Application of Taqman RTi-PCR Assay in Specific Detection of Klebsiella pneumoniae from Surface Waters

Year 2018, Volume: 44 Issue: 1, 95 - 106, 30.04.2018

Esen Tutar , Kübra Sueda Akıncı İsmail Akyol

Abstract

Klebsiella pneumoniae is an opportunistic
pathogen causing nosocomial infections. The normal habitat of K. pneumoniae is the human intestines
and the bacterium causes no infection in normal flora. Although, many Klebsiella infections are
hospital-acquired infections, K. pneumoniae
may also be transferred from environmental sources due to its widely
distribution in nature. The environmental isolates of K. pneumoniae additionally pose a risk to humans as clinical
isolates. The present study aims to investigate the potential of K. pneumoniae in surface waters by using
PCR and RTi-PCR assays. We have optimized PCR and RTi-PCR assays with high
sensitivity and specificity for K.
pneumoniae. Surface waters samples were collected from different regions
and analyzed by using PCR and RTi-PCR assays. The results indicated that all
tested water samples are contaminated with K.
pneumoniae at different levels. The RTi-PCR findings were confirmed by
conventional PCR.

Keywords

K. pneumoniae, Taqman assay, RTi-PCR, surface waters

Yüzey Sularında Bulunan Klebsiella pneumoniae'nin Özgül Olarak BelirlenmesindeTaqman RTi-PCR Yönteminin Uygulanması

Abstract

Klebsiella pneumoniae, nozokomiyal enfeksiyonlara neden olan fırsatçı bir patojendir. Klebsiella pneumoniae'nin normal habitatı insan bağırsaklarıdır ve bakteri normal florasında hiçbir enfeksiyona neden olmaz. Çoğu Klebsiella enfeksiyonu hastane kaynaklıdır ancak K. pneumoniae doğada yaygın olarak bulunmasından dolayı çevresel kaynaklardan da transfer edilebilir. Ayrıca, K. pneumoniae'nin çevresel izolatları, klinik izolatlar gibi insanlar için bir risk oluşturmaktadır. Bu çalışma ile PCR ve RTi-PCR analizlerini kullanarak yüzey sularında K. pneumoniae'nin potansiyelini araştırmak amaçlanmıştır. Klebsiella pneumoniae için yüksek hassasiyet ve özgüllük ile PCR ve RTi-PCR analizleri optimize edilmiştir. Yüzey suları örnekleri farklı bölgelerden toplanmış ve PCR ve RTi-PCR deneyleri kullanılarak analiz edilmiştir. Sonuçlar, test edilen su örneklerinin K. pneumoniae ile farklı seviyelerde kontamine olduğunu göstermiştir. RTi-PCR bulguları geleneksel PCR ile teyit edilmiştir.

Keywords

K. pneumoniae, Taqman assay, RTi-PCR, Yüzey suları

References

• Aboh EA, Giwa FJ, Giwa A (2015). Microbiological assessment of well waters in Samaru, Zaria, Kaduna, State, Nigeria. Annals of African Medicine, 14: 32‑38.
• Barati A, Ghaderpour A, Chew LL, Bong CW, Thong KL, Chong VC, Chai LC (2016). Isolation and characterization of aquatic-borne Klebsiella pneumoniae from tropical estuaries in Malaysia. International Journal of Environmental Research and Public Health, 13.
• Chen L, Chavda KD, Findlay J, Peirano G, Hopkins K, Pitout JDD, Kreiswirth BN (2014). Multiplex PCR for identification of two capsular types in epidemic KPC-producing Klebsiella pneumoniae sequence type 258 strains. Antimicrobial Agents and Chemotherapy, 58: 4196‑4199.
• Clegg S and Murphy CN (2016). Epidemiology and Virulence of Klebsiella pneumoniae. Microbiology spectrum, 4: 1‑17.
• Deleo FR, Chen L, Porcella SF, Martens CA, Kobayashi SD, Porter AR, Kreiswirth BN (2014). Molecular dissection of the evolution of carbapenem-resistant multilocus sequence type 258 Klebsiella pneumoniae. Proc Natl Acad Sci U S A, 111: 4988‑4993.
• Delibato E, Fiore A, Anniballi F, Auricchio B, Filetici E, Orefice L, De Medici D (2011). Comparison between two standardized cultural methods and 24 hour duplex SYBR green real-time PCR assay for Salmonella detectionin meat samples. The new microbiologica, 34: 299‑306.
• Ding T, Suo Y, Zhang Z, Liu D, Ye X, Chen S, Zhao Y (2017). A Multiplex RT-PCR Assay for S. aureus, L. monocytogenes, and Salmonella spp. Detection in Raw Milk with Pre-enrichment. Frontiers in Microbiology, 8: 1‑11.
• Dong D, Liu W, Li H, Wang Y, Li X, Zou D, Yuan J (2015). Survey and rapid detection of Klebsiella pneumoniae in clinical samples targeting the rcsA gene in Beijing, China. Frontiers in Microbiology, 6: 1‑6.
• Fukushima H, Kawase J, Etoh Y, Sugama K, Yashiro S, Iida N, Yamaguchi K (2010). Simultaneous Screening of 24 Target Genes of Foodborne Pathogens in 35 Foodborne Outbreaks Using Multiplex Real-Time SYBR Green PCR Analysis. International journal of microbiology, 2010.
• Gadsby NJ, McHugh MP, Russell CD, Mark H, Conway-Morris A, Laurenson IF, Templeton KE (2015). Development of two real-time multiplex PCR assays for the detection and quantification of eight key bacterial pathogens in lower respiratory tract infections. Clinical Microbiology and Infection, 21: e788.
• Gierczyński R, Jagielski M, Rastawicki W, Kałuzewski S (2007). Multiplex-PCR assay for identification of Klebsiella pneumoniae isolates carrying the cps loci for K1 and K2 capsule biosynthesis. Polish journal of microbiology / Polskie Towarzystwo Mikrobiologów = The Polish Society of Microbiologists, 56: 153‑6.
• He P, Zhu G, Luo J, Wang H, Yan Y, Chen L, Chen Z (2016). Development and Application of a One-Tube Multiplex Real-Time PCR with Melting Curve Analysis for Simultaneous Detection of Five Foodborne Pathogens in Food Samples. Journal of Food Safety, 0: 1‑7.
• June M, Paul O, Kiplagat K (2016). Bacteriological Quality of Water from Selected Water Sources in Samburu South – Kenya, 9: 310‑316. Kong RYC, Lee SKY, Law TWF, Law SHW, Wu RSS (2002). Rapid detection of six types of bacterial pathogens in marine waters by multiplex PCR. Water Research, 36: 2802‑2812.
• Kurupati P, Chow C, Kumarasinghe G, Poh CL (2004). Rapid Detection of Klebsiella pneumoniae from Blood Culture Bottles by Real-Time PCR Rapid Detection of Klebsiella pneumoniae from Blood Culture Bottles by Real-Time PCR. Journal of clinical microbiology, 42: 8‑12.
• Li B, Zhao Y, Liu C, Chen Z, Zhou D (2014). Molecular pathogenesis of Klebsiella pneumoniae. Future Microbiology, 9: 1071‑1081.
• Miah B, Majumder AK, Latifa GA (2016). Evaluation of microbial quality of the surface waters of Hatirjheel in Dhaka City. Stamford Journal of Microbiology, 6: 30‑33.
• Nadkarni MA, Martin FE, Jacques NA, Hunter N (2002). Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology (Reading, England), 148: 257‑66.
• Pitout JDD, Nordmann P, Poirel L (2015). Carbapenemase-producing Klebsiella pneumoniae, a key pathogen set for global nosocomial dominance. Antimicrobial Agents and Chemotherapy, 59: 5873‑5884.
• Podschun R, Pietsch S, Höller C, Ullmann U (2001). Incidence of Klebsiella Species in Surface waterss and Their Expression of Virulence Factors Incidence of Klebsiella Species in Surface waterss and Their Expression of Virulence Factors, 67: 1‑4.
• Ramalingam N, Rui Z, Liu HB, Dai CC, Kaushik R, Ratnaharika B, Gong HQ (2010). Real-time PCR-based microfluidic array chip for simultaneous detection of multiple waterborne pathogens. Sensors and Actuators, B: Chemical, 145: 543‑552.
• Samuel O, Florence N, Ifeanyi O (2016). Microbial Quality Assessment of Commercial Bottled Water Brands in Major Markets in Awka , Nigeria. Universal Journal of Microbiology Research, 4: 1‑5.
• Seo KH and Brackett RE (2005). Rapid, specific detection of Enterobacter sakazakii in infant formula using a real-time PCR assay. J Food Prot, 68: 59‑63.
• Shannon K E, Lee DY, Trevors J T, Beaudette LA (2007). Application of real-time quantitative PCR for the detection of selected bacterial pathogens during municipal wastewater treatment. Science of The Total Environment, 382: 121‑129.
• Struve C and Krogfelt KA (2004). Pathogenic potential of environmental Klebsiella pneumoniae isolates. Environmental Microbiology, 6: 584‑590.
• Tabassum A, Saha ML, Islam MN (2015). Prevalence of multi-drug resistant bacteria in selected street food and water samples. Bangladesh J. Bot., 44: 621‑627.
• Vuotto C, Longo F, Balice M, Donelli G, Varaldo P (2014). Antibiotic Resistance Related to Biofilm Formation in Klebsiella pneumoniae. Pathogens, 3: 743‑758.
• Xiao X, Zhang L, Wu H, Yu Y, Tang Y, Liu D, Li, X (2014). Simultaneous Detection of Salmonella, Listeria monocytogenes, and Staphylococcus aureus by Multiplex Real-Time PCR Assays Using High-Resolution Melting. Food Analytical Methods, 7: 1960‑1972.