efficacy and a favorable safety profile against MRSA in mice.

Graphical abstract

Highlights

• •The Streptomyces tuirus used is the first reported from the Lebanese rhizosphere.
• •The crude extract of S. tuirus showed potent antibacterial activity against MRSA.
• •The crude extract restored hematological and biochemical parameters to normal.
• •The treatment with crude extract lowered CRP levels, suggesting anti-inflammatory activity.
• •Liver and kidney tissues showed normal histology after crude extract treatment.

1. Introduction

Antimicrobial resistance (AMR) arises when microorganisms lose their sensitivity to antimicrobials, resulting in more challenging infections that lead to increased morbidity and mortality rates. The growing prevalence of AMR is a pressing concern, as it poses a complicated challenge to the effective management of serious infections that do not respond to existing antibiotics.¹ A major contributor to this growing problem is that the inappropriate and excessive applications of human medicine and agricultural practices have facilitated the global transmission of multidrug-resistant (MDR) bacteria and resistance genes that are currently regarded as emerging contaminants in the environment.²

In response to the growing global health threat, the World Health Organization (WHO) introduced the Global Priority Pathogen List (PPL) in 2018, designed to provide vital support to researchers focused on the innovation and development of new antibiotics. Among the pathogens of concern, Staphylococcus aureus is of particular importance in the context of antimicrobial resistance due to its widespread infections.³ Statistics from the European Center for Disease Control and Prevention reveal the severe consequences of hospital-acquired infections (HAI), impacting around 3 million patients each year, with estimated deaths of 50,000. Notably, MDR strains of S. aureus are among the primary causes of these infections.⁴

S. aureus is classified as a Gram-positive bacterial pathogen, capable of causing infections that may result in soft tissue damage and various other diseases. The severity and intensity of these effects can differ based on the site of infection.⁵ Importantly, certain strains of S. aureus possess the mecA gene within their genome, which enables resistance to beta-lactam antibiotics through the synthesis of penicillin-binding protein (PBP) known as PBP2a.⁶ Typically, these resistant strains, referred to as methicillin-resistant S. aureus (MRSA), account for a considerable proportion of nosocomial infections reported in medical facilities and emergency care settings. The most notable antibiotics for the treatment of MRSA are vancomycin, daptomycin, ceftaroline, and linezolid.⁷ However, the prevalent use of vancomycin as the first-line MRSA therapy has resulted in an increase in resistant strains, particularly vancomycin intermediate-resistant S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA), which generally emerge under prolonged selective antibiotic pressure.⁸

In light of the increasing challenges associated with the development of new antibiotics, researchers are redirecting their focus toward exploring alternative antibiotics that present a lower risk of contributing to AMR. While synthetic compounds are often regarded with high expectations for their antimicrobial efficacy, there is growing interest in natural products that have emerged as a promising alternative.¹ İn this regard, a variety of research studies have been undertaken to discover new drugs originating from microbial sources.9, 10, 11 Furthermore, it is estimated that 70% of secondary metabolites are derived from microbial sources.¹²

Actinobacteria represent a phylum of filamentous bacteria that are Gram-positive, spore-forming, and widely distributed in various environments. This phylum includes one of the largest genera, Streptomyces, which plays a crucial role in the production of numerous naturally occurring antimicrobial compounds.¹³ These organisms are particularly important due to their ability to synthesize bioactive secondary metabolites that are candidates for drug discovery, including antibacterial, antiviral, antifungal, antiparasitic, and antitumor activities, as well as vitamins and enzymes.¹⁴ Such metabolites play an essential role in combating infectious diseases and are vital in the progress of the pharmaceutical industry.¹⁵

Generally, Actinobacteria are considered the foremost producers of antibiotics. More than 80% of the antibiotics that are currently in use, including chloramphenicol, tetracycline, macrolides, and nucleosides, are synthesized by these organisms.¹⁶ We have previously shown that the ethyl acetate crude extract of Streptomyces tuirus exhibited potent antibacterial activity against MRSA in vitro.17, 18 However, despite these promising findings, the therapeutic efficacy of this crude extract has not been validated under in vivo conditions. This validation is necessary to determine its applicability in clinical or preclinical use. Thus, the assessment of the effectiveness of antimicrobials in vivo is deemed essential. This study focused on the in vivo validation of the anti-MRSA capabilities and toxicity of ethyl acetate crude extract obtained from S. tuirus in a MRSA-infected mouse model, thereby presenting the first in vivo evidence of its effectiveness and expanding our previous in vitro results.

2. Methodology

2.1. Strain

S. tuirus was isolated from the rhizosphere of Origanum majorana that was grown in Sidon, South Lebanon, as described in our previous publication.¹⁷

2.2. Production and extraction of secondary metabolites

The fermentation conditions of the actinobacterial isolate, and the ethyl acetate extraction of secondary metabolites were performed as previously described.17, 18 The extracted metabolites were dissolved in 5% dimethyl sulfoxide (DMSO) to a final concentration of 50 mg/ml.

2.3. Animals and ethics statement

A total of 36 healthy male BALB/c mice, aged 6–8 weeks, weighing 23 ± 1.2 g, were acquired from the animal house at Beirut Arab University (BAU). Male mice were selected to minimize variability associated with hormonal fluctuations and sex dependent differences in immune response, thereby ensuring consistency in infection and treatment outcomes.

The mice were housed in a climate-controlled room where the temperature was maintained between 22 and 25 °C, with a light cycle of 12 h of light and 12 h of darkness. They were given a week to acclimatize before the start of the experimental procedures. Throughout this period, all animals were housed under standard conditions, with continuous access to a standard pellet diet and water. The experimental procedures were conducted in accordance with the regulations outlined by the Institutional Review Board (IRB) (code number: 2025-A-0062-S-P-0723). All laboratory animals were treated with human care in accordance with the guidelines established by the National Institutes of Health (NIH).

2.4. Inoculum preparation for infection

A single colony of the MRSA bacterium was cultured overnight in nutrient broth at 37 °C with shaking. The resulting culture was centrifuged, and the bacterial pellet was resuspended in sterile saline. The suspension was then adjusted to approximately 10⁸ CFU/ml in accordance with the 0.5 McFarland standard.

2.5. Animal experimental grouping

A total of 36 mice were divided into 6 distinct groups of 6 mice each. Groups I and II were designated as negative control groups that received an intraperitoneal (i.p) injection of 0.2 ml sterile saline or DMSO, respectively. Group III was treated exclusively with the ethyl acetate crude extract obtained from S. tuirus at 10 mg/kg to assess and confirm the safety of the crude extract and monitor any treatment-related effects independent of infection. Infection was established in Groups IV, V, and VI with 0.1 ml of the MRSA isolate at 10⁸ CFU/ml. Following infection, mice were observed for a two-hour period for the development of early clinical signs, including reduced activity and ruffled fur, to confirm successful infection before treatment.

After this period, treatments were administered as follows: Group IV received no treatment. Group V was treated with the ethyl acetate crude extract at a dosage of 10 mg/kg once daily for 7 days.¹ Mice of Group VI were intraperitoneally treated (i.p) with vancomycin, the standard antibiotic treatment, at 150 mg/kg for 7 days,¹⁹ as the MRSA isolate used in this study was confirmed to be susceptible to vancomycin based on prior antibiogram testing. Treatment efficacy was monitored throughout the experimental period by daily observation of clinical signs and recording of survival rates.

Regarding dose selection, the administered dose of vancomycin (150 mg/kg) was based on previously reported in vivo therapeutic regimens and while that of the crude extract (10 mg/kg) was based on preliminary studies and was chosen as an initial concentration to evaluate efficacy while minimizing potential toxicity.

2.6. Blood collection and measurements

Following a period of 7 days of treatment, blood samples were obtained for hematological and biochemical analysis.

Blood samples were collected in ethylenediaminetetraacetic acid (EDTA) tubes for hematological analysis from the different groups on the seventh day following infection. Complete blood count with differential (CBCD) test was performed at the Professional Healthcare Diagnostics (PHD) laboratory in Sidon, Lebanon, utilizing the Sysmex CBC-D machine. The results provided included key parameters such as the red blood cell (RBC) count, hemoglobin level, hematocrit value, platelet count, white blood cell (WBC) count, and differential WBC. Blood collection was performed under anesthesia by i.p injection of ketamine (50 mg/kg) and xylazine (10 mg/kg) to minimize animal pain and distress.

The serum was collected from the blood sample post centrifugation at 3000 rpm for 15 min. Then, the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and the concentrations of C-reactive protein (CRP) and creatinine were determined using the Roche c111 analyzer at the same laboratory.

2.7. Histopathological examinations

Mice were anesthetized as previously described, after which euthanasia was performed, and the liver and kidney were dissected. The organs were then preserved in 10% formalin and sent to the INP Institute National de Pathologie laboratory in Beirut, Lebanon, for histological examination. After fixation, the samples were dehydrated in progressively higher concentrations of alcohol, cleared with xylene, and embedded in liquid paraffin wax. Longitudinal Sections measuring 5 to 6 mm were obtained with the aid of a microtome. Hematoxylin and eosin (HE) staining was applied to the sections, and all the slides were examined at BAU using a light microscope. The images were obtained through visualization software, ensuring optimal brightness and contrast, at magnifications of 10 and 40×.

2.8. Statistical analysis

All experiments were performed in triplicate. The results are expressed as mean ± standard error of the mean (SEM). A one-way ANOVA was employed to assess statistical differences among the groups, followed by the Tukey test. A P-value of <0.05 was considered significant. Statistical significance was determined using IBM SPSS 2022 software.

3. Results

3.1. Impact of the crude extract on the hematological parameters

The hematological parameters of the different groups in response to MRSA infection and treatment with the extract are summarized in Table 1. Treatment with the crude extract was associated with an improvement in these parameters compared to the untreated infected group, likely reflecting a reduction in infection severity.

Table 1.

Hematological analysis of BALB/c mice in response to MRSA infection and treatment.

	Group I (Saline)	Group II (DMSO)	Group III (Extract)	Group IV (Infected)	Group V (Treated)	Group VI (Antibiotic)
RBCs (106/μL)	9.98 ± 0.30	9.77 ± 0.22	10.08 ± 0.21	7.08 ± 0.41⁎⁎	8.99 ± 0.37#	8.35 ± 0.31
HGB, (g/dL)	15.14 ± 0.13	14.87 ± 0.17	15.25 ± 0.19	11.94 ± 0.41⁎⁎⁎	14.22 ± 0.33##	13.90 ± 0.27
HCT, (%)	46.17 ± 0.40	46.48 ± 0.52	47.09 ± 0.43	37.08 ± 0.50⁎⁎⁎⁎	44.86 ± 0.37###	43.74 ± 0.62
PLT, (103/μL)	892 ± 16.10	907 ± 11.79	885 ± 20.82	697 ± 14.80⁎⁎⁎	855 ± 24.01##	842.33 ± 8.76
WBC (109/L)	6.45 ± 0.28	6.68 ± 0.40	6.12 ± 0.21	11.97 ± 0.48⁎⁎⁎	7.94 ± 0.36##	8.19 ± 0.48
NEU, (%)	21 ± 2.08	22.33 ± 1.45	23 ± 0.58	43.67 ± 1.19⁎⁎⁎	30.67 ± 1.20##	32 ± 1.15
LYM, (%)	71 ± 1.15	71.33 ± 1.86	69.67 ± 0.88	53 ± 0.94⁎⁎	63 ± 1###	62.67 ± 0.88
MON, (%)	6.33 ± 0.67	5.33 ± 0.88	6.67 ± 0.88	2.33 ± 0.27⁎⁎	4.67 ± 0.33##	3.67 ± 1.20
EOS, (%)	1.67 ± 0.33	1.00 ± 0.00	0.67 ± 0.33	1.00 ± 0.00	1.67 ± 0.33	1.67 ± 0.67
BASO, (%)	0	0	0	0	0	0

RBCs (Red Blood cells), HGB (Hemoglobin), HCT (Hematocrit), PLT (Platelets), WBC (White blood cells), NEU (Neutrophils), LYM (Lymphocytes), MON (Monocytes), EOS (Eosinophils), BASO (Basophils). Data are expressed as the mean ± SEM. One-way ANOVA was employed to compare the values among the groups. * represents a significant difference compared to the saline control group; # represents a significant difference between the infected and treated groups. #: P ≤ 0.05; ** or ##: P ≤ 0.01; *** or ###: P ≤ 0.001; ****: P ≤ 0.0001.

3.1.1. Impact of crude extract on RBCs, hemoglobin, and platelet levels

The infected group (IV) exhibited a significant decrease in RBC count (29.06%), hemoglobin (21.14%), and hematocrit levels (19.69%) compared to the saline control Group I (P-values < 0.01). However, treatment with the crude extract post MRSA infection was associated with a significant increase in RBC count (26.80%), hemoglobin (19.10%), and hematocrit (20.98%) (P-values < 0.05).

The platelet counts in the MRSA-infected Group IV significantly decreased (21.86%) compared to the control Group I (P-value 0.0008). Upon treatment post MRSA infection with the crude extract (Group V) or with the positive control vancomycin (Group VI), a marked increase in platelet count was observed, 22.67% (P-value 0.004) and 20.80% (P-value 0.001), respectively.

A direct comparison between the extract-treated group (V)and the antibiotic-treated group (VI) revealed closely comparable effects in erythrocyte-related parameters. Notably, the extract-treated group exhibited slightly higher values, with no statistically significant differences between the groups, RBC count (P-value 0.25), hemoglobin (P-value 0.47), hematocrit (P-value 0.21), and platelet counts (P-value 0.63) compared to the vancomycin-treated group, indicating a marginally improved recovery of erythrocyte and platelet indices.

3.1.2. Impact of crude extract on WBC profile

The total WBC count in the MRSA-infected Group IV significantly increased (85.58%, P-value 0.001) with a significant increase in neutrophils (107.95%, P-value 0.0008) compared to the saline control Group I. On the other hand, treatment with the crude extract (Group V) or vancomycin (Group VI) post-infection resulted in a significant reduction in WBC count, of 33.67% (P-value 0.004) and 31.58% (P-value 0.007), respectively. Additionally, these treatments resulted in a significant decrease in neutrophil count of 29.77% (P-value 0.002) with the crude extract (Group V), and 26.72% (P-value 0.003) with vancomycin (Group VI).

A comparison between the two treatments revealed closely comparable effects on leukocyte-related parameters. The crude extract-treated group (V) showed a slightly greater reduction with no statistically significant differences in both WBCs (P-value 0.68) and neutrophil counts (P-value 0.82), compared to the antibiotic-treated group (VI), although these differences were minimal. Overall, both treatments effectively reduced infection-associated leukocytosis and neutrophilia.

Concerning lymphocyte count, a notable decrease of 25.35% was observed in the infected group (Group IV) when compared to the saline control Group I (P-value 0.01). Conversely, a significant improvement (P-value 0.0003) in this count was recorded after the treatment with the crude extract (Group V) or vancomycin (Group VI) of 18.87% and 18.25%, respectively.

3.2. Impact of crude extract on biochemical parameters

3.2.1. Impact of crude extract on liver function

The AST and ALT serum activity were significantly elevated in the MRSA-infected Group IV compared to the saline control Group I, with a 120.73% increase (P-value < 0.0001) in AST and a 170.69% increase (P-value 0.0006) in ALT (Fig. 1a and b, respectively). Following treatment with the crude extract, a significant reduction in the activity of these enzymes was observed. In contrast, treating the infected mice with vancomycin (Group VI) still maintained high activity of these enzymes. The AST and ALT activities significantly decreased post-treatment with the crude extract by 42.94% (P-value 0.001) and 43.33% (P-value 0.006), respectively, compared to the untreated Group IV.

Fig. 1.

Serum biochemical analysis of BALB/c mice in response to MRSA infection and treatment. (a) Aspartate aminotransferase (AST) levels; (b) Alanine aminotransferase (ALT) levels; (c) C-reactive protein (CRP); (d) Creatinine levels. Data are expressed as the mean ± SEM. ns, not significant, *, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001; ****, P ≤ 0.0001.

In contrast, vancomycin-treated mice (Group VI) showed persistently elevated enzymatic activity, which remained higher than that observed in the crude extract-treated group (V). Direct comparison between both treated groups revealed that the crude extract was significantly more effective than vancomycin in reducing AST and ALT activities (P = 0.0003 and P = 0.0006, respectively), suggesting a comparatively improved hepatic profile under the experimental conditions.

As for the control groups, Group II and III, no significant changes were observed in AST and ALT levels compared to the saline control group (I), implying their safety on the liver (Fig. 1a and b, respectively).

3.2.2. Impact of crude extract on inflammatory marker

The CRP levels in the MRSA-infected mice were also significantly elevated by 588.38% compared to the saline control group (I) (P-value < 0.0001), as shown in Fig. 1c. However, following treatment with the crude extract or vancomycin, a significant reduction in CRP levels was observed, with a decrease of 83.86% in the crude extract-treated group (P-value < 0.0001).

In contrast, comparison between the crude extract and vancomycin-treated groups revealed no significant difference in CRP levels (P = 0.41), indicating comparable anti-inflammatory effects of both treatments in reducing CRP levels.

3.2.3. Impact of crude extract on kidney function

The level of creatinine exhibited a significant increase of 56.41% in the infected Group IV in comparison to the saline control Group I (P-value 0.0003). The treatment with the crude extract was associated with a significant reduction in creatinine levels by 32.79% (P-value 0.0003) compared to the untreated one. Conversely, the treatment with vancomycin continued to sustain elevated levels of creatinine (Fig. 1d), implying its nephrotoxicity. Direct comparison between the crude extract- and vancomycin-treated groups confirmed that the crude extract was significantly more effective in reducing creatinine levels than vancomycin (P = 0.0016).

Concerning the control groups, Group II and III, no substantial changes in creatinine levels were noted in comparison to the saline control group (I), suggesting their renal safety.

3.3. Histological analysis

3.3.1. Impact of the crude extract on the liver structure

The histological examination of livers (Fig. 2) from the saline control Group I, DMSO Group II, crude extract Group III, and the extract-treated mice post MRSA infection Group V exhibited normal histo-morphological characteristics, including well-formed hepatic cords, central veins, and portal areas. However, the liver of the MRSA-infected mice, Group IV, showed numerous regions of necrosis, accompanied by localized abscesses primarily consisting of neutrophils. Similarly, the antibiotic-treated Group VI demonstrated areas of periportal inflammation, which are characterized by lymphocytic infiltration.

Fig. 2.

Hematoxylin and eosin-stained section of livers from different groups. The livers from the saline control (1), DMSO control (2), crude extract (3), infected (4), crude extract-treated (5), and antibiotic-treated (6) were examined under a light microscope at a magnification of 10×. Black arrow: necrosis; Blue arrow: neutrophil infiltration; Red arrow: Periportal infiltration with lymphocytes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.3.2. Impact of the crude extract on the kidney structure

The histopathological examination of the kidneys (Fig. 3) from the saline control Group I, DMSO Group II, crude extract Group III, and the extract-treated mice post MRSA infection Group V showed well-preserved parenchyma characterized by clearly defined glomeruli, intact tubular epithelial cells, and no signs of inflammation, fibrosis, or other abnormalities. Conversely, histopathological abnormalities were observed in both the MRSA-infected mice, Group IV, and the antibiotic-treated Group VI, where infiltration of inflammatory cells accompanied by marked vacuolar degeneration of the epithelial cells was shown, in addition to areas of necrosis, presence of casts within the lumina of renal tubules seen in the antibiotic-treated Group VI.

Fig. 3.

Hematoxylin and eosin-stained section of kidneys from different groups. The kidneys from the saline control (1), DMSO control (2), crude extract (3), infected (4), crude extract-treated (5), and antibiotic-treated (6) were examined under a light microscope at a magnification of 40×. Brown arrow: inflammatory cells; Blue arrow: vacuolar degeneration; Black arrow: necrosis; Red: casts and shedding cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

4. Discussion

The rise of antibiotic resistance poses a significant challenge to global health, with bacterial resistance levels continuing to rise. The inappropriate use of antibiotics has accelerated the emergence of antibiotic resistance, resulting in more challenging treatment scenarios.²⁰ The need for new and effective antibiotics against MDR pathogens is a major concern in the field of infectious disease treatment. Researchers are focusing on the exploration of new bioactive compounds from Streptomyces species to effectively combat these MDR pathogens.²¹ The current research focused on assessing the antimicrobial activity of ethyl acetate crude extract derived from S. tuirus, a bacterium that was isolated and identified from the rhizosphere of O. majorana, against MRSA-infected mice.

Hematological parameters serve as important biomarkers for diagnosing injuries to organs or tissues. Well-established reference values are essential for interpreting variations induced by different diseases.⁵ In this study, MRSA infection resulted in a reduction in RBC, hemoglobin, and platelet count. This observed phenomenon may be due to RBC lysis during infection or a decrease in iron levels within the body, which indirectly affects the production of erythrocytes.²² These results were in agreement with the previous report that revealed a significant decrease in RBC, hemoglobin, and platelet count in mice infected with MRSA.²³ On the other hand, treatment with the crude extract significantly enhanced the previously mentioned hematological parameters, indicating its potent efficacy in combating MRSA infection.

A high WBC and neutrophil count was observed post-MRSA infection. It serves as an indicator of both physical and mental health, demonstrating that the immune system is actively fighting an infection.²⁴ This is in agreement with a previous study showing that leukocytosis and neutrophilia were two conditions that were linked with Escherichia coli O:157: H7 infections in both humans and mice.²⁵ Conversely, treatment with the crude extract of S. tuirus reduced infection-associated elevations in WBC and neutrophil counts, further supporting the effectiveness of the crude extract in managing infection.

The evaluation of biochemical properties provides essential data concerning clinical health, nutritional status, and the metabolic activities of various organs and tissues.⁵ Typically, each organ releases distinct enzymes in response to injury. An elevated level of enzymes is regarded as a diagnostic indicator for several medical conditions.²⁶ The assessment of the kidney function indicator showed an increase in creatinine levels post-MRSA infection. It is well known that infectious diseases resulting from pathogens, including viruses, bacteria, and protozoa, can disrupt the normal function of the kidneys.²⁷ Similarly, Abdelaziz et al., demonstrated that on day 7 postinfection, both S. aureus and Klebsiella pneumoniae-infected mice showed an increase in creatinine levels.¹ This increase may reflect MRSA-induced renal impairment associated with infection, while treatment with crude extract may exert a protective effect on renal function.

In addition, the evaluation of parenchymal cell injury is primarily conducted through the measurement of AST and ALT, which are more sensitive than alternative serum enzymes.²⁸ These enzymes are present in hepatocytes and are also distributed across different tissues and organs. Their release into the bloodstream occurs as a result of cellular necrosis and alterations in cell membrane permeability.²⁹ Our investigation indicated that infecting mice with MRSA caused a significant rise in AST and ALT activity. These findings align with those of Wang et al., who demonstrated a significant elevation in these parameters among the infected group by Vibrio parahaemolyticus.³⁰ This elevation suggests MRSA-induced hepatic injury, likely due to infection- associated tissue damage and membrane disruption, while treatment may confer hepatoprotective effects.

CRP is an acute-phase protein that is produced by the liver in response to inflammation or infection.³¹ Our results showed a significant increase in CRP levels post MRSA infection, and these results are consistent with a report that indicated a significant increase in the CRP level at 0.5, 1, 3, and 21 days in BALB/c mice infected with MRSA.³² On the other hand, our results revealed that CRP values in the vancomycin-treated group (VI) and crude extract group were found to be lower than those in the infected group, highlighting their effective impact in managing infection.

Concerning the histopathological modifications in the livers and kidneys post MRSA infection, histopathological examinations revealed necrosis, inflammatory cell infiltration, and neutrophil-rich abscess formation. Grosz et al., reported in their study that the i.p injection of S. aureus led to a predominant distribution of the bacteria within hepatic and splenic tissues, which subsequently resulted in the formation of liver abscesses.³³ On the other hand, the crude extract played a crucial role in restoring the normal physiological state in mice, as verified by the hematological, biochemical, and histopathological assessments. Similarly, Abdelaziz et al.,¹ declared that the bioactive metabolites of S. misakiensis effectively treated S. aureus-infected mice.

Regarding vancomycin treatment of MRSA infection, elevated AST and ALT activities as well as creatinine levels were observed, along with histopathological abnormalities in both liver and kidney tissues, highlighting the complications associated with this antibiotic, as also shown by Takigawa et al.,.³⁴ On the other hand, our S. tuirus extract at 10 mg/kg showed no toxicity to either the liver or the kidneys, and these results were validated histologically. Similar results were obtained by Abdelaziz et al., who reported that the bioactive metabolites of S. misakiensis had no clear indicators of toxicity in the organs treated.¹

The enhanced antibacterial activity observed in this study may be linked to the recognized ability of Streptomyces species to produce a broad range of secondary metabolites. Streptomyces are known to synthesize numerous structurally unique compounds, including polyketides, non-ribosomal peptides, terpenoids, and alkaloids, many of which demonstrate significant activity against Gram-positive pathogens such as MRSA.³⁵ These metabolites can interact with cellular targets, including inhibition of cell wall biosynthesis, disruption of protein synthesis, and alteration of membrane integrity.³⁶ These multitarget mechanisms are beneficial in addressing MDR organisms, as they decrease the probability of resistance development and enhance therapeutic efficacy.³⁷ Consequently, the observed in vivo activity in this study may be due to the synergistic effects of various bioactive components found in the crude extract.

Overall, the crude extract demonstrated significant effects against MRSA infection. However, a limitation of this study is the absence of quantitative bacterial load determination, which would provide a more precise evaluation of antimicrobial efficacy.

5. Conclusion

The findings of this study revealed that the ethyl acetate crude extract obtained from S. tuirus effectively treated MRSA infection in mice, as demonstrated by improved hematological and biochemical parameters. Additionally, the biochemical and histopathological evaluations confirmed the safety of the crude extract, which showed no toxicity to the liver or kidneys. The crude extract from S. tuirus demonstrates its potential as a promising candidate for a new antimicrobial agent targeting MRSA infections. Further characterization of the crude extract is crucial for identifying the bioactive compounds that are responsible for its antibacterial properties, enabling subsequent evaluation of their pharmacokinetic behavior. Additionally, future research will focus on the long -term effectiveness, safety, survival outcomes, and dose-response relationships using multiple extract doses to better understand the therapeutic potential of the crude extract.

CRediT authorship contribution statement

Mona Tfaily: Writing – original draft, Methodology, Formal analysis. Miriam Al Battal: Writing – review & editing, Methodology. Jamilah Borjac: Writing – review & editing, Supervision, Methodology, Conceptualization.

Funding

This research didn't receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.