Introduction
Mastitis, a major disease in lactating goats, is caused by Staphylococcus aureus (S. aureus) due to its production of various toxins, various resistance proteins and biofilm formation (Ijaz et al., Reference Ijaz, Ghumman, Javed, Ahmed, Rasheed, Anwaar, Sabir and Muzammil2024; Javed et al., Reference Javed, Ijaz, Ahmed, Rasheed, Sabir and Jabir2024). It results in high economic losses due to reduced milk production, adverse effects on milk quality, higher incidence of animal culling and increased treatment costs (Ijaz et al., Reference Ijaz, Ghumman, Javed, Ahmed, Rasheed, Anwaar, Sabir and Muzammil2024). Many strains of S. aureus in causation of mastitis are bounded by slime layer that facilitates in attachment and settlement of organism to udder tissue’s epithelium (Ahmed et al., Reference Ahmed, Ijaz, Khan and Anjum2022). The proteins facilitating the production of polysaccharide intercellular adhesion and capsular polysaccharide/adhesion in most of Staphylococcal species are encoded by intercellular adhesion (ica) locus that is made up of icaA, D, B and C genes. Among ica genes, icaA and icaD play a significant role in biofilm formation, as the icaA gene encodes the enzyme acetylglucosaminyltransferase, which is involved in synthesizing N-acetylglucosamine oligomers from UDP-N-acetylglucosamine (Ahmed et al., Reference Ahmed, Ijaz, Khan and Anjum2022). The development of biofilm formation mainly governed by ica (A,D) genes in S. aureus is one of most defensive mechanisms that aids in bacterial evasion from immune system of host and causing persistent udder problems in lactating animals (Ahmed et al., Reference Ahmed, Ijaz, Khan and Anjum2023). Moreover, biofilm-forming S. aureus is also associated with decreased susceptibility to various antibiotics due to lower antibiotics penetration through the biofilm matrix, reduced metabolic rate of organism inside biofilm layer and transfer of various antimicrobial resistance (AMR) genes via horizontal pattern (Ahmed et al., Reference Ahmed, Ijaz, Khan and Anjum2023). This is a molecular study in Pakistan aimed at investigating the prevalence of biofilm-producing S. aureus in goats’ subclinical mastitis (SCM) along with the phylogenetic analysis of the icaA gene in S. aureus. Furthermore, antibiotic resistance profiling of biofilm-positive and -negative S. aureus was also carried out to evaluate its ability to induce intramammary infections in goats.
Materials and methods
This study was carried out in different goat flock of different sizes (small, medium and large) in various areas with latitude and longitude of 29.3544°N, 71.6911°E, Punjab, Pakistan, where livestock rearing is common among people. An overall 384 goat milk samples were collected from December 2022 to February 2024, across all seasons (summer, winter, spring and autumn) using a convenient sampling method. Initial milk samples were screened by the California mastitis test a CMT score of 2 was considered as the threshold for screening of SCM in goats followed by microbiological analysis (Javed et al., Reference Javed, Ijaz, Durrani and Ali2023). The confirmed S. aureus isolates were further subjected to different biofilm assays, i.e. phenotypic methods (Congo Red agar [CRA], microtitre plate) and genotypic assay (presence of icaA gene) to evaluate the biofilm production by S. aureus isolates. The molecular confirmation of biofilm production was carried out by polymerase chain reaction (PCR) as the primers (F: 5′-CCTAACTAACGAAAGGTAG- 3′ and R: 5′-AAGATATAGCGATAAGTGC- 3′) reported by Ahmed et al. (Reference Ahmed, Ijaz, Khan and Anjum2023). After sequencing the positive isolates, the icaA gene sequences were aligned using BioEdit software, and phylogenetic analysis was conducted with MEGA-X software to examine the identity of the study isolates with those reported from different countries (Fig. 1). The sequences were first BLAST against the NCBI database to determine the maximum similarity index and query coverage. The sequences with 100% identity with each other were selected for further analysis, and representative sequences were chosen for phylogenetic analysis with global isolates. The in vitro antibiotic susceptibility pattern of all S. aureus isolates (icaA positive and icaA negative) was checked by Kirby–Bauer’s disc diffusion method (CLSI, 2019). The prevalence of biofilm-producing S. aureus was calculated by dividing the number of biofilm-positive samples by the total number of S. aureus isolates. The data regarding the drug susceptibility trial were conducted by descriptive statistics.
Phylogenetic tree of icaA-positive S. aureus isolates. Red colour showing the study isolates.
Results
The current study reported the overall prevalence of SCM of 56.25% (216/384), while 53.24% (115/216) of the isolates were positive for the growth of S. aureus genotypic basis (Table 1). The findings of biofilm production by S. aureus isolates was determined by both phenotypic as well as genotypic methods in which 44 and 31 isolates were confirmed biofilm producers on basis of CRA and microtitre plate methods, respectively. The findings of molecular detection of PCR targeting icaA gene showed that 114 strains (55.34%) of S. aureus were declared biofilm producers, while 92 strains of S. aureus were found negative declared as non-biofilm producers. Molecular characterization of the isolates Goat.1, Goat.2, Goat.4 and Goat.8 revealed that Goat.1 closely resembled an Argentina isolate (JQ244772) and clustered with it in the phylogenetic tree. The isolates Goat.4 and Goat.8 showed more similarity to each other than to icaA isolates from other countries, such as Denmark, France, India and the USA. Furthermore, Goat.1 and Goat.2 exhibited more similarity with each other and with Argentina isolate than that of the other two local isolates. The Indian isolate (JX298873) formed a separate clade, and the China isolate (CP119571) displayed the least evolutionary relationship, forming an outgroup and the comparative analysis emphasized the distinctiveness of our study isolates compared to those from other regions. The results of antibiogram profiling showed that biofilm-producing S. aureus showed high resistance 82.09% towards cefoxitin and was declared as methicillin-resistant S. aureus, while 47.76% of biofilm-producing S. aureus strains were called as vancomycin-resistant S. aureus on phenotypic basis. The resistance pattern showed towards other antimicrobial drugs was oxytetracyline > gentamicin > amikacin > trimethoprim + sulphamethoxazole. Out of the tested antibiotic groups, high-sensitive antibiotics were fusidic acid > levofloxacin > linezolid (Table 2). Among the total tested isolates, 45.22% (52/115) isolates showed resistance to at least three different antibiotic groups simultaneously and were declared as multiple drug resistant (MDR) isolates as the multiple antibiotic resistance index (MARI) of these isolates was greater than 0.2. The remaining 52.17% (60/115) of the S. aureus strains depicted the MARI less than 0.2, while 03 isolates did not show resistance to any of the antibiotics (Table 2).
Prevalence of subclinical mastitis, S. aureus and biofilm-positive S. aureus in goats
In vitro antimicrobial susceptibility profiling of biofilm-positive and biofilm-negative S. aureus (n = 115)
CFT, cefoxitin; AK, amikacin; CN, gentamicin; VA, vancomycin; TY, tylosin; TMP, trimethoprim + sulphamethoxazole; T, oxytetracycline; CIP, ciprofloxacin; LEV, levofloxacin; FA, fusidic acid; LNZ, linezolid.
Discussion
The overall prevalence of SCM was recorded to be 56.25% which in similar to the findings of Hussein et al. (Reference Hussein, Fouad, KA, AM, D'Ambrosio, Scaloni and Gomaa2020) but higher than the previous studies regarding SCM in goats (Altaf et al., Reference Altaf, Ijaz, Ghaffar, Rehman and Avais2019; Muzammil et al., Reference Muzammil, Saleem, Aqib, Ashar, Mahfooz, Shoaib, Naseer, Sohrani and Ahmad2021). The current study reported a 48.37% prevalence of S. aureus which is higher than reported by Hussein et al. (Reference Hussein, Fouad, KA, AM, D'Ambrosio, Scaloni and Gomaa2020) and Muzammil et al. (Reference Muzammil, Saleem, Aqib, Ashar, Mahfooz, Shoaib, Naseer, Sohrani and Ahmad2021) who cultured 43% and 31.7% of S. aureus population from goat milk samples, respectively. Much higher prevalence was justified due to presence of exopolysaccharides (slime) production by this bacterium (Altaf et al., Reference Altaf, Ijaz, Ghaffar, Rehman and Avais2019). Biofilm formation is considered an integral element in causing the increased prevalence of mastitis as it provides protection from harsh environments and makes the treatment strategies ineffective (Ahmed et al., Reference Ahmed, Ijaz, Khan and Anjum2023). This study confirmed the occurrence of icaA gene in 114 isolates (55.34%) of S. aureus which differs from the previous study of Ahmed et al. (Reference Ahmed, Ijaz, Khan and Anjum2023) and Javed et al. (Reference Javed, Ijaz, Durrani and Ali2023). Another study by Andrade et al. (Reference Andrade, Laranjo, Costa and Queiroga2021) stated the prevalence of icaA gene in five isolates out of seven tested S. aureus strains of goat origin. The increasing trends of AMR in staphylococci isolates from mastitis cases may be due to imprudent and abuse of antimicrobial agents (Altaf et al., Reference Altaf, Ijaz, Ghaffar, Rehman and Avais2019). The results depicted that the highest percentage of resistance pattern of biofilm-positive isolates was found towards cefoxitin (82.09%), oxytetracycline (79.10%) and gentamicin (71.64%) which lines with the results of Altaf et al. (Reference Altaf, Ijaz, Ghaffar, Rehman and Avais2019), Ahmed et al. (Reference Ahmed, Ijaz, Khan and Anjum2023) and Javed et al. (Reference Javed, Ijaz, Durrani and Ali2023). The isolates showed high sensitivity patterns towards linezolid, levofloxacin and fusidic acid which favours the current study findings with the outcomes of Ahmed et al. (Reference Ahmed, Ijaz, Khan and Anjum2023). The findings of MARI show that 45.22% of the isolates exhibited MDR pattern which is similar to findings of Javed et al. (Reference Javed, Ijaz, Durrani and Ali2023). AMR in S. aureus isolates is rapidly increasing due to number of factors including self-medication, lack of quality antimicrobial agents, using antibiotics without proper consultation, development of new antimicrobials and lack of diagnostic facilities for antibiotic susceptibility testing (Javed et al., Reference Javed, Ijaz, Durrani and Ali2023). This study provides first molecular evidence of biofilm-producing S. aureus isolated from goat milk, highlighting the discrepancies between phenotypic and genotypic identification of the antibiotic-resistant S. aureus strains with emphasis on icaA gene in S. aureus as the virulence factor in causing goat mastitis. The study also suggested strategies such as drug repurposing and active surveillance to combat AMR.
Acknowledgements
The authors sincerely acknowledge project no. PHEC/PIRCA/20150/4 and Medicine Research Laboratory (MRL), Department of Veterinary Medicine, University of Veterinary and Animal Sciences, Lahore, for their valuable laboratory facilities and technical assistance.
