DETECTION OF INTRACELLULAR ADHESION GENE (icaA and icaD)AND BIOFILM FORMATION STAPHYLOCOCCUS AUREUS ISOLATES FROM MASTITIS MILK OF SHEEP AND GOAT

In the present study, a total of 150 mastitis milk samples were collected from sheep and goat (75 for each one) and were analyzed for the presence of S.aureus.. The obtained results indicate that this bacterium observed in 20% of these samples (21.33% from sheep and 18.66% from goat) .The study of antibiotic susceptibility test to 9 different antibiotics showed that S. aureus was 100% resistant to penicillin and 100% sensitive to vancomycin, gentamycin , clarithromycin and chloramphenicol . whereas for cefoxitin (alternative to methicillin) resistance was 47%.There were a variable sensitivity percentage for the rest of antibiotics: Tetracycline (70%), Ciprofloxacin (80%) , Clindamycin (83%). The biofilm-forming ability of S. aureus was evaluated via microtiter plates and the result revealed that, all the studied isolates were either moderate biofilm producer or weak biofilm producer while the non-biofilm producer and strong biofilm producer were not detected among the tested isolate.The relationship between biofilm formation and resistance to methicillin showed there as no significant differences (P>0.05) in the percentage of weak and moderate biofilm producers between MRSA and MSSA isolates . PCR analysis was applied to DNA extracted from S.aureus isolates from milk samples .The results of PCR assay revealed that all S.aureus isolates gave positive results for both icaA and icaD genes (100%) with Product size 151 and 211 bp, respectively. study indicate that biofilm producing S.aureus have a major role player on the occurrence of mastitis .In addition, there was high prevalence of MRSA isolates (47%) in mastitic milk at the study area


INTRODUCTION
Mastitis means inflammation of the udder and is a common disease among dairy animals worldwide. It is often associated with bacterial intramammary infections (IMI),influence milk quality and yield negatively, therefore, mastitis is of major economic concern for the farmer (1,2).
Staphylococcus aureus is generally regarded as one of the major etiologic agents of mastitis in dairy animals (3,4,5). This pathogen has the potential to develop resistance to almost all the antimicrobial agents used for the management of the disease (3,5,6). S. aureus is also well known for its tolerance to a wide range of adverse circumstances. This tolerance is related to diverse genetic capabilities including the ability to form biofilms in the host, which contributes to the resistance of this microorganism against antibiotics (7,8).
Four proteins including IcaA, IcaD, IcaB and IcaC encoded by the icaADBC operon are associated with the production of PNAG. IcaA and IcaD are the most important proteins for the production of PNAG (11). Carriage of the ica operon is a characteristic of most clinical S. aureus strains (12) Production of the extracellular polysaccharide in S. aureus is currently the best understood mechanism of biofilm development, this ica operon can be further differentiated to the icaA, icaD, icaB and icaC loci each responsible for relevant pathogenic and virulent factors involved in polysaccharide intercellular adhesin synthesis (13).
This study aimed to determine the isolation rate of S. aureus from sheep and goat mastitis cases, potential of these isolates to carriage ica operon and it is phenotypic evaluation of antibiotic susceptibility and biofilm formation.

Samples collection
A total 150 milk sample were collected from clinical and subclinical mastitis of sheep and goat (75 for each one). The samples were collected after cleaning the udder by a piece of cloth then using cotton moistened by alcohol 70% and removing the first flowage of milk and collecting 10 ml in sterile tube, transported with ice box. The subclinical mastitis was confirmed with California mastitis test according to (14). From each sample, 1 ml of milk was pipetted into sterile microcentrifuge tubes and centrifuged at 5000 rpm for 5 min at room temperature. The supernatant was then discarded and the pellet was directly inoculated onto plated of mannitol salt agar (14).

Staphylococcus aureus isolation and identification.
Milk samples were inoculated on mannitol salt agar and incubated at 37°Cfor 24hrs. All colonies from primary cultures were purified by subculture on brain-heart infusion (BHI) agar and then inoculated onto MSA and incubated at 37°C for 24 h. (15).

Bacterial "DNA extraction" and PCR Method:
PCR technique was performed for detection ,icaA gene and icaD gene in "Staphylococcus aureus" isolated from mastitis milk samples by following steps:-

1-DNA extraction:
Genomic DNA of S.aureus isolates were extracted by using Genomic DNA Kit ( Geneaid . U S A) according to manufacturing instructions

2-Nano drop:
The extracted DNA was estimated by "nanodrop device" at 260 /280 n m, and then kept at deep freezer until used in PCR method.

3-Primers:
The PCR primers that used in this study for detection icaA and icaD genes were design by (20).These primers were provided by (Bioneer company, Korea) ( Table   2).

6-PCR product analysis:
The PCR products (151 b p and 2011 b p) were examined by electrophoresis in a 1.5% "agarose gel" using "1X TBE buffer", stained with "ethidium bromide", and conceive under "gel documentary".

Bacterial isolation and identification
According to the results of isolation and identification there were ٣٠ ( 2٠%) isolates ofS.aureus( Table 4). The percentage of S.aureus isolates observed in sheep was 21.33% while in the goat (18.66%). There as no significant differences (P>0.05) in the percentage of S.aureus isolates between these mastitc milk samples .  The identification was confirmed with automated VITEK-2 compact system using GP cards with ID massage confidence level as excellent (probability percentage from 95-99).

Antibiotics susceptibility test
After the identification of S. aureus, susceptibility test was performed for all S. aureus (30 isolates) by disk diffusion method to examine 9 different antibiotics as clarified in table (5). The results showed that, the highest resistant rate was against pencillin (100%) followed by cfoxitine (50%), tetracycline (30%), clindamycin , ciprofloxacin (22%) and clarithromycin (4%) .On the other hand, all the tested isolates showed 100% sensitivity to A B vancomycin, gentamycin and Chloramphenicol. There was a significant difference among the antibiotics resistancy (P< 0.01).

Biofilm formation assay by micro titer plat.
The ability of S. aureus sterilized 96-well polystyrene microtiter plates and then absorbance was determined at 580 nm in an ELISA reader for the determination of the degree of biofilm formation for studied isolates that adhered on the surface of represented the degree of the biofilm thickness that formed by the studied isolates on the surface of the microtiter well. All and the results obtained are categor biofilm forming capacity: weak or non producers (OD490nm 0.064 of the present study revealed that, all the tested isolates were found to be biofil producer at different level (F (20%) isolates were moderate biofilm producer and the rema weak producer,.There were significant differences (P<0.05) in the percentage of weak and moderate biofilm formation between sheep and goat isolates. no significant differences (P>0.05) in the percentage of weak and moderate biofilm formation between MRSA and MSSA isolates ( Table 7). study revealed that, all the tested isolates were found to be biofil producer at different level (Fig 2). As shown in (table 6), out of a total 30 tested isolate, 6 %) isolates were moderate biofilm producer and the remaining isolates 24 (80 ak producer,.There were significant differences (P<0.05) in the percentage of weak moderate biofilm formation between sheep and goat isolates.Moreover, There were no significant differences (P>0.05) in the percentage of weak and moderate biofilm ation between MRSA and MSSA isolates (Table 7).

Detection of icaA and icaD gene.
The PCR analysis was applied to DNA extracted from S.aureus isolates from milk samples and the results of PCR assay revealed that all S.aureus isolates gave positive results for both icaA and icaD genes (100%) with Product size 151 and 211 bp, respectively (Fig 3 and 4). In the present study the isolation rate of S.auresus froim mastitis cases in ewes was 21.33%.This finding in agreement with the previous study (21) in which the isolation rate On the other hand ,the current result may appear higher than the result of(23-25), who isolate S.aureus from ovine mastitis in percentage 6.6%, 11.8% and 6.2 % respectively, and lower than results of (26,27), who recorded a percentage 90%, 40%, respectively.

Antibiotic susceptibility test
All the S.aureus isolates were resistance to penicillin and sensitive to vancomycin ,gentamicin ,clarithromycin and chloramphenicol .
The high sensitivity rate twoard vancomycin and chloramphenicol in the current result may belong to low rate of usage in the animals and human host.
In the present study, cefoxitin was used for detection MRSA strains. According to (18)  Methicillin resistance is clinically the most important, since single genetic element can convers resistance to most commonly prescribed class of antimicrobials-the beta lactam antibiotics, which include penicillins, cephalosporin and carbapenems (44,45).
The reason behind continuous increasing in resistant to β-lactam antibiotics is caused by the overuse or misuse of these antibiotics and by the use of poor quality antibiotics. It also results from natural genetic changes, or mutations, within the organisms that cause The results of the present study showed that the susceptibility of animals isolate against chloramphenicol, gentamicin,clarithromycin and ciprofloxacin were 100% while, the sesnsitivity against tetracycline 70%Ciprofloxacin 80% and clindamycin83% .This finding in agreement with the previous study (21) who found the sensitivity of S.aureus isolated from dairy animals to clarithromycin , ciprofloxacin ,gentamicin were 100% ,100% and 94.1%.Moreover ,the present result were compatible with local study of (35)who found the sensitivity rate of S.aureus was 100% to gentamicin and ciprofloxacin , 94.5% to erythromycin and clindamycin and 89.9% to doxycycline . However , the local study of (47) showed slightly higher rate of resistance to gentamicin,clarithromycin and ciprofloxacin in a percentage of 29% ,12% ,9% ,respectively.

Biofilm Formation
The isolated S. aureus were evaluated for biofilm formation capability using phenotypic screening as well as molecular detection of icaA and ica D genes. Microtiter plate (MTP) showed that, 30/30 isolates (100%) were able to form biofilm .In addition,all S.aureus isolates were investigated for biofilm associated genes, icaA and icaD.
Molecular investigation revealed that both icaA and icaD genes were present in the 100% of isolates.
These data are in accordance with those reported by (48)who detected icaA and icaD in all S. aureus isolates by PCR techniques. Similar results were obtained by (49) who found that all the isolates were biofilm producing and contain ica locus .
The current results also were compatible with the studies of (50), (51).Whom found all clinical isolates of S. aureus were biofilm producer and positive for both icaAand icaD genes .In addition ,the present study are in line with local study of (8)who found 94.117%of biofilm production in strains of S. aureus isolates from bovine mastitis. On the other hand , slightly lower percentage of biofilm production were reported by the study of (35) reported that, 80.6% of S.aureus isolates were biofilm positive when tested by MTP method.
The present result close to many worldwide studies such as (52) who found all strains tested were biofilm producer by MTP and 97% of them harboring icaA and icaD gene. (53) who found all 32 S. aureus isolates harbored the icaA and icaD genes.
However, our results are in contrast with the data reported by (54), who detected icaA and icaD genes in only (12.5%) of 23 S. aureus isolates and (55) who detected icaA and icaD genes in 70% of S. aureus isolates. The variations in the presence of icaAD genes from different studies might be due to the heterogeneity in the genetic origins of S.aureus (55) In the present study, a high percentage of agreement (100%) was observed between the genotypes and phenotypes of isolates, determined by PCR and MTP, respectively.
Broad applicability, reliability and high reproducibility of the MTP were previously verified for bacterial biofilms (56).
On the other hand , failure of S. aureus strains that possess the ica locus to form biofilm has been reported in vitro (53) and biofilm producing S.aureus that lack ica operon also reported by many studies such as (57,58).These results suggest that biofilm production is regulated by the interaction of different regulatory mechanisms and the expression of ica genes is strongly influenced by environmental factors such as glucose, temperature, osmolarity, and growth in anaerobic conditions (59). Indeed, transcriptional regulation of the ica operon is complex, involving the interdependent and independent activity of several activators and repressors. Differential transcriptional regulation of the locus and/or putative ica-independent biofilm mechanisms can influence biofilm production phenotype (60). Insertional inactivation and point mutations in the ica locus were reported as other plausible mechanisms to give rise to biofilm-negative variants in S.aureus (61). Thus, the difference between phenotypic and genotypic characterization may be due to the heterogeneity in the genetic origins, and not because of the presence or absence of genes required for the biofilm formation.Therefore, a combination of phenotypic and genotypic assays should be employed for improved confidence in identifying biofilm-producing S. aureus isolates.
In the present study the statistical analysis showed there was no significant differences (P>0.05) in the percentage of weak and moderate biofilm formation between MRSA and MSSA isolates.The current result agreed with the study of (62) who reported there is no significant differences in biofilm production between MRSA and MSSA. in contrast, biofilm producing ability was higher in MRSA isolates according to the study of (63).on the other hand, lower level of biofilm production in MRSA than MSSA were reported by (64).The lower level of biofilm production in MRSA strain may be due to the biofilm