Biofilm Producing Staphylococcus aureus and Bovine Mastitis: A Review  

Ali Raza1 , Ghulam Muhammad1 , Sumaira Sharif2 , Asia  Atta2
1. Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad, Pakistan
2. Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan
Author    Correspondence author
Molecular Microbiology Research, 2013, Vol. 3, No. 1   doi: 10.5376/mmr.2013.03.0001
Received: 23 Jan., 2013    Accepted: 08 Feb., 2013    Published: 10 Apr., 2013
© 2013 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Raza et al., 2013, Biofilm Producing Staphylococcus aureus and Bovine Mastitis: A Review, Molecular Microbiology Research, Vol.3, No.1 1-8 (doi: 10.5376/mmr.2013.03.0001)


Mastitis is one of the major problematic diseases of dairy animals throughout the world associated with etiological agents but Staphylococcus aureus is one of the major causes which are responsible for this dairy scourge. Biofilm production by the microorganisms is considered an important virulence factor responsible for adhesion of these microorganisms with living or non-living surfaces. Staphylococcus aureus isolates which produce biofilm lead to chronic mastitis in dairy animals. The intramammary infection due to biofilm producer S. aureus is difficult to treat even with intra-mammary antibiotics so proper considerations should be given to the infections produced by biofilm producing bacteria. Development of an effective vaccination against the bacteria which produce biofilm may provide success to control such type of perilous infections.

Dairy animals; Mastitis; Staphylococcus aureus; Biofilm production; Antibiotic resistance

1 Introduction
Mastitis is one of the most crucial diseases of cattle and buffalo because it causes innumerable problems to milk production, milk processing and quality of milk & milk products which results in huge economic losses to the dairy industry. The physical, chemical, bacteriological and other qualities of milk are affected by mastitis. Mastitis as a dairy scourge represents an impediment to the development of dairy industry. In as much as, the milk of infected animals contains pathogenic organisms and their toxins, the disease is also important from consumers stand point (Munro et al., 1984). The use of antibiotics in mastitis is practiced on large scale which may create problems after emergence of resistant strains of bacteria in result of excessive treatment with these drugs and then entrance of the resistant bacteria in food chain may lead to serious consequences in future life (White and Mc Dermot, 2001). The spread of zoonotic organisms although rare in the presence of modern processing techniques like pasteurization but in case of unpasteurized milk products and failure of pasteuri- zation presents a serious threat to human health.

Biofilm is a structural community of bacterial population in which they are enclosed and composed of self-created polymeric matrix. These are adhesive to inert and free living surfaces that enhance the protection of their growth in the environment. The biofilm producer microbes start certain mechanism of adhesion to a surface and then formation of micro-colonies resulting into a three dimensional structure of mature biofilm (Prakash et al., 2003).

The purpose of this article is to comprehensively review the role of biofilm producing S. aureus in bovine mastitis and impact of biofilm as a virulence factor on pathogenesis of mastitis caused by S. aureus.

2 Staphylococcus Aureus and its Significance in Mastitis
Mastitis can be caused by a whole consortium of pathogenic microorganisms. Nonetheless, Staphylococcus aureus is considered to be the number one mastitis pathogen (Athar, 2007). Although mastitis can be caused by 137 microorganisms (Ranjan et al., 2006), other microorganisms which may be responsible for mastitis include Strep. agalactiae, Strep. uberis, Enterobacter aerogenes, Actinomyces pyogenes, E. coli, Klebsiella spp., certain fungi and yeasts (Gruet et al., 2001).

Contagious pathogens like S. aureus and Strepto- coccus agalactiae (Str. agalactiae) are generally considered the most predominant organisms respon- sible for mastitis in the dairy herds lacking an effective mastitis control program (Kheirabadi et al., 2008; Radostits et al., 2007).

These etiologic agents and environmental opportunistic pathogens like Escherichia coli (E. coli) are not unusual to the countries with poor standards of dairy farming system. It is suggested that even in currently developed countries, before the adoption of mastitis control strategies like antiseptic teat dipping and dry period antibiotic therapy, these three pathogens were the main problem in relation to mastitis in dairy cows (Bramely and Dodd, 1984).

The importance of contagious mastitis is further reflected by a review authered by Allore (1983) wherein she concluded that contagious pathogens (S. aureus & Str. agalactiae) and E. coli are responsible for > 75% of the clinical cases of mastitis and S. aureus is the major one among these. These bacterial species tend to interact with each other and mammary gland itself which can further aggravate the conditions. It is evident from the reports of clinical mastitis by E. coli after blitz therapy (Whole herd treatment for removing Strep. agalactiae infections) with anti- microbials. There might be accidental entrance of environmental micro-organisms at the time of intra mammary infusions (Boyer, 1997).

Staphylococci were first discovered by the Scottish surgeon, Sir Alexender Ogston (1880) and since then it has been found associated with a myriad of human and animal diseases. He named the round microorganism in infected tissue as “Staphylococcus” (Greek staphyle means bunch of grapes; kokkos means berry). Nocard identified staphylococci from mastitis in sheep in 1887 and then in 1890, Guillebeau stated that these organisms were responsible for mastitis in cattle (Jonsson and Wadstorm, 1993).

Staphylococcus aureus is one of the significant causes of udder infection in dairy animals (Sargeant et al., 1998). Intramammary infections (IMI) with this pathogen may lead to clinical and sub-clinical mastitis and is usually associated with the increase in somatic cell number (SCC). Staphylococcus aureus is a problem in a variety of locations and under different management styles. Due to small herd size in Pakistan, dairy cows and buffaloes are hand milked. Contagious mastitis pathogens, in particular S. aureus are reportedly a problem in hand milked dairy herds (Oliver et al., 1975).

A perusal of the literature depicted that S. aureus is one of the most infectious pathogenic bacteria causing bovine mastitis. This organism attacks very quickly to all types of cells in mammary glands so its control is challenging. Mastitis associated with S. aureus lean towards sub-clinical and chronic form that leads to low response to orthodox antibiotic therapy due to several causes including intracellular localization of the organism in mammary gland epithelial cells. This stubborn infection was associated to absenteeism of the body defense mechanism in which various host and bacterial dynamics are involved (Oviedo-Boyso et al., 2007). The chronicity of S. aureus infection is attributed partly at least to the ability of this bacterium to produce biofilm. Staphylococcus aureus is reportedly responsible for more than 80 percent of subclinical bovine mastitis with associated pecuniary loss of US $ 300 per animal per year (De Graves and Fetrow, 1993; Wilson et al., 1997; Karahan et al., 2011).

Staphylococcus aureus usually colonizes in the teat canal initially. After colonization, the bacteria adhere to the epithelium of ducts and alveoli in the gland and starts toxin production. The adherence of bacteria then stimulate macrophage stimulation and migration of neutrophils from blood into the milk which will lead to high somatic cell number (SCC), swelling of the mammary gland, damage to the host defense system and epithelial cells (Cucarella et al., 2004). Mostly, the protracted infections are related with microbial growth as adhesive colonies enclosed by a large exopolysaccharide matrix, establishing a biofilm (Costerton et al., 1999).

3 Biofilm and its Determination
Biofilm is a structural complex of bacteria in which they are enclosed and composed of self-made polymeric matrix. These make connections to inert and free living surfaces which enhance the secure growth in the environment (Prakash et al., 2003). Biofilms are causing more and more problems in daily life like production of different diseases due to contamination of medical equipment, food industry and environ- mental sets because of biofilm’s particular charac- teristics like resistance to UV light, antibiotics, biocide chemicals, amplification of genomic change, changed biodegradability and increased secondary metabolite production (Costerton et al., 1987). As aggregation of cells leads to clump formation due to production of biofilm, bacteria are not susceptible to macrophage facilitated engulfment and become resistant to some antibiotics (Monzon et al., 2002).

Poly-N-acetyl β 1, 6 glucosamine (PNAG), a surface polysaccharide is a key component of the staphylococcus biofilm matrix which is synthesized by proteins encoded by the intercellular adhesion ica operon and it can elicit a protective immune response (Maira-Litran et al., 2004). Another biofilm consti- tuent among mastitis isolates is the Bap protein but it is not so much common (Cucarella et al., 2001).

Dhanawade et al. (2010) investigated the role of biofilm production by S. aureus as vital virulence factor by determining the biofilm contents adopting different phenotypic and genotypic methods. They collected 102 S. aureus samples from subclinical bovine mastitic cases. Frequency of biofilm producers was 48.03% when identified by Congo red agar (CRA) method while it was 36.27% using tube method. In tissue culture plate method (TCP) without and with de-staining, the frequency of this trait was 19.60% and 29.41%, respectively. By using standardized polymerase chain reaction (PCR), 102 S. aureus isolates were investigated for the detection of intracellular adhesion genes, ica A and ica D responsible for biofilm formation. Out of the pool, both genes were present in 36 (35.29%) strains. Considering PCR as a standard test, CRA and TCP without de-staining were the most sensitive and specific tests, respectively. PCR technique was standardized to detect the ica A and ica D genes is trustworthy for identifying biofilm forming potential of S. aureus leading to quick determination of biofilm producing staphylococci.

Mathur et al. (2006) compared the three methods of biofilm detection. To detect the biofilm trait, 152 clinical isolates of Staphalococci were curtained by tissue culture plate (TCP), tube method (TM) and Congo red agar (CRA) method. Of the 152 staphylococcal isolates, 57.8% (88) exhibited biofilm production when TCP method was used and strains were further categorized as high (n= 22; 14.47 %) and intermediate (n= 60; 39.4 %) biofilm producer organisms while 46.0 % (70) isolates were weak or non-biofilm producers. While using TM, it was difficult to discriminate between weak biofilm producers from biofilm negative isolates. The study showed that CRA method does not show a relationship with either of the other two methods for detecting biofilm formation. The results showed that the TCP method was the most profound, precise and reproducible method to detect the biofilm production by staphylococci.

Vasudevan et al. (2003) conducted a study to evaluate in vitro slime production, biofilm formation and presence of genes associated with biofilm production i.e. ica A and ica D in S. aureus isolated from bovine mastitic samples. They used CRA method and 32 out of 35 tested isolates produced slime while only 24 of the microbes were biofilm producer in vitro. However, all of the 35 sequesters contained the ica locus i.e. ica A and ica D genes. The experiment showed the ica genes among S. aureus mastitis isolates were highly prevalent and their presence was not always accompanied with in vitro formation of slime or biofilm. They suggested that phenotypic and genotypic tests may be used in combination for determination of biofilm formation in S. aureus.

Ammendolia et al. (1999) reported that slime-producing strains were amongst coagulase-negative staphylococci in equal proportion as reported in other studies. Unexpectedly, a great proportion of S. aureus strains were able to produce this extracellular substance. But in the latter case, this trait was resilient when there was addition of carbohydrate source in the growth medium. Manifestation of the slime-associated antigen appeared to be species specific characteristic and limited to the S. epidermidis isolates. Its strong relationship with the ability to produce intense biofilm contents showed slime-associated antigen as a possible virulence indicator for S. epidermidis.

Transmission and scanning electron microscopes were used by Christensen et al. (1982) for the detection of slime production by S. epidermidis. The results of this study showed that slime producing strains were enclosed in an adhesive layer on the catheter surface. Perversely, the non-slime producers were not enclosed. It was concluded that slime associated attachment may be a perilous feature in the pathogenesis of S. epidermidis infections associated with implantation of medical devices (e.g. prosthetic cardiac valves, cerebrospinal fluid shunts, orthopedic appliances and intravascular catheters).

4 Biofilm and Disease Production
It has been shown by direct observation of bacteria in natural settings that they usually grow adhered to surface-liquid or liquid- air interfaces and embedded in a self-produced extracellular polymeric matrix (Costerton et al., 1999). It is suggested that firstly the bacteria attached to the surface of ducts and alveoli in the mammary glands and start production of toxins. These attached bacteria enhance macrophage instigation and neutrophils movement from the blood into the milk (resulting in an increase in the somatic cell count), mammary gland swelling, host defense impairment and the epithelial cell damage. The bacteria will reach the basal sub-epithelial cell layers, fix fibrinogen along with other host receptor proteins and establish the infection that eventually becomes chronic (Foster and Hook, 1998).

Mostly long-lasting infections are associated with bacterial growth in the form of adhesive colonies surrounded by a large exopolysaccharide matrix, creating a biofilm (Costerton et al., 1999). Biofilms are resistant to macrophage mediated phagocytosis due to their aggregate size and also become resistant to some antibiotics (Monzon et al., 2002). The extracellular matrix has complex structure that varies between different bacterial species and even within the same species in different environmental circum- stances (Maira-Litran et al., 2004).

Regardless of their heterogeneous composition, exopolysaccharides are important component of biofilm matrix and provide the framework for microbial cells to be inserted into it (Branda et al., 2005). The most common exopolysaccharides are cellulose and β-1,6 linked N-acetylglucosamine. They are the most common components of the biofilm matrix of many different bacteria. In addition to exopolysaccharides, exterior proteins also play an essential role in biofilm formation. Many of the external proteins intricate in biofilm production have several mechanical and practical features in common and therefore, the existence of a group of external proteins has been proposed. Biofilm associated protein (Bap) was explained in a S. aureus bovine mastitis isolate as the first member of this group (Lasa and Penades, 2006).

Maturation of biofilm provides further protection to bacterial cells due to production of another slime layer, glycocalyx. The chemical structure of these slime films is still unknown but evidence suggested that it is principally composed of hydrated polysaccharides. The nutrient supply inside the biofilm becomes limited which results in decreased growth potential of the bacterial biofilms and discrete drift through channels across the biofilm aim to preserve profusion (Stoodley et al., 2002). Some other dynamics like oxygen profusion, carbon source, osmolarity and internal pH control the biofilm maturation. When the biofilm attained a serious mass, a vibrant stability is reached then the outer most layers begin to produce free living organisms. These bacteria are free to sneak away the biofilm and to inhabit other surfaces (Dunne, 2002).

5 Biofilm and Antibiotic Resistance
The bacteria carrying this typical peculiarity are highly resilient to antibiotics. This type of resilience can involve different reasons including (a) exopoly- saccharides (EPS) produced by the biofilm producing organisms which provide them physical/ chemical barrier and prevent the adsorption of antibodies or different antibiotics. EPS also bind the antibiotics that are attempting to reach across the biofilm because these are negatively charged and act as ion-exchange resin, (b) bacteria embedded in biofilm results in decreased growth rate of the bacteria and smaller size of the cells which make the cells less pervious to antibiotics, as all antimicrobial drugs are more operative in destroying the fast growing cells (Thien and O’ toole, 2001), (c) antibiotic degrading enzymes are also trapped in the biofilm structure and inactivate the incoming antibiotic molecules effectively, like in Pseudomonas aeruginosa β-lactamase is 32-folds higher in biofilm producing cells than the same strain grown planktonically and (d) the cell wall protein structure of the bacteria in biofilm is transformed up to 40 % from that of free living bacteria (Potera, 1999).

The membrane of the bacteria in biofilm have greater tendency to propel out the antibiotics before they impair or even targets of the antibiotics on cell surface may disappear (Potera, 1999); most of the antibiotics are deactivated by the reactive oxidants such as hypochlorite and H2O2 which are produced by oxidative burst of phagocytic cells. This mechanism of deactivation is more in the outer layers of biofilm than inner ones because the oxidants have poor penetration across the biofilm layers that might be the reason of failure of the phagocytic cells to extinguish biofilm microorganisms (Thien and O’ toole, 2001; De Beer et al., 1994); biofilms also provide an ideal place for the interchange of extra-chromosomal DNA which is accountable for antibiotic resistance, virulence dynamics and environmental persistence competences at enhanced rates leading to an ideal environment for development of drug resistance (Ghigo, 2001).

Ciftci et al. (2009) conducted a study for the determination of methicillin resistance and slime production of S. aureus in bovine mastitis. A triplex PCR was targeting 16S rRNA, nuc and mecA genes for recognition of staphylococcus species, Staphy- lococcus aureus and MRSA, singly. For determination of slime production, a PCR test targeting ica A and ica D genes was performed. In the expermient, a total of 59 strains were confirmed as Staphylococcus aureus by both conservative tests and PCR, while 13 of them were found to be methicillin resistant (MR) and mec A gene was present in only 4 (30.7%) isolates. Even though out of 59, 22 (37.2%) S. aureus isolates were positive for slime production in CRA while in PCR study, only 15 were positive for both ica A and ica D genes. Sixteen and 38 out of 59 strains were positive for ica A and ica D genes, respectively. Only 2 out of 59 strains were positive for MR and mucus (slime) production, phenotypically, suggesting lack of corre- lation between MR and slime production in these isolates. In conclusion, the optimized triplex PCR in this study was useful for rapid and reliable detection of methicillin resistant S. aureus. It also showed that only PCR targeting ica A and ica D may not be sufficient to detect the slime production and further studies targeting other ica genes should be conducted for accurate evaluation of slime production characters of S. aureus strains.

Melchoir et al. (2006) reviewed role of biofilm in recurrent mastitis. They suggested that production of mammary gland infection where symptoms are trailed by subclinical disease is relatively parallel to the signs described for infective maladies in humans. They also reviewed that phase variation between biofilm production and planktonic bacteria is caused by genomic alteration which makes bacteria less hostile and more resistant to host’s immune response as well as resistant to effective antimicrobials.

Turkyilmaz and Eskiizmirliler (2006) conducted a trial to determine the production of slime factor and antibiotic resistance in staphylococcal isolates sequestered from different clinical samples of animal origin. They collected 180 staph spp. (90 positive for coagulase production and 90 staphylococci negative for coagulase production). They determined the slime production by Congo red agar method (CRA), microplate method (MP) and standard tube (ST) method. The rate of slime production by all staphy- lococci scrutinized by CRA, MP and ST methods was 61.1%, 55.5% and 50.5%, correspondingly. The presence of antibiotic resistance was evaluated by the agar disk diffusion technique. The proportion of resistance against penicillin, methicillin, ampicillin and gentamycin in slime-producing (SP) staphulococci was 49.0, 24.5, 23.6 and 13.6%, individually. For non-slime producers (NSP) it was 42.9%, 15.7%, 14.2% and 12.9%, respectively. The correlation showed that SP isolates had more resistance to antibiotics as equated to those of NSP. All the strains were prone to vancomycin. The fallouts of the trial revealed that there was no statistically substantial variance concerning the tests applied (CRA, MP and ST tests; X2 = 0.28).

6 Vaccination against Staphylococcus aureus mastitis
The current practices to control mastitis seem to be ineffective and usually result in persistent and chronic infections with pockets of contagion in the herd for long time. Vaccination against S. aureus appears to be rational for the control of disease (Pellegrino et al., 2010).

To determine the effectiveness against Staphylococcal mastitis in ruminants, various vaccination tactics including surface polysaccharides have been assessed like, inactivated bacteria and toxoid (Opdebeeck and Norcross, 1984; Athar, 2007), S. aureus bacterin- toxoid adjuvanted with MontanideTM (Yousaf, 2009), bivalent S. aureus and Strep. agalactiae bacterin- toxoid adjuvanted with aluminium hydroxide (Ahmad and Muhammad, 2008), contagion encased by a mucoid material (likely a biofilm matrix) named a pseudo-capsule (Watson and Devies, 1993), different types of capsular polysaccharide (CP) like CP5, CP8 and CP336 associated to protein carriers (Von Eiff et al., 2007) and a combination of mucus (slime) in liposomes, toxoid and various deactivated pathogens (Amorena et al., 1994). Numerous field experiments with these and further vaccines together with crude extract of encapsulated bacteria in aluminium hydroxide (Giraudo et al., 1997) have also been conducted. Recently, a considerable protection response was observed using bacterin-toxoid prepared from biofilm producing isolate of S. aures in dairy cattle (Rashid, 2011) and in rabbits (Raza, 2012). Vaccines showed a reasonable degree of fortification against S. aureus mastitis (Pereira et al., 2011).

7 Conclusion
Mastitis is classified under one of the most common problems of dairy animals throughout the world and many etiological agents are responsible for this disease, Staphylococcus aureus is one of the major causes which are associated with mastitis. Biofilm production by the microorganisms is deliberated as a significant virulence factor responsible for adhesion of these microorganisms with living or non-living surfaces. Biofilm contents make a community of bacteria and help them to survive in unfavorable environmental conditions. Staphylococcus aureus isolates which produce biofilm lead to chronic mastitis in dairy animals as it makes the bacteria resistant to most of the antibiotics and natural defense of the body (phagocytosis). The intramammary infection due to biofilm producer S. aureus is difficult to treat even with intra-mammary antibiotics so proper conside- rations should be given to the infections produced by biofilm producing bacteria. Development of an effective vaccination against the bacteria which produce biofilm may provide success to control such type of perilous infections.

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