Alsaadi et al (2020): Prevalence of mecA gene in MRSA isolates August 2020 Vol. 23 Issue 12 Study the prevalence of mecA gene in methicillin resistance staphylococcus aureus (MRSA) isolated from different clinical specimens and their antibiotic resistance profile Basima Q. Hasan AlSaadi1 , SehamH. Mohaisen1, ZainabT. Kazaal1, Zeena N. Abdulla1 1. Institute of genetic engineering and biotechnology for post graduate studies/ university of Baghdad/ Iraq *Corresponding author: [email protected] (ALSaadi) Abstract: Methicillin-resistant S aureus has progressed into an important pathogen of humans and is endemic in hospitals worldwide. MRSA strains carry multiple antibiotic-resistant genes. Recent work aim to investigate the prevalence of mecA gene among clinical isolates of MRSA .Fifty isolates of Staphylococci were demonstrated as methicillin resistancestaphylococcus aureus from various clinical specimens ( urine ,blood ,wounds ,and burns )after confirming their identity using morphological and biochemical tests ,as well as diagnosis by VITEK2 system. Also antibiotic sensitivity testing performed for all isolates by disc diffusion methods.Fifty isolates of S. aureus classified as MRSA taken from different clinical cases.mecA gene was detected by PCR technique to amplify 533bp using specific primers for the gene. The results showed that MecA gene was detecting in 39(78%) of Staphylococcus aureus isolates.Most of MRSA isolates were multiresistant to three antibiotic classes beta-lactams and multidrug resistant to other common antibiotics, such as aminoglycosides, therefore this work emphasis that there are other possible resistance mechanism may interact with mec A gene and causes development of methicillin resistance staphylococcus aureus Keywords: mecA gene; methicillin resistance; toxicity; risky; health; clinical specimens How to cite this article: Alsaai BQH, Mohaisen SH, et al (2020): Studying the prevalence of mecA gene in methicillin resistance Staphylococcus aureus (MRSA) isolated from different clinical specimens and their antibiotic resistance profile , Ann Trop Med & Public Health; 23(S12): SP2312023. DOI: http://doi.org/10.36295/ASRO.2020.231223 Introduction: Infection due to methicillin- resistant staphylococcus aurous ( MRSA) is a major problem world- wide and infection have been associated with a variety of clinical manifestations including skin infection , pneumonia ,cellulitis , impetigo and soft tissue abscesses and many serious diseases such as osteomyelitis ,and endocarditis ( Reddy, et al 2017), andassociated with infections acquired in the hospital ( nosocomial infection ) (Nasution, et al 2018). Infectionscaused by these bacteria are more difficult to treat with commonstandard antibiotics, so they are a potential risk for the humanhealth (Askarinia, et al 2018). Prevalence of MRSA inhospitals in different parts of the world diverse, ranging between 2-70%, 20% in average (Bell and Turnidge ,2002). In Iran, the prevalence of MRSA in healthcare workers was 16% - 35% (Denis, et al 2004). The MRSA rate was 10.1% among healthcare workers from Jordan (Aqelet al ,2015), 73% among healthcare workers from Saudi Arabia (Lyer ,et al,2014), and 22.5% in healthcare workers from Iraq (Hussein, et al,2017, Hawraa,et al,2014) Mec A gene is within range of a particular chromosome in staphylococcus – cassette chromosome ( SCCmec) , and methicillin resistance is conferred by mecA gene which codes for penicillin- binding protein2a( PBP2a)causing decreased binding affinity for B- lactam antibiotics , including the Penicillinase- resistant penicillin (Memmi,et al ,2008) .PBP is a group of enzymes in the cell membrane of S. aureus that catalyzes the trans-peptidation for peptidoglycan chain formation .(Hussein,et al 2019) .The affinity of PBP2a is highly slow that s. aureous stays alive in high concentration of antibiotics substances .(Elhassan,et al 2015) Detection of mec A by PCR is a gold standard and it’s a rapid and simple assay for detection of s. aurous isolated from various clinical specimens in Baghdad hospitals. Materials and Methods Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2020.231223 Alsaadi et al (2020): Prevalence of mecA gene in MRSA isolates August 2020 Vol. 23 Issue 12 - Samples collection : A total of (50) fifty isolates of Methicillin Resistance Staphylococcus Aurous (MARSA) were included in this study. They were isolated from different clinical specimens blood (12), urine (14), wounds(12),burn( 10 ) and ear swab ( 2 ) .Samples were collected from patients with different clinical infections attending to various hospital of Baghdad province , in the period from September to the end of December (2018) .Specimens were cultured on MacConkys` and blood agar and then primary sub cultured on Mannitol Salt Agar processed using the standard microbiology technique(Murray,etal,1999). Themannitol salt agar (HIMEDIA, India) was used forinoculation and the agar plates were then incubated at 350Cfor 18-24 hours in aerobic atmosphere (Kumury,et al 2015). Identification of Staphylococcus aureus and MRSA Depending on culture characteristics, by using Grams` stain as initial identification of Staph spp.( Hussein, et al 2019),then colonies were identified by using biochemical tests including ᵦ - hemolysis on blood agar, catalase 3%,oxidase and urease, for confirmation of identified bacteria the VITEK2compact system was used ( BioMerieux, France ) - Antibacterial Susceptibility Testing: Test was performed for all the fifty (50) S. aurous isolates using the following antibiotics: Penicillin G ,Methicillin,Ampicillibn ,Piperacillin ,Oxacillin ,Kanamycin ,Levofloxacin ,Ciprofloxacin ,Rifampin , Vacomycin ,Erythromycin ,Tetracyclin , Chlomromphenicol ,Gentamicin , Tobramycin ,Clindamycin and Amikacin , byusing Kirby-Bauer disk diffusion as recommendedby the CLIS guidelines(2017) moreover , Mueller – Hinton Agar ( Oxoid ) was used for antibiotic sensitivity testing . Molecular Analyses Extraction of DNA: DNA was extracted from all pure bacterial Isolates using Genome DNA Purification Kit (DSB1, China) and extraction was done according to the manufacturing company. Then concentration and purity of DNA were estimated using Nanodrop (Bio group,USA)(Sambrook and Russell ,2001) Detection of mecA by Polymerase Chain Reaction: For searching for MecA gene in the S. aureous strains using PCR technique according to (Pournajat ,et al 2014 ). Amplification of mecAgene was performed using specific primer MecA – F: AAAAAA GGT GGT GAT TGG C and MecA –R: AGT GCA GTA CCG GAT TTG C (Kumurya , et al ,2015) these produce a PCR amplicon of 533 base pair . the PCR program was adopted in optimal annealing temperature of PCR reaction was find out after several try to be 55°C with a total volume of 25µl.).Which consist of 12.5 µl GOTaq® Green Master Mix , 1µl of forward and revers primer for each ,8.5µl nuclease free water and 2 µl bacterial DNA . PCR was performed under the optimal conditions shown in Table -1- Table 1- The optimal conditions for amplifying MecA gene PCR steps TempCᶜ Time Cycles` No. Denaturation 95 3 mint. 1 Annealing 55 30 sec. 30 cycles Extension 72 60sec. Extended extension 72 6 mint. 1 PCR product was visualized in mini gel (2 %) stained with 10mg /ml ethidium bromide in 1 xTBE buffer( Promega ,USA), DNA ladder (100-1500bp)(Promega ,USA) used as a marker of DNA size and documented in UV transilluminator, Documentation system for observation of PCR products under 320 nm UV light (Lee , et al ,2012). Results and Discussion In the present study, fifty (50) S. aureus isolates from various clinical samples were identified as shown in Table ( 2) . High prevalence of bacteria were found in urine 14/ 28 % ,whereas blood and wound swab were 12/ 24% isolates respectively , followed by burns obtain 10/20% isolates and finally ear swab were obtained 2 /4 % isolates . Table 2- Prevalence of S aureous isolates from different clinical specimens Clinical specimens Samples No. Prevalence% Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2020.231223 Alsaadi et al (2020): Prevalence of mecA gene in MRSA isolates August 2020 Vol. 23 Issue 12 Urine 14 28 Blood 12 24 Wound swabs 12 24 burns 10 20 Ear swab 2 4 Total 50 100 Infection due to MARSA remain worldwide problems and infection rate have been associated with a variety of clinical manifestation including pneumonia ,sepsis ,and skin infection (Manjunath and Padma, 2012) .Studies carried in Kurdistan showed the prevalence of MRSA was found to be 4.2%(Hussein ,et al 2019).Also a study conducted by Abulreesh ,etal 2017) revealed low number of MRSA isolatesfrom different clinical samples (22%,N=50),while Rahemia and Kirime (2016) shown that the prevalence of MRSA 32.6%in Iran .This variation of the prevalence could be due to in part to different population ,geographical location and quality of samplings . The result of antibacterial sensitivity profiles of MARSA isolates were presented in (Table 2) Table2- Antimicrobial resistance profile No. antibiotics Sensitive Intermediate Resistance Penicillin G 16% 0 84% Methecillin (ME) 0 0 100% Ampicillin(AM) 0 0 100% Piperacillin(PRL) 4% 0 96% Oxacillin(OX) 0 0 100% Kanamycin(K) 66% 24% 10% Azthromycin(ATM) 16% 8% 76% Levofloxacin(LEV) 94% 0 6% Ciprofloxacin(CIP) 94% 0 6% Rifampin( RA) 92% 0 8% Vancomycin(VA) 84% 0 16% Eryhromycin(F) 12% 18% 70% Tetracyclin(TE) 34% 0 66% Chloremphenicol(C) 66% 2% 12% Gentamicin(GM) 76% 0 24% Tobramycin(TOB) 82% 2% 18% Clindamycin(CD) 56% 6% 38% Amikacin(AK) 42% 24% 32%