Randomly amplified polymorphic DNA analysis of Staphylococcus chromogenes isolated from bovine and bubaline mastitis in Karnataka

Background and Aim: In recent times, non-aureus staphylococci (NAS) have emerged as the major organisms isolated from mastitis cases in dairy animals, with a predominance of Staphylococcus epidermidis and Staphylococcus chromogenes. As compared to Staphylococcus aureus, much less is known about the molecular types or the spatiotemporal epidemiology of these NAS species. In the present study, randomly amplified polymorphic DNA (RAPD) was employed to detect genetic polymorphisms, intraspecies diversity, and epidemiology of S. chromogenes strains (n=37) isolated from bovine and bubaline mastitis cases in the state of Karnataka. Materials and Methods: Thirty-seven S. chromogenes isolates (14 from bovines and 23 from bubaline) isolated from subclinical mastitis cases, from organized and unorganized sectors, were subjected to RAPD typing. Further, methicillin resistance was determined by cefoxitin disk diffusion method. Results: The amplified DNA fragments ranged from 150 to 3000 base pairs and yielded several RAPD profiles. Further analysis using Digital Image Correlation Engine correlation coefficient and UPGMA method showed that the 37 isolates could be classified into 12 distinct RAPD types (A to L) at 62% similarity (D=0.889). Four of the most predominant RAPD types, B, A, C, and E, in that order, and together, represented 65% of the isolates. High diversity was observed among the isolates both within farms and between geographic locations. Most of the isolates exhibited methicillin resistance. This is the first such report from India. Conclusion: In the absence of defined multilocus sequence type protocols or sufficient sequences available in the public domain, RAPD can be employed to determine genetic diversity of S. chromogenes isolates.


Introduction
Bovine mastitis is an important disease which affects productivity in dairy animals, thereby directly or indirectly affecting farmers' income and the economy of the country [1]. In India, Staphylococcus species constitute the main etiological agents of mastitis in cattle and buffaloes [2]; however, of late, non-aureus staphylococci (NAS) have been increasingly isolated [3]. The most predominantly reported NAS causing persistent intramammary infections and high somatic cell counts (SCCs) is Staphylococcus chromogenes [4][5][6][7][8].
Molecular epidemiological studies can contribute to the understanding of source, transmission routes, disease causation and outcome, and prognosis as well as to the understanding of mechanisms of host adaptation of pathogens. For Staphylococcus aureus, several typing methods have been used, and these include ribotyping, random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, spa typing, and multiple locus variable number tandem repeat analysis [9]. For NAS species, strain typing methods such as PFGE, amplified fragment length polymorphism, RAPD [10,11], and whole-genome sequencing [12] have been employed. The polymerase chain reaction (PCR)based DNA fingerprinting method, RAPD, which is a rapid, discriminative, easily performable, and interpretable analytical assay, has been successfully used to detect polymorphisms of both S. aureus and NAS strains isolated from bovine mastitis [11,[13][14][15][16][17].
There is very little information on the genotypic profiles and antimicrobial resistance of mastitis-associated S. chromogenes isolates from bovine mastitis cases in India. Hence, the present work was undertaken to investigate the intraspecies diversity and their genetic relationship using RAPD as well as methicillin resistance of mastitis-associated S. chromogenes isolates.

Ethical approval
Ethical committee approval was not required as the study did not involve any animal experimental work.

Study area and period
The bacterial isolates comprised those isolated previously from subclinical mastitis (SCM) cases in organized and unorganized sectors, from samples collected from different geographical locations [in and around Bengaluru, (Tumakuru, Chennapatna, Ramanagara, near Hosur), Dharwad, and Bidar] between June 2009 and January 2013 (Table-1) from bovines and bubaline. Species confirmation by PCR was done and then RAPD analysis on S. chromogenes isolates were carried out in the month of september 2019.

Bacterial isolates used in the study
Following aseptic precautions, a pooled quarter milk sample was collected after discarding the initial few squirts of milk from each animal. Each animal was only sampled once. The milk was subjected to SCC, electrical conductivity (EC), California mastitis test, and bromothymol blue strip test. For declaring SCM in cows, the cutoff values used for SCC and EC were 5×10 5 cells/mL and 6.5 mS/cm, respectively [18]. In buffaloes, the values were 2×10 5 cells/mL and 3.8 mS/ cm, respectively [3]. NAS were isolated following standard microbiological methods and had been maintained in the Department of Veterinary Microbiology, Karnataka Veterinary Animal and Fisheries Sciences University, Bengaluru.

Extraction of genomic DNA
The genomic DNA of Staphylococcus isolates was extracted from an overnight culture, using a bacterial DNA extraction kit (Uniflex TM DNA Isolation Kit , Genei TM , Bengaluru, India), following the manufacturer's instructions. The integrity of genomic DNA was assessed by 2% agarose gel electrophoresis and visualizing the bands through UV transillumination. The DNA samples were frozen at −20°C.

Identification of the species of the isolates
The isolates were confirmed as S. chromogenes based on colony morphology (size and color), hemolytic pattern, Gram's staining, and catalase and coagulase tests. Further confirmation was accomplished by PCR. For S. chromogenes, 303 base pairs (bp) region of the sodA gene [18] was amplified using the primer pairs 5'-CGTGACTAAGTTAAACGATGCAG-3' (forward) and 5'-CCATTATTTACAACGAGCCATG-3' (reverse). The reaction mixture (25 µL) contained 2.5 µL of 10× Taq buffer, 1 U of Taq DNA polymerase, 25 mM of each of the dNTPs, 10 pmoles/µL of each primer, 3 µL of template DNA, and 16.3 µL of nuclease-free water. The reaction was carried out with an initial denaturation step at 94°C for 5 min, followed by 30 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, with a final extension step at 72°C for 10 min. The products were resolved by electrophoresis on 2% agarose gel, stained with ethidium bromide, and imaged using a gel documentation system (Gelstan 4×, The Medicare Scientific Supplies, India). The PCR products were sequenced (Eurofins) and subjected to bioinformatics analysis for species confirmation. In this study, 37 isolates (14 from bovines and 23 from bubaline) identified and confirmed as S. chromogenes were selected and subjected further to antimicrobial susceptible testing and RAPD analysis.

Antimicrobial susceptibility testing to determine methicillin resistance
All the isolates were tested for methicillin resistance using cefoxitin disks (30 µg), by employing the Kirby-Bauer disk diffusion method [19], and the isolates were classified either as resistant or sensitive according to the guidelines recommended by the Clinical Laboratory Standards Institute [20].
The RAPD patterns were analyzed using Gel compare II version 2.5 (Applied Maths, St-Martens-Latem, Belgium). The similarity of profiles was calculated based on Digital Image Correlation Engine correlation coefficient and clustered using the unweighted pair group method with arithmetic mean (UPGMA) method. The discriminatory index (D) was calculated according to Hunter and Gaston [21]. The relationship between clustered groups was expressed as percentage similarity and presented as a dendrogram.

Confirmation of S. chromogenes isolates by PCR
All the 37 isolates subjected to species-specific PCR yielded the expected 303 bp amplicon, and Available at www.veterinaryworld.org/Vol.14/January-2021/37.pdf nucleotide sequencing confirmed the isolates to be S. chromogenes.

Determination of methicillin resistance of S. chromogenes isolates
Fourteen of the 37 S. chromogenes isolates were found to be sensitive to methicillin while the remaining were resistant. All the isolates from Dharwad were resistant. Isolates from Bengaluru and Hosur were predominantly sensitive, whereas isolates from Bidar and Channapatna were predominantly resistant. In Ramanagara and Tumakuru, isolates were equally distributed between sensitive and resistant types. The proportion of resistant isolates was 50% (n=7) in bovines and 70% (n=16) in bubaline.

RAPD and clustering analyses for S. chromogenes
From the 37 isolates, the RAPD-PCR generated 7-19 fragments with DNA size ranging from 150 to 3000 bp (Figure-1). Analysis of RAPD profiles at 62% similarity revealed 12 types (A through L). A dendrogram generated based on the similarity matrix is presented ( Figure-2 and Table-2). The most predominant profile type was B followed by A, C, and E, and they together represented 65% (24/37) of the isolates. Type B was characterized by the presence of 8-19 amplimers ranging from 320 to 2800 bp. Type A contained 9 or 11 amplimers ranging from 160 to 3000 bp. Type C consisted of 11-17 amplimers ranging from 210 to 2700 bp (Table-2  Bovine isolates clustered in the RAPD types F, J, K, and L while bubaline isolates clustered under B, C, D, G, H, and I types. Types A and E consisted of five isolates each from bovine and bubaline herds (Table-2 and Figure-2).
Region-wise clustering of RAPD types revealed that types A and C contained isolates from three geographical locations, whereas type E contained five isolates from four locations. Type A contained five isolates from bovine herds of Tumakuru and bubaline herds of Channapatna and Dharwad. Type B consisted of nine isolates, all from bubaline herds of Channapatna and Dharwad. About 50% of the isolates (n=4) from Dharwad clustered under type B (Table-1 and Figure-2). Type C consisted of five isolates    and Bengaluru (n=4 each) clustered into three types. In Bengaluru, all the four isolates from a single farm clustered in three different RAPD types, whereas less diverse types were found in Bidar, Dharwad, and Ramanagara. In Bidar, all three bovine isolates from organized farms clustered under a single type (J) and two of the three isolates from unorganized farms clustered under a single type (E) ( Table-2 and Figure-2).

Discussion
Among the NAS, S. chromogenes is the most predominant pathogen associated with mastitis in India and other countries [4][5][6][7][8]. In the present study, genetic diversity of bovine and bubaline mastitis-associated S. chromogenes was evaluated by RAPD typing, a method which has been shown earlier to be useful in the discrimination of S. chromogenes isolates [17]. In the current study, 62% of S. chromogenes isolates were found to be resistant to methicillin, with a preponderance (70%) in bubaline isolates, whereas resistant and sensitive isolates were almost equally distributed in bovines. This proportion is much higher than 0% or 8.2% reported for bovine mastitis in Dutch dairy cows [5,22,23]. On the other hand, while no prevalence data have been presented, two mecA + isolates, each of S. chromogenes and Staphylococcus Epidermidis, were reported from samples (n=150) of raw bovine milk, and ovine and caprine cheese in Northwest Spain [24].
The RAPD typing has been employed before for characterizing bovine mastitis-associated S. aureus isolates. In a study conducted in the USA and the Republic of Ireland [25], majority of S. aureus isolates from cows in Ireland had RAPD profiles similar to those identified for the isolates recovered from cows in the USA. While these data suggest that bovine mastitis is caused by a few clones of S. aureus which have a broad geographic dissemination, different RAPD types were identified in S. aureus isolates both within the herds and among different farms in China [16]. In any case, RAPD has not been popularly applied for molecular fingerprinting of livestock S. aureus isolates because of the wider usage of other typing methods.
The 37 S. chromogenes obtained from seven geographic regions in Karnataka clustered into 12 groups (A-J). Among these, 65% of the isolates clustered in four predominant RAPD types, namely, B, A, C, and E. Similar observations were made with large Chinese dairy farms in which eight distinct RAPD types (A-H) were observed, with 73% of the isolates being represented by the most prevalent types A and F [17]. This also corroborates the highly conserved patterns observed by PFGE in S. chromogenes isolates obtained from cows and pigs [26]. Analysis of genetic heterogeneity and diversity can be used to track the movement of pathogens as well as to associate with pathogenesis. A study with S. chromogenes isolates (n=28) causing bovine intramammary infections showed limited genotypic heterogeneity by RAPD both within and between Flemish dairy herds, reflecting high genetic conservation within the species [11]. Importantly, it was found that isolates from the environment displayed largely the same genotypes as those from milk, suggesting that they were more udder adapted. On the other hand, the isolates studied by us were more diverse both within the farms and between the locations, except for the bubaline isolates from Ramanagara where both of them belonged to type C, and in Dharwad, where 50% of the isolates belonged to type B (Table-1). Diversity was also observed with 66 S. chromogenes isolates obtained from 30 animals from three dairy herds of the USA, where 33 different PFGE patterns were observed indicating genetic diversity in strains even when multiple sampling was done [10]. However, it is difficult to generalize the results or compare the RAPD or PFGE types between distant geographical locations due to the small number of studies relating S. chromogenes.
Most of the isolates from the predominant RAPD types (A, B, C, and E) in the present study were methicillin-resistant except for isolates Bo26 in type A, Bu74 and Bu75 in type B, Bu105 in type C, and Bo23 and Bo139 in type E, all of which were sensitive.

Conclusion
This is the first study from India with respect to molecular fingerprinting of mastitis-associated S. chromogenes isolates. In the absence of protocols for other typing methods or the availability of sufficient genome sequences, RAPD-PCR can be employed as a tool to determine the diversity of S. chromogenes isolates. However, a large number of isolates both from the environment and milk need to be analyzed to track transmission and dissemination events.