Molecular identification of diarrheal Aeromonas using immuno magnetic polymerase chain reaction (IM-PCR) technique: a comparative study with conventional culture method

Background Aeromonas are ubiquitous bacteria causing many clinical conditions including acute diarrhea. Diarrheagenic Aeromonas harbors aerolysin gene secreting virulent enterotoxin, aerolysin. Objectives To develop a molecular and immunological based method for detection of Aeromonas. Methods Diarrheal Aeromonas strains were identified from stool samples using culture, enterotoxicity testing using mice model. During immune magnetic polymerase chain reaction IM-PCR protocol, aerolysin specific antibodies were bound with immuno magnetic binding. Sensitivity and specificity tests for IM-PCR were conducted. Results There was high detection of Aeromonas using IM-PCR (12.4 %) technique when compared to low isolation with culture (5.1%). Our study confirmed that some strains of enterotoxic Aeromonas strains were uncultivable. Enterotoxicity tests on culture isolates revealed many strains were negative. IM-PCR detected high, (62/500) rate of identification of Aeromonas with aerolysin toxin gene. Aeromonas species identified after IM-PCR were A. hydrophila (40.3% ), A. veronii (17.7 %), A. caviae (14.5 %), A. trota (11.2 %), A. jandei (9.6 %) and A. schuberti (6.4%). All A. trota strains were undetected by cultivation. Conclusion High sensitivity and specificity of IM-PCR are due to preparation of aerolysin antibodies and immuno magnetic binding, prior to PCR. Since diseases due to Aeromonas are increasingly reported, IM-PCR is recommended for detection from clinical specimens.


Introduction
The members of the genus Aeromonas are Gram negative bacilli similar to Vibrionaceae 1,2 . Importance of aeromonad diarrhea has been established by many microbiologists [3][4][5][6] . In developing nations, numerous food and water related epidemics due to Aeromonas were reported [7][8][9] . Even in developed countries variety of infections due to Aeromonas have been noted [10][11][12][13] . Aeromonas is also known to cause other infections ranging from African Health Sciences Vol 19 Issue 2, June, 2019 © 2019 Subbaram et al. Licensee African Health Sciences. This is an Open Access article distributed under the terms of the Creative commons Attribution License (https://creativecommons.org/licenses/BY/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

African
Health Sciences meningitis, pneumonia, wound associated sepsis to ocular disease [14][15][16] . It was noticed that aeromonads can cause diseases in healthy individuals and very severe infections in immune compromised patients 17,18 . Aeromonad diarrhea in humans is caused by six species of Aeromonas consisting of A. hydrophila, A. veronii (biotype Sobria and Veronii), A. caviae, A. trota, A. jandei and A. schuberti 19 . Many virulent determinants of this bacterium such as pili adhesins, enterotoxin (aerolysin), cytolytic toxin, hemoytic toxin, lipases,and proteases have been studied 20,21 . Various bacteriological data revealed that aerolysin toxin gene is similar to cholera toxin (CT) gene and Aeromonas strains containing aerolysin toxin gene are highly associated with diarrhea 22 . This article brings out our study based on immuno magnetic polymerase chain reaction (IM-PCR) technique to detect pathogenic Aeromonas strains harboring aerolysin gene from diarrheal stool samples. Current diagnostic methods in diarrheal Aeromonas in laboratories and research organizations include microscopy, staining, culture using selective media, biotyping and serotyping. These routinely used procedures are time consuming, inaccurate and insensitive in detection. Current proposed method is novel and unique because it uses both immunological based binding and molecular based PCR set up for the diagnosis of diarrheal Aeromonas.

Materials and methods
Cultivation of Aeromonas using conventional culture techniques A total of 500 diarrheal stool specimens were analyzed over a period of twelve months in our Diarrheal Active Surveillance Unit (DASU), Jimma University, Ethiopia. Isolation of Aeromonas was performed using alkaline peptone water and ampicillin sheep blood agar (ASBA) for 24 hours at 37˚C2 3 .

Enterotoxic potential of isolated Aeromonas strains
Aeromonas cultures grown in peptone water were centrifuged at 6000 x g for 10 minutes to obtain cell free supernatant. Aerolysin present in the supernatant was furtherconcentrated and purified by sterile filtration (0.22 µm filters, Millipore, Billerica, USA). 50 μL doses of purified aerolysin toxin were injected through sub-plantar route into paw of Swiss male mice (7 week old with weight 30 -35 g). Severity of inflammatory responses and edema were recorded starting from 1 hour up to 96 hours 24 .
Immunization and preparation of aerolysin specific antibodies Rabbits were immunized with aerolysin recombinant protein (aerA, MyBioSource, Canada) in divided, increasing doses over a period of 1, 2, 3 and 6 months.
Immuno magnetic binding of aerolysin immunoglobulins 50 μL of aerolysin specific immunoglobulins were allowed to bind with anti-immunoglobulin coated on magnetic chromium oxide for 30 minutes at 25˚C in a shaker.

Immune magnetic binding of aerolysin secreting
Aeromonas in stool Stool specimens were reacted with magnetic particles containing aerolysin specific immunoglobulins at 25˚C for 1 hour. Magnetic particles were centrifuged at 1000 g for 5 minutes. Clear supernatant was subjected to PCR.

Molecular detection using Immuno magnetic PCR technique (IM-PCR)
Using AeroFr -CCAAGGGGTCTGTGGCGACA-(forward primer) and AeroRv-TTTCACCGGTAACAG-GATTG-(reverse primer) (Toyoba, Japan) a 209 bp part of aerolysin gene was amplified using thermal cycler (Perkin Elmer, US). Denatured DNA sample was obtained by heating the supernatant for 15 minutes and treating it with ice. Reaction mixture contained 2.0 μl of 2 mM dATP, dGTP, and dCTP, 15 nM of denatured DNA and 2.0 μl of 10 X amplification buffer. PCR thermal conditions were set as 32 cycles at 93º C for 1 minute, 54ºC for 1minute and 74ºC for 10 minutes.

Specificity and sensitivity of Immuno magnetic PCR (IM-PCR)
An overall number of 150 stool specimens from diarrheal cases were included for assessment of the specificity and sensitivity of Immuno magnetic PCR. Specimens from various sources were included. Statistical analysis were conducted using IBM -SPSS version 21.0.

Comparison of Aeromonas isolates with other established pathogens using conventional culture techniques
Our results showed that the isolation rate of Aeromonas using culture was low when compared with established pathogens. (Figure 1). A. trota was not isolated by culture method. (Table 1).

Immune magnetic binding of aerolysin secreting Aeromonas in stool
Immune diffusion method showed specific antigen-anti-body reaction suggesting antibodies produced in rabbits was highly specific. This recommended the application of aerolysin specific antibodies in immune magnetic binding. Molecular detection of all aerolysin genes containing aeromonads in stool were detected using IM-PCR technique.

Identification of pathogenic Aeromonas by IM-PCR
The total number of Aeromonas strains identified using this technique was 62/500 (12.4%) (Figure 2). In addition, species of Aeromonas detected after IM-PCR were, A.

Discussion
Developing countries employ culture based identification strategy which is time consuming and results may delay the initiation of appropriate treatment 25 . There were some molecular based detection systems are developed for detection of aeromonad infections but unfortunately these protocols were found to be unaffordable to all diagnostic laboratories 26,27 . To overcome these disadvantages, our team has developed a novel and rapid PCR based method for detecting diarrhea causing Aeromonas directly from stool samples. Our IM-PCR based detection method is one of the first of its kind to detect Aeromonas directly from stool by coupling antigen -antibody based followed by PCR protocol.
Routine culture and identification for established pathogens along with Aeromonas resulted in high percentage of isolation rates for E. coli (31.4%), Entamoeba histolytica (29.3%) and Shigella (11.4%). It was observed that the isolation rate for Aeromonas was only 5.1%. In addition we have found that the undetected pathogens were also high with 12% (Table 1). Reasons for low isolation rate of Aeromonas and inability to detect pathogens may be attributed to possible presence of antibiotics in the samples, ampicillin sensitivity of some Aeromonas strains, significantly low number of pathogens in the samples or otherstressful factors that may influence the retardation of growth of pathogens in culture media. These issues during conventional culture methods were already faced by many microbiologists 23,28,29 . PCR based IM-PCR method detected significantly high,  (Table  1).
IM-PCR method expressed sensitivity of 100% and specificity of 99%. IM-PCR detected one V. cholerae as positive and may be due to genetic similarity between cholera toxin (CT) gene and aerolysin gene 22 . Moreover two environmental samples (fresh water sources) have also become positives for IM-PCR and this suggests that environment is the main reservoir and source for pathogenic Aeromonas [30][31][32][33] .
When compared to culture methods, IM-PCR method showed high accuracy, efficacy, quality, ease in application and rapidity. Increased sensitivity and specificity of IM-PCR method may be due to its novel procedures like, preparation of aerolysin specific antibodies and Immuno magnetic binding of aerolysin immunoglobulins, prior to actual PCR protocol. Due to vast number of aeromonad cases are increasingly reported in humans, it is suggested that IM-PCR may be useful in the diagnosis.

Conflict of interest statement
We declare that we have no conflict of interest.