INTRODUCTION:

Staphylococci are Gram-positive microorganisms belongs to the family Staphylococcaceae, order Bacillales, class Bacilli in the phylum Firmicutes. Thirty-eight species and thirteen subspecies of coagulase negative Staphylococci CNS have been accurately described [1]. Amongst these, the species group that includes oxidase-positive (i.e. Staphylococcus sciuriand Staphylococcus lentus) are deliberated of excessive interest, together phylogenetically and clinically, because of these strains are mostly signifies with environmental and wild animal origins that have not been submitted to selective pressures due to human influence.

Coagulase-negative staphylococci (CNS) mainly cause nosocomial infections, such as catheter-related bloodstream infections, prosthetic valve endocarditis, central nervous system shunt infections and prosthetic joint infection [2]. The omnipresent dissemination of S. sciuri surely makes this species the most famous group illustrative of the oxidase positive staphylococci [3]. Staphylococcus haemolyticusis a member of CNS that constitute most of the typical human skin vegetation. Be that as it may, worries over S. haemolyticus as a developing human pathogen are expanding, as it is currently the second most much of the time secluded life form from blood diseases among CNS after S. epidermidis [4,5]. However, the taxonomic validity of such delineation in the absence of genome analysis is currently subjected to controversial debates [6]. CNS have truly being portrayed as kindhearted commensal creatures [7,8]. Today, CNS speak to one of the major nosocomial pathogens in human and animal medicine [9]. Staphylococcal Cassette Chromosome (SCC) mec is easily transmitted to CNS, CNS play an important role in spreading and horizontal transfer of drug resistance gene [10,11].

Staphylococcal superantigens (SAgs) including staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin-1 (TST1) [12]. have been classified as member of the pyrogenic superantigen toxins family for the reason that of their biological actions and structural relatedness [13]. This study aimed to determine the presence of superantigen toxin genes in some Methicillin resistant of clinical coagulase-negative staphylococci isolates.

MATERIALS AND METHODS:

Sample Collection:

A total of 251 samples of seminal fluid infections, septicemia, tonsillitis and wound infections were obtained from patients (12-45 years old), hospitalized at Al-Musayyib General Hospital in Babylon, Iraq for the period of September to December 2017. Conventional methods, standardized biochemical methods; catalase test, oxidase test, coagulase test, mannitol fermentation [14,15] and Vitek 2 system ( Biomerieux, USA) were used to identify the bacterial isolates.

Phenotypic Detection of Methicillin Sensitivity:

The isolates were experienced for cefoxitin (30μg) and cefepime (30μg) with the disc diffusion method according to the Clinical and Laboratory Standards Institute guidelines [16]. Zones of inhibition were measured and compared with the CLSI guideline.

DNA Extraction:

Genomic DNA was extracted by using Geneaid GBB100, Taiwan kit. Extraction of DNA was done according to manufacturer's instructions. Nanodrop framework Promega (USA) was utilized to assess DNA concentration and purity. DNA was stored at -20 ºC for PCR assay.

Primers Selection:

The sequence of oligonucleotide forward and reverse primers( Alpha DNA, Canada) which were used to detect sea, seb, seg, seh, sei, selp, tst1 and mecA are listed in the table 1. The volumes of primers were between 1.1 to 2.3 µl which were added to reaction mix with a final concentration 0.2 µM of each primer.

Table ( 1):List of the primers

Genes	Primers	Oligonucleotides sequence 5′ ……..……….. 3′	PCR products (bp)	References
sea	SEA-3	CCTTTGGAAACGGTTAAAACG	127	17
	SEA-4	TCTGAACCTTCCCATCAAAAAC		17
seb	SEB-1	TCGCATCAAACTGACAAACG	477	17
	SEB-4	GCAGGTACTCTATAAGTGCCTGC		17
seg	SEG-1	AAGTAGACATTTTTGGCGTTCC	287	18
	SEG-2	AGAACCATCAAACTCGTATAGC		18
seh	SEH-1	GTCTATATGGAGGTACAACACT	213	19
	SEH-2	GACCTTTACTTATTTCGCTGTC		19
sei	SEI-1	GGTGATATTGGTGTAGGTAAC	454	18
	SEI-2	ATCCATATTCTTTGCCTTTACCAG		18
selp	SEP-3	TGATTTATTAGTAGACCTTGG	396	18
	SEP-4	ATAACCAACCGAATCACCAG		18
tst1	TST-3	AAGCCCTTTGTTGCTTGCG	447	17
	TST-6	ATCGAACTTTGGCCCATACTTT		17
mecA	GMECAR-1	ACTGCTATCCACCCTCAAAC	163	20
	GMECAR-2	CTGGTGAAGTTGTAATCTGG		20

Multiplex PCR:

Multiplex PCR of each basis set was performed with GoTaq Green Master Mix PCR Kit as demonstrated by producer's rules. Each reaction, blend 50μl involved 25 μl of 2X PCR Master Mix, 2μl of each primer and 2μl (10– 100 ng) of DNA template and 7μl of nucleus free water. DNA enhancement was finished with the going with warm cycling: an underlying denaturation of DNA at 95°C for 15 min was trailed through 35 cycles of intensification (95°C for 30s, 57°C for 1.5 min, and 72°C for 1.5 min), completing through a last augmentation at 72°C for 10 min. The products of PCR were settled by electrophoresis in 2% agarose gel in 1X TBE buffer for 60 min. colored by 5μl/100ml of Red Safe and imagined on a transilluminator [21].

RESULTS AND DISCUSSION:

Isolation and Identification:

Out of 251 clinical samples only 60 isolates were CNS, distributed as following: S. sciuri 19 (31.66%) isolates, S. haemolyticus 15(25%) isolates, S. saprophyticus 14 (23.33%) isolates and S. lentus 12 (20%) isolates depending on colony morphology, biochemical tests and Vitek2 system as shown in figure (1). Svecet al. (2016) collected a group of S. sciuri (n=62) isolates obtained from human, while Kawamura et al. [22] found that S. haemolyticus (12.2%) and S. saprophyticus (3.6%).

S. sciuri was the most common isolate (31.66%) while S. haemolyticus was the second most common isolate (25%). A total of 178 (70.91%) samples from males while 73 (29.08 %) from female patients. Sangwan and Kumari [23] reported that the most common isolate in India was S. epidermidis (38.33%) while S. saprophyticus (35%) was the second recorded common isolated afterward S. haemolyticus (15%). The identification of CNS species and the characterization of the genetic diversity of the strains constitute a first step towards CNS safety assessment [24].

Screening for Methicillin-Resistant Isolates:

A-Phenotypic method:

Disc diffusion method was used to detect methicillin resistanct isolates , two types of antibiotics were used; cefoxitine and cefepime antibiotic discs [25]. All isolates showed resistance to cefoxitin and cefepime. The emergence of CNS not only as human pathogens but also as reservoirs of antibiotic resistance determinants requires the deployment and development of methods for their rapid and reliable identification [26].

B-Genotypic method:

Additional way to detect methicillin resistanct isolates was done depending on a mobile genetic element mecA, multiplex PCR assay was intended for direct detection of methicillin resistance gene mecA [21, 27-28]. Based on the specific amplifications of the mecA gene, the multiplex PCR procedure permitted the specific identification of isolates and the determination of its susceptibility to β-lactam antibiotics. The results showed that 25% of isolates contained mecA gene figure (2). While Krediet et al. [29] established that 92% of all CNS isolates were mecA positive.

The mecA gene is the essential gene located in the chromosome that encodes the modified penicillin binding protein PBP2, which has a lower affinity for beta-lactams due to a distorted active site [30]. A study by Ghaznavi-Rad et al. [31] in Iran was found high level of SCCmec genetic variety amongst MRCoNS isolates and all of the (70 MR-CoNS) isolates were contained mecA gene. isolates under study were defined as Methicillin Resistant if resistant by cefoxitin and cefepime or if mecA positive [32].

Distribution of Superantigenic Toxin Genes among the Isolates:

Genes amplification by multiplex PCR technique was used for detection of sea, seb, seg, seh, sei, selp and tst1 genes. Results showed that only S. sciuricarry sea, seb genes, S. lentuscarry seh gene, S. saprophyticuscarry seg, sei, tst1 genes, S. haemolyticus avoid any of these genes as shown in figure (2). Orden et al. [33] also found three CNS strains produce tst1, SEs and tsst1 are exotoxins originally identified in S. aureus, but they are also detected in CoNS [34].

All isolates were devoid from selpgene, S. sciuri, S. haemolyticus, S. saprophyticus and S. lentusare a reservoir of genes that, after horizontal transfer, facilitate the potential of S. aureus to colonize, survive during infection, or/and resist antibiotic treatment, Otto [35] have provided additional examples supporting the hypothesis that S. epidermidis and other CoNS have an important function as providers of genes that contribute to the survival of S. aureus during infection and as a commensal.

The enterotoxins that generated by the sea gene generate more severe immunological responses and subsequently more tissue damages compared with other enterotoxins. However, SEs are similar in structural characteristics and biological activities, but they are different in their mechanisms of actions [36]. The enterotoxin genes are encoded in mobile genetic elements, such as prophages, plasmids, and pathogenic islands. Those mobile genetic elements are responsible for the horizontal transfer of virulence or antibiotic resistance genes. Staphylococcal toxins are designated as SE with demonstrated emetic activity, while staphylococcal-like toxins showed no emetic activity in primate models [37,38].

Table (2): Prevalence of superantegenic toxin genes and mecA gene among the isolates

Genes	Number of Isolates
Genes	*S. sciuri*	*S. haemolyticus*	*S. saprophyticus*	*S. lentus*
*sea*	10 (16.66%)	0	0	0
*seb*	4 (6.66%)	0	0	0
*seg*	0	0	4(6.66%)	0
*seh*	0	0	0	5 (8.33%)
*sei*	0	0	6(10%)	0
*selp*	0	0	0	0
*tst1*	0	0	5(8.33%)	0
*mecA*	19 (31.66%)	15(25%)	14(23.33%)	20 (33.33%)

ACKNOWLEDGEMENTS:

The authors would like to thank Mustansiriyah University (www.uomustansiriyah.edu.iq) Baghdad- Iraq for its support in the present work.

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Received on 11.02.2019 Modified on 02.03.2019

Research J. Pharm. and Tech 2019; 12(9):4480-4484.

DOI: 10.5958/0974-360X.2019.00771.6