DETECTION OF BIOFILM FORMATION AND ITS ASSOCIATED GENES IN COAGULASE NEGATIVE STAPHYLOCOCCI ISOLATED FROM MILK OF COWS AFFECTED WITH SUBCLINICAL MASTITIS

Assiut University web-site: www.aun.edu.eg

DETECTION OF BIOFILM FORMATION AND ITS ASSOCIATED GENES IN COAGULASE NEGATIVE STAPHYLOCOCCI ISOLATED FROM MILK OF COWS AFFECTED WITH SUBCLINICAL MASTITIS

AMAL AWAD

Bacteriology, Mycology and Immunology Department, Faculty of Veterinary Medicine,

Mansoura University, Mansoura 35516, Egypt.

Received: 28 June 2017;       Accepted: 30July2017

INTRODUCTION

Bovine mastitis is a multifactorial disease that affects dairy cows and causes serious economic losses in dairy animals worldwide (Bradley et al., 2002). Staphylococci are considered as a major cause of mastitis in dairy cows worldwide. Coagulase negative staphylococci are usually considered to be minor pathogens and this group is heterogenic which contains more than 50 species and subspecies. However, CNS have become the most common mastitis causing agents in many countries (Dieser et al., 2014 and Vanderhaeghen et al., 2014). CNS are found on normal skin of the udder teat and on milkers' hands which form part of the normal flora. This may cause opportunistic infection via penetrating secretory tissues. They usually isolated from subclinical mastitis but have also been found in clinical mastitis (Pyörälä and Taponen 2009; Vanderhaeghen  et al.,  2014).  This  may be  resulted

Corresponding author: Dr. AMAL AWAD

E-mail address: amalabdo@mans.edu.eg

Present address: Bacteriology, Mycology and Immunology Department, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt

in tissue damage and decrease in milk production. (Schukken et al., 2009). Furthermore, CNS may cause insistent bovine intramammary infections that exist for long period especially in the absence of mediation (Gillespie et al., 2009).

A biofilm is an organized community of bacteria that are surrounded by a self-created, polymeric matrix that stick to either living or inorganic surface and creates a protected method of growth that permits its existence in a unfavorable condition of growth (Costerton et al., 1999). Whereas biofilms might be not affect the severity of the disease (Tremblay et al. 2013 and Osman et al., 2015), they may facilitate the adhesion and colonization of CNS on the mammary gland tissues, which may leads to persistent infections (Tremblay et al., 2013). Moreover, biofilm-formation could possibly inhibit antimicrobial treatment as it makes CNS strains growing inside biofilms more resist to the frequently used antimicrobials. (Tremblay et al., 2014).

Polysaccharide intercellular adhesin (PIA) is the primary determinant of the accumulation phase in biofilm formation. PIA Production is assisted by the ica ADBC operon, and strains contain this cluster are considered as biofilm producers (Cramton et al., 1999). Biofilm associated protein (Bap) which is encoded by the bap gene may also responsible for formation of biofilms. Biofilm associated proteins have been recognized in staphylococcus species (Götz 2004). Bap shared in biofim formation as it involved in the primary attachment stage and responsible also for cell-to-cell aggregation with PIA (Cucarella et al., 2001). The overall aims of this study, were to conduct a phenotypic characterization of coagulase negative staphylococci isolated from bovine mastitis, testing the ability of CNS to form biofilm in vitro in addition genotypic characterization of some biofilm associated genes (icaA, icaD, bap).

MATERIALS AND METHODS

Samples collection

160 individual quarter milk samples were collected from cows with subclinical mastitis during the first half of 2016 from two different dairy farms located in Dakahlyia provinces, Egypt. The numbers of the cows in each farm ranged from 300 to500 animals. California masttis test (CMT) was used for screening the milk samples for the existence of subclinical mastitis (CMT positive) following the protocol described by NMC (1990) and Quinn et al. (2002). All samples were collected following the standard milk sampling techniques (NMC 1990). In brief, hands were washed with clean water and detergents, udder and teats were cleaned with water and dried. The teat on the far side of the udder is cleaned first and then those of the near side (Nibret et al., 2011). The teat ends subsequently were cleaned with a piece of cotton soaked in 70% ethyl alcohol. After discarding the first 2-3 milking streams, of milk (10mL) was collected from subclinical mastitic cases into sterile Falcon tubes. The samples were transferred in an icebox to the laboratory where they kept at 4ºC until examination within 24h.

Bacteriological examination

Bacterial examination was done according to Quinn et al. (2002). Briefly, loopful of milk sample was streaked on the surface of blood agar base supplemented with7% sheep blood (Oxoid, UK). The inoculated blood agar plates were incubated at 37 ºC for 24 hr under aerobic condition. The growing colonies were examined for their culture characters including, size, shape, color of colony, and their hemolytic activity. Staphylococcus presumptive colonies were subcultured onto the surface of nutrient agar plate (Oxoid) for farther investigation.

Identification of CNS

Gram stain was used for morphological characterization of the suspected colonies. Subsequently, the suspected staphylococcus colonies were subjected to biochemical tests including, catalase and coagulase tests. In addition, a group of biochemical reactions were used for differentiation of Staphylococci from Micrococci including, oxidation and fermentation of glucose, resistance to bacitracin (0.04 U) and susceptibility to furazolidone (100 µg) (Baker 1984). For CNS identification, A set of biochemical reaction were used following the reference method proposed by Kloos and Schleifer (1975) and Bannerman (2003), including, utilization of sugar (sucrose, mannitol, arabinose, lactose, fructose, ribose and mannose xylose, xylitol,  maltose, trehalose), urease production, haemolysis on blood agar, nitrate reduction, ornithine decarboxylase and novobiocin resistance. Furthermore, staphylococcus isolates were confirmed by API Staph system (bioMérieux, Marcy l’Etoile - France).

Detection of biofilm formation in vitro

For detection of biofilm formation in vitro by CNS species using polysterne surface of microtiter plates, a 96 well microtiter plates were used following the protocol provided by Stepanović et al. (2007).

DNA extraction

For DNA extraction, QIAamp DNA Mini kit (Qiagen, Germany, GmbH) was used following the manufacturer’s instruction with minor modification. In brief, 200 µl of the sample was incubated at 56^OC for 10 min with 200 µl of lysis buffer and 10 µl of proteinase K. Subsequently, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged and nucleic acid was eluted with 100 µl of elution buffer.

PCR on biofilm associated genes.

Biofilms associated genes (icaA, icaD, bap) were evaluated by PCR. Oligonucleotide primer sets (Metabion, Germany) used in this study are listed in table (1). PCR reaction was conducted in a final volume of 25µl consists of 12.5 µl of Dream Taq Green PCR Master Mix (2X) (Thermo Scientific), 5 µl of DNA template,1 µl of each primer of 20 pmol concentration and 5.5 µl of nuclease free water. PCR reactions were conducted in 96 well Applied Biosystem thermal cycler. PCR products were separated by electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH). The gel was visualized under UV and photographed.

RESULTS

In this study, a total of 160 quarter milk samples were collected from cows with subclincal mastitis after screening with CMT, the overall prevelance of CNS was 43.12% (69/160). The most frequently isolated species was S. chromogenes (17/69, 24.63%) followed by S. epidermidis (15/69, 21.72%) S. xylosus (14/69, 20.28%), S.haemolyticus (10/69, 14.49%), S. hominis (7/69, 10.14%), S. simulans (4/69, 5.79%) and S. saprophyticus (2/69, 2.89%) (Table 2).

The ability of CNS to form biofilms was evaluated by microtiter plate assay. The frequency of biofilm formation by CNS was 81.15% (56/ 69). Among CNS isolates, 53.57% (30/56) were strong, 32.14% (18/ 56) were moderate and 14.28% (8/56) were week biofilm producers. S. chromogenes had the highest ability to form biofilms (28.57%) followed by S. epidermidis and S. xylosus (19.64%), S. haemolyticus (16.07%), S.hominis (8.92%), S. simulans (5.35%) and S. saprophyticus (1.78%) (Table2). S. chromogenes was also the predominate strong biofilm producer (33.33%), followed By S.epidermidis (26.67%), S.xylosus (20%), S.haemolyticus (13.33%) and S. homins (6.67%).

Concerning the distribution of biofilms associated genes, the frequency of icaA was 36.23% (25/69) while, icaD frequency was 47.82% (33/69) and both of them were identified in 20 (28.98%) isolates. In the current study, all icaD positive genes were also able to produce biofilms in vitro. In addition, there are 7.25% (5/69) of icaA-positive failed to form biofilms. Finally, bap gene couldn’t be identified in all CNS (Table 3).

Photo 1: Agarose gel electrophoresis showing the amplification of icaA gene at 1315bp. Lane neg.: negative control. Lane L: 100 bp DNA ladder.

Photo 2: Agarose gel electrophoresis showing amplification of icaD gene at 381bp. Lane Neg. negative control, Lane Pos: positive control, Lane L: 100 bp DNA ladder.

Table 1: Primers sequences, target genes, amplicon sizes and cycling conditions.

Table 2: Distribution of CNS species and their biofilms production.

Table 3: Patterns of in vitro biofilms formation and biofilms associated genes.

DISSCUSION

CNS are considered as the most commonly recovered bacteria from intramammary infections in several countries (Gentilini et al., 2002; Sampimon et al., 2009). For instance, In Argentina, CNS are the most prevalent pathogens (52.1%), followed by S. aureus (21.3%) (Dieser et al., 2014). In this study, the prevalence rate for CNS was 43.12% (69/160). Compering to our findings, the prevalence rate for CNS in Eastern Ethiopia was 34.2% (Zeryehun and Abera, 2017) from mastitis. The most frequently isolated species was S. chromogenes (17/69, 24.63%) (Table 2). In agreement with the obtained results, S. chromogenes has been recorded as a predominant species in several studies conducted worldwide, in Canada (48.27%; Condas et al., 2017), in Argentina (41.4%; Felipe et al. 2017 and 44.4%, Srednik et al. 2017), in Belgium (46.4%; Supré et al., 2011), USA (36.3%; Sawant et al., 2009) and the Netherlands (36%; Sampimon et al., 2009).

Biofilm formation plays an important role in the protection of bacterial species from harsh environmental condition, which leads to their persistence, and results in long lasting infection. (Cucarella et al., 2004). Biofilms protect the bacteria from the action of the immune system components and hindering the action of phagocytes (Fox et al., 2005), as well as, biofilm acts as a barrier against antimicrobial agents (Stewart, 1996). In this study, out of 69 CNS isolates, 56 isolate able to form biofilms in vitro (81.15%). These findings were in agreement with several studies conducted worldwide (Tremblay et al., 2013; Felipe et al., 2017; Srednik et al., 2017). While, a lower percentages (31.3%) were recorded by Simojoki et al. (2012).The diversity in the results of invitro biofilms formation by CNS may be attributed to the differences in the type of the used growth medium and its chemical composition as well as the condition of growth which might be influence the expression of bacterial genes and accordingly biofilm formation of staphylococci (Fredheim et al., 2009). In this study, S. chromogenes had the highest ability to form biofilm and also S. chromogenes was the predominate strong biofilm producers (33.33%). The same results were recorded by Felipe et al. (2017), while, Srednik et al. (2017) found that S. haemolyticus was the most common biofilm producer strains, and Tremblay et al. (2013) found that S. xylosus had the highest ability to form biofilm. The diversity in the results may be attributed to the differences in the distribution of CNS species in different countries and the intraspecies variations (Tremblay et al., 2013; Oliveira et al., 2015). Additionally, intraspecies variations in biofilm formation was explained by Ajitkumar et al. (2013) who identify three different genotypes within S. chromogenes.

Biofilm formation by Staphylococcus species has been associated with several genes (Simojoki et al., 2012; Tremblay et al., 2013). In this study, the frequency of icaA and icaD were 36.23% (25/69) and 47.82% (33/69) respectively, and both of them were identified in 20 (28.98%) isolates (Table 3). Compering to the obtained results, Simojoki et al. (2012) and Srednik et al. (2017) found very low frequency for icaA. While, Felipe et al. (2017) observed amplification for icaA and icaD in 73.2% of the isolates. In Egypt, in a study conducted by Darwish and Asfour (2013) on a total of 68 CNS isolates, the prevalence of icaA, icaD genes was 5.9%, 47.1% respectively. The percentage of biofilm positive/ ica- negative strains was 33.33% (23/69) (Table 3). However, it was very high (96.6%) with Srednik et al. (2017) and very low (13.8 %) with Osman et al. (2015). In this study, all icaD positive genes were also capable of producing biofilm in vitro which in agreement with Liberto et al. (2009) and its presence may confirm the role of ica in adhesion mechanism. However, in this study, there are 7.25% (5/69) of icaA-positive failed to form biofilm in vitro (Table 3). These finding were in agreement with Ruzicka et al. (2004) who demonstrated that 20% of strains with ica genes did not express invitro biofilm formation. The absence of phenotype inspite of the presence of ica could be attributed to  several reasons which may affects ica expression such as, insertion of sequence elements or point mutations (Götz, 2004), or the action of the ica Rrepressor which inactivate the ica operon (Conlon et al., 2002).

Regarding bap gene, non of CNS isolates harbored bap gene in this study which was in agreement with the findings of Arciola et al. (2003) and Serray et al. (2016). While, Srednik et al. (2017) identified bap gene in a few isolates (n=3), Felipe et al. (2017) identified bap gene in 13.4% (11/82) and Darwish and Asfour (2013) identified bap gene in 4.4% of the examined CNS isolates. Also, Tremblay et al. (2013) found that there was a difference in the distribution of the bap gene among CNS species, as they found that bap gene was confined to S. xylosus with a percentage of 92%. The discrepancies between studies may be attributed to geographical differences and the distribution of CNS species in the examined samples.

CONCLUSION

CNS species may persist in the mammary gland due to biofilm production and consequently, resulted in long lasting infection. In this study, 81.15% of CNS were capable of producing biofilms invitro and there is an association between biofilm-producing strains and presence of ica genes which suggests that ica genes plays an important role in the pathogenesis of infection and as a result revealed their important role of ica genes as a virulence determinates for staphylococci.

CONFLICT OF INTERESTS

No conflict of interests is declared.

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