VIRULENCE POTENTIAL OF S. aureus ISOLATED FROM IMPORTED AND LOCAL CHICKEN DEPENDING ON THE PHENOL SOLUBLE MODULINS ( PSMMEC ) IN DUHOK PROVINCE, KURDISTAN REGION OF IRAQ

Staphylococcus aureus is considered as one of the major foodborne pathogens in human and animals which can lead to a wide range of diseases, including food poisoning. The toxins of S. aureus play an important role in disease pathogenesis, contributing to both injury of the host tissues and the immune response. One of these toxins is phenol-soluble modulin ( PSM ) peptides which has the ability of immune invasion and considered as a cytolytic toxin. Commonly, mobile genetic elements (MGE) of S. aureus that carrying antibiotic resistance gene do not carry the virulence genes, however, PSMmec has been identified within the methicillin resistance staphylococcus-encoding MGE SCCmec. This study was conducted for six months, over-all 200 whole chicken carcasses were collected including (100) local chicken and (100) imported one from supermarkets in Duhok province. The samples for S. aureus were cultured on mannitol salt agar and then were confirmed using colony morphology, biochemical test like, catalase test and coagulase test, in addition to the species specific primer ( nuc gene) for PCR. The PCR positive samples were selected and used in this study. The aim of this study was to evaluate the virulence potential of S. aureus isolated from imported and local chickens depending on PSMmec -complex PCR (spanning PSM , xylR and mecR genes) and mecA . The results of this study shows that 46 isolates out of 57 from imported chickens were carried mecA (methicillin resistant isolates) from


INTRODUCTION
Staphylococcus aureus is the most common foodborne pathogen distributed all over the world.These microorganism carriers a virulence gene called phenol-soluble modulins (PSMs), comparing with other virulence factors, this gene is α-helical peptides and has the ability to play a role as pro-inflammatory cytolytic toxins (1).Antibiotic resistance genes are normally located on mobile genetic elements (MGEs) like plasmids, genomic islands and, transposons (2).The methicillin resistance gene (mecA) is specifically situated in the staphylococcal cassette chromosome-mec (SCCmec).At least four main and many sub-types of SCCmec elements are introduced, their sizes ranging from 21 to 67 kb, these elements are characterized by two important gene complexes (mec and ccr) and transposons as an accessory gene loci (2).PSM gene has been described by (3), In methicillin-resistant S. aureus (MRSA).Kaito et al., 2008, found that the PSM-mec, partly overlaps the open reading frame of a putative virulence-modulating, "fudoh" (4).It was determined that PSM-mec is found within SCCmec elements genes which include the genes responsible for mecA, recombinase genes, regulatory elements and inconsistently other resistance genes (5)(6)(7).In these elements, the merely known related virulence factor is PSM-mec.This gene has been identified in S. aureus SCCmec types II, III and VIII, SCCmec types II and III in S. epidermidis as well as found in S. cohnii, S. saprophyticus, S. sciurii and S. vitulinus (3).The virulent of S. aureus infection is mostly determined by the ability of S. aureus toxins to make an infection.For example, S. aureus may produce phenol-soluble modulins (PSMs), toxic shock syndrome toxin-1, enterotoxins, leukocidins, a-toxin and other toxins (8)(9)(10).Many of them have the ability to destroy immune cells, thus contributing significantly to the immune evasion capacity of S. aureus.Many S. aureus toxins are encoded on the bacterial core genome such as a-toxin and PSMs (1,11).
This study was aimed to screen S. aureus isolated from imported and local chicken for the presence of PSM-mec complex associated with mecA in order to obtain more data on its distribution and to determine the virulence potential of these isolates as this gene is considered as a virulence determinant that connects the transcriptional regulation, virulence in staphylococci.

MATERIAL AND METHODS
This study was conducted for six months from March, 2019 to September 2019.

Sample collection
A total of 200 whole chickens were scanned for the detection of S. aureus (100 of local chickens and 100 of imported chickens).Local fresh chicken were collected from the Duhok chicken abattoir in Summel, Duhok province.While, the whole imported chicken carcasses were collected from different supermarkets in Duhok city (Turkish origin).These carcasses were collected and transferred immediately in a cold box to the Microbiology Laboratory at College of Veterinary Medicine for microbiological analysis.

Isolation and identification of S. aureus
The whole chicken was transferred to a sterile plastic bag then 400 ml of buffer peptone water (BPW) was added to the bag.The carcass was rinsed and washed thoroughly for about 2 minutes.For isolation, 10 ml of chicken rinse were collected aseptically and mixed with 90 ml of BPW.The broth was then incubated for 24 h at 37°C.A loop of the broth culture was streaked onto the mannitol salt agar (MSA) and then incubated aerobically for 18-24 h at 37°C. S. aureus colonies were firstly examined based on colonial morphology.The suspected S. aureus colonies were then selected and streaked onto MSA to get pure colonies of S. aureus.Gram-staining and catalase test were then applied for colonies that have a typical morphological feature.Gram and catalase positive isolates were further confirmed for S. aureus biochemically using coagulase test and also using species-specific primer for PCR.

Ttotal DNA extraction
DNA was extracted from S. aureus isolates according to (12).Shortly, DNA was prepared by resuspending 2-3 colonies in 100 µl of deionized autoclaved water and mixed very well.
Bacterial suspensions were boiled in a heat block for 20 min.The suspension was then centrifuged at 10,000 xg for 1 min.The supernatant was collected to new eppendorf tubes and then used as the DNA template for subsequent tests.DNA quality and concentrations were assessed using NanoDrop 2000C spectrophotometer (Theremo Scientific).The ratio 260/280 nm was in the range of 1.55 to 2.3 for all DNA samples used.The DNA samples were stored at -20°C for further analysis.

PCR technique
Details of the nucleotide sequences of the primes used for PCR amplification are provided in Table (1).Approximate gene and primer localizations, based on positions within staphylococcus genomes can be seen in (13).Current study all PCR reactions were achieved in (0.2 ml) PCR tubes, crystal hot start master mixes 2x was used for this PCR (Jena Bioscience, Germany).
A total of 20 µl reaction volumes were prepared according to the manufacturer's instructions.Shortly, each reaction involved the following components: 10 µl of master mix (2X), 1.5 µl of each forward and reverse primer (10pmol/µl), and template of DNA (~ 2 µl) according to the concentration (50ng/µl) and dH2O was added to be completed to 20 µl.PCR amplification was carried out in a PCR System 9700 Thermocycler (Applied Biosystems).All the isolates used in this study were further been confirmed using thermo-stable nuclease gene (nuc) in detection of S. aureus (14).
PCR primers used in this study shows in (Table1).First PCR Reaction conditions of PSMmec primer included an initial denaturation (2 min at 96°C) followed by 35 cycles (20 sec at 96°C, 20 sec at 70°C and 20 sec at 72°C).A second PCR covered the region from xylR to PSMmec (PSMmec and xylR), while the third one spanned the region from (PSMmec to mecR).
Both second, third and MecA PCR reactions included denaturation for (2 min at 96°C) followed by 35 cycles (20 sec at 96°C, 20 sec at 55°C and 70 sec at 72°C), the details can be seen in Table 2 (16).

Gel electrophoresis technique:
PCR products were established by electrophoresis in a 1.5% (w/v) agarose gel using 1X TAE buffer with nucleic acid dye Syber® Safe (Invitrogen).Seven microliters of each PCR product were loaded into each well.The size of the PCR products was showed by loading 7 µl of 100bp DNA ladder (Jena Bioscience, Germany).The fragment sizes were then visualised and photographed under UV light figure (1).

RESULTS AND DISCUSSION
From 200 (100 local and 100 imported chicken) whole chicken samples were examined for the present of S. aureus, 108 isolates were found to be positive for staphylococcus by culture method (80 from imported frozen chicken and 28 from local chicken), however, only 68 from imported and 22 from local chicken were noticed to be S. aureus species depending on the coagulate test.Moreover, these samples were further been confirmed using   3), while none of the local isolates were give positive results for the third PCR (mecR/PSMmec), (Table 3).The PCR results are shown in Figure ( 1) Table3: Description of PCR results of PSM complex *According to the genes tested the S. aureus isolated for imported chickens have SCC elements SCCMECI, II (2A) and IID, while the local chickens have just SCCmec IID.This classification is done depending on the study results of (3,6,7) , however, this must be confirmed by sequencing of SCCs in the future study.
On the basis of available MRSA genome sequences, the PSMmec gene is found mostly within SCCmec determinants of types II and III (18).It has been noticed that CC12-MRSA, WA-MRSA-59 harbored SCCmec elements containing mecR but missing xylR.However, this study revealed that some of the imported chickens isolates may carries SCCmec type (IIA) and IID elements having xylR but lacking mecR this classification depends on the outcome of (3,13,16).The PSMmec was not found in SCCmec types I, IIC, IIE, IV, V or XI and in SCC elements missing the mec complex (13,16).Accordingly, this study also revealed that 13 isolates of S. aureus from imported chicken were just carries mecA, this is suggests that these isolates may carry either SCCmec type I or II.Some S. aureus isolates of the current study were missing PSMmec, this is an indication of either absent of SCCmec elements, or carrying SCCmec types I, IIC, IIE, IV and V as stated by (13,16).Virulence factors association with mobile genetic elements (MGEs) deliberating drug resistance detected in E. coli (2,19) and enterococci (20), however, the connection of PSMmec to SCCmec in staphylococci, is rather unique (21).The αtype PSM peptide has a cytolytic and proinflammatory action and plays an important role in MRSA infection.In a study conducted by (22), found that 7.6% of all MRSA strains carried the psm gene from the isolates collected from blood, sputum, wound, and trachea.Joo and coworkers (2011) (23) state that PSMs have cytolytic activities on the neutrophils, red blood cells, and white blood cells and impact on pathogenesis of bacteremia, skin and soft tissue infections.According to their surfactant like properties, PSMs have also been suggested to influence biofilm maturation and detachment (21)(22)(23)(24).

It was found that the
The presence of PSMmec in MRSA strains can be a key factor for the pandemic spread and abundance success of SCCmec elements.Therefore, this study suggest that the S. aureus isolated from imported chicken may be more virulent than the isolates from local chicken due the present of PSMmec complex which were from types I, II(2A) and IID as listed in Table (3).
Isolates resistance to methicillin with PSM contribute to staphylococcal virulence.
Accordingly, this study shows that this pattern is more obvious in imported chicken isolates rather than local chickens.The role of methicillin resistance groups in the pathogenicity of staphylococcal has also studied by (3), they identified that the majority of serious antibiotics and virulence resistance elements may be associated in staphylococcal MGEs.Our study shows that in similarity to the previous belief, S. aureus can handle both resistance and virulence factors on MGEs, consequently, this combination will help to transfer of two important factors for causing human disease in one genetic episode.

Table 1 :
Nucleotide sequences of primers used in this gene (nuc gene) by conventional PCR, from these only 57 and 18 were indicated as S. aureus in both imported and local chicken, respectively, these results have been published in a separate study(14), the same isolates have been used to implement this study.Identification of the Staphylococcus cassette chromosome mec (SCCmec) of this study isolates will mainly depend on the previous studies outcomes(3,13,16,17), inorder to recognise the possible classification of MGEs of S. aureus isolated in this study.In present study, a PCR reaction that covering the area from xylR to PSMmec indicate that, out of 57 imported isolates only 31 isolates were found to be positive for this region, and 2 out of 18 isolates from the local chickens were give positive results for this locus.In 32 isolates PSMmec, were found negative (20 from imported and 12 from local) isolates, from these xylR was also absent.Results of the third PCR mecR/PSMmec were only found in 15 imported isolates, in addition to the present of mecA, PSMmec/xylR and PSMmec (Table
(5,7,13)s associated with mecI and/or xylR, in another word it can be found in the isolates harboring Classification of Staphylococcal Cassette Chromosome mec (SCCmec) types II, II-A, II-B, II-D, III, and VIII or types II-A/B and III as irregular elements related to SCCmec.However, PSMmec was not limited just in S. aureus.The other staphylococcus species such as S. pseudintermedius, S. epidermidis, S. hominis S. fleuretti, S.saprophyticus, S. vitulinus and S. simulansmay may also carry PSMmec.Present of this gene is not only restricted in one host species, it was noticed in different isolates from different host like in cattle, turkeys, pigs, goats, sheep, cattle, cats and human(5,7,13).