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Vet Res Commun (2010) 34:8189 DOI 10.

1007/s11259-009-9326-0 SHORT COMMUNICATION

Detection of intercellular adhesion genes and biofilm production in Staphylococcus aureus isolated from bovine subclinical mastitis
Nirmala B. Dhanawade & Dewanand R. Kalorey & R. Srinivasan & Sukhadeo B. Barbuddhe & Nitin V. Kurkure

Accepted: 25 October 2009 / Published online: 10 November 2009 # Springer Science + Business Media B.V. 2009

Abstract Biofilm production by Staphylococcus aureus, an important virulence factor was investigated employing phenotypic and genotypic methods. A total of 102 S. aureus isolates from bovine subclinical mastitis cases were included in the study. Maximum number of biofilm producing strains were detected by Congo red agar (CRA) method (48.03%) followed by tube method (36.27%). Tissue culture plate method (TCP) without and with destaining identified 19.60 and 29.41% of S. aureus as biofilm producers, respectively. A polymerase chain reaction for detection of intercellular adhesion genes, icaA and icaD, responsible for biofilm formation was standardized. Of the 102 S. aureus isolates investigated, 36 (35.29%) strains revealed presence of both the genes. Considering polymerase chain reaction as a standard test, CRA and TCP without destaining were the most sensitive and specific, respectively. PCR technique standardized for detection of the icaA and icaD genes is reliable for identifying biofilm producing potential of S. aureus which may help in rapid detection of biofilm-producer Staphylococci. This would allow the early application of control measures. Keywords Staphylococcus aureus . Biofilm production . Mastitis . icaA . icaD Abbreviations CMT California Mastitis Test CRA Congo red agar
N. B. Dhanawade : D. R. Kalorey : N. V. Kurkure Nagpur Veterinary College, Maharashtra Animal and Fishery Sciences University, Nagpur 440 006, India R. Srinivasan Madras Veterinary College, Tamilnadu University of Veterinary and Animal Sciences, Chennai 600 006, India S. B. Barbuddhe (*) ICAR Research Complex for Goa, ELA, Old Goa 403 402, India e-mail: barbuddhesb@yahoo.com

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MTCC PCR SCM TCP icaA

Microbial Type Culture Collection Polymerase chain reaction Subclinical mastitis cases Tissue culture plate method Intercellular adhesion gene A

Introduction Staphylococcus aureus represents a major agent of contagious bovine mastitis. It commonly spreads from infected to non-infected cows at milking in absence of proper hygienic and managemental practices (Annemuller et al. 1999; Cuteri et al. 2004; Leitner et al. 2003). The pathogenesis of mastitis caused by S. aureus is attributed to the combined effect of extracellular factors and toxins, together with the invasive properties of the strain such as adherence, biofilm formation and resistant to phagocytosis (Cramton et al. 1999). A slime layer surrounds the majority of S. aureus strains causing mastitis, which helps in adherence and colonization of the organism on the mammary gland epithelium (Aguilar et al. 2001; Baselga et al. 1993). Many virulence factors have been investigated from S. aureus isolated from bovine subclinical mastitis cases (SCM) (Salasia et al. 2004; Kalorey et al. 2007). The ability of S. aureus to form biofilm is considered to be an important virulence determinant influencing its pathogenesis in mastitis (Cifrian et al. 1994). Biofilm is an exopolysaccharide, a slime matrix around multiple layers of cells. The ability of S. aureus to form biofilm helps the bacterium to survive hostile environments within the host. It suggests that biofilm production is possibly important virulence factor in the establishment of staphylococcal infection (Costerton et al. 1999). The implication of biofilms in infections and drug resistance has triggered an increasing interest in the characterization of the genes involved in biofilm formation. The intercellular adhesion (ica) locus consist of the genes icaADBC and among the ica genes, icaA and icaD have been reported to play a significant role in biofilm formation in S. aureus and S. epidermidis (Gotz 2002). Recently, the ica locus has been detected in majority of the mastitic S. aureus isolates indicating its potential role as a virulence factor in the pathogenesis of mastitis in ruminants (Vasudevan et al. 2003). India, the largest milk producer of milk in the world, also has the highest number of cattle in the world. Among the various diseases, bovine mastitis continues to be the most important and costly disease in dairy animals. In India, monetary losses due to subclinical mastitis are substantial (FAO 2003). Biofilm production by Staphylococcus of mammary origin has been reported from various parts of world (Cucarella et al. 2001; Vasudevan et al. 2003), from human skin (Costerton et al. 1999; Lopez et al. 2002, Mathur et al. 2006; Jain and Agarwal 2009) and by other microbes (Kaur et al. 2009). Little information is available regarding genotypic characterization of S. aureus of intramammary origin with reference to intercellular adhesion genes and its association with phenotypic characters in this part of the world. The purpose of this study was to determine the occurrence of the icaA and icaD genes in a collection of S. aureus isolates from bovine SCM. Slime forming ability was evaluated by Congo red agar, tube and tissue culture plate methods.

Materials and methods Staphylococcus aureus MTCC 1430, S. aureus MTCC 96, Klebsiella pneumoniae MTCC 423 Pseudomonas aeruginosa MTCC 1610, Proteus mirabilis MTCC 425 and P. vulgaris

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MTCC 426 were procured from Institute of Microbial Technology and Gene Bank, Chandigarh, India. S. epidermidis ATCC 35984 and S. epidermidis ATCC 35983 were kindly provided by Ranbaxy Limited, New Delhi, India. These strains have been used as reference strains. A total of 102 Staphylococcus aureus isolates were included in the study. The isolates were recovered from cows suffering from subclinical mastitis (SCM) from five organized dairy farms in central (2 farms, 28 isolates) and southern India (3 farms, 74 isolates). The strains were selected randomly from each of the farm proportionate to the total number of isolates recovered from the particular farm. The animals were confirmed for SCM by California Mastitis Test (CMT). The isolates were identified as S. aureus using classical microbiological methods, biochemical tests and the tube coagulase test (Cowan and Steel 1970; Cruickshank et al. 1975). Qualitative detection of biofilm production in S. aureus strains was performed by cultivation on Congo red agar (CRA) as described earlier (Freeman et al 1989). The medium composed of brain heart infusion (BHI) broth (37 gms/L), sucrose (36 gms/L), agar powder (30 gms/L) and Congo red dye (0.8 gms/L). Inoculated CRA plates were incubated at 37C for 24 h followed by subsequent storage at room temperature. Colonies on CRA were observed at 48 and 72 h. Along with S. aureus test strains, known biofilm positive strains (S. aureus MTCC 1430 and S. aureus MTCC 96) served as positive controls. Strains producing black colonies with a rough, dry and crystalline consistency were considered biofilm producers. Strains producing red colonies with rough, dry and crystalline consistency or smooth colonies were classified as biofilm non-producers. A qualitative assessment of biofilm formation was determined by tube method as described (Christensen et al. 1982) with some modification. Briefly, 5 ml trypticase soya broth (TSB) with 0.25% glucose was inoculated with loopful of broth culture and incubated for 24 h at 37C. The tubes were decanted and washed for three times with sterile phosphate buffer saline (PBS, pH 7.4) and dried. After drying, tubes were stained with 0.1% safranin. Excess stain was removed and tubes were washed with sterile distilled water. Tubes were dried in an inverted position and observed for biofilm formation. Biofilm formation was considered positive when a visible stained film lined the wall and bottom of the tube. Ring formation at the liquid interface was not considered an indicative of biofilm formation. Tubes were examined and the amount of biofilm formation was scored as 0-absent, 1+ moderate and 2+ strong. The ability of S. aureus isolates to produce biofilm was determined (Cucarella et al. 2004) with slight modification. Briefly, S. aureus isolates were inoculated in TSB individually and grown overnight at 37C. Cultures were diluted 1:40 in TSB containing 0.25% glucose, and 200 l of bacterial cell suspension was added in duplicate to wells of sterile 96 well-flat bottom tissue culture (TC) plates (Tarsons, Kolkata, India). TSB with glucose (200 l) served as negative control. The TC plates were incubated at 37C., Medium was replaced after 12 h of incubation and the content of each well was gently removed by tapping the plates at 24 h. The wells were washed three times with 0.2 ml of sterile PBS (pH 7.4) to remove free floating planktonic bacteria. After washing, TC plates were dried in an inverted position and biofilms formed by adherent sessile organisms in plate were stained with 200 l safranin (0.1% w/v) for 30 s. Wells were rinsed thrice with sterile distilled water and kept for drying. After drying, the concentration of safranin was determined by measuring the optical density (OD) value at 490 nm using microplate ELISA reader (Thermo Labsystem, USA). Then, the plates were destained with 200 l of 95% absolute alcohol and the concentration of safranin was determined (OToole and Kolter 1998). Uninoculated wells containing TSB

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with glucose served as blanks. To correct background staining, OD values of blank were averaged and subtracted from all test OD values. Corrected absorbance values were used for reporting biofilm production. Quantitative classification of biofilm production based on OD values was carried out (Mathur et al. 2006). Standardization of polymerase chain reaction (PCR) for amplification of intercellular adhesion genes, icaA and icaD was carried out employing standard strains of S. aureus (MTCC 1430 and MTCC 96). The primers for detection of intercellular adhesion genes (icaA and icaD) used in this study were synthesized from Genetix Biomed Asia, New Delhi, India. The primer sequences for the icaA gene were, forward 5-CCT AAC TAA CGA AAG GTA G-3 and reverse 5-AAG ATA TAG CGA TAA GTG C-3 and for the icaD gene, forward 5-AAA CGT AAG AGA GGT GG-3 and reverse 5-GGC AAT ATG ATC AAG ATA C-3 (Vasudevan et al. 2003). The primers were designed to generate products for the icaA and icaD genes as 1315 bp and 381 bp, respectively. The genomic DNA of S. aureus isolates was extracted as per the method described earlier (Wilson 1987). PCR conditions described by Vasudevan et al. (2003) were suitably modified for the detection of intercellular adhesion genes namely icaA and icaD. Briefly, PCR was set for 50 l reaction volume. Reaction mixture for PCR was optimized as follows5 l of 10X PCR buffer (consisting of 100 mM Tris-HCl, pH 8.3; 500 mM KCl; 15 mM MgCl2 and 0.01% gelatin), 0.75 l dNTP mix (10 mM, with a final concentration of 0.2 mM), and 1 M of forward and reverse primer of each set (final concentration 0.1 M each), 1.25 unit of Taq DNA Polymerase, 2 l of DNA and sterilized milliQ water to make up the reaction volume. The reaction was performed in Px2 Thermal cycler (Thermo Hybaid, USA) with a pre-heated lid. The cycling conditions included an initial denaturation at 94C for 3 min. followed by 40 cycles each of 45 s denaturation at 92C, 45 s annealing at 49C and 1 min. extension at 72C. It was followed by final extension of 7 min. at 72C. The presence and size of the amplified products were confirmed by electrophoresis on 1.5% agarose gel. The specificity of the standardized PCR was tested by screening the standard strain of bacterial species. Considering PCR as gold standard test, sensitivity and specificity of test employed for detection of biofilm producing S. aureus was worked out (Thapliyal and Mishra 1996). Sensitivity% True positive 100 True Positive False positive True negative 100 True negative False negative

Specificity%

Results The ability of S. aureus isolates to produce biofilm was studied employing CRA, TM and TCP methods. A total 102 strains were selected for studying the biofilm producing ability. Overall, 49 (48.03%) strains of S. aureus produced black rough colonies on CRA and characterized as biofilm producers (Table 1). Out of these 12 and 37 strains originated from farms located in central and southern India, respectively. The isolates from all the farms showed biofilm producing ability. Out of 102 S. aureus isolates, 37 (36.27%) isolates identified as biofilm producers by tube method, 23 isolates were strong and 14 isolates were moderate biofilm producers. Considering OD without destaining, 16 and 4 isolates were

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strong and moderate biofilm producers, respectively. However, after destaining 17 and 13 isolates, respectively were strong and moderate biofilm producers. The PCR was standardized for detection of intercellular adhesion genes namely, the icaA and icaD, responsible for biofilm formation. The primer sets for icaA and icaD genes allowed positive amplification of 1315 bp and 381 bp, respectively (Figs. 1 and 2). Each of the primer was found to be specific to the target gene as it specifically amplified the gene from S. aureus (MTCC 96 and MTCC 1430) strains. The icaD gene was also detected in two standard strains of S. epidermidis. These genes were not detected in other bacterial species tested for specificity of the standardized PCR employed in present study. Overall by PCR, 36(35.29%) S. aureus isolates were observed to be positive for both the icaA and icaD genes. In this study, out of 49 strains positive by CRA, 30 were positive for both the icaA and icaD genes, and 19 were negative (Table 1). Six strains positive for icaAD genes were negative by CRA. Out of 37 strains producing biofilm by tube method, 22 were positive for icaAD and 15 were negative. Of the 36 strains positive for icaAD, 16 were positive by TCP method without destaining and 21 by TCP with destaining.

Discussion Bovine mastitis caused by S. aureus remains a substantial problem for milk producers worldwide. S. aureus is an adaptable opportunistic pathogen whose ability to persist and multiply in a variety of environments causes a wide spectrum of diseases in both humans and animals. The pathogenesis of S. aureus strains is attributed to a combination of extracellular factors and properties such as adherence and biofilm formation (Cramton et al. 1999; Vancraeynest et al. 2004). The ability of S. aureus to form biofilms helps the bacterium to survive hostile environments within the host, and is considered to be responsible for chronic or persistent infections (Costerton et al. 1999). Biofilm has been found to facilitate the adherence and colonisation of Staphylococci on the mammary gland epithelium, also contributing to the evasion of the immunological defences and to the difficulty of pathogen eradication, often resulting in persistent infections (Cucarella et al. 2002, 2004; Zadoks et al. 2002; Vasudevan et al. 2003; Brouillette et al. 2005; Fox et al. 2005; Melchior et al. 2006). It has been proposed that biofilm, like capsule, protects S. aureus against phagocytosis. In addition, it may help S. aureus to resist antibiotic therapy (Cucarella et al. 2004). Information of biofilm formation by coagulase positive S. aureus of

Table 1 Biofilm production by S.aureus isolated from bovine SCM as detected by different methods and their evaluation considering PCR as a standard test Test Biofilm producing strains 49 (48.03) 37 (36.27) 20 (19.60) 30 (29.41) Strains positive for icaAD 30 (61.22) 22 (59.45) 16 (80.00) 21 (70.00) Strains negative for icaAD 19 (38.77) 15 (40.54) 4 (20.00) 9 (30.00) Sensitivity (%) 83.33 61.11 44.44 58.33 Specificity (%) 71.21 77.21 93.93 86.36

CRA Tube method TCP method (without destaining) TCP method (with destaining)

Figures in parenthesis indicate percentage

86 Fig. 1 Amplification of icaA gene of S. aureus of bovine subclinical mastitis origin. Lane MDNA molecular weight marker. Lanes 14amplification of icaA (1315 bp) Lane 1: Positive control Staphylococcus aureus MTCC 1430; Lane 2, 3 & 4 Field strains. Lane 5Negative control Klebsiella pneumoniae MTCC 423

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intramammary origin is largely lacking. In present study, out of 102 strains of S. aureus isolated from bovine subclinical mastitis, 48.03% strains were considered biofilm producer by CRA method. Strains producing black colonies with a rough, dry and crystalline consistency were considered biofilm producers. In contrast to this criterion, S. aureus producing black rough and smooth colonies were considered as biofilm producers (Mathur et al. 2006). Prevalence of biofilm producing S. aureus from bovine mastitis has been reported earlier (Vasudevan et al. 2003). The biofilm-forming ability of Staphylococci field isolates from bovine subclinical mastitis was evaluated by (Oliveira et al. 2006). Fox et al. (2005) reported biofilm production by 41.4% of S. aureus from milk samples as compared to 24.7 and 14.7% of the isolates collected from skin and liners. The isolates were associated with intramammary infection. Biofilm formation by clinical isolates of S. aureus originated from blood, infected devices and skin surfaces of human beings has been reported earlier (Arciola et al. 2001; Catalanotti et al. 2005; Yazdani et al. 2006; Mathur et al. 2006). In another study, Kaur et al. (2009) demonstrated biofilm formation in group B streptococci (GBS) isolated from different sources in the north Indian community. Results of biofilm assessment in TCP method after destaining with alcohol were better. It might be due to destaining of biofilm
Fig. 2 Amplification of icaD gene of S. aureus of bovine subclinical mastitis origin. Lane MDNA molecular weight marker. Lanes 15amplification of icaD (381 bp) Lane 1: Positive control Staphylococcus aureus MTCC 1430; Lane 2, 3, 4 & 5 Field strains. Lane 6 Negative control Klebsiella pneumoniae MTCC 423

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formed on wall of wells after addition of alcohol increasing the accuracy of result. Present study indicated prevalence of the icaA and icaD genes in 35.29% of S. aureus mastitogens. High prevalence of intercellular adhesion genes namely, icaA and icaD from S. aureus of bovine mastitic origin were reported earlier (Cucarella et al. 2001; Vasudevan et al. 2003). Intercellular adhesion genes (icaA and icaD) were reported from S. aureus recovered from human medical devices (Cramton et al. 1999; Arciola et al. 2001; Catalanotti et al. 2005; Yazdani et al. 2006). This indicated an important role of biofilm in pathogenesis of infections caused by S. aureus. In present investigation, frequency of biofilm producing S. aureus as detected by various in vitro methods differed from that of genotypic method. Similar variation between phenotypic (CRA) and genotypic methods (PCR) for detection of biofilm producing S. aureus has been reported in respect of CRA (Yazdani et al. 2006) and TCP (Vasudevan et al. 2003). Vasudevan et al. (2003) earlier compared the PCR protocols described with CRA and the quantitative OD method. Previously tube method results have been compared with the quantitative OD method (Christensen et al. 1985). It has been reported that failure of staphylococcal strains that possess the ica locus to form biofilm in vitro could possibly be due to point mutations in the locus and/or other yet unidentified factors that negatively regulate polysaccharide intercellular adhesion synthesis or influence biofilm formation (Cramton et al. 1999). There is some experimental evidence to support the development of new clones referred to as biofilm-negative with both the icaA and icaD genes (Arciola et al. 2001). Expression of the icaADBC operon was shown to be a highly variable factor modulated by phase variation and genome rearrangements. It was suggested that the variable biofilm expression contributes to adaptation of the bacterium in changing the environmental conditions of incubation (Costerton et al. 1999; Rachid et al. 2000). Findings of the present study indicated variable prevalence of biofilm producing S. aureus employing phenotypicin vitro qualitative and quantitative and genotypic methods. It has been previously demonstrated that phenotypic expression of biofilm formation is highly susceptible to in vitro conditions (Baselga et al. 1993) and hence can be detected variably by different methods. Therefore, a combination of various methods (phenotypic and genotypic) would be useful for identifying biofilm producing S. aureus. Considering the role of biofilms in the adherence and colonisation of Staphylococci on the mammary gland epithelium, evasion of the immunological defences, resistance to antibiotic therapy and to the difficulty of pathogen eradication the findings have high significance.

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