Alsaadi et al (2020): Prevalence of mecA gene in MRSA isolates August 2020 Vol. 23 Issue 12

Study the prevalence of mecA gene in resistance aureus (MRSA) isolated from different clinical specimens and their 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 isolates.Most of MRSA isolates were multiresistant to three antibiotic classes beta-lactams and multidrug resistant to other common , 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 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 - 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%

Recent study revealed that all the isolates were resistant to Ampicilin, Oxacillin and Methecillin which are beta –lactam antibiotics , high resistance were shown to Pipracillin(96%),Penicillin G(84%),zthromicin (76%)and Erythromycin (70%)and some isolates were show sensitivity to Levofloxacin, Ciprofloxacin 94% for each , Rifampin92%,Vancomycin84%and Tobromycin82%. A study in Saudi Arabia (Al Ruaily and Khalil ,2011) showed that s. aureus were resist to Cephalosporin (95%)Ciprofloxacin87%,Vancomycin100% and Penicillin 100% ((Al Ruaily and Khalil ,2011) .Another study in Sudan by Alhassan et al (2015) showed that all isolates were resist to Methicillin and Ampicillin ,While Nasution (2018) in Indonesiashowed that all isolates were resist to Amoxicillin,Ampicillin/Sulbactam and Cephalosporin group also high resistant to Ciprofloxacin(97.5%),Gentamycin(87.5%)and Levofloxacin97.5% and some isolates still sensitive to Vancomycin82.5%, Trimethoprim/ Sulfamethxazole 72.5%, and Tigecycline100%. The bacterial isolates considerMRD when they exhibit resistance to at least 3 or more un related agents , ,that MDR has been an escalating global threat in modern medicine, In this work high percentage of MRSA which had also revealed multiple resistance to various drugs they are resistance to oxacillin, ampicillin ,methicillin ,and piperacillin .Since multiple- antibiotics resistant S. aureus strain are the major health problem, on the other hand they cause several nosocomial and community acquired infections , therefore ,fast and accurate detection of resistant isolates are critical goal of clinical microbiology . Moreover isolates which collected from various clinical samples were investigated for carriage of mecAgene. Genotypic analysis revealed that MecA gene was detecting in 39(78%) of S. aureusisolates with PCR product 355bp as shown in (figure1), Table (3),which show distribution of mecA gene in different clinical isolates .Thatfinding of mecA gene is the major evidence for thedetection of MRSA 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 isolate. Study conducted in Kurdistan shown that 55 of 109 (50.4%) isolates were MRSA carrying the mecA gene (Husseinet al 2019)another study bySiddiqui et, al (2018) thatmecA positive isolates were 36/104 (35%), mecA gene is located withinChromosome in a structure called Staphylococcal Cassette Chromosome (SCCmec) encodes mutant PBP2a or PBP2' of 76 kDa (Jain, et al 2008).

Figure 1: The size of PCR products 533 bp. The PCR product was electrophoresis on 2% concentration agarose gel at 70 voltage for 90 minute, stained with Ethidium Bromide then visualized under U.V light, L: DNA ladder (100-3000), Lane (1-10) PCR products of the mecA gen

Table 3- shown distribution of MecA gene according to clinical isolates No. of isolates Presence of MecA gene Clinical specimens S1 + Wound swab S2 + Wound swab S3 + Wound swab S4 + Wound swab S5 + Wound swab S6 + Wound swab S7 - Ear swab S8 - Ear swab S9 + Wound swab S10 + Urine S11 + Urine S12 + Urine S13 + Urine S14 + Urine S15 + Urine S16 + Urine S17 - Burns S18 - Burns S19 - Burns S20 + Blood S21 + Blood S22 + Blood S23 + Blood S24 + Urine S25 + Urine S26 + Urine S27 - Wound swab S28 - Wound swab S29 + Blood 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

S30 + Blood S31 + Blood S32 + Wound swab S33 + Wound swab S34 + Wound swab S35 + Burns S36 + Burns S37 + Burns S38 + Burns S39 + Blood S40 + Blood S41 + Blood S42 - Urine S43 - Urine S44 - Urine S45 - Urine S46 + Blood S47 + Blood S48 + Burns S49 + Burns S50 + Burns

The presence of mecA gene indicates the potential resistance to b- lactam group and may uses as a marker to identified MARSA. Studies conducted by (Hafezet al, 2009; Meshref and Omer 2011; Al- Zu’bi et al, 2004) showed high prevalence of MecA gene among their S.aureus isolates and may exhibit that mecA gene was prevalent among their isolates at a higher rate than that gained by current study and the high prevalence of mec A gene in S.aureusisolates represents potential reservoir for the spread of this gene. Concluded from these findings that there are mechanisms other than the presence of mec A gene responsible for B- lactam resistance of MRSA and need more investigation

References: 1- Reddy,P.N.;Sharma,K.andDirisala,V.R.(2017) An update on clinical Burden, Diagnostic Tools and Therapeutic Options of Staphylococcus aureus .Infectious Diseases :Research and Treatment.10:1-15

2- Nasution,G.S. ; Suryanto, D. and Kusumawati,R.L.( 2018) .Detection of mecagene from methicillin resistant staphylococcus aureus isolates of north sumatera.Earth and Environmental Science 130(2018) 012026:1-7

3- Askarinia,M.;Ghaedi,M.;Manzouri,L.;Khoramrooz,S.S.;Asghar,S..et al (2018). The effect of Cu- BPDCA-TY on Antibacterial Activity and the Expression of mecA gene in clinal and standard strains of Methicillin Resistant Staphylococcus aureous. Jundishapur Journal of Microbiology .11(3):1-7

4-Bell,J.and Turnidge ,J.D.(2002) High prevalence of Oxacillin –resistant Staphylococcus aureous isolates from hospitalized patients in Asia –pasific and South Africa . results from sentry antimicrobial surveillance program , 1998-1999.Antimicrob Agents CHemother. 46(3):879- 81

5Denis,O;Deplano,A.;Nonhoff,C.Deryck,R;Demendonce,R;Rottiers,S.;Vanhoof,R.andStruelens,M.J.( 2004) National surveillance of methicillin –resistant Staphylococcus aureous in Belgian Hospitals indicates rapid diversification of epidemic clones .Antimicrob Agents Chemother .48(9):3625-3629

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6-Aqel, A.A.;Alzoubi, H.M.; Vickers, A.;Pichon,B.and Kearns, A.M.(2014) Molecular epidemiology of nasal isolates of methicillin-resistant Staphylococcusaureus from Jordan. J. Infect. Public. Health. 8(1):90–7

7- Iyer, A.;Kumosani, T.;Azhar, E.; Barbour, E. andHarakeh, S.( 2014) High incidence rate of methicillin-resistant Staphylococcus aureus (MRSA) among Healthcare workers in Saudi Arabia. J. Infect. Dev. Ctries;8(3):372–8.

8-Hussein, N.R.;Assafi,M.S.andIjaz, T.(2017). Methicillin-resistant Staphylococcus aureus nasal colonisation amongst healthcare workers inKurdistan region, Iraq. J Glob Antimicrob Resist. 2017;9:78–81.

9- Hawraa,W.A.;AL-Dulaimi,T.and AL-Marzoqi,A.H.A( 2014).Phenotypic detection of resistance in Staphylococcus aureous isolates : detection of (mecA and femA ) gene in methicillin resistance Staphylococcus aureous (MRSA) by polymerase chain reaction .Journal of Natural Sciences Research .14(1):112-118 10-Memmi, G.; Filipe, S.R.;Pinho, M.G.; Fu Z and Cheung, A. (2008) Staphylococcus aureus PBP4 is essential for beta-lactam resistance in community-acquired methicillin-resistant strains. AntimicrobAgents Chemother. 52(11): 3955-66.

11- Hussein, N.; Salih,R.S.andRasheed ,N.A.(2019).Prevalence of Methicillin –Resistant Staphylococcus aurous in Hospitals and Community in Duhok, Kurdistan Region of Iraq . Int. J.Infection .6 (2) e89636:1-4

- 12-Elhassan, M.M. ;Ozbak, H.A. ;Hemeg, H.A.;Elmekki, M.A. and Ahmed, L.M. (2015) Absence of themecA Gene in Methicillin Resistant Staphylococcus aureus Isolated from Different Clinical Specimens in Shendi City, Sudan. BioMed Research International. 1-5

13- Brenner, D.J.; Krieg, N.R. and Staley, J.T.(2005) Bergeyʼs Manual of Systematic Bacteriology. Volum Two. Part B. 2nd ed. USA: Spriger;.1106 p.

14- Kumurya ,A.S.; Gwarzo, M.Y., and Uba, A. (2015) One Step PCR for Detection of Staphylococcus aureus specific Sequence gene and mecA gene. International Journal of Advanced Materials Research. 1(3):73-79

15- CLSI. Performance Standard for Antimicrobial Susceptibility Testing.(2017) 27th ed. CSLI supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute.; p.32-41.

16-Sambrook, Jand Russell. D(2011) .Mollecular Cloning ,Alaboratory Manual.4th.ed.Newyourk :Cold Spring Harbor Laboratory Press

17-Pournajat, A.; Ardebili, A.; Goudarzi, L.; Khodabandeh, M.; Narimani, ,T. and Abbaszadeh, H. (2014) PCR based identification of Methicillin-resistant Staphylococcus aureus strains and their antibiotic resistance profiles. Asian Pacific Journal of Tropical Biomedicine. 4: S293- S297.

18- Lee, P.Y.;Costumbrado, J. H. C, and Kim, Y.H. ( 2012). Agarose Gel Electrophoresis for the Separation of DNA Fragments. J.Vis. Exp. 62: 3923

19- Manjunath, S. and Padma ,T.(2012) .Methicillin Resistant Staphylococusaureus: Resistance Genes and Their Regulation.International Journal of Pharmacy and Pharmaceutical science. 4, (Suppl 1): 658-667 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

20-Abulreesh, H.H.;. Organji,R.S ;;. Osman, E.H.G.; Elbanna,K. ;Almalki,H.K.M and Ahmad, I. (2017). Prevalence of antibiotic resistance and virulencefactors encoding genes in clinical Staphylococcus aureus isolates in Saudi Arabia. Clinical epidemiology and global health, 5: 1 9 6 – 2 0 2

21-Rahimi1,F.and Karimi1,M.(2016).Characteristics of Virulence Factors in Methicillin-Resistant Staphylococcus aureus Strains Isolated From a Referral Hospital in Tehran, Iran .Arch Clin Infect Dis.11 (1): e33220.

22-Al-Ruaily, M.A. and Khalil, O.M. (2011) Detection of (mecA) gene in Methicillin Resistant Staphylococcus aureus (MRSA) at Prince A/ Rhmansidery hospital, Al Jouf, Saudi Arabia. Journal of Medical Genetics and Genomics. 3(3): 41-45.

23- Siddiqui1,T.; MuhammadN.I.; Khan,N.M.; Fatim,S.; Alam ,N.------et, al.(2018). Prevalence of mecA: Genotyping screening of community acquired MRSA isolates in Karachi, Pakistan.Pakistan journal of pharmaceutical sciences.31,.5(Suppl),: 2091-2094

24-Jain ,A.; Agarwal, A. and Verma, R.K. (2008) disc diffusion test for detection of meticillin-resistant staphylococci. Journal of Medical Microbiology. 57: 957–961.

25- Hafez,E.E.;Sohaimy,S.A. and Saadani,M.E( 2009)“The effect of the mecA gene and its mutant form on the response of S.aureus to the most common antibiotics,”International Journalof Immunological Studies, 1(1): 106–122

26-Meshref, A.A. and Omer,M.K(2011). “Detection of (mecA) gene in methicillin resistant Staphylococcus aureus (MRSA) at Prince A/RhmanSidery,” Journal of Medical Genetics and Genomics, 3(3):41–45

27- Al-Zu’bi,E.;Bdour,S. and Shehabi,A.A(2004) “Antibiotic resistance patterns of mecA-positive Staphylococcus aureus isolates from clinical specimens and nasal carriage,” Microbial Drug Resistance, 10,( 4):. 321–324,

Acknowledgments We would like to thank the department of Genetic engineering and theappreciation goes to the entire staff of medical microbiology laboratories of the various health institutions for their assistance in the collection of the clinical specimens as well as to the staff of molecular biology of the department for their technical assistance.

Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2020.231223