Molecular characterization of genes responsible for biofilm formation in Staphylococcus aureus isolated from mastitic cows

Abstract Background and Aim: Mastitis is considered a significant disease of lactating animals. There are new attitudes for recognizing genes responsible for causing this disease to overcome and change the manipulation of this problem. This study aimed to isolate and identify Staphylococcus aureus strains from mastitic bovine animals and detect some specific biofilm-forming genes (icaA, icaD, and biofilm-associated protein [bap] genes clfA, fnbA, agrI, agrII, agrIII, agrIV, and cna). Materials and Methods: A total of 121 mastitic milk samples were analyzed using biochemical tests (catalase test, oxidative-fermentative test, and coagulase test) and Gram stain. Multiplex polymerase chain reaction was applied to characterize biofilm genes (icaA, icaD, bap, clfA, and fnbA) in addition to (agrI, agrII, agrIII, agrIV, and cna). Results: Among the 121 milk samples, 35 staphylococci isolates were derived with an incidence of 28.92% (35/121); among them, 19 are coagulase positive. Ninety percent of the isolates had ica genes (icaA and icaD) while bap gene was not recognized in any isolate. In addition, the incidence of fnbA, can, and clfA was 89.5% each. The prevalence of agr specific groups (agrI, agrII, agrIII, and agrIV) was 78.9%, 52.6%, 10.5%, and 15.8%, respectively. Conclusion: This study concluded that S. aureus has variant mechanisms of pathogenicity to form biofilm devoid of carrying a specific gene.


Introduction
Mastitis in bovine is the most widespread disease among dairy cattle. Consequently, it influences its production due to a combination of different factors such as host, climate, and infectious agents and reduces the quantity and quality of produced milk [1]. The primary cause of bovine mastitis is Staphylococcus aureus, responsible for a dramatic decrease in both quality and quantity of milk production, resulting in significant economic consequences in the dairy farms [2].
S. aureus strains have immense pathogenicity (e.g., encoding virulence factor genes and antibiotic resistance) and several ways to infect humans, such as through foodstuffs, skin infection during the milking process, and direct contact with contaminated fomites. Subsequently, animals producing food, especially cows, are a major route for S. aureus to enter the food chain [3]. Thus, identifying the virulence factors and mechanisms of this pathogen involved in such criteria is important. Furthermore, how they assist in adhering and colonizing the mammary glands' epithelium cells, leading to persistence, successful foundation, and continuance in the host tissue. The most common virulence factors that aid staphylococci in adhering and colonizing the epithelium of the mammary gland are related to their capacity to form biofilms, resulting in loss of immunological defenses and frequent infections. Furthermore, staphylococcal adhesions have been shown to be necessary for binding host cells [4]. Biofilm formation may lead to continual contamination or infection because biofilm cells are highly resistant to hygiene measures, the effect of antimicrobial agents, and host immunity [5]. The process of biofilm formation by Staphylococcus spp. requires the contribution of different genes and proteins [6]. First, adherence of bacterial cells to a surface is initiated by a capsular antigen polysaccharide/adhesin (PS/A). Following that, growth occurs to create a multi-layered biofilm that induces polysaccharide intercellular adhesin (PIA) production. The intercellular adhesion operon is responsible for PIA and PS/A synthesis in staphylococcal species (ica), formed by the icaA, icaB, icaC, and icaD encoding genes as well as regulatory gene, icaR, carrying icaA, icaB, icaC, and icaD proteins [7]. Furthermore, S. aureus has many adhesins that play an important role in the onset of pathogenicity through the binding of host tissues that are considered essential factors of virulence. These adhesins include fibronectin-binding proteins (fnbA and fnbB), clumping factors (clfA and clfB), collagen-binding protein (cna), biofilm-associated protein (bap) [8], and collagen-binding protein (cna) [9], which are considered essential virulence factors in binding host cells, colonization, and invasion [10]. The accessory gene regulatory (agr) system is fundamental in S. aureus virulence gene expression. The agr operon, which includes the genes agrA, agrB, agrC, and agrD, controls more than 70 genes in S. aureus, 23 of which regulate infectivity [11]. Furthermore, S. aureus genes could be divided into four groups of (agr I, agr II, agr III, and agr IV) genes. They are different in their characteristics and occurrence in various geographical areas. Thus, it is necessary to determine the main types in each region [12].

Ethical approval
This study was approved (no. 12020232/2019) by Ethical Committee for Medical Research at the National Research Centre, Egypt.

Study period and location
The study was conducted from January to March 2020. The study was conducted at National Research Center, Dokki, Egypt. The samples were processed at the National Research center, Veterinary Research Division, Microbiology and Immunology Department Laboratory.

Collection of samples
One hundred and twenty one affected quarter milk samples (quarter selected based on physical examination; appearance of inflammation as redness and swelling) were obtained from 40 cows suffered from mastitis and did not receive any medical treatment for 7-10 days in private farms in Giza Governorate, Egypt. Those farms did not implement the required hygienic measures to control mastitis and other infectious diseases. The milking process was performed using a traditional method.
Before the milk collection, the animals did not receive antibiotic treatment for at least 1 month. The collection of milk samples was conducted under complete aseptic conditions according to Oliver et al. [13].

Isolation and identification of S. aureus
Every milk sample was cultured on two plates: Columbia Agar base with 5% defibrinated sheep blood (Oxoid, UK) and mannitol salt agar (Oxoid). The tested plates were incubated at 37±1°C for 24-48 h. According to colony morphology, Gram staining, in addition to catalase reaction, and oxidative-fermentative test, all isolates were identified as staphylococci. Coagulase test was used to characterize all S. aureus strains [14].

DNA extraction
DNA milk samples were extracted using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) with some modifications (temperature adjustment at 25°C and pH set at 4.0) to the manufacturer's instructions.

PCR products
PCR products were separated by electrophoresis at room temperature (25°C) on 1.5% agarose gel (AppliChem, Germany, GmbH) in 1× Tris-borate-EDTA buffer with gradients of 5 V/cm. A gel documentation system (Alpha Innotech, Biometra, Germany) was used to photograph the gel (Gel documentation system; Biometra, Germany) and the data were evaluated using a computer software (Genesys image capture, Biometra, Germany).

Results and Discussion
Bovine mastitis is a significant disease in lactating herds worldwide [20]. S. aureus is considered the most common causative agent, leading to more virulent mastitis in cows. It possesses the greatest risk in dairy production in many countries [21].
In the current study, a total of 35 staphylococci isolates have been isolated with a prevalence of 28.92% (35/121). The identified prevalence of S. aureus and Coag-ve staphylococci other than S. aureus was 57.14% (20/35) and and 42.85% (15/35). All staphylococci isolates were confirmed using 16S rRNA gene, as shown in Figure-1.
S. aureus isolated from subclinical mastitis cow was 36% [22] and less than the prevalence (6.5%) detected by Haltia et al. [23]. According to this study, the high prevalence of S. aureus may result from contaminated milk utensils and Milker's hands.
The possibility of S. aureus infection occurring is related to its capacity to release different factors of virulence that contribute to the invasion of the bacteria [24]. The formation of biofilm increased the virulence of S. aureus. In addition, strains can create biofilms that possess higher antibiotic tolerance, antiseptics, and poor environmental conditions [25]. The genes of ica are responsible for slime formation in S. aureus by controlling PIA production. It can also determine the ability of S. aureus strains to generate biofilm.
These results almost agree with other findings as ica genes were identified in all isolates [3]. While Gowrishankar et al. [28] detected those isolates of S. aureus in India carry ica genes in a percentage of 84.13%. In Mexico, Avila-Novoa et al. [29] identified the genes in 52.3% of isolates.
The bap gene implicates biofilm formation by promoting primary attachment and adhesion to inert and live surfaces [30]. This study showed that all the tested strains (100%) were negative for the bap gene (Figure-4).    Available at www.veterinaryworld.org/Vol.15/January-2022/26.pdf According to Vautor et al. [31], the absence of bap indicates that the ica-dependent pathway is predominantly responsible for adhesion and biofilm development in strains. Bissong et al. [32] found that the occurrence of the bap gene was limited (12,15.6%). Li et al. [33] identified bap gene in 43.9% of S. aureus strains biofilm producers, proving the significance of bap gene in biofilm production. Our results are in agreement with Xu et al. [34], who were unable to detect bap gene in S. aureus recovered from subclinical mastitic cow. Khoramrooz et al. [35] and Darwish and Asfour [36] detected expression of bap gene in 5% and 2.5% among obtained isolates, respectively. Our results showed that the ica-gene process could sometimes be essential for attachment and formation of biofilm among isolated strains; this can be a possible explanation for our finding.
The fnbA genes appear to be necessary for bacterial invasion and adhesion, and they may be associated with their ability to form biofilms. The incidence of expression of the surface protein genes for S. aureus (fnbA, can, and clfA) was 89.5% for each gene as reported in Table-2; these results demonstrated resemblance and minor variations to earlier studies; Peerayeh et al. [37] revealed that clfA and fnbA ( Figures-5 and 6) encoding genes had been found in each of the tested isolates (20 strains), with can gene are being found in 20% of identified    isolates (Figure-7). In contrast, Ote et al. [38], and Ikawaty et al. [39] observed a higher prevalence of can gene (31.9% and 49%, respectively).
S. aureus strains have been divided into four categories, agrI to agrIV, [18] based on differences in their agr genes. The agr-encoded quorum-sensing system's important function in virulence regulation makes it an appropriate target for antimicrobial drug development. According to Table-2, the prevalence of agr specificity groups (agrI, agrII, agrIII, and agrIV) were 78.9%, 52.6, 10.5%, and 15.8%, respectively. According to Figures-8 to 11, our results showed that agrI was the most common type found in isolated S. aureus. Javdan et al. [40], and Cheraghi et al. [41], stated that the most dominant type was agr Type I. It is said that definite groups of agr in S. aureus are implicated in certain diseases, such as isolates that possess agrI    are related to bacteremia and persistent diseases [42]. The development of biofilm is a complex process involving numerous factors such as poor hygienic measures and poor management of milking practices.

Conclusion
It can be concluded that the variant mechanisms of pathogenicity induced by S. aureus to form biofilm without needing a specific gene. It is necessary to identify the presence of genes related to biofilm production as formation of biofilm results in attachment to glandular udder tissue and biomaterials, thus increasing the virulence of bacteria. Besides, the biofilm's existence increases the bacterial resistance to antibiotics, consequently complicating its treatment. This study presents preliminary results for additional in-depth prospect studies. Finally, good hygienic measures and habits, in addition to good management of milking practices, can reduce the incidence of S. aureus mastitis.

Authors' Contributions
RHE and EGS: Collected the samples, applied the practical work including isolation of S. aureus and identification of obtained isolates. ESI, WHE and AAA: Planned the work, applied the practical work including isolation of S. aureus and identification of obtained isolates, PCR and revised the manuscript. SMD: Carried out PCR and drafted the manuscript. NSA: Revised the manuscript. All authors read and approved the final manuscript.