Development of duplex-PCR for identification of Aeromonas species

Carina Lucena Mendes-Marques Ernesto Hofer Nilma Cintra Leal About the authors

Abstract

Introduction

The number of reports of intestinal infections caused by Aeromonas spp. has increased significantly in recent years. In most clinical laboratories, identification of these bacteria is carried out by general phenotypic tests that sometimes do not accurately differentiate Aeromonas and Vibrio.

Methods

A duplex-polymerase chain reaction (PCR) was developed directed to 2 targets identifying Aeromonas spp. pathogenic to humans.

Results

The duplex-PCR results were reproducible and specific for Aeromonas spp. pathogenic to humans.

Conclusions

This method will allow differentiation between Vibrio and Aeromonas spp. in patients with in cholera-like symptoms and can also be used in water quality monitoring.

Aeromonas ; Identification; PCR


Aeromonas spp. are gram-negative aquatic bacteria involved in infections such as pneumonia, sepsis, hemolytic uremic syndrome, septic arthritis, and more recently they have been reported causing intestinal infections11. Janda MJ, Abbott SL. The genus Aeromonas: Taxonomy, pathogenicity and infection. Clin Microbiol Rev 2010; 23:35-73..

Aeromonas diagnosis in most clinical laboratories, especially in developing countries, is based on phenotypic methods. The oxidase test is used for differential diagnosis from Enterobacteriaceae and a series of other biochemical tests are used for differential diagnosis from Vibrio and Plesiomonas 22. Guenguesh KS, Ahmed SF, El-Khalek RA, Al-Gendy A, Klena J. Aeromonas-associated infections in developing countries. J Infect Dev Ctries 2008; 2:81-98.. The results are imprecise and Aeromonas is often misclassified, being mainly misidentified as Vibrio, which similarly grows on thiosulfate citrate bile salts sucrose (TCBS) and is oxidase positive33. Abbott SL, Seli LS, Catino M Jr, Hartley MA, Janda MJ. Misidentification of unusual Aeromonas species as members of the genus Vibrio: a continuing problem. J Clin Microbiol 1998; 36:1103-1104.. Commercial systems for bacterial identification such as API20E and Vitek have proven useless for Aeromonas identification33. Abbott SL, Seli LS, Catino M Jr, Hartley MA, Janda MJ. Misidentification of unusual Aeromonas species as members of the genus Vibrio: a continuing problem. J Clin Microbiol 1998; 36:1103-1104.,44. Park TS, Oh SH, Lee EY, Lee TK, Park KH, Figueras MJ, et al. Misidentification of Aeromonas veronii biovar sobria as Vibrio alginolyticus by the Vitek system. Lett Appl Microbiol 2003; 37:349-353.. Hence, the role of Aeromonas as an etiologic agent of infection remains underestimated22. Guenguesh KS, Ahmed SF, El-Khalek RA, Al-Gendy A, Klena J. Aeromonas-associated infections in developing countries. J Infect Dev Ctries 2008; 2:81-98..

Several molecular methods for genotypic identification of Aeromonas spp.55. Figueras MJ. Clinical relevance of Aeromonas species. Rev Med Microbiol 2005; 16:145-153. are now available66. 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; 50:2069-2073.99. Yu CP, Farrell SK, Robinson B, Chu KH. Development and application of real-time PCR assays for quantifying total and aerolysin gene-containing Aeromonas in source, intermediate, and finished drinking water. Environ Sci Technol 2008; 42:1191-1200.. However, most of them are species-specific and targeted to potential virulence genes. Hence, they are unable to recognize non-virulent Aeromonas species.

Here we describe a duplex polymerase chain reaction (PCR) that provided timely and accurate identification of medically important Aeromonas spp. by amplification of genes encoding glycerolphospholipid: cholesterol acyltransferase (gcat) and small subunit (16S) recombinant DNA (rRNA).

Preliminary tests were performed using reference strains of Aeromonas spp. most commonly involved in human diseases (A. caviae, A. hydrophila, A. jandaei, A. media, A. veronii, and A. trota) as well as Vibrio species of major medical importance (V. cholerae, V. alginolyticus, V. fluvialis, V. furnissi, V. mimicus, V. parahaemolyticus and V. vulnificus). We also tested 40 strains of Aeromonas spp. that were isolated from feces of patients with diarrhea.

Bacterial cultures were provided by the Bacterial Culture Collection of Health Importance/IOC/FIOCRUZ. Aeromonas strains were identified genotypically by restriction fragment length polymorphism (RFLP)66. 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. and Vibrio isolates were typed by biochemical and serological methods. Extraction of chromosomal DNA from the cultures was performed as previously described1010. Leal NC, Sobreira M, Leal-Balbino TC, Almeida AM, Silva MJ, Mello DM, et al. Evaluation of a RAPD-based typing scheme in a molecular epidemiology study of Vibrio cholerae O1, Brazil. J App Microbiol 2004; 96:447-454..

Some authors1111. Chacón MR, Castro-Escarpulli G, Soler L, Guarro J, Figueras MJ. A DNA probe specific for Aeromonas colonies. Diagn Microbiol Infect Dis 2002; 44:221-225.,1212. Castro-Escarpulli G, Figueras MJ, Aguilera-Arreola G, Soler L, Fernández-Rendón E, Aparício GO, et al. Characterization of Aeromonas spp. isolated from frozen fish intended for human consumption in Mexico. Int J Food Microbiol 2003; 84:41-49. have suggested that all Aeromonas spp. harbor gcat, but others report that some do not77. Nam IY, Joh K. Rapid detection of virulence factors of Aeromonas isolated from a trout farm by hexaplex-PCR. J Microbiol 2007; 45:297-304.,1313. Guerra IMF, Fadanelli R, Figueiró M, Schreiner F, Delamare APL, Wollheim C, et al. Aeromonas associated diarrhoeal disease in south Brazil: prevalence, virulence factors and antimicrobial resistance. Braz J Microbiol 2007; 38:638-643.,1414. Nawaz M, Khan SA, Khan AA, Sung K, Tran Q, Kerdahi K, et al. Detection and characterization of virulence genes and integrons in Aeromonas veronii isolated from catfish. Food Microbiol 2010; 27:327-331.. Therefore, we included the second primer targeted to the 16S gene. Primer sequences gcat-f: 5′-ctcctggaatcccaagtatcag-3′ and 5′-gcat-r ggcaggttgaacagcagtatct-3′ were previously described1515. Soler L, Figueras MJ, Chacón MR, Vila J, Marco F, Martínez-Murcia AJ, et al. Potential virulence and antimicrobial susceptibility of Aeromonas popoffii recovered from freshwater and seawater. FEMS Immunol Med Microbiol 2002; 32:243-247., and 16S-f 5′-acgcaggcggttggataagt-3′ and 5′-16S-r ggcaacaaaggacaggggt-3′ were designed for the present study. For primer design, Aeromonas spp. 16S gene sequences were collected from the European Molecular Biology Laboratory (EMBL) database (accession nos. X60411, X60412, X60415, X60416, FJ998417, HM007582, AB034760, AJ224309, and FJ998415) and aligned by MegAlign (DNAstar), and the conserved regions within the gene were selected.

Duplex-PCR reactions were prepared in a total volume of 25 µL containing 50 mM KCl, 10 mM Tris-HCl, 2.5M MgCl2, 400 mM of each dNTP, 40 pmol GCAT primers, 20 pmol 16S primers, 1U Taq DNA polymerase (Promega), and 20 ng DNA. The amplifications were performed in a Biometra T-3000 Genetic Analyzer thermal cycler programmed for 35 cycles of 1 min at 94°C, 1 min at 54°C, 1 min at 72°C and a final 5 min extension at 72°C. Ten microliters of PCR products were electrophoresed in a 1% agarose gel containing SYBR Safe DNA gel stain (Invitrogen) at 100 V for 1 h, visualized on an ultraviolet (UV) transilluminator, and photographed using the Kodak 1D image analysis version 3.5 (Digital Kodak Science).

Duplex-PCR reproducibility was assessed by quadruplicate assays with 4 Aeromonas reference strains (A. hydrophila ATCC 7966T, A. veronii ATCC 35624T bio veronii, A. caviae ATCC 15468T, and A. hydrophila IOC 11036), and specificity was assessed employing 6 reference Aeromonas and 7 Vibrio spp. isolates. As noted, 40 clinical strains of Aeromonas spp. were also included in the tests. Although there are more than 30 Aeromonas species described, only 6 are commonly found to be involved in human infections11. Janda MJ, Abbott SL. The genus Aeromonas: Taxonomy, pathogenicity and infection. Clin Microbiol Rev 2010; 23:35-73., and differential diagnosis is clinically challenging. These clinically important species were among those included in the present study.

The 2 target segments of gcat (237 bp) and 16S (∼ 600 bp) were amplified in all Aeromonas reference strains tested ( Figure 1) (Figure 2, lanes 1-8) as well as in the 40 clinical isolates mentioned (data not shown). The gcat gene was not amplified in any Vibrio species tested (Figure 2, lanes 9–16). However, faint bands corresponding to 16S were seen with V. cholerae non-O1/non-O139, V. alginolyticus, V. mimicus, and V. parahaemolyticus (Figure 2, lanes 10, 11, 14, and 15, respectively).

FIGURE 1
Duplex-PCR reproducibility. Lanes: M: 100 bp molecular marker; 1: Aeromonas caviae ATCC 7966; 2: Aeromonas veronii ATCC 35624; 3: Aeromonas caviae ATCC 15468; 4: Aeromonas hydrophila IOC 11036; 5: negative control.

FIGURE 2
Duplex-PCR specificity. Lanes: M: 100 bp molecular marker; 1: Aeromonas caviae; 2: atypical Aeromonas caviae; 3: Aeromonas hydrophila; 4: Aeromonas jandaei; 5: Aeromonas media; 6: Aeromonas veronii; 7: atypical Aeromonas veronii; 8: Aeromonas trota; 9: Vibrio cholerae O1; 10: Vibrio cholerae non-O1/non-O139; 11: Vibrio alginolyticus; 12: Vibrio fluvialis; 13: Vibrio furnissi; 14: Vibrio mimicus; 15: Vibrio parahaemolyticus; 16: Vibrio vulnificus; 17: positive control; 18: negative control.

Vibrio spp. were included because of their biochemical and serological similarities to Aeromonas, which, as noted, have previously made differentiation difficult. Although the 16S gene was amplified in some of the Vibrio species, it did not hinder the efficacy of the test, which recorded samples positive for Aeromonas only when both of the targeted genes were amplified.

The duplex-PCR method introduced here showed high reproducibility and specificity for Aeromonas spp. Therefore it should be useful as an alternative to phenotypic methods for identifying these bacteria and allowing a presumptive differentiation between the Aeromonadaceae and the Vibrionaceae that are commonly involved in human infections.

Rigorous validation of the technique should be sought by increasing testing with clinical Aeromonas isolates and other gram-negative oxidase positive bacteria strains. However, we consider publication of these preliminary results necessary because they indicate that laboratory identification of Aeromonas spp. can be improved with the duplex-PCR method we describe.

If validated, this duplex-PCR method can be employed to more effectively evaluate the incidence of Aeromonas in human enteric disease during routine diagnosis versus the traditional phenotypic procedures. It will allow a better understanding of the emerging role Aeromonas species in the pathogenesis of enteric infections and assist in guiding appropriate control measures.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

FINANCIAL SUPPORT

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

The authors are thankful to Dr. Alzira Almeida for critical review and to the Bacterial Culture Collection of Health Importance/IOC/FIOCRUZ for providing bacterial cultures.

REFERENCES

  • 1
    Janda MJ, Abbott SL. The genus Aeromonas: Taxonomy, pathogenicity and infection. Clin Microbiol Rev 2010; 23:35-73.
  • 2
    Guenguesh KS, Ahmed SF, El-Khalek RA, Al-Gendy A, Klena J. Aeromonas-associated infections in developing countries. J Infect Dev Ctries 2008; 2:81-98.
  • 3
    Abbott SL, Seli LS, Catino M Jr, Hartley MA, Janda MJ. Misidentification of unusual Aeromonas species as members of the genus Vibrio: a continuing problem. J Clin Microbiol 1998; 36:1103-1104.
  • 4
    Park TS, Oh SH, Lee EY, Lee TK, Park KH, Figueras MJ, et al. Misidentification of Aeromonas veronii biovar sobria as Vibrio alginolyticus by the Vitek system. Lett Appl Microbiol 2003; 37:349-353.
  • 5
    Figueras MJ. Clinical relevance of Aeromonas species. Rev Med Microbiol 2005; 16:145-153.
  • 6
    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.
  • 7
    Nam IY, Joh K. Rapid detection of virulence factors of Aeromonas isolated from a trout farm by hexaplex-PCR. J Microbiol 2007; 45:297-304.
  • 8
    Sen K, Rodgers M. Distribution of six virulence factors in Aeromonas species isolated from US drinking water utilities: a PCR identification. J Appl Microbiol 2004; 97:1077-1086.
  • 9
    Yu CP, Farrell SK, Robinson B, Chu KH. Development and application of real-time PCR assays for quantifying total and aerolysin gene-containing Aeromonas in source, intermediate, and finished drinking water. Environ Sci Technol 2008; 42:1191-1200.
  • 10
    Leal NC, Sobreira M, Leal-Balbino TC, Almeida AM, Silva MJ, Mello DM, et al. Evaluation of a RAPD-based typing scheme in a molecular epidemiology study of Vibrio cholerae O1, Brazil. J App Microbiol 2004; 96:447-454.
  • 11
    Chacón MR, Castro-Escarpulli G, Soler L, Guarro J, Figueras MJ. A DNA probe specific for Aeromonas colonies. Diagn Microbiol Infect Dis 2002; 44:221-225.
  • 12
    Castro-Escarpulli G, Figueras MJ, Aguilera-Arreola G, Soler L, Fernández-Rendón E, Aparício GO, et al. Characterization of Aeromonas spp. isolated from frozen fish intended for human consumption in Mexico. Int J Food Microbiol 2003; 84:41-49.
  • 13
    Guerra IMF, Fadanelli R, Figueiró M, Schreiner F, Delamare APL, Wollheim C, et al. Aeromonas associated diarrhoeal disease in south Brazil: prevalence, virulence factors and antimicrobial resistance. Braz J Microbiol 2007; 38:638-643.
  • 14
    Nawaz M, Khan SA, Khan AA, Sung K, Tran Q, Kerdahi K, et al. Detection and characterization of virulence genes and integrons in Aeromonas veronii isolated from catfish. Food Microbiol 2010; 27:327-331.
  • 15
    Soler L, Figueras MJ, Chacón MR, Vila J, Marco F, Martínez-Murcia AJ, et al. Potential virulence and antimicrobial susceptibility of Aeromonas popoffii recovered from freshwater and seawater. FEMS Immunol Med Microbiol 2002; 32:243-247.

Publication Dates

  • Publication in this collection
    10 May 2013

History

  • Received
    13 Sept 2011
  • Accepted
    20 Oct 2011
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