In vitro studies of Searsia lancea leaf extracts against multi-drug resistant bacterial isolates from clinical cases of bovine mastitis

Abstract

Introduction Bovine mastitis, a common inflammatory disease affecting mammary glands of dairy cattle, has severe implications for milk quality and production. It is a critical pathology in global dairy herds, and poses a significant economic burden on the dairy industry, leading to substantial financial losses due to decreased production and increased culling. Bovine mastitis is a significant problem in the dairy industry due to antibiotic-resistant pathogens forming biofilms via quorum sensing. Pathogenic microorganisms, particularly Streptococcus species, Staphylococcus species and Escherichia (E.) coli, are the major aetiological agents. Managing mastitis typically involves antibiotics, but the continuous use of conventional antimicrobial agents has led to antimicrobial resistance and treatment failures. The ability of bacteria to form biofilms is associated with resistance, while quorum sensing plays a vital role in biofilm formation. This complex interplay between quorum sensing (QS) and biofilm formation challenges mastitis management, necessitating the development of alternative therapeutics to combat this microbial threat effectively. In addition, managing inflammation with its accompanying oxidative stress in mastitis is a holistic approach in managing the disease. Plants have been shown to demonstrate antioxidant and anti-inflammatory activities, and show promise in restoring drug sensitivity and enhancing host immunity, therefore, to address this issue, researchers are exploring plant-derived antimicrobial agents. This study, therefore, aimed to investigate the antibiotic resistance profile of major bacterial isolates from mastitis cases, and to determine the antibacterial, antibiofilm and anti-quorum sensing, antioxidant and anti-inflammatory activities and cytotoxicity of the extracts and fractions of selected South African plants. This will serve to contribute to the development of alternative treatments for bovine mastitis caused by resistant bacteria. The selection of plants (Searsia lancea, Erythrina caffra, Antidesma venosum, and Indigofera frutescens) was based on reported minimum inhibitory concentration (MIC) of less than 0.1 mg/mL against staphylococcal bacteria of mastitis origin (Akinboye et al., 2023). The antimicrobial susceptibility of the bacterial strains was determined using a disc diffusion method, while the biofilm forming ability (BFA) of bacterial isolates in Brain Heart Infusion broth (BHI) and Tryptic Soy broth (TSB) was determined. Furthermore, the antibacterial, antibiofilm and anti-quorum sensing activities of the extracts and fractions were determined against a panel of mastitis pathogens using a two-fold serial microdilution assay. In addition,

v their antioxidant and anti-inflammatory activities as well as their effects on mRNA expression of their pro-inflammation cytokine genes were investigated using standard methods, while their cytotoxicity was determined against Vero cells and bovine dermis cells. Among 32 clinical bacterial isolates tested, 84.38% (27) exhibited antibiotic resistance, with 55.56% (15) being multidrug-resistant. These include 100% (7) of the E. coli isolates, 50% (2) of the staphylococcal isolates, 42.86% (3) of Streptococcus (Str.) uberis, and 14.29% (1) each of Str. agalactiae and Str. dysgalactiae strains. These results reveal a concerning trend of high

antibiotic resistance among clinical isolates, with a significant portion being multidrug- resistant. Remarkably, E. coli isolates showing 100% resistance, along with varying resistance

in other strains, pose challenges for conventional antibiotic effectiveness and highlight the

necessity for targeted strategies due to specific strain resistances, especially in cases of E. coli- associated mastitis in dairy cattle.

Moreover, all plant extracts exhibited antibacterial activity against all the bacterial isolates, but S. lancea demonstrated better activity compared to other plants. The MIC values obtained ranged between 0.01 - 2.50 mg/mL, with the narrowest range obtained with the acetone extract of S. lancea (MIC = 0.01 – 0.57 mg/mL). The overall MIC range for all the extracts against the E. coli isolates was 0.01 – 0.31 mg/mL, which showed that the extracts exhibited good to moderate activities against these multidrug resistant (MDR) E. coli isolates. The lowest MIC observed (0.01 ± 0.00 mg/mL) was demonstrated by acetone and ethanol extracts of S. lancea. Interestingly, the acetone extract of S. lancea also showed good activity against 80% (24) of the 30 organisms tested, followed by the ethanol extract (60%). Though, the acetone extract of S. lancea demonstrated the highest total antibacterial activity (TAA) against E. coli, the ethanol extract of S. lancea demonstrated higher efficacy (TAA) against three of the four species of isolates. Also, when considering individual isolates, the ethanol extract of S. lancea exhibited an exceptional TAA value of 15 790 mL/g against ECO4, and TAA values above 1 000 mL/g against 83.33% (25 out of 30) of the organisms which is more than 56.67% (17) by its acetone extracts. This suggests the ethanol extract is more efficacious than the acetone extract. The dichloromethane (DCM) and ethyl acetate fractions of ethanol extracts of S. lancea had good antibacterial activity against the isolates with MIC values as low as 0.001 and 0.01 mg/mL, respectively. In comparison to the reference antibiotic, ciprofloxacin, the DCM and ethyl acetate fractions demonstrated better antibacterial activity, highlighting the potential of these fractions as alternatives to commonly used antibiotics against mastitic pathogens. Interestingly, the crude ethanol extract demonstrated better antibacterial activity against Str. agalactiae, Str.

vi uberis, E. coli and Sta. aureus isolates than all the fractions, while in sharp contrast, all the fractions demonstrated better activities than the extract against Str. dysgalactiae isolates. This suggests species-specific additional, synergistic or antagonistic antibacterial activities between the compounds in the extract, and this infers fractionation of the extracts potentiates or attenuates the effects of the compounds in the extract against the bacteria. Moreover, the extracts, particularly from S. lancea, demonstrated low cytotoxicity toward BD and Vero cells, crucial for ensuring safety in potential therapeutic applications. Remarkably, S. lancea displayed the highest mean selectivity index value of 25.70, indicating its selectivity in targeting bacterial pathogens while being less harmful to host cells. The fractions exhibited minimal cytotoxic effects on bovine dermis (BD) cells. The selectivity index values of S. lancea extracts and DCM fraction were remarkably high, reaching 100 and 90, respectively, further emphasizing their safety and selectivity in targeting bacteria while being non-toxic to mammalian host cells. Out of the 29 isolates tested, 93.10% (27) and 68.97% (20) demonstrated BFA in TSB and BHI, respectively. Compared to BHI, TSB appeared to enhance BFA of the bacteria except for Str. uberis strains. These findings highlight a significant prevalence of BFA among isolates, emphasizing potential biofilm-related challenges in bovine mastitis management. Moreover, the varying BFA between different growth media suggests a crucial influence of the medium on biofilm formation. Additionally, the strain-specific behaviour, notably seen in Str. uberis strains, underscores the need for nuanced, tailored approaches when managing different bacterial strains causing mastitis. Furthermore, biofilm inhibition and eradication tests revealed varying activities of extracts and fractions, with S. lancea displaying the most potent antibiofilm activity at varying sub-MIC concentrations. All the plants demonstrated good biofilm disruption ability (BDA) against 24 h preformed biofilms of the isolates except for E. caffra, while S. lancea displayed good BDA against all the 48 h preformed biofilms of the bacteria. Generally, the plants' antibiofilm activities appeared to improve as the biofilm matured, with few exceptions. In addition, DCM and ethyl acetate fractions generally exhibited good inhibition at various concentrations against most of the bacterial strains tested, but the ethyl acetate fraction had the best antibiofilm and preformed biofilm disrupting activities. The differing abilities of extracts and fractions to inhibit and eradicate biofilms highlight the complexity of managing biofilm-related infections in mastitis. Searsia lancea stands out with potent antibiofilm activity at sub-MIC

vii concentrations, suggesting its potential as a key player in combating biofilm-forming bacteria associated with mastitis. While most plants demonstrated good BDA against 24 h preformed biofilms, S. lancea displayed consistent effectiveness against even longer matured 48 h biofilms of bacteria. This suggests a promising ability to disrupt established biofilms, a crucial aspect in managing chronic or recurrent mastitis cases. The ethanol extracts of S. lancea demonstrated a minimum quorum sensing inhibition concentration (MQSIC) at 2.50 mg/mL, and the lowest MQSIC50 value (< 0.08 mg/mL), demonstrating its quorum quenching ability. This indicates its potential to interfere with bacterial communication systems, disrupting their ability to coordinate and cause infections, which could be pivotal in controlling mastitis. Remarkably, the DCM and water fractions demonstrated the lowest MQSIC value, with DCM having the lowest MQSIC50 value of 0.01 mg/mL, which suggests that the fractions have the potential to modulate virulence factors by specifically inhibiting the formation of the violacein pigment indicating inhibition of quorum sensing at sub-MIC levels. Lastly, S. lancea demonstrated better antioxidant and anti-inflammatory activities compared to other extracts, and was able to downregulate the expression of iNOS gene by 99% while upregulating the expression of COX-2 and IL-6 in LPS-stimulated RAW 264.7 macrophages. This indicates its potential to tackle both oxidative stress and inflammatory expression of iNOS as well as enhance immune response in immunocompromised cows. In conclusion, this research highlights the urgency of tackling AMR in livestock management, and demonstrates the promising therapeutic potential of these plants, particularly S. lancea, in treating bovine mastitis. The study also showed that the extracts exhibited significant antibacterial activity against these drug-resistant strains, as S. lancea had the most potent antibacterial activity and appeared promising as a broad-spectrum antibacterial agent against mastitis-related bacteria. Importantly, it had a moderate cytotoxicity profile, particularly the ethanol extracts and its fractions. This suggests that S. lancea extracts could serve as a valuable resource for developing novel treatments. Moreover, this study underscores the potential of fractions of the S. lancea extract as valuable resources in combating antimicrobial resistant mastitis pathogens. Further investigation of their mechanisms, identification of active compounds, and thorough in vivo assessments are essential steps toward their development as effective preventative and treatment options for microbial infections such as bovine mastitis. Furthermore, the ability of these plants, especially S. lancea, to inhibit QS and biofilms at various developmental stages may play a pivotal role in managing mastitis infections and curbing the emergence of antimicrobial resistance. Nonetheless, further research is needed to elucidate the precise mechanisms underlying the inhibition of quorum signalling and biofilms, and to identify the specific compounds responsible for the observed activities. Overall, the study highlights S. lancea as a promising candidate alternative for treatment of infection, oxidative stress and inflammation in bovine mastitis, underlining the importance of further research on the plant and the active compounds within its fractions, with the intent of developing S. lancea into a global South African product in the management of bovine mastitis worldwide.