Mastitis is a disease of major economic importance in dairy herd, because of the reduction of farm profitability, decreased milk production, discarded milk, treatment costs and culling (Grohn et al., 2005). Group B streptococcus (GBS) also known as Streptococcus agalactiae is an important bovine pathogen and is frequently involved in clinical and subclinical mastitis. Infections due to S. agalactiae also have major public health consequences and may cause bacterial sepsis, pneumonia and meningitis in neonates and localized infections such as subcutaneous abscesses, urinary tract infection or arthritis in adults.

S. agalactiae is an obligate pathogen of the epithelium and tissues of ruminant mammary glands. Treatment with intra-mammary infusion of antibiotics is the main approach to deal with the infection and number of studies on in vivo and in vitro trials to assess the antibiotic sensitivity/resistant pattern has been documented. Frequent use/misuse of antibiotics in animals in general or for treatment of mastitis has resulted in development of resistance among the pathogens. Resistant determinants identified in S. agalactiae includes mefA and ermB genes encoding resistance to erythromycin (Marimon et al., 2005), tetM, tetO, tetL and tetK conferring resistance to tetracycline (Lopardo et al., 2003) and pbp2b gene contributing to penicillin resistance (Charpentier and Tuomanen 2000).

A variety of virulence factors contribute to the pathogenicity of S. agalactiae. Several surface proteins and polysaccharide capsules have been identified within this species. scpB gene (encodes for surface enzyme) ScpB (C5a peptidase), the bca gene (a-protein), the lmb gene (laminine-binding protein) and bac (b-antigen), cyl (b-hemolysin), glnA (glutamine synthetase), cfb (Christie-Atkins-Munch-Peterson (CAMP) factor and scaA (aggregation factor) (Dmitriev et al., 2002).

Not much work has been reported from the region regarding occurrence of group B streptococci in bovine mastitis. Therefore, the present study was envisaged to study the phenotypic and genotypic antibiotic resistance patterns and genetic diversity among group B streptococci isolated from mastitis milk of bovines from the Punjab state.

Milk samples were collected from 182 lactating bovines (130 cattle and 52 buffalo). The samples were streaked onto 5% sheep blood agar and incubated for at 37°C for 24-48 h. for isolation of S. agalactiae. The isolates were identified on the basis of cultural, morphological, biochemical characteristics and CAMP test.
 
Antibiotic sensitivity test
 
All the confirmed S. agalactiae isolates were subjected to antibiotic sensitivity test by disc diffusion method using antibiotic discs (HiMedia Laboratories Ltd. Mumbai) against 11 antibiotics viz. ampicillin (10 mcg), ceftriaxone (30 mcg), ciprofloxacin (5 mcg), co-trimoxazole (25 mcg), erythromycin (15 mcg), gentamicin (10 mcg), meropenem (10 mcg), ofloxacin (5 mcg), tetracycline (30 mcg), penicillin (10 mcg) and vancomycin (30 mcg). Zone of inhibition was interpreted as sensitive, intermediate and resistant as per the CLSI 2016 guidelines.
 
Molecular characterization of S. agalactiae
 
Confirmation of S. agalactiae isolates by PCR
 
DNA was extracted from the S. agalactiae spp. isolates using HiPurA bacterial genomic DNA purification kit (Himedia, Mumbai) as per manufacturer instructions.Confirmation of S. agalactiae isolates was done by PCR using the primers (STR-AG-I'5-TAGTTTTGAGAGGTCTTGTGG-3' and STR-AG-II  '5-ATATTCACAGCGTTTTCG-3') as described by Goli et al., 2012. Amplification reaction mixture was prepared in a total volume of 25 μL consisting of Green Taq master mix (12.5 μL), forward primer (20 pmol/μl), reverse primer (20 pmol/μl), template DNA (3 μL) and nuclease free water (7.5 μL). PCR was performed in a thermo cycler with the conditions: initial denaturation (94°C for 1 min) followed by 30 cycles each of denaturation (94°C for 1 min), annealing (54.2°C for 1 min) and extension (72°C for 1 min) and one cycle of final extension (72°C for 10 min).
 
Molecular detection of antibiotic resistance genes
 
Primers used to amplify ermA, ermB, mefA and mefE genes are listed in Table 1. PCR was performed with the conditions: initial denaturation (94°C for 10 min) followed by 35 cycles, each of denaturation at 94°C for 1 min, Annealing temperature (as per Table 1) and extension at 72°C for 1 min and final extension at 72°C for 10 min.


 
Detection of virulence genes by PCR
 
Primer sets used to amplify five major virulence genes (cyl, glnA, cfb, hylB and scaA) genes are listed in Table 1. PCR was performed with the conditions: initial denaturation (5 min at 94°C) followed by 35 cycles each of denaturation (94°C for 30 sec), annealing (52°C for 30 sec) and extension (72°C for 90 sec) and a final extension (72°C for 10 min).
 
Analysis of PCR product
 
Amplified products were separated by electrophoresis at 70V for 1 h in a 1.5% agarose gel and visualized by UV transillumination. A 100 bp DNA ladder (Gene ruler, MBI fermentas) was used in as molecular size standards for each of the reactions described above. A negative control, consisting of the same reaction mixture but with water instead of template DNA was included in each run.
 
Characterization of Streptococcus agalactiae strains by randomly amplified polymorphic DNA analysis
 
Random amplified polymorphic DNA (RAPD) is easy and sensitive technique to characterize the genotypic diversity of Streptococcus spp. isolates (Zadoks et al., 2003). In this technique arbitrary primers are used to amplify polymorphic segments of DNA to evaluating the genotypic diversity of S.agalactiae (Martinez et al., 2000). The genetic diversity of the six S. agalactiae isolates was carried out as per protocol described by (Chatellier et al., 1997). Eight primers were selected with the properties that they are 9 or 10 nucleotides in length, between 40 and 77% GC in composition and contained no palindromic sequence. The sequences of primers used for RAPD are indicated in Table 2.



DNA extracted from S. agalactiae isolates using HiPurA bacterial genomic DNA purification kit (Himedia Mumbai) was used in the reaction. Amplification reaction mixture was prepared in a total volume of 25 μL consisting of Green Taq master mix (12.5 μL), primer 1 μL (20 pmol/μL), DNA 1 μL (25 ng) of extracted DNA) and 10.5 μL nuclease free water. PCR was performed in a thermocycler (ABI thermo) with the conditions: initial denaturation (4 min at 94°C) followed by 45 cycles each of denaturation at 94°C for 1 min, annealing at 36°C for 1 min and extension at 72°C for 1 min and final extension at 72°C for 10 min. Amplified products were separated by electrophoresis at 50V for 4 h in a 1.2% agarose gel and visualized by UV transillumination. A 1-kb DNA ladder (Gene ruler, MBI fermentas) was used as molecular size standards. RAPD banding pattern was analyzed and phylogenetic tree constructed by PyElph software (Pavel and Vasile, 2012).

A total of 182 (130 cattle and 52 buffalo) milk samples were subjected to CMT, of which 134 (112 cattle and 22 buffalo) were found positive for mastitis and were inoculated on 5% sheep blood agar. All organisms were presumptively identified as streptococci by colony morphology and Gram staining. Among a total of 8 streptococcal isolates, 6 strains were confirmed as S.agalactiae as they gave positive results as indicated by production of arrow head hemolytic pattern on CAMP test. CAMP test has been routinely used for confirmation of S. agalactiae by Ahmadi et al. (2010) and Amosun et al. (2010).
 
Antibiotic resistance among S. agalactiae isolates
 
All the isolates were sensitive to meropenem, vancomycin, ofloxacin, ciprofloxacin and erythromycin. Highest resistance was found for co-trimoxazole (66.66%), tetracycline (66.66%) and ampicillin (50%) followed by gentamycin and ceftriaxone (16.66% each). High degree (72.5% and 82.6%) of resistance to tetracycline among S. agalactiae isolates has also been reported by Gao et al., (2012) and Gizachew et al. (2019), respectively. In present study, 33.33% of the isolates were resistant to penicillin, whereas Nakamura et al., (2011) observed all isolates were susceptible to penicillin and Gizachew et al., (2019) reported 33.6% resistance to penicillin among S.agalactiae. Although penicillin serves as a primary antimicrobial drug for clinical mastitis and has been used for decades in veterinary clinics, the results indicate that penicillin should be used discretely in the treatment of bovine S.agalactiae infection. Besides beta-lactams, erythromycin seemed to be the most active antimicrobial agent since all the isolates were sensitive to erythromycin. This finding is similar to those of previous reports from Sarah and Salah (2014). Masoud et al., (2016) found 35.5% resistance for erythromycin in S. agalactiae.
 
Detection of tetracycline resistance genes
 
Four isolates out of 6 (66.66%) were positive for the presence of tetM gene (Fig 1). All the four isolates had been phenotypically observed as tetracycline resistant. Sarah and Salah (2014) reported high prevalence to the presence of the tetM gene (99%) in tetracycline resistant streptococci. Three out of the six tested isolates (50%) revealed amplification of tetO gene (Fig 2), of the four isolates showing phenotypic resistance to tetracycline, only three isolates harbor tetO gene whereas one isolate showing resistance to tetracycline, was negative for tetO gene. tetM gene is the most prevalent resistance determinant accounting for tetracycline resistance in Gram-positive bacteria (Roberts, 1996). We observed high rate of tetracycline resistance as was described in other study of Gao et al. (2012) reported 52.9% of tetM gene and 17.6% tetO gene. High rates of tetracycline resistance in tetracycline resistant isolates correlated with the presence of the tetM gene.



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Detection of erythromycin resistance gene
 
Out of 6 isolates tested for identification of erythromycin resistance genes (ermA, ermB, mefA and mefE), none were found positive. However occurrence of erythromycin resistance genes in S agalactiae isolates have been reported by Boswihi et al. (2012) who reported ermA (5.5%), mefA (5.5%) and mefE (11%).

In present study, phenotypically all isolates were highly sensitive for erythromycin. It can be inferred that there was a definite pattern observed between the antibiotic resistance by the phenotypic and genotypic methods.

The details of occurrence of different antibiotic resistance genes in individual isolates have been shown in Table 3.


 
Detection of virulence associated genes
 
The PCR amplification results for the five virulence genes are depicted in (Fig 3) (cyl, glnA) and (Fig 4) (cfb, scaA, hylB) and Table 3. cyl gene of S. agalactiae is required for the production of hemolysin. It codes for beta-hemolysin, which is responsible for tissue injury and the systemic spread of the bacteria and lead to meningitis (Doran et al., 2003). We found 83.33% isolates were positive for cyl, these results are in accordance with those of Dmitriev et al. (2002) they found cyl gene in all the isolates while Spellerberg et al. (2000) reported that 23% isolates were harbouring the cyl gene. Gene glnA (glutamine metabolism) have significant role in the virulence and involved in nutrition and metabolism of various bacterial pathogens (Hendriksen et al., 2008). In present study, 66.66% isolates found to contain the glnA gene, these results are in accordance with those of Ding et al. (2016), according to their study, gene glnA was discovered only in S. agalactiae at incidences of 46.9% whereas Dmitriev et al., (2002) reported in all the isolates. The CAMP factor (cfb) is a pore-forming protein (protein B) secreted by S. agalactiae that potentiates the action of staphylococcal sphingomyelinase (beta toxin) (Jain et al., 2012). In the present study cfb gene was discovered at 66.66%, which is similar to Shome et al., (2012) reported 85.7% prevalence of cfb gene among S. agalactiae isolates. Still the significance of CAMP factor in pathogenicity of S. agalactiae is not properly know, hence it is not putative virulence factor (Lasagno et al., 2011). The hylB gene codes for hyaluronate lyase which help to break hyaluronic acid, N-acetylglucosamine and glucuronic acid (components of extracellular matrix) It also known as spreading factor which helps to the host tissue invasive (Duran-Reynals, 1942). In present study we found 83.33% isolates were positive for hylB, these results are in accordance with those of Gunther et al., (1996), according to their 72% of the GBS were hylB positive whereas Dmitriev et al., (2002) reported hylB gene in all the isolates studied. In the present study, 66.66% isolates were positive for scaA. Occurrence of scaA gene in 45.7 per cent and 100 percent of S. agalactiae isolates has been reported by Ding et al., (2016) and Dmitriev et al., (2002), respectively.

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Molecular characterization of group B streptococci by random amplification of polymorphic dna (rapd)
 
Out of 8 primers tested, only 3 primers (OPS11, OPA3 and AP42) with GC content of 40 to 70% gave reproducible patterns comprising fragments with a large size range and a small number of low-intensity bands. They gave the best differentiation of the 6 isolated strains. The reproducibility of the RAPD patterns obtained with these three primers was verified by repeating experiments under the same conditions. Each strain was tested at least twice times.

Dendrogram of S.agalatciae by OPS11 (Fig 5a) revealed that isolates S5 and S4, S6 and S2 evaluate with similar distance but S3 forming near out group with all other isolates. Sample1 is evaluating totally different from other isolates. OPA3 revealed sample 5 and 4 showed same genetic distance from other isolates (Fig 5b). Isolates 5, 4, 2, 3, 1 formed two clades and sample 6 was having entirely different level of evolution. According to AP42 primer S6, S3, S5 forming one group and Samples 4, 1, 2 forming another group (Fig 5c). In this two groups sample 5 and 2 forming outgroup with other isolates. Primers AP42 and OPS11 yielded similar type of pattern but OPA3 primer giving different kind of banding pattern. The isolates of S.agalactiae showed a great intraspecific diversity; various workers also reported high genetic diversity among bovine isolates (Martinez et al., 2000 and Baseggio et al., 1997). RAPD is simple and fast technique which is used for characterization of S. agalactiae strains. In cluster analysis, RAPD method identifies the same virulent families and able to discriminate strains inside each cluster and thus is more sensitive for identifying intraspecific diversity among isolates (Wang et al., 1993).

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S. agalactiae (Group B streptococci) isolated from six out of a total of 182 milk samples indicated a prevalence of 3.3%. The antibiogram of six isolates revealed higher resistance against oxytetracycline (66.66%) and co-trimoxazole (66.66%) followed by ampicillin (50%), penicillin (33.33%). The antibiotic resistant isolates were carrying genes for tetracycline resistance (tetM and tetO). Two of the isolates carrying all five virulence associated genes viz. cyl, scaA, hylB, glnA and cfb. The isolates of S. agalactiae showed a great intraspecific diversity.