Document Type : Research Paper

Authors

1 University of Anbar/College of Science

2 University of Anbar/Desert Studies Center

10.37652/juaps.2015.124384

Abstract

This study includes collected of 399 samples from different sources included clinical and non clinical samples. 293 clinical samples (urine, wounds, burns, stool, sputum). Non- clinical samples included 106 samples (water, soil) from November 2014 through February 2015. 51 samples were diagnosed as K. pneumoniae using phenotypic, biochemical and cultural diagnosis features and definitely diagnosed with API20E and vaitek test. Results of antibiotic resistance against different antibiotics showed that K. pneumoniae isolated from clinical and non- clinical samples varied in their resistance to these antibiotics and all isolates were sensitive 100% to Imipenem. A number of specific primers were designed to detect bacteria by using distinct gene and also to detects resistance to antibiotics using multiplex PCR technology for the presence or absence of 8 genes(magA, rmpA, htrA, Uge-1, gent-2,blaCTX, terA-1,StrA-2) that encodes for main virulence factors to diagnose K. pneumoniae. The result showed that that 45 isolates had rmpA which might be used as detection marker, in the same reaction of multiplex PCR detection of 4 genes related with resistance to different antibiotic groups(StrA,terA,blaCTX,gent-2) were done all K,pneumoniae contained StrA gene. According to this study we can concluded that multiplex PCR can use to distinguish isolated bacteria from different sources more correctly and also to determine its antibiotic resistance that are widely used in short time and little effort without need to routine testing.

Keywords

Main Subjects

[1]Manikandan C and Amsath A.(2013) Antibiotic susceptibility pattern of KlebsiellaPneumoniae isolated from urine samples.International Journal of Current Microbiology. APP.Sci Vol.2 No.8 PP.330-337.
[2]Brisse S, GrimontF,andGrimont P.A.D(2006) The Genus KlebsiellaProkaryotes 6:159-196 CHAPTER3.3.8
[3]Hackstein H, Kranz S, LippitschA,Wachtendorf A, Kershaw O,  Gruber A.D, Michel G, Lohmeyer J, Bein G, Baal N and Herold S.(2013) Modulation of respiratory dendritic cells during KlebsiellaPneumoniae infection . Respiratory Research :14:91 PP. 1-11 http:// respiratory-research.com/ content/14/1/91.
[4]Fang C.T., Shan Y.L.,Wen C.Y., PoR. H., Kao L.L.(2010). The Function of wzy-k1 (maga) the Serotype K1 polymerase Gene in klebsiella pneumonia CPS Gene cluster. The Journal of Infections Diseases. 201:1268-1269. 
[5]Al-Jailawi M. H, Zedan T.H and Jassim K.A, (2014). Multiplex PCR Assay for Identification of Klebsiella pneumonia. Int.J.Pharm .Sci. Rev .Res .,26(1) No.18 Pages: 112-117.
[6]Aher, T, Roy ,A. and Kumar P( 2012). Molecular Detection of Virulence Genes Associated with Pathogenicity of Klebsiella spp. Isolated from the Respiratiry Tract of Apparently Healthy as well as Sick Goats. Israel Journal of Veterinary Medicine Vol. 67(4) .PP. 249-252.
[7]Abdul RazzaqM.S,Trad J.K and Al-Maamory E .H .KH. (2013). Genotyping and Detection of Some Virulence Genes of Klebsiella pneumonia Isolated from clinical Cases. Medical Journal of Babylon Vol.10,No.2,PP.387-399.
[8]Zamani A, Mashouf R.Y, Namvar A.M.E and Alikhani M. Y.(2013). Detection of magA Gene in Klebsiella spp. Isolated from Clinical Samples .Iranian Journal of Basic Medical Sciences ;16(2),PP. 173-176.
[9]Turton J.F, Perry C, Elgohari S and Hampton C.V(2010).PCR characterization and typing of Klebsiella pneumonia using capsular type- specific, variable number tandem repeat and virulence gene targets. Journal of Medical Microbiology, 59;PP.541-547.
[10]Fevre C, Passet V, Detetoile A, Barbe V, Frangeul L, Almeide A.S, SansonettiPh, Tournebize R and Brisse S(2011).PCR-Based Identification of KlebsiellaPneumoniae Subsp. Rhinoscleromatis, the Agent of Rhinoscleroma . PLOS NEGLECTED TROPICAL DISEASES VOL.5,Issue 5,e1052 pp. 1-7.
[11]Ahmed O.I, EL-Hady S.A, Ahmed T.M and Ahmed I.Z (2013). Detection of blaSHV and blaCTX-M genes in ESBL producing Klebsiella pneumonia isolated from Egyptian Patients with suspected nosocomial in fections .The Egyptian Journal of Medical Human Genetics ,14, PP.277-283.
[12]Cortes G, AstorzaB.d, Benedi V .J and AlbertiS(2002). Role of the htrA Gene in Klebsiella pneumonia Virulence.INFECTION AND IMMUNITY.Vol.70 ,No.9 PP. 4772-4776.
[13]Ibrahim Y.M (2013) Attenuated HtrA-mutant of streptococcus pneumonia induces protection in murine models of pneumococcal pneumonia and bacteraemia.African Journal of Microbiology Research Vol.7(3), PP. 237-244
[14]Bergeron M. G and Kee D.(2001). New DNA-Based PCR Approaches For Rapid Real-Time Detection and Prevention of GBS Infection In New borns and Pregnant women. J.Molec.Medi.;3:1423.
[15]Macfaddin, J.F.(2000).Biochemical Tests for Identification of Medical Bacteria. 3rd ed. Lippincott,Williams and Wilkins. Philadelphia .London.
[16]Prescott ,L.M.;Harly, J.P.and Klein, D.A. (1996). Microbiology, 3rd ed., The McGraw-Hill Companies, Inc., U.S.A.
[17]Vandepitte J, Engback K., Piot,P. and Heuck C.C.(1991).Basic Laboratory Procedures in clinical Bacteriology. WHO, Geneva.,PP: 78- 110.
[18]Holt , J.J., Krieg , N.R., Sneath , B.H.A., Staley ,J.T. and Williams ,S.T. (1994).Bergey’s manual determinative bacteriology. 9th Ed. Williams and Wilken , Baltimore , PP.175-248.
[19]CLSI, (Clinical and laboratory standars in statute) (2007). Performance standard for antimicrobial susceptibility testing; seventeenth informational supplement M100-S17. Vol. 27 No (1).
[20]Woodford, N.; ward, M.E.; kanfmann, M.E.; Fagan, J.; James, D.J.; Johnson, A.D., warner, R.; pearson, A.; Leach, J.S.; warren, M and Livemore, D.M. (2006).Molerular characterization of Escherichia coli isolates producing CTX-M-5 extended spectrum B-Lactamases (ESBLs)in the united kingdom. Health protection Agency., 57: 154-155.
[21]Petite, p.l., schneeberger, p., lidala, V., Butter, M., Wamola, IA. (1991). Bacteriology of infections in aural tropical area of Kenya: isolates and antibiotic Susceptibility. (abs). East. Afr. Med. J., 68 (7): 500-6.
[22]Cortés G., de Astorza B., Benedi V.J andAlberti S. (2002).Role of the htrA Gene in Klebsiellapneumoniae Virulence.Infection and Immunity; 70(9) pp 4772- 4776.
[23]Regue, M., Hita, B., Pique, N., Izquierdo, L., Merino, S., Fresno, S., Benedi, V. J., and Tomas J. M.: A Gene, uge, Is Essential for K.pneumoniaVirulence. Infect. Immun. 54–61, 2004
[24]Lin H. An ,HuangYa. Li, Yeh K. M, Siu L.K, Lin J.Ch and Chang F.Y,(2014) Regulator of the mucoid phenotype A gene increases the virulent ability of extended- spectrum beta-lactamase- producing serotype non-K1/K2 Klebsiella pneumonia .Journal of Microbiology ,Immunology and infection (2014) xx,1-8.
[25]Gonza´ lez-Zorn, B., Catalan, A., Escudero, J. A., Dominguez, L,Teshager,T.,Porrero,C.andMoreno,M.A.(2005).Genetic basis for dissemination of armA.JAntimicrob Chemother.56,583-585.
[26]Galimand, M., Courvalin, P. & Lambert, T. (2003). Plasmid-mediatedhigh-level resistance to aminoglycosides in Enterobacteriaceae due to16S rRNA methylation.Antimicrob Agents Chemother 47, 2565–2571.
[27]Yan, J. J., Wu, J. J., Ko, W. C., Tsai, S. H., Chuang, C. L., Wu, H. M., Lu,Y. J. & Li, J. D. (2004). Plasmid-mediated 16S rRNAmethylasesconferring high-level aminoglycoside resistance in Escherichia coliandKlebsiellapneumoniaeisolates from two Taiwanese hospitals. JAntimicrobChemother 54, 1007–1012.
[28]Yang Zhang,1 Hua Zhou,1 Xiao-qiang Shen,2 Ping Shen,1 Yun-song Yu1and Lan-juan Li. Plasmid-borne armAmethylase gene, together with blaCTX-M-15 and blaTEM-1, in a Klebsiellaoxytocaisolate from China. Journal of Medical Microbiology (2008), 57, 1273–1276. 
[29]Lin,Y.T., Yuan,Y. J., Chen ,T. L., Chang,P. F. (2010). Bacteremic community-acquired pneumonia due to Klebsiellapneumoniae: Clinical and microbiological characteristics in Taiwan, 2001-2008. BMC Infectious Diseases, 10:307.
[30]Li B., Yi Y., Wang Q., Woo P.C.Y., Tan L., Jing H., Gao G.F and Liu C.H. (2012). Analysis of Drug Resistance Determinants in Klebsiellapneumoniae Isolates from a Tertiary-Care Hospital in Beijing, China. PLoS ONE 7(7): e42280