Phenotypic and Serotypic Characterization of Staphylococcus aureus Strains from Subclinical Mastitis Cattle

Staphylococcus aureus is known as a major causative agent of mastitis in dairy cattle. In the present study, 104 isolates of Staphylococcus originated from subclinical mastitis cattle characterized for the phenotypic properties and the presence of Staphylococcal protein A (Spa). Some bacteria were resistances against several antibiotics were also studied, such as erythromycin, streptomycin, tetracycline, cefepime, nitrofurantoin, amikacin, chloramphenicol, and ciprofloxacin. About 78% of the isolated were moderately sensitive to nitrofurantoin, while 89% were highly resistant to cefepime and ciprofloxacin. Using the various mammals’ sera, seven isolates out of 104 revealed the presence of Spa.


INTRODUCTION
Staphylococcus aureus is a major opportunistic pathogen in humans and one of the most important pathogenic Staphylococcus species in veterinary medicine (Peton and Loir, 2014).The emergence of antibiotic resistant strains of S. aureus has driven renewed interest in better defining mechanisms by which virulence factors interact with host proteins to support S. aureus virulence (Flick et al., 2013).Naturally, S. aureus is a commensally bacterium of the skin and mucosa of human, dairy cattle, and other warm-blooded animals.
However, it is also often found to responsible for several severe infections.S. aureus is of particular interest due to special capacity to acquire resistance (Hoffmann et al., 2015).Furthermore, circumstances such as the increasing prevalence of multidrug resistant S. aureus continue to press the growing international concern and need for the latest situation in farmed setting.It shows that S. aureus differs from other pathogens in that it has evolved as a commensally (Rasigade and Vandenesch, 2014).
In ruminants, S. aureus is a major causal agent of mastitis.These infections can be relatively mild, yet serious, while life-threatening infections can be resulted from the expression of staphylococcal virulence factors that are regulated by several virulence factors (Pragman and Schlievert, 2004).S. aureus has capability of producing a variety of exoproteins that able to its ability to colonize the mammary gland such as hemolysins, coagulase, slime, and protein A (Coelho et al., 2011).Staphylococcus has accessory gene regulatory D (AgrD) functioned as precursor for auto inducing peptide in a quorum-sensing system regulating virulence phenotype of the preeminent pathogen Staphylococcus sp.(Gonzalez et al., 2014).The Agr system produces cytotoxins that act differently on the erythrocyte and Staphylococcus itself secretes these virulence factors including alpha, beta and delta hemolysins (Oscherwitz et al., 2014).Beta and alpha hemolysins are the most important in pathogenesis of the intramamarian infections (Park et al., 2004).These hemolysins can be expressed on sheep blood agar as different type of hemolysis occurred.Among above exoproteins, Staphylococcal protein A (Spa) is an important virulence factor) due to its high affinity for antibody fraction crystalizable (Fc) domains of immunoglobulin G (Fridy et al., 2015) and plays a role in opsonophagocytosis during S. aureus infection in mammalian hosts.The Spa, a cell wall anchored protein of S. aureus, has the ability to interact with several host components in mammalian cell, possibly indicating a virulence factor in S. aureus infections (Palmqvist et al., 2002).In poultry isolates, the Spa is absent due to the Immunoglobulin Y, the avian equivalent of IgG, does not bind with Spa since the IgY does not have Fc region (Lowder and Fitzgerald, 2010).
While it shares some virulence factors, S. aureus can also turned to be resistant to antimicrobial drugs, especially to â-lactam antibiotics (McAdam et al., 2012).The antibiotic therapy of mastitis caused by S. aureus is generally unsuccessful due to the resistance.Furthermore, S. aureus has been reported to frequently cause multiple antimicrobial resistance patterns, particularly to amikacin, streptomycin, and chloramphenicol (Margariti et al., 2014).
A number of immunological techniques including ELISA and Western blot have been described to detect and quantify Staphylococcal proteins (Nguyen et al., 2010).The Staphylococcal protein A restriction fragment length polymorphism (RFLP) has been applied extensively to differentiate isolates of S. aureus with protein A (Morandi et al., 2009).The methods required well-trained personal and considerably sophisticated laboratory equipment.Considering that protein A is a specific product of S. aureus and about 99% of the bacterial strains contain this protein (Huang, 2007), therefore we use the Spa to study the virulence of S. aureus isolated from subclinical mastitis cattle.In this study, the isolates were identified at the species level and the Spa was characterized by serum soft agar (SSA) technique.The isolates were also evaluated for phenotypic activities such as the presence of coagulase and hemolytic activity.In addition, the S. aureus strains were tested for resistance to cefepime, amikacin, streptomycin, ciprofloxacin, nitrofurantoin, and chloramphenicol.

Bacterial Isolates
Isolates of 104 Staphylococcus isolated from bovine subclinical mastitis were the samples of the study.All isolates had been identified previously as Staphylococcus sp. and as a collection of Microbiology Laboratory in Veterinary Medicine Faculty, Bogor Agricultural University.S. aureus Cowan 1 was used as positive control while S. epidermidis as negative control.

Identification of S. aureus
Standard microbiological methods for the identification of Staphylococcus aureus strains were applied.Coagulase-positive staphylococci was determined used Baird-Parker Agar (Oxoid) Egg Yolk Tellurite Enrichment.The bacteria were inoculated onto the agar surfaces and incubated aerobically for 24 h at 37°C.Typical colonies of coagulase-positive staphylococci are black, shiny, and convex, surrounded by clear zones of approximately 2-5 mm.All isolates were inoculated onto manitol salt agar and incubated at 37°C.After incubation, suspect colonies were examined by Gram staining.The colonies with morphologies compatible with Staphylococcus spp.were transferred to Tryptic Soy Agar (TSA) (Oxoid).After growth, staphylococci were identified on the basis of colony characteristics, Gram staining, pigment production, hemolysis and the following biochemical reactions, catalase activity, coagulase test (rabbit plasma) and amikacin (30 µg/disk), streptomycin (10 ìg/disk), ciprofloxacin (10 ìg/disk), nitrofurantoin (300 ìg/ disk) and chloramphenicol (30 ìg/disk) (Oxoid).Isolates were categorized as susceptible and resistant based upon interpretative criteria developed by Clinical and Laboratory Standards Institute (CLSI, 2007).

RESULTS AND DISCUSSION
The identification for all isolates gave primary morphological and biochemical of the S. aureus.From a total of 104 isolates, 8 (7.7%) were identified as S. aureus and 96 (92.3%) to non-S.aureus.Twenty-eight isolates (16.9%) have the ability to produce free coagulase.
Coagulase positive and double hemolysis are used to distinguish S. aureus from other Staphylococcal species.Coagulase plays as virulent factor by its ability to coaggulate blood plasma.The clotted of plasma in the coagulase test occurred caused by the converted prothrombin coagulase complex (staphylocoagulase).Trough coagulation, staphylococci captured within a fibrin meshwork, enables this pathogen to disseminate lesions and to resist opsonophagocytic clearance by host immune cells.The Staphylococcus isolates presented different hemolysis type on sheep blood agar plates.Sixty-one (58.7%) isolates expressed double hemolysis, 37(35.6%)isolates had beta hemolysis and 6(5.7%) isolates praduced gamma hemolysis.The distribution of â-hemolysis type in this study was lower compare to previous results (Morandi et al., 2009) that found hemolysin 66 of 122 S. aureus isolates from dairy products.Most of the Staphylococcus isolated derived from bovine subclinical mastitis showed double hemolysis a typical hemolisis for S. aureus.The distribution of double hemolysis of Staphylococcus isolates in this study agreed with manitol fermentation, finally aerobic and anaerobic utilization of glucose by FDA (2005).

Determination of Some Virulence Factors
Coagulase Test.Coagulase test is based on the ability of S. aureus to produce a protein product called coagulase (Mohammed and Abuelghait, 2014).Tube coagulase (TC) technique is chosen in this study.Free coagulase activity was determined by the method described by FDA (2005).Suspect S. aureus colonies were transferred into small tubes containing 0.2 mL BHI broth and emulsified thoroughly.BHI culture suspensions were incubated for 24 h at 37°C.0.5 mL coagulase plasma with Na citrate was added to the BHI culture, mixed thoroughly, incubated at 37°C and examined over 6 h for clot formation.Only firm and complete clot that stays in place when tube is inverted is considered positive for S. aureus.
Hemolysin Production.Alpha and betahemolysin were evaluated by plating strains on 5% sheep blood agar.The plates were incubated for 24 h at 37°C and then overnight at 4°C, when positive strains showed a wide zone of incomplete hemolysis with sharp edges.Non-hemolysis on 5% sheep blood agar was evaluated as gamma hemolysis (FDA 2005).

Detection of Protein A with Soft Agar (SA) and Serum Soft Agar (SSA)
The Presence of protein A was detected using soft agar technique (Wibawan et al., 2009) with the addition of various mammals' serum.Bacterial suspension (1 loop) was inoculated into 10 mL of soft agar (Brain Heart Infussion (BHI) (Oxoid)+0.15%)and or into 10 mL of serum soft agar (BHI+0.15% of bacto agar+100 µL rabbit/ canine/feline/sheep/chicken serum), agitated using a vortex vigorously and incubated at 37°C for 24 h. the strain containing protein A will show the changes of colony formation from diffuse in SA to compact in SSA, except SSA containing chicken serum, which was set diffuse.The negative strains will remain as diffuse colonies in SA as well as in SSA. S. aureus Cowan 1 is used as positive control since this bacteria known could produce high protein A. As negative control, Staphylococcus epidermidis is used since these bacteria cannot produce protein A

Antibiotic Resistance
Antibiotic susceptibility was determined by agar diffusion test of Muller-Hinton (Oxoid) using the following disks: cefepime (30 µg/disk), SSA (Figure 1).The compacted colony in SSA formed by S. aureus isolated from subclinical mastitis could partly explain the persistence and chronicity of staphylococcal subclinical mastitis.This colony is formed by growth inhibition of S. aureus due to IgG cover the S. aureus cell and this mechanism resulted the colony looks globular or compact.This form showed mechanism used by S. aureus to evade host defenses.It can be considered that protein A as virulence factor allows S. aureus to adhere of host cells and host tissues, invade of host cells, spread and manipulate of the immune response.
previous studies (Stephan et al., 2001).Six out of 104 γ (5.7%) of our Staphylococcus isolates were c-hemolysis (Table 1).It is indicate that these six isolates are not belonging to S. aureus.
The presence of protein A can be detected in seven out of eight S. aureus isolates (Table 2).From the tested S. aureus isolates, we found S. aureus mutant that is showed by sample of 49, which does not express protein A. The main factor influences the release protein A to the cell wall envelope of S. aureus are the presence of sortase A (srtA).srtA is a transpeptidase that attaches surface proteins to the cell wall.It cleaves between Gly and Thr of LPXTG (Leu-Pro-any-Thr-Gly) motif and catalyses the formation of an amide bond between the carboxylgroup of threonine and the amino-group of the cell-wall peptidoglycan (Mazmanian et al., 2001).This mechanism causes its peptidoglycan to be split and surface proteins are displayed over the staphylococcal surface.S. aureus lacking the srtA gene will fail to anchor and display some surface protein such as protein A. This finding can be considered that not all S. aureus was able to produce protein A to the surface of S. aureus.Moreover, the samples of 7 and 9 have less protein A than sample of 54, 58, 76, 80 and 83.However, only sample of 76 has a same pattern with Cowan 1.It indicates that sample of 76 is rich of protein A.
Staphylococcal protein A can be expressed in SSA as compact formation, which appeared globular (Figure 1).The shape related with the change of colony formation from diffuse to compact after the presence of mammals' sera in  However, the gene-encoded protein A, Spa, has host specificity and Spa is absent in poultry (Lowder et al., 2009).It has been showed from this study that S. aureus tested in soft agar containing chicken sera cannot form compact colony (Figure 1).As mentioned previously, Immunoglobulin Y does not bind Protein A (Lowder and Fitzgerald, 2010), which this trait can show protein A only can bind to Fc region of immunoglobulin G and plays a role in opsonophagocytosis only during S. aureus infection in mammalian hosts.
In this study, isolates tested showed different results in each animal's serum (Table 2) that furthermore this distribution of colony form indicates the species-differences of IgG ability to bind protein A. In this study, serum of rabbit showed poor affinity to the cell while it is surprising given the rabbit serum is often used in SSA test (Djannatun 2002).Meanwhile, in our study, the canine serum and sheep serum gave the best affinity in SSA test.Therefore, the canine and sheep serum may be also advisable for further SSA test.
In this study, all of the nine S. aureus strains studied were tested for resistance to antibiotics.The antibiotics selected for the study regarded as the most commonly used antibiotic in the medical and veterinary fields.The results of the sensitivity or resistance of S. aureus were presented in Table 3.The results support the possibility of the low efficacy of antibiotic treatment for mastitis caused by staphylococci is considered multiple resistant.The results of antibiotic sensitivity test (Table 2) indicated that the pathogen, S. aureus showed resistant to cefepime (89%), ciprofloxacin (89%), and amikacin (44%) in a declining order.Our result was then contrary to the previous study by (Patnaik et al., 2014) who had observed that S. aureus were sensitive to ciprofloxacin (100%) and amikacin (85.72%), while Margariti et al. (2014) reported that S. aureus was sensitive to amikacin (20%).However, in the other hand, S. aureus was moderately sensitive to nitrofurantoin (78%) and low sensitive to streptomycin (45%) and chloramphenicol (45%).Therefore, we found that most of S. aureus isolates were susceptible to nitrofurantoin.This may be due to that nitrofurantoin is not used routinely for the treatment of bacterial disease in domestic ruminants in Indonesia.On the other hand, our staphylococcal isolates showed high level of resistance particularly to cefepime and ciprofloxacin, which are commonly used in treatment for bacterial infections.This high rate of resistance to the particular antibiotics in our staphylococcal collection is likely due to selective pressure from misuse (Adwan, 2006), or exacerbated by frequent usage of intra mammary infusions (Kateete et al., 2013).If cefepime and ciprofloxacin were used indiscriminately, it probably will further complicate mastitis if the causative agent happens to be resistant to these antimicrobials (Ranjan et al., 2010).Furthermore, the resistance of these antibiotics has significance to public health since the transmission of resistant bacteria to the environment or dairy products could cause failure antibiotics treatment in human.

CONCLUSION
Our results confirm the wide variety of phenotype and serotype diversity of S. aureus from subclinical mastitis cases.Also, our study revealed that not all S. aureus strain could express protein A on the surface of these bacteria.SSA observation conducted one isolate revealed protein A that gave same result compare to S. aureus Cowan 1. S. aureus strains isolated from subclinical mastitis of cows has presented resistance to cefepime, ciprofloxacin, and amikacin.

SUGGESTION
Molecular assay is needed to detect the presence of SaP gene in sample showed negative result in SSA assay.

Table 1 .
Production of different types of hemolysis by Staphylococcus strains

Table 2 .
Detection of protein A with SSA

Table 3 .
Susceptibility of Staphylococcus aureus to antibiotics