Development of duplex-PCR for identifi cation of Aeromonas species

Introduction: The number of reports of intestinal infections caused by Aeromonas spp. has increased signifi cantly in recent years. In most clinical laboratories, identifi cation 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 specifi c 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 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 infections 1 .
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 2 .The results are imprecise and Aeromonas is often misclassifi ed, being mainly misidentifi ed as Vibrio, which similarly grows on thiosulfate citrate bile salts sucrose (TCBS) and is oxidase positive 3 .Commercial systems for bacterial identification such as API20E and Vitek have proven useless for Aeromonas identifi cation 3,4 .Hence, the role of Aeromonas as an etiologic agent of infection remains underestimated 2 .
Several molecular methods for genotypic identifi cation of Aeromonas spp. 5 are now available [6][7][8][9] .However, most of them are species-specifi c 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 identifi cation of medically important Aeromonas spp.by amplifi cation 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. fl uvialis, V. furnissi, V. mimicus, V. parahaemolyticus and V. vulnifi cus).We also tested 40 strains of Aeromonas spp. that were isolated from feces of patients with diarrhea.

REFERENCES
The authors declare that there is no confl ict of interest.

ACKNOWLEDGMENTS FINANCIAL SUPPORT
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 fi nal 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 100V for 1h, 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 7966 T , A. veronii ATCC 35624 T bio veronii, A. caviae ATCC 15468 T , and A. hydrophila IOC 11036), and specifi city 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 infections 1 , and differential diagnosis is clinically challenging.These clinically important species were among those included in the present study.
Vibrio spp.were included because of their biochemical and serological similarities to Aeromonas, which, as noted, have previously made differentiation diffi cult.Although the 16S gene was amplifi ed in some of the Vibrio species, it did not hinder the effi cacy of the test, which recorded samples positive for Aeromonas only when both of the targeted genes were amplifi ed.
The duplex-PCR method introduced here showed high reproducibility and specifi city 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 identifi cation 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.
Mendes-Marques CL et al -Duplex-PCR for Aeromonas identifi cation