In the dairy industry, mastitis is considered as the most common and economically important disease worldwide, due to reduced milk quality and production, milk discard, involuntary culling, veterinary services, treatment costs and increased labor costs (de Souza Santos et al., Reference de Souza Santos, de Lima, Abad, de Oliveira, da Silva, de Moura and Mota2020). Although mastitis is a multifactorial disease, involving interrelationships between microorganisms, host and environmental factors, microorganisms have been playing an important role in the emergence and dissemination of disease (Singha et al., Reference Singha, Ericsson, Chowdhury, Nath, Paul, Hoque and Rahman2021). Among microorganisms, in particular, bacteria are the most common cause of mastitis cases, and staphylococci are the main etiological agents isolated from mastitic milk samples (Gomes et al., Reference Gomes, Saavedra and Henriques2016).
The increasing trend of antimicrobial resistance presents a growing burden for the prevention and treatment of mastitis due to widespread misuse of antimicrobials. The emergence of antimicrobial resistance among mastitis pathogens is also a concern for public health because resistant bacteria can also be transmitted to humans through the food chain (Oliver and Murinda, Reference Oliver and Murinda2012).
Biofilm forming ability of Staphylococcus spp. is one of the important virulence factors that facilitate adhesion and colonization on the mammary gland epithelium, leading to recurrent or persistent infections (Melchior et al., Reference Melchior, van Osch, Lam, Vernooij, Gaastra and Fink-Gremmels2011). Staphylococcal biofilm formation is a two-step process involving cell attachment and the formation of an extracellular matrix. For cell attachment, staphylococci can express a variety of bacterial surface molecules that interact with host tissues. These molecules are known as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs: Foster and Höök, Reference Foster and Höök1998).
The purpose of the current study was to determine the prevalence of subclinical mastitis, to obtain detailed data on staphylococci associated with subclinical mastitis, to determine the antimicrobial susceptibilities of the isolates, to evaluate the biofilm-forming ability of the isolates and finally to investigate biofilm-related genes and MSCRAMMs by polymerase chain reaction (PCR).
Materials and methods
A detailed description of materials and methods is provided in the online Supplementary File. The study was approved by the Animal Ethical Committee of the International Center for Livestock Research and Training, Lalahan, Ankara (30.05.2018/152).
Study area and sample collection
This study was carried out among 200 lactating Anatolian buffalo breeds over six months (from June 2018 to September 2018) in the province of Çorum, which is located in the Central Anatolia region of Turkey. All farms (n = 42) included in the study were family-sized, and the herd size of the selected farms varied from 5 to 30 water buffalos. The milk yield of the buffalos ranged from 3 to 7 kg per day, with on average 1160 kg of annual milk yield. The lactation period was between 230 and 240 d. All animals were subjected to clinical examination and California mastitis test (CMT), then the CMT positive milk samples were microbiologically examined.
Isolation and identification of staphylococci
Isolation of staphylococci was conducted as previously described (Quinn et al., Reference Quinn, Carter, Markey and Carter1999). The identification of staphylococci at the species level was performed using a MALDI Bruker Biotyper system (Bruker Daltonics Inc., Billerica, MA, USA).
Antimicrobial susceptibility testing
Antimicrobial susceptibilities of the isolates were determined using the disc diffusion method and the results were interpreted according to Clinical and Laboratory Institute (CLSI, 2022) guidelines.
Determination of biofilm formation
The biofilm-forming ability of the isolates was investigated by two different methods: Congo Red Agar (CRA) Method and Microtiter Plate (MTP) Method. All methods are detailed in the online Supplementary File.
Results and discussion
In this study, 24% (48) of the water buffaloes were diagnosed with subclinical mastitis. Özenç et al. (Reference Özenç, Vural, Şeker and Uçar2008) reported a prevalence rate of 16.8% in Afyon province in Turkey. While Sharif and Ahmed (Reference Sharif and Ahmad2007) found the prevalence of sub-clinical mastitis as 37.75% in Pakistan, Elhaig and Selim (Reference Elhaig and Selim2015) reported the prevalence of subclinical mastitis as 43.3% in Egypt.
Out of 160 milk samples, 107 (66.9%) Staphylococcus spp. including 28 S. aureus and 79 coagulase-negative staphylococci (CoNS) were isolated. Özenç et al. (Reference Özenç, Vural, Şeker and Uçar2008) also reported that staphylococci were the most common bacteria with a rate of 52.95%. Preethirani et al. (Reference Preethirani, Isloor, Sundareshan, Nuthanalakshmi, Deepthikiran, Sinha, Rathnamma, Nithin Prabhu, Sharada, Mukkur and Hegde2015) reported staphylococci as major pathogens isolated from subclinical bovine mastitis cases of buffaloes in India.
As can be seen in online Supplementary Tables S1 and S2, among the staphylococci, S. aureus showed a higher resistance rate to penicillin, ampicillin, amoxicillin-clavulanic acid (53.6–67.9%), and showed low resistance rates to trimethoprim-sulfamethoxazole, ciprofloxacin, cefoxitin (3.6–14.3%), while no resistance was observed to gentamicin and chloramphenicol. On the other hand, CoNS displayed intermediate resistance to penicillin and ampicillin (41.8–51.9%), low resistance to trimethoprim-sulfamethoxazole (16.5%) and least resistance to chloramphenicol, cefoxitin, erythromycin, tetracycline, amoxicillin-clavulanic acid (1.3% to 7.6%). The CoNS were all sensitive to gentamicin and ciprofloxacin. The high resistance for beta-lactams was not surprising, because β-lactams are widely prescribed agents in Turkey. These are consistent with previous reports of β-lactam resistance rates in staphylococci isolated from buffalo mastitis (Medeiros et al., Reference Medeiros, França, Krewer Cda, Peixoto Rde, de Souza, Cavalcante, da Costa and Mota2011; Preethirani et al., Reference Preethirani, Isloor, Sundareshan, Nuthanalakshmi, Deepthikiran, Sinha, Rathnamma, Nithin Prabhu, Sharada, Mukkur and Hegde2015).
The results for biofilm forming ability obtained by the CRA method and MTP method are comparatively given in online Supplementary Table S3. While the bap gene was not detected in any staphylococci, various biofilm-associated and MSCRAMM gene profiles was observed among staphylococci (online Supplementary Table S4). The frequency of genes associated with adhesion and biofilm formation in S. aureus and CoNS strains isolated from mastitis cases vary according to the geographical area studied (Felipe et al., Reference Felipe, Morgante, Somale, Varroni, Zingaretti, Bachetti, Correa and Porporatto2017). Overall, 70.9% (n = 56) and 97.5% (n = 77) of the isolates were determined to be biofilm producers by the CRA method and MTP, respectively. The contradictory results between phenotypic methods and genotypic methods have been reported in previous studies (Ciftci et al., Reference Ciftci, Findik, Onuk and Savasan2009; Darwish and Asfour, Reference Darwish and Asfour2013). Biofilm forming ability of the isolates that were negative for icaA and icaD genes could be attributed to ica gene independent control of slime production/adhesion mechanism (Liberto et al., Reference Liberto, Mater, Quirino, Lamberti, Capicotto, Puccio, Barreca, Focà, Cascio and Focà2009). In contrast, the inability to form biofilm of the isolates having icaA and icaD genes could be due to point mutations in the ica locus or any other unidentified factors that negatively regulate polysaccharide intercellular adhesion synthesis or influence biofilm formation (Cramton et al., Reference Cramton, Gerke, Schnell, Nichols and Götz1999). Some researchers also indicated that the process of biofilm formation is complex involving many genes or many unknown variables (Darwish and Asfour, Reference Darwish and Asfour2013; Tremblay et al., Reference Tremblay, Lamarche, Chever, Haine, Messier and Jacques2013). The results obtained from this and other studies indicated the importance of other mechanisms of biofilm-forming independent of the ica operon. Biofilm-associated and MSCRAMM genes observed among staphylococci are given in Table 1.
Biofilm-associated and MSCRAMM genes among staphylococci
a The isolates were found to be biofilm-positive by MTP.
In conclusion, the prevalence of subclinical mastitis cases in buffalos was found to be 24% in the family-sized farms where no preventive control programs for mastitis were applied. The findings of the study also showed that staphylococci isolated from buffalo mammary glands have the ability to develop biofilm with different degrees despite the presence or absence of biofilm and MSCRAMMs genes. Finally, despite high rates of resistance to beta-lactam antibiotics, staphylococci generally exhibited low resistance to other individual antibiotics, indicating that prudent use of antibiotics is necessary to prevent the emergence and dissemination of antibiotic-resistant bacteria in buffalo mastitis cases.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029923000080.
Acknowledgements
The authors would like to thank Hatay Mustafa Kemal University Research Fund (Project Number: 18.M.060) for financially supporting this study.
