Staphylococcus aureus (S. aureus) is a major contagious pathogen responsible for both the clinical and subclinical mastitis in dairy cattle all over the world. The ability of S. aureus to form biofilm is considered to be a major virulence factor influencing its pathogenesis. In addition, it often creates intricacy in treatment of bovine mastitis using conventional antibiotics and produce recalcitrant drug resistant infections. This indeed demands urgent remedial measures as outbreaks of livestock associated methicillin resistant S. aureus (LA-MRSA) and community associated methicillin resistant S. aureus (CA-MRSA) are on the rise and currently being implicated as a rapidly emerging cause of numerous life threatening and therapy refractory human infections as well. In this study, a total of 22 S. aureus isolates from bovine clinical mastitis were evaluated for their ability to form biofilm by using phenotypic and genotypic methods. Using Congo red agar method, 9.09 per cent and 22.72 per cent of S. aureus were considered as strong and intermediate biofilm producers respectively. The presence of icaA and eno genes associated with biofilm formation was confirmed by using polymerase chain reaction (PCR). This study indicates a high prevalence of the ica (63.63 per cent) and eno (100 per cent) genes among S. aureus isolates from bovine mastitis.
HIGHLIGHTS
The mere presence of biofilm associated genes is not always associated with in vitro biofilm formation.
A combination of phenotypic and genotypic tests is recommended for the investigation of biofilm formation
MastitisbiofilmCongo red agaricaAeno genes
India is an agricultural country, where the dairy industry is closely interwoven with the socioeconomic fabric of the rural farming community. Mastitis, often exacerbated by antimicrobial resistance is the prime challenge faced by dairy farmers all over the world. Staphylococcus aureus being a ubiquitous bacterium, acts as a significant contagious pathogen responsible for persistent and recurrent intramammary infections with minimal response to the antimicrobial therapy.
The success of this pathogen could be attributed to the multitude of its virulence factors, extensive resistance mechanisms that enable them to withstand the currently available antibiotic repository, and perhaps due to the dissemination of various multidrug resistant (MDR) strains. However, the biofilm formation is yet another multi-layered defense mechanism that augments the success of staphylococci. The possibility of biofilms as an important virulence determinant and a multi-layered defense mechanism that could result in a loss of sensitivity to virtually all class of antibiotics, has recently garnered the attention of many researchers.
Biofilms are organised sessile or motile aggregates of microorganisms encased in a self-produced polymeric matrix composed of exopolysaccharides, DNA and proteins (Hoiby et al., 2010; Melchoir et al., 2006). It is the result of expression of a complex network of signal molecules orchestrated under the coordinated expression of certain genes and bacterial quorum sensing (QS) system (Aricola et al., 2012).
How to cite this article: Vijayakumar, K. and Jose, K.R. (2021). Phenotypic and genotypic characterization of Staphylococcus aureus isolated from bovine mastitis for biofilm formation. J. Anim. Res.,11(2): 249-255. Source of Support: None; Conflict of Interest: None
Biofilm formation in bacteria is a two- step process which involves the bacterial adhesion followed by tissue invasion and multiplication (Vasudevan et al., 2003). An exopolysaccharide, intercellular adhesion/ polymeric N-acetyl-glucosamine (PIA/PNAG) that are regulated by proteins coded by the intercellular adhesion (ica) locus consisting of icaADBC genes regulates the bacterial adhesion (Fitzpatrick et al., 2005; Hennig et al., 2007). Among the ica genes, icaA and icaD have been reported to a play a significant role in biofilm formation in S. aureus and S. epidermidis. The icaA gene encodes N-acetylglucosaminyl transferase, the enzyme involved in the synthesis of the polysaccharide intercellular adhesin (PIA) and capsular polysaccharide/ adhesin (PS/A) that mediates the bacterial adhesion. (Arciola et al., 2001). On the otherhand, the eno gene encodes the laminin binding protein that facilitates adhesion and ultimately helps in tissue invasion.
The decreased antimicrobial susceptibility of bacteria in biofilms is caused by multiple factors such as the nutritional limitation, genetic basis, composition and interactions among organisms of biofilms, occurrence of persister cells and biofilm-specific protective stress responses in bacteria (Hall and Mah, 2017). The presence of biofilm matrix reduces the antimicrobial penetration and paves way to the tolerance and resistance of the organism towards the particular antibiotic. This protects the pathogen and lead to recurrent and chronic intramammary infections (Melchoir et al., 2007) ultimately aggravating the antimicrobial resistance crisis which is emerging as a silent pandemic.
Hence, early and rapid detection of the pathogen and the dynamics of biofilm formation is an absolute necessity for addressing the challenges of antimicrobial resistance. Therefore, the present study was envisaged to investigate the biofilm forming potential of S. aureus isolated from bovine mastitis using congo red agar method and also to determine the presence of icaA and eno genes associated with biofilm formation. This could help in developing crucial strategies and therapeutic concepts to effectively combat and mitigate the spread of these omnipresent pathogen which has tremendous impact on livestock production, animal welfare, global food safety and public health.
MATERIALS AND METHODSSample collection
The present study was conducted on 83 cross bred lactating dairy cows with clinical mastitis in and around Thrissur district, Kerala. The midstream lacteal secretions of each animal were collected aseptically in sterile screw capped vials.
Isolation and identification of Staphylococcus aureus
Isolation of bacteria was attempted by direct streaking of a loopful of milk sample on to brain heart infusion agar (BHIA- M211, Himedia), followed by incubation of the plates at 37oC for 12 to 18 h. The isolates were identified based on morphological characteristics on Gram’s staining. The presumptively identified S. aureus isolates were further verified by direct streaking and subsequent incubation at 37°C for 24 h on mannitol salt agar (MSA-M118, Himedia) and hichrome staph selective agar (SSA-M1931, Himedia) which was prepared according to the manufacturer’s instruction. Biochemical characterisation of the isolates were done based on positive catalase and coagulase tests and negative oxidase test as per Barrow and Feltham (1993) and Quinn et al. (2013).
Isolation of genomic DNA
The DNA was extracted from the presumptively identified S. aureus isolates using snap chill method (heat lysis method) as described by Junior et al. (2016) with slight modifications. For this, approximately three millilitres of overnight grown S. aureus culture in mannitol salt broth (MSB-M383, Himedia) was centrifuged at 10,000 rpm for 10 min at 4oC (Hispeed Centrifuge, KEMI). The pellet formed was resuspended in one milliliter of sterile nuclease (DNAase & RNAase) free milli-Qwater (ML064, Himedia), followed by centrifugation at 10,000 rpm for 10 min at 4oC and the supernatant was discarded. The pellet obtained was further resuspended in 100μl tris EDTA buffer with pH 8, mixed in a vortex and kept in boiling water bath for 15 min. Then snap chilled on crushed ice for 30 min and centrifuged at 10,000 rpm for 10 min, at 4oC. After that the supernatant containing DNA was collected in sterile nuclease free centrifuge tubes and further checked for concentration and purity using a Nanodrop Spectrophotometer (NanodropTM 1000 Spectrophotometer). The DNA samples with 260/280 ratio greater than 1.8 were selected and used as template for the polymerase chain reaction (PCR).
Genotypic characterisation of Staphylococcus aureus and its biofilm formation
Molecular confirmation of the presumptively identified S. aureus isolates were done by PCR targeting the 16SrRNA for identification of Staphylococcus spp. and 23SrRNA for identification of the S. aureus. The genotypic analysis of bacterial biofilm formation was carried out by PCR targeting the icaA (intercellular adhesion gene A) and eno (encoding lamininbinding protein) genes respectively.
The PCR was performed in a total volume of 25 µl reaction mixture by combining the reagents as shown in table 1 using the programmable S1000 Thermal cycler, (BioRad, USA). The details of the primers used and the PCR protocol are shown in table 2 and 3 respectively. PCR products were analysed on 1.2 per cent agarose gel stained with ethidium bromide, visualised under the ultraviolet transilluminator ((GeneiTM, Bengaluru) and photographed using GelDoc apparatus (DocTM Gel EZ imager, BIORAD, USA).
Components of PCR reaction mix (Source: Original)
Primers used for genotypic characterisation of S. aureus and its biofilm formation (Source: Original)
PCR protocol for amplification of various genes (Source : Original)
Phenotypic characterization of biofilm formation
The biofilm formation of each Staphylococcus aureus isolate were evaluated qualitatively by culturing the organism on congo red agar (CRA) as described by Mathur et al. (2006). The CRA was composed of brain heart infusion broth (BHI - 37g/l) (M210, Himedia), sucrose (50g/l) (GRM 601, Himedia), agar (10g/l) (GRM 026, Himedia) and congo red dye (0.8g/l) (GRM 927, Himedia). Congo red stain was prepared as concentrated aqueous solution and autoclaved at 121° for 15 min. Then, it was added to autoclaved BHI agar with sucrose at 55°. Plates were inoculated with test organism and incubated aerobically at 37° for 24 to 48 h. Positive result was indicated by black colonies with a dry crystalline consistency. Weak slime producers usually remained pink, though occasional darkening at the centres of colonies was observed. A darkening of the colonies with the absence of a dry crystalline colonial morphology indicated an indeterminate result.
Greenish blue coloured colonies of Staphylococcus aureus on Hichrome Staph selective agar (Source: Original)
RESULTS AND DISCUSSION
In the present study, isolation of bacteria from bovine mastitic milk was achieved by standard microbiological culture techniques. Upon initial inoculation of 83 mastitic milk samples on to BHIA, 26 samples (31.33 per cent) did not produce any colonies whereas, 57 samples yielded bacterial growth (68.67 per cent), among which S. aureus was isolated from 22 samples (26.5 per cent). The bacterial isolates were identified based on their morphology, colony characteristics on selective media and biochemical characteristics (Barrow and Feltham, 1993; Quinn et al., 2013).
Agarose gel electrophoresis of 16S rRNA specific PCR of Staphylococci spp. (Source : Original)
Agarose gel electrophoresis of 23S rRNA specific PCR of Staphylococcus aureus (Source : Original)
Agarose gel electrophoresis of icaA gene specific PCR of Staphylococcus aureus (Source : Original)
Agarose gel electrophoresis of eno gene specific PCR of Staphylococcus aureus (Source : Original)
The CRA method was often used as an indirect indicator of biofilm formation. (Darwish and Asfour, 2013). Out of the 22 S. aureus isolates, 9.09 per cent, 22.72 per cent and 68.19 per cent were strong, intermediate and negative biofilm producers, respectively. Similar findings were reported by Bose et al. (2009) who reported a total of 32.96 per cent of S. aureus isolates to be biofilm producers by CRA method. In contrast, Darwish and Asfour (2013), reported that 32.5 per cent and 35 per cent of the S. aureus isolates from bovine mastitis to be strong and intermediate biofilm producers where they used a plant based culture medium - Tryptose soya broth when compared to the animal based brain heart infusion broth that was used in our study. Hence, this could not be extrapolated to our study as the phenotypic expression of biofilm in bacteria may vary based on the difference in chemical composition of the culture media.
Though there are numerous low cost, easily adaptable, phenotype based tools for the qualitative and quantitative assessment of biofilm formation (Notowich et al., 2018) several previous studies of S. aureus have reported in vivo biofilm formation eventhough they were phenotypically non biofilm producers. This might be either due to the heterogeneous nature of the phenotypic expression of biofilms (Darwish and Asfour, 2013) or may be due to the fact that the data obtained from most phenotypic typing tools are not guided by the host and mammary tissue factors involved in biofilm formation. This had limited the practical utility of various phenotypic assays in the diagnosis and containment of biofilm formation.
Colonies of Staphylococccus aureus on Congo red agar. (A) Black coloured dry crystalline colonies indicative of strong biofilm producers; (B & C) Rough, dry red coloured colonies indicative of weak biofilm producers; (D) Indeterminate result
Molecular approaches, such as those focused on PCR, were therefore regarded as a method of choice, for the rapid and reliable diagnosis as well as for epidemiological investigations of outbreaks involving biofilm forming strains. It also eliminates the need for the cumbersome cultural procedures and combats for the heterogeneous nature of the phenotypic expression of biofilm formation. Therefore, in the current study apart from the in vitro congo red agar method, PCR was employed to assess the biofilm formation of S. aureus isolates.
Numerous PCR-based systems targeting different genes such as those enoding the intercellular adhesion genes - icaA and icaD, bap gene encoding biofilm associated protein, eno gene encoding laminin binding protein, aap encoding an accumulation associated protein, embP encoding fibronectin adhesion, fbe encoding a fibrinogen adhesion, atlE gene encoding an adhesion and autolysin etc. have been documented in literature (Darwish and Asfour, 2013; Srednik et al., 2017) for the detection of biofilm formation. In the current study, molecular characterisation of biofilm formation was done by PCR targeting the icaA and eno genes. The results indicated that eno gene had the highest prevalence (100 per cent) followed by the icaA gene which was detected in 63.63 percent of the isolates. The presence of eno genes was analogous to the study conducted by Darwish and Asfour (2013) who reported 75 per cent and 92.5 per cent prevalence of eno genes among the S. aureus and coagulase negative staphylococci isolated from bovine mastitis.
A comparison of the phenotypic and genotypic methods for assessment of biofilm formation revealed that the mere presence of icaA and/or eno genes does not always result in phenotypic expression of biofilms. The inability of S. aureus isolates that were positive for icaA and /or eno genes to produce biofilm in vitro can be due to point mutations in the locus and/or any other unidentified factors that negatively regulates and influence the biofilm formation (Darwish and Asfour, 2013). Another probable reason behind this disparity might be the phenomenon of phase variation in response to the in vitro conditions (Baselga et al., 1993).
Thus, it can be concluded from the present study that the S. aureus isolated from bovine clinical mastitis exhibited a greater ability to form biofilms, with different degrees of production using the CRA method. The higher prevalence of the icaA and eno genes responsible for biofilm formation must be considered as an alarming situation since biofilms are increasingly being recognized as a bacterial defense mechanism, that could result in loss of sensitivity to virtually all class of antibiotics, which are considered to be the holy grail of mastitis therapy. Therefore, novel ways of effective treatment, prophylaxis & control of such infections in dairy farms is of paramount importance. The study also highlights the need for modification of in vitro antimicrobial susceptibility tests, which selects antibiotics that are effective only in inhibiting the planktonic microbial population, ignoring the bacteria in biofilms that resist and survive treatment and provides materials for further growth leading to the recurrence or chronicity. Further research involving genomic, transcriptomic and proteomic approaches is required to unravel the complexities of bacterial biofilm formation and related antimicrobial resistance that could reverse a century of medical revolution.
ACKNOWLEDGEMENTS
The authors were thankful to the Kerala Veterinary and Animal Sciences University (KVASU) and the Department of Veterinary Epidemiology and Preventive medicine, College of Veterinary and Animal Sciences, Mannuthy for providing the facilities needed for carrying out the research work.
Arciola, C.R., Campoccia, D., Speziale, P., Montanaro, L., Costerton, J.W.2012. Biofilm formation in Staphylococcus implant infections. A review of molecular mechanisms and implications for biofilm-resistant materials. Biomaterials,33: 5967-5982.Baselga, R.A.F.A.E.L., Albizu, I.N.A.K.I., De La Cruz, M.O.N.T.S.E.R.R.A.T., Del Cacho, E.M.I.L.I.O., Barberan, M., Amorena, B.1993. Phase variation of slime production in Staphylococcus aureus: implications in colonization and virulence. Infect. Immun., 61: 4857-4862.Barrow, C.I., Feltham, R.K.A.1993. Cowan and Steel’s Manual for the Identification of Medical Bacteria. (3rd Ed.). Cambridge University Press, Great Britain, pp. 231-260.Bose, S., Khodke, M., Basak, S., Mallick, S.K., 2009. Detection of biofilm producing staphylococci: need of the hour. J. Clin. Diagn. Res., 3: 1915-1920.Ciftci, A., Findik, A., Onuk, E.E., Savasan, S., 2009. Detection of methicillin resistance and slime factor production of Staphylococcus aureus in bovine mastitis. Brazz. J. Microbiol.,40: 254-261.Darwish, S.F., Asfour, H.A.2013. Investigation of biofilm forming ability in Staphylococci causing bovine mastitis using phenotypic and genotypic assays. Sci. World J.,1-9.El-Razik, A.K.A., Abdelrahman, K.A., Ahmed, Y.F., Gomaa, A. M., Eldebaky, H.A., 2010. Direct identification of major pathogens of the bubaline subclinical mastitis in Egypt using PCR. J. Anim. Sci.,10: 652-660.Fitzpatrick, F., Humphreys, H., O’Gara, J.P.2005. Evidence for icaADBC-independent biofilm development mechanism in methicillin-resistant Staphylococcus aureus clinical isolates. J. Clin. Microbiol., 43: 1973–6.Hall, C.W., Mah, T.F.2017. Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in pathogenic bacteria. FEMS Microbiol. Rev.,41: 276-301.Hennig, S., Nyunt Wai, S., Ziebuhr, W.2007. Spontaneous switch to PIA-independent biofilm formation in an icapositive Staphylococcus epidermidis isolate. Int. J. Med. Microbiol., 297: 117–22.Høiby, N., Bjarnsholt, T., Givskov, M., Molin, S., Ciofu, O.2010. Antibiotic resistance of bacterial biofilms. Int. J. Antimicrob. Agents, 35: 322-332.Junior, J.C.R., Tamanini, R., Soares, B.F., de Oliveira, A.M., de Godoi Silva, F., da Silva, F.F., Augusto, N.A., Beloti, V.2016. Efficiency of boiling and four other methods for genomic DNA extraction of deteriorating spore-forming bacteria from milk. Semina: Ciências Agrárias, 37: 3069-3078Kot, B., Sytykiewicz, H., Sprawka, I., 2018. Expression of the biofilm-associated genes in methicillin-resistant Staphylococcus aureus in biofilm and planktonic conditions. Int. J. Mpl. Sci.,19:3487.Mathur, T., Singhal, S., Khan, S., Upadhyay, D.J., Fatma, T., Rattan, A.2006. Detection of biofilm formation among the clinical isolates of staphylococci: an evaluation of three different screening methods. Indian J. Med Microbiol.,24: 25.Melchior, M.B., Vaarkamp, H., Fink-Gremmels, J.2006. Biofilms: a role in recurrent mastitis infections?. Vet. J.,171: 398-407.Notcovich, S., DeNicolo, G., Flint, S.H., Williamson, N.B., Gedye, K., Grinberg, A., Lopez-Villalobos, N.2018. Biofilm-Forming Potential of Staphylococcus aureus Isolated from Bovine Mastitis in New Zealand. Vet. Sci.,5: 8.Quinn, P., Markey, B., Carter, M., Carter, G.R.2013. In: Clinical Veterinary Microbiology. (2nd Ed) Mosby, St. Louis, pp. 125-292.Srednik, M.E., Tremblay, Y.D., Labrie, J., Archambault, M., Jacques, M., Fernández Cirelli, A., Gentilini, E.R.2017. Biofilm formation and antimicrobial resistance genes of coagulase-negative staphylococci isolated from cows with mastitis in Argentina. FEMS Microbiol. Lett.,364: fnx001fnx008.Vasudevan, P., Nair, M.K.M., Annamalai, T., Venkitanarayanan, K.S.2003. Phenotypic and genotypic characterization of bovine mastitis isolates of Staphylococcus aureus for biofilm formation. Vet. Microbiol.,92: 179-185.