Comparison of multiple locus variable number of tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis methods for typing Staphylococcus aureus isolates

Abstract

In this study, the performance of multi locus variable number tandem repeat analysis (MLVA) method to distinguish pulsed-field gel electrophoresis (PFGE) types, known as the gold standard, for Staphylococcus aureus (S. aureus). It was tried to determine to what extent the results obtained from PFGE method for S. aureus can be obtained with MLVA method. In this way, the possibility of displacement of PFGE method, which is a challenging method in routine use, with MLVA, which is an easier and low cost method has been investigated. MLVA method was performed using a polymerase chain reaction (PCR) method for sdrCDE, clfA, clfB, sspA and spa after a rapid DNA isolation method. The PFGE method was performed with genomic DNA fragments obtained using the SmaI enzyme. The banding model was analyzed both visually and with the Bionumerics software. While isolates were divided into 2 clusters and 16 subtypes with PFGE, the same izolates were divided into 31 subtypes without forming a cluster with MLVA method. With this PCR- based method, it was seen that the strains indistinguishable by PFGE method could be distinguished. Therefore, it was concluded thet MLVA can be used as an alternative, efficient molecular typing method with high epidemiological resolution to beter identify strains unrelated to the same PFGE patterns or to verify the close genetic composition of related isolates.

Keywords

• Multiple-locus variable-number tandem-repeat analysis (MLVA),Pulsed-field gel electrophoresis (PFGE),discrimination power,molecular typing,Staphylococcus aureus

Staphylococcus aureus izolatlarının tiplendirilmesinde çoklu lokus değişken sayı tekrar analizi (MLVA) ve darbeli alan jel elektroforez (PFGE) yöntemlerinin karşılaştırılması

Abstract

Bu çalışmada, Staphylococcus aureus (S. aureus) bakterisi için, çoklu lokus değişken sayı tekrar analizi (MLVA) yönteminin, altın standart olarak bilinen moleküler tiplendirme yöntemi darbeli alan jel elektroforezi (PFGE) ile ayırt edebilme performansı araştırıldı. S. aureus için PFGE yönteminden elde edilen sonuçların MLVA yöntemi ile ne oranda sağlanabileceği belirlenmeye çalışıldı. Bu sayede rutin kullanımlarda zorlu bir yöntem olan PFGE yönteminin, daha kolay ve düşük maliyetli bir yöntem olan MLVA ile rutin de yer değiştirmesinin olanağı araştırıldı. MLVA yöntemi hızlı bir DNA izolasyon yöntemi sonrası, sdrCDE, clfA, clfB, sspA ve spa lokusları için polimeraz zincir reaksiyonu (PCR) yöntemi kullanılarak gerçekleştirildi. PFGE yöntemi ise SmaI enzimi kullanılarak elde edilen genomik DNA fragmanları ile gerçekleştirildi. Bantlama modeli, hem görsel olarak hem de Bionumerics yazılımı ile analiz edildi. PFGE ile izolatlar 2 küme ve 16 alt tipe ayrılırken, MLVA ile aynı izolatlar, küme oluşturmaksızın 31 alt tipe ayrıldı. PCR temelli bu yöntem ile, PFGE yöntemi ile ayırt edilemeyen alt tiplerin ayırt edilebildiği görüldü. Bu sebep ile MLVA yönteminin aynı PFGE paternleri ile ilgisiz suşları daha iyi tanımlamak veya ilişkili izolatların yakın genetik kompozisyonunu doğrulamak için yüksek epidemiyolojik çözünürlüğe sahip, alternatif, verimli moleküler tipleme yöntemi olarak kullanılabileceği sonucuna varıldı.

Keywords

• Çoklu lokus değişken sayı tekrar analizi (MLVA),darbeli alan jel elektroforezi (PFGE),ayırım gücü,moleküler tiplendirme,Staphylococcus aureus

References

1. Ruegg, P.L., A 100-year review: mastitis detection, management, and prevention, Journal of Dairy Science, 100, 10381–97, (2017).
2. Malachowa, N., Sabat, A., Gniadkowski, M., Krzyszton-Russjan, J., Empel, J., Miedzobrodzki, J., Kosowska-Shick, K., Appelbaum, P.C., Hryniewicz, W., Comparison of multiple-locus variable-number tandem-repeat analysis with pulsed-field gel electrophoresis, spa typing and multilocus sequence typing for clonal characterization of Staphylococcus aureus isolates, Journal of Clinical Microbiology, 43, 7, 3095-3100, (2005).
3. Schouls, L.M., Spalburg, E.C., Luit, M., Huijsdens, W., Pluister, G.N., Santen-Verheuvel, M.G., Heide, H.G.J., Grundmann, H., Heck, M.E.O.C., Neeling, A.J. Multiple- locus variable number tandem repeat analysis of Staphylococcus Aureus: Comparison with pulsed-field gel electrophoresis and spa-typing, Plos one, 4, 4, e5082, (2009).
4. de Neeling, A.J., van den Broek, M.J., Spalburg, E.C., van Santen-Verheuvel, M.G., Dam-Deisz, W.D., Boshuizen, H.C., van de Giessen, A.W., van Duijkeren, E., Huigsdens, X.W., High prevalence of methicillin resistant Staphylococcus aureus in pigs. Veterinary Microbiology, 122, 366–372, (2007). de Neeling, A.J., van Leeuwen, W.J., Schouls, L.M., Schot, C.S., van Veen-Rutgers, A., Beunders, A.J., Buiting, A.G., Hol, C., Ligtvoet, E.E., Petit, P.L., Sabbe, L.J., van Griethysen, A.J., van Embden, J.D., Resistance of staphylococci in The Netherlands: surveillance by an electronic network during 1989–1995. Journal of Antimicrobial Chemotherapy, 41, 93–101, (1998).
5. Holmes, A., Edward, G.F., Girvan, E.K., Hannant, W., Danial, J., Fitzgerald, J.R., Templeton, K.E., Multilocus variable- number tandem-repeat methods and pulsed field gel electrophoresis for differentiating highly clonal methicillin-resistant Staphylococcus aureus isolates, Journal of Clinical Microbiology, 48, 10, 3600-3607, (2010).
6. Tenover, F.C., Vaughn, R.R., McDaugal, L.K., Fosheim, G.E., McGowan J.E., Multiple-locus variable- number tandem- repeat assay analysis of meticillin-resistant Staphylococcus aureus strains, Journal of Clinical Microbiology, 45, 7, 2215-2219, (2007).
7. Rasschaert, G., Vanderhaeghen, W., Dewaele, I., Janez, N., Huijsdens, X., Butaye, P., Heyndrickx, M., Comparison of fingerprinting methods for typing methicillin-resistant Staphylococcus aureus sequence type 398. Journal of Clinical Microbiology. 47, 10, 3313-3322, (2009).
8. Sener, K., Saraclı, M.A., Açıkel, C.H., Dogancı, L. Türkiye Klinikleri Mikrobiyoloji Enfeksiyon, 3, 1-6, (2004).

9. Arbeit, R.D., Laboratory procedures for epidemiologic analysis of microorganisms. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH eds. Manual of Clinical Microbiology, 7th ed. Washington DC: ASM Pres, 116-37, (1999).
10. Francois, P., Huyghe, A.,. Charbonnier, Y., Bento, M., Herzig, S., Topopski, I., Fleury, B., Lew, D., Vaudaux, P., Harbarth, S., van Leeuwen, W., van Belkum, A.,. Blanc, D.S., Pittet, D., Schrenzel, J., Use of an automated multiple-locus, variable-number tandem repeat-based method for rapid and high-throughput genotyping of Staphylococcus aureus isolates. Journal of Clinical Microbiology, 43, 3346–3355, (2005). Luczak-Kadlubowska, A., Sabat, A., Tambic-Andrasevic, A., Payerl-Pal, M., Krzyszton-Russjan, J.,. Hryniewicz, W., Usefulness of multiple-locus VNTR fingerprinting in detection of clonality of community- and hospital acquired Staphylococcus aureus isolates. Antonie van Leeuwenhoek, 94, 543– 553, (2008).
11. Malachowa, N., Sabat, A., Gniadkowski, M., Krzyszton-Russjan, J., Empel, J., Miedzobrodzki, J., Kosowska-Shick, K., Appelbaum, P. C., Hryniewicz, W., Comparison of multiple-locus variable-number tandem-repeat analysis with pulsed-field gel electrophoresis, spa typing, and multilocus sequence typing for clonal characterization of Staphylococcus aureus isolates. Journal of Clinical Microbiology, 43, 3095–3100, (2005).
12. Milheirico,C., Oliveira, D.C., Lencastre, H., Multiplex PCR strategy for subtyping the staphylococcal cassette chromosome mec type IV in methicillin-resistant Staphylococcus aureus: ‘SCCmec IV multiplex.’ Journal of Antimicrobial Chemotherapy, 60, 42–48, (2007).
13. Farlow, J., Smith, K.L., Wong, J., Abrams, M., Lytle, M., Keim, P., Francisella tularensis strain typing using multiple-locus, variable-number tandem repeat analysis. Journal of Clinical Microbiology, 39, 3186–3192, (2001).
14. Pourcel, C., Vidgop, Y., Ramisse, F., Vergnaud, G., Tram, C., Characterization of a tandem repeat polymorphism in Legionella pneumophila and its use for genotyping. Journal of Clinical Microbiology 41, 1819–1826, (2003).
15. Schouls, L.M., van der Heide, H.G., Vauterin, L., Vauterin, P., Mooi, F.R. Multiple-locus variable-number tandem repeat analysis of Dutch Bordetella pertussis strains reveals rapid genetic changes with clonal expansion during the late 1990s. Journal of Bacteriology, 186, 5496–5505, (2004)
16. Ausubel, F.M., Kingston, R.E., Brent, R., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. Current protocols in molecular biology. Greene Publishing Associates & Wiley Interscience, New York (1991).
17. Moser, S.A., Box, M.J., Patel, M., Amaya, M., Schelonka, R., Waites, K.B., Multiple-locus variable number tandem- repeat analysis of methicillin-resistant Staphylococcus aureus discriminates within USA pulsed-field gel electrophoresis. Journal of Hospital Infection, 71, 4, 333-339, (2009).
18. Sabat, A., Krzyszton-Russjan, J., Strzalka, W., Filipek, R., Kosowska, K., Hryniewicz, W., Travis, J., Potempa, J., New method for typing Staphylococcus aureus strains: multiple-locus variable-number tandem repeat analysis of polymorphism and genetic relationships of clinical isolates. Journal of Clinical Microbiology. 41,1801–1804, (2003).
19. Mulvey, M.R., Chui, L., Ismail, J., Louie, L., Murphy, C., Chang, N., Alfa, M., Development of a Canadian standardized protocol for subtyping methicillin-resistant Staphylococcus aureus using pulsed-field gel electrophoresis. Journal of Clinical Microbiology, 39, 3481–3485, (2001).
20. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, Swaminathan, B., Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. Journal of Clinical Microbiology, 1995; 33 (9), 2233-9, (1995).
21. Kjeldsen, M.K., Torpdahl, M., Nielsen, E.M., Development and comparison of a generic multiple-locus variable-number tandem repeat analysis with pulsed-field gel electrophoresis for typing of Salmonella enterica subsp. enterica. Journal of Applied Microbiology, 119, 1707-1717, (2015).
22. Boxrud, D., Pederson-Gulrud, K., Wotton, J., Medus, C., Lyszkowicz, E., Besser, J. and Bartkus, J.M., Comparison of multiple-locus variable-number tandem repeat analysis, pulsed-field gel electrophoresis, and phage typing for subtype analysis of Salmonella enterica serotype Enteritidis. Journal of Clinical Microbiology, 45, 536–543, (2007)
23. Liebana, E., Garcia-Migura, L., Clouting, C., Cassar, C.A., Clifton-Hadley, F.A., Lindsay, E.A., Threlfall, E.J., Chappell, S.A., Davies, R.H., Investigation of the genetic diversity among isolates of Salmonella enterica serovar Dublin from animals and humans from England, Wales and Ireland. Journal of Applied Microbiology, 93, 732–744, (2002).
24. Top, J., Banga, N.M.I., Hayer, R., Willens, R.J., Bonten, M.J.M., Hayden, M.K., Comparison of multiple-locus var,able-number tandem repeat analysis and pulsed-field gel electrophoresis in a setting of polyclonal endemicity of vancomycin-resistant Enterococcus faecium, Clinical Microbiology and Infection, 14, 363-369, (2008).
25. Song, M., Shi, C., Xu, X., Shi, X., Molecular typing and virulence gene profiles of enterotoxin gene cluster (egc-positive Staphylococcus aureus isolates obtained from various food and clinical specimens. Foodborne Pathogen and Disease, 13, 11, (2016).