1887

Journal of Medical Microbiology logo

Volume 67, Issue 9

Development of a species-specific PCR-RFLP targeting rpoD gene fragment for discrimination of Aeromonas species Free

Abstract

Purpose. The taxonomy of Aeromonas keeps expanding and their identification remains problematic due to their phenotypic and genotypic heterogeneity. In this study, we aimed to develop a rapid and reliable polymerase chain reaction-restriction fragment length polymorphism assay targeting the rpoD gene to enable the differentiation of aeromonads into 27 distinct species using microfluidic capillary electrophoresis.

Methodology. A pair of degenerate primers (Aero F: 5′-YGARATCGAYATCGCCAARCGB-3′ and Aero R: 5′-GRCCDATGCTCATRCGRCGGTT-3′) was designed that amplified the rpoD gene of 27 Aeromonas species. Subsequently, in silico analysis enabled the differentiation of 25 species using the single restriction endonuclease AluI, while 2 species, A. sanarelli and A. taiwanensis, required an additional restriction endonuclease, HpyCH4IV. Twelve type strains (A. hydrophila ATCC7966, A. caviae ATCC15468, A. veronii ATCC9071, A. media DSM4881, A. allosaccharophila DSM11576, A. dhakensis DSM17689, A. enteropelogens DSM7312, A. jandaei DSM7311, A. rivuli DSM22539, A. salmonicida ATCC33658, A. taiwanensis DSM24096 and A. sanarelli DSM24094) were randomly selected from the 27 Aeromonas species for experimental validation.

Results/key findings. The twelve type strains demonstrated distinctive RFLP patterns and supported the in silico digestion. Subsequently, 60 clinical and environmental strains from our collection, comprising nine Aeromonas species, were used for screening examinations, and the results were in agreement.

Conclusion. This method provides an alternative method for laboratory identification, surveillance and epidemiological investigations of clinical and environmental specimens.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): Aeromonas , micro-fluid chip , molecular typing , PCR-RFLP and rapid identification
© 2018 The Authors
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.000796
2018-07-19
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/jmm/67/9/1271.html?itemId=/content/journal/jmm/10.1099/jmm.0.000796&mimeType=html&fmt=ahah

References

  1. Janda JM, Abbott SL. The genus Aeromonas: taxonomy, pathogenicity, and infection. Clin Microbiol Rev 2010; 23:35–73 [View Article][PubMed]
     [Google Scholar]
  2. Lee WS, Puthucheary SD. Retrospective study of Aeromonas infection in a Malaysian urban area: a 10-year experience. Singapore Med J 2001; 42:57–60
     [Google Scholar]
  3. Puah SM, Puthucheary SD, Liew FY, Chua KH. Aeromonas aquariorum clinical isolates: antimicrobial profiles, plasmids and genetic determinants. Int J Antimicrob Agents 2013; 41:281–284 [View Article][PubMed]
     [Google Scholar]
  4. Chen PL, Wu CJ, Chen CS, Tsai PJ, Tang HJ et al. A comparative study of clinical Aeromonas dhakensis and Aeromonas hydrophila isolates in southern Taiwan: A. dhakensis is more predominant and virulent. Clin Microbiol Infect 2014; 20:O428–O434 [View Article][PubMed]
     [Google Scholar]
  5. Skwor T, Shinko J, Augustyniak A, Gee C, Andraso G. Aeromonas hydrophila and Aeromonas veronii predominate among potentially pathogenic ciprofloxacin- and tetracycline-resistant aeromonas isolates from Lake Erie. Appl Environ Microbiol 2014; 80:841–848 [View Article][PubMed]
     [Google Scholar]
  6. Yano Y, Hamano K, Tsutsui I, Aue-Umneoy D, Ban M et al. Occurrence, molecular characterization, and antimicrobial susceptibility of Aeromonas spp. in marine species of shrimps cultured at inland low salinity ponds. Food Microbiol 2015; 47:21–27 [View Article][PubMed]
     [Google Scholar]
  7. Latif-Eugenín F, Beaz-Hidalgo R, Silvera-Simón C, Fernandez-Cassi X, Figueras MJ. Chlorinated and ultraviolet radiation –treated reclaimed irrigation water is the source of Aeromonas found in vegetables used for human consumption. Environ Res 2017; 154:190–195 [View Article][PubMed]
     [Google Scholar]
  8. Khor WC, Puah SM, Koh TH, Tan J, Puthucheary SD et al. Comparison of clinical isolates of Aeromonas from Singapore and Malaysia with regard to molecular identification, virulence, and antimicrobial profiles. Microb Drug Resist 2018; 24:469–478 [View Article][PubMed]
     [Google Scholar]
  9. Figueras MJ, Alperi A, Beaz-Hidalgo R, Stackebrandt E, Brambilla E et al. Aeromonas rivuli sp. nov., isolated from the upstream region of a karst water rivulet. Int J Syst Evol Microbiol 2011; 61:242–248 [View Article][PubMed]
     [Google Scholar]
  10. Aravena-Román M, Beaz-Hidalgo R, Inglis TJ, Riley TV, Martínez-Murcia AJ et al. Aeromonas australiensis sp. nov., isolated from irrigation water. Int J Syst Evol Microbiol 2013; 63:2270–2276 [View Article][PubMed]
     [Google Scholar]
  11. Martínez-Murcia A, Beaz-Hidalgo R, Svec P, Saavedra MJ, Figueras MJ et al. Aeromonas cavernicola sp. nov., isolated from fresh water of a brook in a cavern. Curr Microbiol 2013; 66:197–204 [View Article][PubMed]
     [Google Scholar]
  12. Beaz-Hidalgo R, Hossain MJ, Liles MR, Figueras MJ. Strategies to avoid wrongly labelled genomes using as example the detected wrong taxonomic affiliation for aeromonas genomes in the GenBank database. PLoS One 2015; 10:e0115813 [View Article][PubMed]
     [Google Scholar]
  13. Figueras MJ, Latif-Eugenín F, Ballester F, Pujol I, Tena D et al. 'Aeromonas intestinalis' and 'Aeromonas enterica' isolated from human faeces, 'Aeromonas crassostreae' from oyster and 'Aeromonas aquatilis' isolated from lake water represent novel species. New Microbes New Infect 2017; 15:74–76 [View Article][PubMed]
     [Google Scholar]
  14. Beaz-Hidalgo R, Alperi A, Buján N, Romalde JL, Figueras MJ. Comparison of phenotypical and genetic identification of Aeromonas strains isolated from diseased fish. Syst Appl Microbiol 2010; 33:149–153 [View Article][PubMed]
     [Google Scholar]
  15. Vincent AT, Rouleau FD, Moineau S, Charette SJ. Study of mesophilic Aeromonas salmonicida A527 strain sheds light on the species' lifestyles and taxonomic dilemma. FEMS Microbiol Lett 2017; 364: [View Article][PubMed]
     [Google Scholar]
  16. Pfeiffer F, Zamora-Lagos MA, Blettinger M, Yeroslaviz A, Dahl A et al. The complete and fully assembled genome sequence of Aeromonas salmonicida subsp. pectinolytica and its comparative analysis with other Aeromonas species: investigation of the mobilome in environmental and pathogenic strains. BMC Genomics 2018; 19:20 [View Article][PubMed]
     [Google Scholar]
  17. Gauthier J, Vincent AT, Charette SJ, Derome N. Strong genomic and phenotypic heterogeneity in the Aeromonas sobria species complex. Front Microbiol 2017; 8:2434 [View Article][PubMed]
     [Google Scholar]
  18. Colston SM, Fullmer MS, Beka L, Lamy B, Gogarten JP et al. Bioinformatic genome comparisons for taxonomic and phylogenetic assignments using Aeromonas as a test case. MBio 2014; 5:e0236 [View Article][PubMed]
     [Google Scholar]
  19. Puthucheary SD, Puah SM, Chua KH. Molecular characterization of clinical isolates of Aeromonas species from Malaysia. PLoS One 2012; 7:e30205 [View Article][PubMed]
     [Google Scholar]
  20. Chen PL, Lee TF, Wu CJ, Teng SH, Teng LJ et al. Matrix-assisted laser desorption ionization-time of flight mass spectrometry can accurately differentiate Aeromonas dhakensis from A. hydrophila, A. caviae, and A. veronii. J Clin Microbiol 2014; 52:2625–2628 [View Article][PubMed]
     [Google Scholar]
  21. Lamy B, Laurent F, Verdier I, Decousser JW, Lecaillon E et al. Accuracy of 6 commercial systems for identifying clinical Aeromonas isolates. Diagn Microbiol Infect Dis 2010; 67:9–14 [View Article][PubMed]
     [Google Scholar]
  22. Borrell N, Acinas SG, Figueras MJ, Martínez-Murcia AJ. Identification of Aeromonas clinical isolates by restriction fragment length polymorphism of PCR-amplified 16S rRNA genes. J Clin Microbiol 1997; 35:1671–1674[PubMed]
     [Google Scholar]
  23. Graf J. Diverse restriction fragment length polymorphism patterns of the PCR-amplified 16S rRNA genes in Aeromonas veronii strains and possible misidentification of Aeromonas species. J Clin Microbiol 1999; 37:3194–3197[PubMed]
     [Google Scholar]
  24. Figueras MJ, Soler L, Chacón MR, Guarro J, Martínez-Murcia AJ. Extended method for discrimination of Aeromonas spp. by 16S rDNA RFLP analysis. Int J Syst Evol Microbiol 2000; 50:2069–2073 [View Article][PubMed]
     [Google Scholar]
  25. Ghatak S, Agarwal RK, Bhilegaonkar KN. Species identification of clinically important Aeromonas spp. by restriction fragment length polymorphism of 16S rDNA. Lett Appl Microbiol 2007; 44:550–554 [View Article][PubMed]
     [Google Scholar]
  26. Soler L, Yáñez MA, Chacon MR, Aguilera-Arreola MG, Catalán V et al. Phylogenetic analysis of the genus Aeromonas based on two housekeeping genes. Int J Syst Evol Microbiol 2004; 54:1511–1519 [View Article][PubMed]
     [Google Scholar]
  27. Küpfer M, Kuhnert P, Korczak BM, Peduzzi R, Demarta A. Genetic relationships of Aeromonas strains inferred from 16S rRNA, gyrB and rpoB gene sequences. Int J Syst Evol Microbiol 2006; 56:2743–2751 [View Article][PubMed]
     [Google Scholar]
  28. Alperi A, Figueras MJ, Inza I, Martínez-Murcia AJ. Analysis of 16S rRNA gene mutations in a subset of Aeromonas strains and their impact in species delineation. Int Microbiol 2008; 11:185–194[PubMed]
     [Google Scholar]
  29. Martínez-Murcia AJ, Figueras MJ, Saavedra MJ, Stackebrandt E. The recently proposed species Aeromonas sharmana sp. nov., isolate GPTSA-6T, is not a member of the genus Aeromonas. Int Microbiol 2007; 10:61–64[PubMed]
     [Google Scholar]
  30. Miñana-Galbis D, Urbizu-Serrano A, Farfán M, Fusté MC, Lorén JG. Phylogenetic analysis and identification of Aeromonas species based on sequencing of the cpn60 universal target. Int J Syst Evol Microbiol 2009; 59:1976–1983 [View Article][PubMed]
     [Google Scholar]
  31. Hu M, Wang N, Pan ZH, Lu CP, Liu YJ. Identity and virulence properties of Aeromonas isolates from diseased fish, healthy controls and water environment in China. Lett Appl Microbiol 2012; 55:224–233 [View Article][PubMed]
     [Google Scholar]
  32. Nagar V, Shashidhar R, Bandekar JR. Characterization of Aeromonas strains isolated from Indian foods using rpoD gene sequencing and whole cell protein analysis. World J Microbiol Biotechnol 2013; 29:745–752 [View Article][PubMed]
     [Google Scholar]
  33. Khor WC, Puah SM, Tan JA, Puthucheary SD, Chua KH. Phenotypic and genetic diversity of Aeromonas species isolated from fresh water lakes in Malaysia. PLoS One 2015; 10:e0145933 [View Article][PubMed]
     [Google Scholar]
  34. Li W, Raoult D, Fournier PE. Bacterial strain typing in the genomic era. FEMS Microbiol Rev 2009; 33:892–916 [View Article][PubMed]
     [Google Scholar]
  35. Nachamkin I, Panaro NJ, Li M, Ung H, Yuen PK et al. Agilent 2100 bioanalyzer for restriction fragment length polymorphism analysis of the Campylobacter jejuni flagellin gene. J Clin Microbiol 2001; 39:754–757 [View Article][PubMed]
     [Google Scholar]
  36. Ho HT, Chang PL, Hung CC, Chang HT. Capillary electrophoretic restriction fragment length polymorphism patterns for the Mycobacterial hsp65 gene. J Clin Microbiol 2004; 42:3525–3231 [View Article][PubMed]
     [Google Scholar]
  37. Hathaway LJ, Brugger S, Martynova A, Aebi S, Mühlemann K. Use of the Agilent 2100 bioanalyzer for rapid and reproducible molecular typing of Streptococcus pneumoniae. J Clin Microbiol 2007; 45:803–809 [View Article][PubMed]
     [Google Scholar]
  38. Lorenz SC, Fischer M, Kase JA. Improved PCR-RFLP method for the identification of Escherichia coli enterohemolysin (ehxA) subtypes. J Microbiol Methods 2014; 100:24–26 [View Article][PubMed]
     [Google Scholar]
  39. Soler-García AA, de Jesús AJ, Taylor K, Brown EW. Differentiation of Salmonella strains from the SARA, SARB and SARC reference collections by using three genes PCR-RFLP and the 2100 Agilent Bioanalyzer. Front Microbiol 2014; 5:417 [View Article][PubMed]
     [Google Scholar]
  40. San Millán RM, Martínez-Ballesteros I, Rementeria A, Garaizar J, Bikandi J. Online exercise for the design and simulation of PCR and PCR-RFLP experiments. BMC Res Notes 2013; 6:513 [View Article][PubMed]
     [Google Scholar]
  41. Puah SM, Chua KH, Tan JA. Virulence factors and antibiotic susceptibility of Staphylococcus aureus Isolates in ready-to-eat foods: detection of S. aureus contamination and a high prevalence of virulence genes. Int J Environ Res Public Health 2016; 13:199 [View Article][PubMed]
     [Google Scholar]
  42. Kluyver AJ, van Niel CB. rospects for a natural system of classification of bacteria. Zentralbl Bakt li Abt 1936; 94:369–403
     [Google Scholar]
  43. Lee C, Cho JC, Lee SH, Lee DG, Kim SJ. Distribution of Aeromonas spp. as identified by 16S rDNA restriction fragment length polymorphism analysis in a trout farm. J Appl Microbiol 2002; 93:976–985 [View Article][PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.000796
Loading
Development of a species-specific PCR-RFLP targeting rpoD gene fragment for discrimination of Aeromonas species
J. Med. Microbiol. 67, 1271 (2018); https://doi.org/10.1099/jmm.0.000796
/content/journal/jmm/10.1099/jmm.0.000796
/content/journal/jmm/10.1099/jmm.0.000796
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error