Molecular characterization of virulence factors in Staphylococcus aureus isolated from bovine subclinical mastitis in central Ethiopia

Staphylococcus aureus (S. aureus) is the most important pathogen involved in bovine mastitis in dairy production. S. aureus produces a spectrum of extracellular protein toxins and virulence factors which are thought to contribute to the pathogenicity of the organism. The aim of this work was to isolate and molecular characterize S. aureus associated with bovine subclinical mastitis in the central part of Ethiopia. A total of 265 lactating dairy cows from various dairy farms in four different geographical locations were screened by the California mastitis test (CMT) for bovine subclinical mastitis. One hundred thirty CMT-positive milk samples were collected and transported to the laboratory. Different biochemical tests and polymerase chain reaction (PCR) were used for the identification of S. aureus isolates. Finally, PCR was performed for molecular detection of virulence genes. From a total of 265 lactating dairy cows screened, 49% (n = 130) were positive for bovine subclinical mastitis. One hundred thirty mastitic milk samples were subjected to bacterial culturing, and one hundred (76%) S. aureus isolates were identified based on phenotypic characters. Sixty-eight confirmed S. aureus isolates were obtained using PCR. The confirmed S. aureus isolates were tested for six virulence genes (tsst-1, hlb, eta, sea, clfA, and icaD) using PCR. Of the six virulence genes screened from all the isolates, only two (clfA and eta) were detected in the isolates. Out of 68 isolates, 25% and 22% were possessed the eta and clfA genes, respectively. The presence of Staphylococcus aureus having virulence genes (eta and clfA) revealed that mastitis is a major concern nowadays affecting animal health, milk quality, and yield. Further genomic study of these isolates will provide broad new insights on virulence.


Background
Mastitis is considered to be the most frequent and most costly production diseases in dairy herds of developed and developing countries including Ethiopia.Mastitis is an in ammatory response of the teat canal as a result of bacterial infection (Song et al., 2020).Staphylococcus aureus (S. aureusisone of the most recognized pathogen causing many serious diseases in humans and animals worldwide, and is the most common causative agent of clinical and subclinical bovine mastitis (Ote et al., 2011).
Mastitis caused by S. aureus is the result of production of several virulence factors that can contribute in different ways of pathogenesis (Vaughn et al., 2020).Virulence factors of S. aureus can be grouped broadly into two major classes which include surface localized structural components that serve as virulence factors and secreted virulence factors, which together help this pathogen to evade the host's defenses and colonize mammary glands (Diep and Otto, 2008).Some of surface localized structural components that serve as virulence factors include membrane-bound factors (collagen binding protein, brinogen binding protein, elastin binding protein and penicillin binding protein ), cell wall-bound factors (protein A, β-Lactamase and protease) and cell surface associated factors (capsule and slime) (Diep and Otto, 2008).Some of the known secretory virulence factors are toxins (staphylococcal enterotoxins, toxic shock syndrome toxin 1, hemolysins and exfoliatin), enzymes (coagulase, staphylokinase, DNAase, phosphatase, lipase and phospholipase).In addition to speci c virulence factor, S. aureus also possesses different mechanisms or traits such as bio lm formation, adhesion to and invasion into mammary epithelial cells and formation of small colony variant (SCV) that enable this pathogen to resist host defense mechanisms.Some of these toxins are known to function as superantigens that cause increased immunological reactivity in the host (Rollin et al., 2015;Bobbo et al., 2017).
The differences in pathogenicity of S. aureus strains could result from geographical distribution and from host-and tissue-related characteristics (van Leeuwen et al., 2005).The numbers and combination of virulence genes may be important contributions to pathogenic potential of S. aureus strains (Zecconi et al., 2006).High number of S. aureus genotypes present in bovine herds worldwide has been studied to develop better strategies of treating mastitis (Kot et al., 2016).The identi cation and characterization of virulence factors of S. aureus causing bovine mastitis will enhance our understanding of the pathogenesis of intramammary infection (Zecconi et al., 2006).In addition, the antibiogram of S. aureus needs to be studied which would indicate the pattern of resistance to various antibacterials contributing to their virulence properties (Graveland et al., 2011).These may in turn contribute to the development of methods to minimize the production losses due to mastitis.Further, the study of evolution of strainspeci c transmission and virulence characteristics including antibiotic resistance in S. aureus isolated from bovine mastitis may help us to understand mechanisms behind emergence of new strains or shifts in mastitis epidemiology in response to control measures, including antibiotic treatment and vaccination (Yu et al., 2012).However, at present few reports has been reported about the occurrence of virulence factors among S. aureus isolated from milk of cows suffering from mastitis but not identi ed by molecular technique in central part of Ethiopia.Furthermore, there is a literature dearth on the prevalence and genetic characterization of virulence determinants in S. aureus in Ethiopia.As to our knowledge, most of the researches in Ethiopia were done in association with prevalence of bovine mastitis cases and its associated risk factors (Abera et al., 2010;Tesfaye et al., 2010) but molecular data on S. aureus causing bovine mastitis in remain scarce.Therefore, the aim of this work was to isolate S. aureus associated with bovine subclinical mastitis and study of molecular characterization of virulence factors in that isolates in central part of Ethiopia.

Methods
Samples and study population Two-hundred and sixty-ve milk samples were collected from lactating dairy cows that showed subclinical mastitis symptoms.Milk samples were collected from intensive production system across different geographical locations (Adaberga, Ambo, Bishoftu and Holeta) in central part of Ethiopia since November 2018 to June 2019.Milk samples were collected and proceeded as described in previous study (Patel et al., 2017).Brie y, udders were wiped with 70% ethyl alcohol and few drops of milk were discarded initially.Simultaneously, CMT was executed on the site and on the basis of CMT score samples were collected (Bhatt et al., 2011;Patel et al., 2017).The study areas were purposively selected based on the agro-ecological differences and abundance of dairy farm milk sheds.The farms included in this study were involved in the production of milk for self-consumption and supplier to milk cooperative.

Bacterial isolation and identi cation
Milk samples were evaluated for mastitis-causing bacteria by bacteriological culture and biochemical tests following the National Mastitis Council Guidelines (Oliver et al., 2004).Briefly, 100μL of milk sample was inoculated onto nutrient broth media (Merck, Germany) with 5% sheep blood (Becton Dickinson Microbiology System, Cockeysville) and incubated at 37°C for 24 hr.Plates were evaluated for bacterial growth, colony morphology and hemolysis after 24 hr.Each pure colony was differentiated by Gramstain.Followed by catalase test.Catalase positive cocci were considered Staphylococcus species and further con rmed by polymerase chain reaction (PCR), and tube coagulase test using rabbit plasma (NVI, Bishoftu, Ethiopia) to differentiate S. aureus from coagulase-negative Staphylococcus species.The resulting culture was used for bacterial DNA extraction and the remaining overnight culture of S. aureus isolate in tryptic soy broth (TSB) (BHI, Merck, Germany) was mixed with equal volume of sterile 85% Glycerol and stored in a -80°C freezer for further molecular work.

Bacterial DNA extraction
Staphylococcus aureus bacteria were sub-cultured on nutrient broth media (NB, Merck, Germany) and incubated at 37°C for 24 hr.Genomic DNA of all phenotypically positive S. aureus isolates was extracted from the culture using the Zymo Research Fungal and Bacterial Genomic DNA MiniPrepTM kit (Zymo Research, Irvine, USA) following the manufacturer's instructions.Purity, quality and quantity of extracted DNA were measured using Nanodrop device (NanoDrop, Thermo Scienti c, USA), gel electrophoresis and spectrophotometer.The extracted genomic DNA was stored at -20°C until next use.
Primers used for the PCR ampli cation were synthesized by Sigma-Aldrich (Bonn, Germany) and master mix synthesized by BioBasic company (BioBasic, Canada).The primers used for molecular identi cation of different virulence-associated genes are indicated in Table 1.Lyophilized primers for the target genes were reconstituted using DNase-RNase free sterile water to obtain 1000µM stock solutions.All primers were stored at -20°C and then nally diluted to working concentration of 10µM.PCR was carried out in a total volume of 25μl containing 12.5μl of 1X Taq PCR Master Mix (Bio Basic, Canada), 1μl of forward primer and 1μl of reverse primer, 3μl of DNA template and 7.5μl sterile nuclease free water.The cyclic polymerase chain reaction conditions of the different primer sets are described on Table 2. PCR products were run on a 1% agarose (w/v) gel using electrophoresis, stained with gel red (Merck, Darmstadt, Germany) at 120 volts for 1hr and visualized under UV light using a BioDoc-itTM imaging system (Cambridge, UK).We used GeneRuler 100bp Plus DNA Ladder (Bioneer).

Statistical analysis
The data generated from the study was arranged, coded and entered to excel spread sheet (Micro oft® o ce excels 2010) and subjected to statistical analysis.The prevalence to every test was calculated as the number of positive cattle divided by the number of examined cases within the speci ed period.The Pearson Chi-square test (χ2) was applied to determine existence of any association between sampling areas and virulence associated genes using SPSS software version 22.0.The signi cance level was set at P-value (0.05) and 95% con dence level.In all cases, 95% con dence level and p-value less than 0.05 was consider as statistical signi cance.

Results
Isolation and identi cation ofS.aureusisolates In this study, of the 265 lactating dairy cows screened, 130 (49%) were positive for bovine mastitis based on CMT.One-hundred and thirty mastitic milk samples were subjected to bacterial culturing, 100 (76%) S. aureus isolates were identi ed based on the morphological and biochemical characters.From a total of 100 phenotypically positive S. aureus isolates, 68(68%) of them were con rmed S. aureus isolates through PCR ampli cation.The presence of 16SrRNA gene (1267bp) was con rmed by PCR in S. aureuspositive isolates (Fig. 1).
Prevalence of virulence genes inS.aureus All 68 PCR con rmed S. aureus isolates were tested for six virulence genes including tsst-1, hlb, eta, sea, clfA and icaD using PCR ampli cation.Of the six virulence genes screened from all the isolates, only two (clfA and eta) were detected.The isolates for the current study were obtained from mastitic bovine milk samples representing four geographical locations (Adaberga, Ambo, Bishoftu and Holeta) in the central parts of Ethiopia.Out of 68 isolates, 17 (25%), 15 (22%) and 6 (8.8%) isolates were possessed eta, clfA and combination of eta and clfA genes, respectively.The large proportion of these isolates which harbor eta and clfA genes were obtained from Holeta (46%, 7/15) and Adaberga (52%, 9/17), respectively.The prevalence of virulence gene was not statistically signi cant between different sampling areas (X 2 = 1.239;P = 0.744).The prevalence rates of the virulence genes were depicted in Fig. 3 below.The expected PCR product sizes obtained from these PCR products were 638 and 676bp for clfA and eta, respectively (Fig. 2).

Discussion
Staphylococcus aureus is one of the major cause of mastitis that leads to reduction of milk production in dairy cattle (Krishnamoorthy et al., 2017).The control of bovine mastitis is vital not only in Ethiopia but also in the world.Therefore, it is essential to investigate the pathogens using molecular techniques as vibrant components to control intra-mammary infections.In dairy industry the mastitis can be reduced by identi cation of exact pathogenesis and virulent factors present in infectious microorganisms.The molecular typing of infectious agents is known to be essential part of infection control strategies and is crucial to track and spread of contagious infections from one region to others or among different herds.Consequently, it is crucial to examine the mastitis causing bacteria using molecular methods as forceful tools to control IMI.Because S. aureus is the most commonly contagious mastitis pathogens worldwide, it is important to reveal virulence factors of these agents to develop effective control strategies against mastitis caused by this pathogen (Khan et al., 2013).In addition, an effective vaccine against IMI is not available, therefore prevention and control of mastitis needs detection of the principal antigenic determinants for the strategy and progress of more pro cient vaccines against mastitis causing bacteria, especially S. aureus.
A number of studies have been conducted in Ethiopia on the prevalence of S. aureus in bovine milk (Abera et al., 2012;Mekonnen et al., 2017).Most of these researches focused on the importance of this pathogen as a cause of clinical and subclinical mastitis, however, its virulence determinants have not been well addressed.To our knowledge, there is no reliable information on molecular data of virulence genes in S. aureus from mastitic bovine milk samples in Ethiopia.Epidemiological studies indicates that S. aureus strains agents of milk produce a group of virulence factor and it is believed that there is a relationship between severity of infection and the virulence factors produced by S. aureus (Almaw et al., 2008).Hence, in this study, the prevalence of certain virulence genes such as sea, eta, hlb, clfA, icaD and tsst-1 for S. aureus was evaluated.
In this study, from a total of 130 CMT positive isolates, S. aureus was the most frequently encountered organism with an isolation rate of 76%.The predominance and primary role of S. aureus isolate in bovine mastitis has also been reported in other studies (Abera et al., 2012;Demissie et al., 2018).Apart from Ethiopia, S. aureus has also been reported as the chief etiological agent of mastitis in cattle by many studies from African and Asian countries (Abebe et al., 2016).Though direct comparisons among studies might be di cult, but in general, the variation in the prevalence between the present and previous studies might be due to differences in detection methods, geographical location of the study sites, and differences in farm management practices in each studied farms.S. aureus is adapted to survive in the udder and usually establishes mild subclinical infection of long duration from which it is shed through milk serving as source of infection for other healthy cows and transmitted during the milking process (Radostits et al., 2007).Hence, the organism has been assuming apposition of major importance as a cause of bovine mastitis.
Out of 100 phenotypically positive S. aureus isolates, 68 % of them were con rmed S. aureus isolates by using PCR ampli cation.The nding of this study was in agreement with (Li et al., 2018b).Regardless of the isolation and identi cation techniques employed, the con rmation of S. aureus in milk using molecular highlights the need for both strict farm management practices and proper sanitary procedures to be implemented during milking operations.
The pathogenicity of S. aureus is closely related to presence of various virulence genes (Kot et al., 2016).
In this study, six virulence factors of the pathogen were screened but only two of them were positive based on PCR ampli cation.Our data showed that 15 out of 68 S. aureus isolates carried exfoliative toxin A (eta) (22.05%) and 17 out of 68 S. aureus isolates contained clfA (25%) genes.Of 68 S. aureus examined, 32 (47.05%) were positive for one or more virulence genes.About half (52.95%) of the isolates did not contain any of the virulence genes tested.The eta and clfA were found at higher frequencies whereas sea, hlb, icaD and tsst-1 were not found in all tested isolates.Five isolates harbored both eta and clfA genes.There has been no published information regarding clamping factor A (clfA) and exfoliative toxin A (eta) in the Ethiopian context.This is the rst investigation regarding to these genes in Ethiopia and there is no other work on these virulence factors.This nding is different from (Srinivasan et al., 2006) who examined 78 S. aureus isolates from the milk of cows with mastitis for 16 enterotoxin genes and found that 73 (93.6%) of the isolates were positive for one or more enterotoxin genes from a similar area.However, (Srinivasan et al., 2006) tested for 16 enterotoxin genes whereas in this study only one enterotoxin genes were tested.This might be the reason for the low prevalence of positive isolates in this study.The presence of the clumping factor gene is considered as Staphylococcus species virulence gene in development and severity of mastitis in cows (Aarestrup et al., 1995).The above results suggested that S.aureus isolates with different genetic background have different ability to acquire mobile genetic elements such as plasmids, phages and pathogenicity islands.

Conclusions
High prevalence of virulence genes (clfA and eta) in S.aureus bacteria were the most important ndings of our study.All of the S. aureus bacteria harbored clfA and eta putative virulence factors which showed that they can use as speci c genetic markers for detection of pathogenic S.aureus bacteria in bovine subclinical mastitis cows.Presence of virulence factors in mastitis causing Staphylococcus aureus is an alarming spot for veterinarians, as several sources are there for spreading of microorganisms to human being.The emergence of different antibiotic resistance and virulence in the last two decades is exerting a lot of pressure in the health sector.Detailed genomic evaluation of particular antibiotic resistant strain with virulent factors may possess a great scope to develop new disease control strategy.Agarose gel electrophoresis of PCR amplicon of clfA and eta genes of representative S. aureus isolates.Lane M is a 1000-bp DNA marker (DNA ladder, Bio Basic); lanes 1 to 6 are test samples.

Table 1 :
Description of the primers used for molecular identi cation of different virulence-associated genes detection in S. aureus isolates

Table 2 :
Cyclic polymerase chain reaction conditions of the different primer sets