Detection of Biofilm Genes (IcaA and IcaD) in Staphylococcus spp

One hundred thirty one clinical bacterial samples were collected from skin, pus, urine and blood from patients at Al-Salam General Teaching Hospital in Mosul city during March until July/ 2012, The present study included identification using API ID 32 STAPH system to the species Staphylococcus. aureus, Staph. lugdunensis, Staph. epidermidis, Staph. hominis, Staph. auricularis and Staph. haemolyticus, Biofilm formation genes at icaA (188 bp.) were found in 35.7% of Staph. aureus, Staph. hominis, and Staph. xylosus but 42.8 % of Staph. aureus, Staph. epidermidis, Staph. hominis, and Staph. xylosus contained icaD (198 bp.) gene only.


INTRODUCTION
Members of the genus Staphylococcus are gram positive cocci that occur singly, in pairs, tetrads, short chains (three or four cells), and irregular grape-like clusters. They are non-motile, non-spore-forming, and usually un-encapsulated or have limited capsule formation. Most species are facultative anaerobes and positive to catalase test (Dworkin et al., 2006).
Staphlyococcus spp. can form a biofilm on nearly any synthetic polymers used as prosthetic devices. In addition, they can bind to blood, matrix proteins, and human cell receptors. Staph. epidermidis is regarded as a leading species in causing chronic polymer-associated clinical infection (Mack et al., 2013).
Colonization of tissues or implants is frequently a first step in infection; and biofilm formation plays a crucial role. One can imagine that the observed genetic variability in biofilm formation, which ranges from super to non-biofilm formers, underlies a well-directed survival and M 1 2 3 4 5 6 7 spreading program that we are only gradually learning to understand. Once a Staphylococcal biofilm is formed, the cells are nearly invulnerable. They are then shielded from the immune system and resist antibiotic treatment. Therefore, it is very important to understand the various aspects of biofilm formation, it is suggested that to develop more specific ways of overcoming Staphylococcal resistance in chronic infections (Pitts et al., 2003 andRomeo, 2008).

MATERIALS AND METHODS Sample Collection:
One hundred thirty one samples were collected clinically from patients (skin, pus, urine and blood) of both sexes sufferings from bacterial resistant to most antibiotics at Al-Salam General Teaching Hospital, in Mosul city from March to July, 2012. Samples were collected by transport media and sterile swabs damped in normal saline. Isolation and identification were carried out using standard microbiological methods all isolates were identified by API ID 32 STAPH system (Bio Merieux).
The program is first 2 min at 94°C , followed by twenty five cycles at 94°C for 1 min, 52°C for 1 min, and 72°C for 2 min and followed with final extension at 94°C for 4 min and cooling 4°C for 3 min. The presence of a PCR bands were confirmed by 1% (wt/vol) agarose gel electrophoresis and visualization with ethidium bromide (Mariana et al., 2009).

RESULTS AND DISCUSSION
The results obtained from API test were as follows: four isolates were Staph. aureus; three isolates were Staph. lugdunensis; two isolates were Staph. epidermidis; two isolates were Staph. hominis; one isolate was Staph. auricularis and one isolate was Staph. haemolyticus, the ica locus which is required for the synthesis of the polysaccharide intracellular adhesion (PIA) of Staphylococcus plays a role in cell to cell interactions during biofilm formation and is predominantly present in clinical isolates (Ando et al., 2004).
Five isolates contain icaA gene. Mariana and coworkers, (2009) stated that Staph. aureus and Staph. epidermidis contained this gene, and Nasr and colleagues, (2012) found icaAD genes in (32.0%) of Staphylococcal isolates, biofilm formation causes catheter associated and nosocomial infections with the presence of icaA and icaD genes (Arciola et al., 2001).

icaD:
The species Staph. hominis, Staph. epidermidis, Staph aureus and Staph xylosus contain the gene of biofilm which form 42.8% (icaD 198 bp.) Fig. (2). Yazdani and their collaegues (2006) and Nasr and their workers (2012) indicated a high prevalence of the icaAD gene among Staph. aureus isolates and their presence is not always associated with in vitro formation of slime or biofilm, Cafiso and coworkers, (2004) recorded that (35%) of Staphylococcus spp. contained icaA and icaD genes and some of them carried icaD gene only, Satorres and Alcaraz, (2007) found that (35.2%) and (48.4%) of Staph. aureus and Staph. epidermidis strains contained icaA and icaD genes respectively, Mariana and colleagues, (2009) recorded that all Staphylococcus spp. under test were containing this gene, Mahmoud Gad and coworkers, (2009) stated that both icaA and icaD genes were parts of one operon and so the entire operon was either present in or absent and they found that all Staphylococci isolated from catheter segments showed a higher extent of biofilm production than those isolated from urine samples.