luxS Gene and Biofilm Formation in Streptococcus uberis Isolated from Bovine Mastitis Cases

S. uberis is a ubiquitous environmental pathogen colonizing and infecting Dairy cattle (De Vos et al., 2009). The dry period is the highly susceptible period to S. uberis infection (Marshall et al., 1986). The incidence of new S. uberis infections can be reduced by prophylactic antibiotic treatment during dry period (Williamson et al., 1995). Steeneveld et al., (2007) reported that S. uberis, a primary environmental pathogen was a major cause of mastitis in dairy cattle. Chronic mastitis caused by S. uberis is extremely costly and difficult to treat.
 
Biofilms are formed as a result of the cell multiplication leading to a mature structure consisting of many layers of cells, connected to each by extracellular polysaccharides (Yarwood and Schlievert, 2003). These exopolysaccharides can be enzymes (Mootz et al., 2013; Tielen et al., 2013) or structural proteins (Cucarella et al., 2001) and other polymers (e.g., lipids) (Davey et al., 2003). Although biofilms formed by other Streptococcus species like S. mutans were well examined and characterised, the ability of S. uberis to form biofilms was only recently described (Crowley et al., 2011). Merritt et al., (2003) identified the luxS gene of S. mutans which was recognized as the enzyme primarily responsible for the production of autoinducer-2 (AI-2) interspecies quorum signals. The luxS-dependent quorum sensing is involved in biofilm formation of S. mutans. The detailed microscopic analyses of luxS mutant S. mutant biofilm indicate a clear difference in mature biofilm structure between the wild type and luxS mutants. Yanping, (2017) stated that the virulence of the luxS mutant strain of S. agalactiae was decreased in the tilapia infection model, exogenous AI-2 molecule and luxS gene complementation with plasmid could complement the deficiencies of function in the luxS mutant strain.

Sample collection
 
A total of 91 milk samples were collected from mastitis cases of cattle and buffaloes from Veterinary Hospitals and Farms in Krishna, Guntur and West Godavari districts, Andhra Pradesh. The details of the Veterinary Hospitals and farms are listed in Table 1. The study was carried out during the period of October 2017 to March 2018, in the Department of Veterinary Microbiology, NTR College of Veterinary Sciences, Gannavaram, Andhra Pradesh.
 

 
Isolation and Identification
 
Approximately 10 ml of milk was collected aseptically from clinical cases into sterile vials. Collected samples from each quarter were transported on ice and immediately cultured or stored at 4°C until cultured/enriched. Milk samples were centrifuged at 2000 g for 10 minutes at 37°C, supernatant was discarded and 5 ml of brain heart infusion (BHI) broth was added to the sediment and incubated at 37°C for 24 hr (Cruickshank et al., 1975). Selective isolation was done by inoculating 0.9 ml of Streptococcus selective (SS) broth with 0.1 ml of culture from the BHI broth and incubated at 37°C in an anaerobic jar for 24 hr. The morphology of the organisms was studied by Gram’s staining. SS broth with gram positive cocci in chain were further inoculated on to Edward’s medium.
 
Phenotypic characterization
 
The cultures showing greyish, pinpointed colonies and/or aesculin hydrolysis on Edward’s medium were tentatively identified as Streptococcus species. The suspected isolates of Streptococcus species were further identified by various biochemical tests viz., catalase test, ninhydrin test, sodium hippurate hydrolysis test and type of haemolysis on 7% sheep blood agar.
 
Biofilm detection
 
The detection of slime production in Streptococcus species isolates was done according to Freeman et al., (1989). CRA was prepared using BHI agar supplemented with 5% sucrose and 0.08% Congo red dye. The dye was prepared as a concentrated aqueous solution and it was autoclaved at 121°C for 15 minutes separately from the other medium constituents. Congo red dye was added when the agar had cooled to 55°C. The plates were inoculated and incubated at 37°C for 24- 48 hr.
 
The ability of S. uberis strains to form biofilms in vitro on an abiotic surface was determined with a method previously described by others (Christensen et al., 1985; Merrit et al., 2005) with minor modifications by Moore, (2009).
        
Quantification of biofilm producing colonies was done according to Milanov et al., (2015). Cut-off OD (ODc) is defined as three standard deviations above the mean OD of the negative control. Strains were classified as follows: Non-biofilm producers (OD ≤ ODc); Weak biofilm producers (ODc<OD ≤ 2 × ODc); Moderate biofilm producers (2 × ODc <OD≤4 × ODc); Strong biofilm producers (OD>4 × ODc).
 
DNA extraction
 
DNA was extracted by High salt method (Anand Kumar, 2009) and re-suspended in 40 μl sterile distilled water and stored at -20°C till use.
 
PCR identification and detection of luxS gene
 
The S. uberis was confirmed by using species specific primers Sub 302/Sub 396 coding for 23S rRNA and luxS gene was detected by specific PCR primer. The primers used in this study and other details are mentioned in (Table 2). The PCR tests were carried out in Proflex PCR system, Applied Biosystems. All the reactions were carried out in a volume of 25 μl in 0.2 ml PCR tubes. The PCR amplicons were analyzed by electrophoresis on 1.7% agarose gel stained with 0.5 μg of ethidium bromide/1 ml in Tris-Borate EDTA (TBE) buffer. The conditions and components of PCR are given in (Table 3 and 4) respectively.
 

 

 

From this study it is suggestable that both phenotypic and genotypic methods should be taken together for determination of biofilm production, which can be influenced by various other factors. Also, the results express that any of the isolates without luxS didn’t produce a strong biofilm and it is concluded that there may be other genes for regulation of biofilm production and/or luxS gene has a regulatory role for one or more genes related to biofilm formation in S. uberis.