ARTICLE INFO

Article Type

Original Research

Authors

Bokaeian   M. (1)
Tahmasebi   H. (*)
Mohammadzadeh   A.R. (2)
Adabi   J. (1)
Sepehri Rad   N. (3)






(*) Microbiology Department, Medicine School, Zahedan University of Medical Sciences, Zahedan, Iran
(1) Microbiology Department, Medicine School, Zahedan University of Medical Sciences, Zahedan, Iran
(2) Microbiology Department, Medicine School, Gonabad University of Medical Sciences, Gonabad, Iran
(3) Infectious Diseases & Tropical Medicine Research Center, Zahedan University of Medical Sciences, Zahedan, Iran

Correspondence

Address: Microbiology Department, Medicine School, Zahedan University of Medical Sciences, Dr. Hesabi Square, Zahedan, Iran. Postal Code: 98167-43463
Phone: +985433295744
Fax: +985433425732
h.tahmasebi87@yahoo.com

Article History

Received:   November  22, 2015
Accepted:   April 16, 2016
ePublished:   June 30, 2016

ABSTRACT

Aims It is very important to detect the coagulase-negative Staphylococci, which produce the hospital infections. Being one of the most expensive and time-consuming stages before the polymerase chain reaction (PCR), DNA extraction is one of the primary stages of PCR. Then, it should be noticed that the elimination of the stage might save time and costs. The aim of this study was to compare two PCR methods including the method with the utilization of the extracted DNA with the extraction kit and the direct PCR method in the detection of the methicillin-resistant genes in the coagulase-negative Staphylococci.
Materials & Methods In the descriptive cross-sectional study, 135 Staphylococcus epidermidis and 88 Staphylococcus saprophyticus samples were studied, separated from blood, wounds, urinary catheter, and urine samples of patients hospitalized in the treatment centers of Zahedan. The direct PCR was done on the Staphylococcus saprophyticus and Staphylococcus epidermidis colonies. PCR with the extracted DNA was done for mecA and 16srDNA genes using the extraction kit, and the results were compared.
Findings In both methods, mecA and 16srDNA genes were successfully amplified in 310bp and 420bp related to Staphylococcus bacteria identifying gene and methicillin resistant gene, respectively. In addition, there were approximately the same band qualities.
Conclusion In order to save time and costs, the direct PCR method can be used to detect methicillin-resistant coagulase-negative Staphylococci.

Keywords: Staphylococcus epidermidis , Staphylococcus saprophyticus , Drug Resistance , Polymerase Chain Reaction ,

CITATION LINKS

[1]Inweregbu K, Dave J, Pittard A, Nosocomial infections. Contin Educ Anaesth Crit Care Pain. 2005;5(1):14-7.
[2]Martin MA, Pfaller MA, Wenzel RP. Coagulase-negative staphylococcal bacteremia: Mortality and hospital stay. Ann Intern Med. 1989;110(1):9-16.
[3]Cheung GY, Otto M. Understanding the significance of Staphylococcus epidermidis bacteremia in babies and children. Curr Opin Infect Dis. 2010;23(3):208-16.
[4]Schwalbe RS, Stapleton JT, Gilligan PH. Emergence of vancomycin resistance in coagulase-negative staphylococci.. N Engl J Med. 1987;316(5):927-31.
[5]Rupp ME, Archer GL. Coagulase-negative staphylococci: Pathogens associated with medical progress. Clin Infect Dis. 1994;19(2):231-43.
[6]Huebner J, Goldmann DA. Coagulase-negative staphylococci: Role as pathogens. Ann Rev Med. 1999;50(1):223-36.
[7]Rupp ME, Archer GL. Coagulase-negative staphylococci: Pathogens associated with medical progress. Clin Infect Dis. 1994;19(2):231-43.
[8]Makki AR, Sharma S, Duggirala A, Prashanth K, Garg P, Das T. Phenotypic and genotypic characterization of coagulase negative staphylococci (CoNS) other than Staphylococcus epidermidis isolated from ocular infections. Invest Ophthalmol Vis Sci. 2011;52(12):9018-22.
[9]Raei F, Eftekhar F. Studying the presence of blaZ gene and betalactamase production in clinical isolates of Staphylococcus epidermidis. Iran J Med Microbiol. 2008;2(2):35-41.
[10]Maskell R. Importance of coagulase-negative staphylococci as pathogens in the urinary tract. Lancet. 1974;1(7867):1155-8.
[11]Uçkay I, Harbarth S, Ferry T, Lübbeke A, Emonet S, Hoffmeyer P, et al. Meticillin resistance in orthopaedic coagulase-negative staphylococcal infections. J Hosp Infect. 2011;79(3):248-53.
[12]Klingenberg C, Aarag E, Rønnestad A, Sollid JE, Abrahamsen TG, Kjeldsen G, et al. Coagulase-negative staphylococcal sepsis in neonates. Association between antibiotic resistance, biofilm formation and the host inflammatory response. Pediatr Infect Dis J. 2005;24(9):817-22.
[13]Mombach Pinheiro Machado AB, Reiter KC, Paiva RM, Barth AL. Distribution of staphylococcal cassette chromosome mec (SCCmec) types I, II, III and IV in coagulase-negative staphylococci from patients attending a tertiary hospital in southern Brazil. J Med Microbiol. 2007;56(10):1328-33.
[14]Park JY, Fox LK, Seo KS, McGuire MA, Park YH, Rurangirwa FR, et al. Comparison of phenotypic and genotypic methods for the species identification of coagulase-negative staphylococcal isolates from bovine intramammary infections. Vet Microbiol. 2011;147(2):142-8.
[15]Zadoks RN, Watts JL. Species identification of coagulase-negative staphylococci: genotyping is superior to phenotyping. Vet Microbiol. 2009;134(1-2):20-8.
[16]Andreson R, Reppo E, Kaplinski L, Remm M. Genomemasker package for designing unique genomic PCR primers. Bio Med Clin Bioinform. 2006;7:172.
[17]Walley AJ. PCR protocols: current methods and applications. J Med Genet. 1994;31(1):87.
[18]Ottens R, Templeton J, Paradiso V, Taylor D, Abarno D, Linacre A. Application of direct PCR in forensic casework. Forensic Sci Int Genet Suppl Ser. 2013;4(1):e47-8.
[19]Rychlik W. Selection of primers for polymerase chain reaction. Methods Mol Biol. 1993;15:31-40.
[20]Shahbazi B. Narenji H. Comparison of four methods of DNA extraction from gram-negative and gram-positive bacteria. Zanko J Med Sci. 2014;15(45):9-16.
[21]Chum PY, Haimes JD, André CP, Kuusisto PK, Kelley ML. Genotyping of plant and animal samples without prior DNA purification. J Vis Exp. 2012;10(67):3791-3844.
[22]Fiebelkorn KR, Crawford SA, McElmeel ML, Jorgensen JH. Practical disk diffusion method for detection of inducible clindamycin resistance in Staphylococcus aureus and coagulase-negative staphylococci. J Clin Microbiol. 2003;41(10):4740-4.
[23]Capoluongo E, Giglio A A, Lavieri MM, Lesnoni-La Parola I, Ferraro C, Cristaudo A, et al. Genotypic and phenotypic characterization of Staphylococcus aureus strains isolated in subjects with atopic dermatitis. Higher prevalence of exfoliative B toxin production in lesional strains and correlation between the markers of disease intensity and colonization density. J Dermatol Sci. 2001;26(2):145-55.
[24]Raeymaekers L. Basic principles of quantitative PCR. Mol Biotechnol. 2000;15(2):115-22.
[25]Borst P. Genetic mechanisms of drug resistance: A review. Acta Oncol. 1991;30(1):87-105.
[26]Montazeri EA, Khosravi AD, Jolodar A, Ghaderpanah M, Azarpira S. Identification of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from burn patients by multiplex PCR. Burns. 2015;41(3):590-4.
[27]Quirk M. First VRSA isolate identified in USA. Lancet Infect Dis. 2002;2(9):510.
[28]Ahsani MR, Shamsaddini BM. Compare of two methods of direct PCR and PCR with DNA extraction in Clostridium Perfringens typing. Iran Vet J. 2012;8(4):5-12.
[29]Mousazade Moghadam M, Babavalian H, Mirnejad R , Shakeri F. Rapid DNA extraction of bacterial genome of Staphylococcus aureus using laundry detergents and assessment of the efficiency of DNA in downstream process using PCR. Med Lab J. 2012;6(1):35-42.
[30]Eickbush TH, Moudrianakis EN. The histone core complex: An octamer assembled by two sets of protein-protein interactions. Biochem. 1978;17(23):4955-64.
[31]Lichtensteiger CA, Steenbergen SM, Lee RM, Polson DD, Vimr ER. Direct PCR analysis for toxigenic Pasteurella multocida. J Clin Microbiol. 1996;34(12):3035-9.
[32]Nakao H, Popovic T. Development of a direct PCR assay for detection of the diphtheria toxin gene. J Clin Microbiol. 1997;35(7):1651-5.
[33]Fode-Vaughan KA, Maki JS, Benson JA, Collins ML. Direct PCR detection of Escherichia coli O157:H7. Lett Appl Microbiol. 2003;37:239-43.
[34]Inglis GD, Kalischuk LD. Use of PCR for direct detection of Campylobacter species in bovine feces. Appl Environ Microbiol. 2003;69(6):3435-47.
[35]van Hal SJ, Stark D, Lockwood B, Marriott D, Harkness J. Methicillin-resistant Staphylococcus aureus (MRSA) detection: comparison of two molecular methods (IDI-MRSA PCR assay and GenoType MRSA Direct PCR assay) with three selective MRSA agars (MRSA ID, MRSASelect, and chromagar MRSA) for use with infection-control swabs. J Clin Microbiol. 2007;45(8):2486-90.
[36]Nishimura N, Nakayamaa H, Yoshizumib S, Miyoshib M, Tonoikea H, Shirasakia Y, et al. Detection of noroviruses in fecalspecimens by direct RT-PCR without RNA purification. J Virol Methods. 2010;163(2):282-6.
[37]Fazaeli A, Fuladi B, Hashemi Shahri M, Sharifi I. Evaluation of a direct PCR in comparison with routine microscopy and in vitro culture for diagnosis of cutaneous leishmaniasis. Iran J Public Health. 2007;36(Suppl 1):1-2.