Phenotypic Characterization, Genetic Analysis and Antibiotic Sensitivity of Aeromonas hydrophila Isolates causing Abdominal Dropsy in Labeo rohita collected from Punjab, Pakistan

Motile Aeromonas Septicaemia (MAS) is a common fresh water ﬁsh disease and major threat to the aquaculture in Pakistan. The present study was carried out on suspected cases of Rohu ( Labeo rohita ) to characterize and genetically analyze local strains of Aeromonas (A.) hydrophila , a key pathogen responsible for the said disease in farmed ﬁsh of Pakistan. A total of ninety suspected samples were collected from ﬁsh farms in various districts of Punjab from June 2018 to April 2019. The samples were processed and A. hydrophila strains were isolated. The primary identiﬁcation of sixty seven isolates was veriﬁed by colony morphology, microscopy and phenotypic characterization with ten biochemical reactions. The A. hydrophila strains of test samples were characterized by polymerase chain reaction (PCR) using 16S rRNA at desired size of 356 bp. The PCR ampliﬁed product was subjected to DNA sequencing and phylogenetic analysis showed homology with related strains of Aeromonas spp. By antibiotic sensitivity test, the isolates were checked for nine antibiotics, in which pathogen was sensitive to four and resistant to ﬁve drugs. Results of molecular characterization conﬁrmed strains as A. hydrophila which are useful to take preventive measures against the said disease.


Introduction
The carps are important source of fish meat in Pakistan and neighboring countries. Indian major carps including Rohu (Labeo rohita), Mori (Cirrhinus mrigala) and Thaila (Catla catla) are considered as potential source of protein in Pakistan. These carps are considered as high-value commercial fish species and being cultured on priority by local farmers 1 . On the other hand, Pakistan aquaculture industry is facing threats from several sources, with disease being the most critical hurdle to more semi-intensive, intensive carp farming and feral systems. This bacteria especially belonging to the genus Aeromonas (family Aeromonadaceae) are widespread in freshwater environment, and have been implicated as fish pathogens 2 , 3 . It is an opportunistic pathogen most likely responsible for Infectious Abdominal Dropsy, Ulcer Disease, Motile Aeromonas Septicemia (MAS), Hemorrhagic Septicemia and Red-Sore Disease at different growth stages leading to high mortality rates in aquaculture 4 , 5 . This bacterium can be found in fresh, salt, marine, estuarine, chlorinated, and un-chlorinated waters and can survive in both aerobic and anaerobic environments 6

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A. hydrophila is rod shaped heterotrophic, gram-negative bacterium ubiquitously present in freshwater environment 7 . It is normally inhabited in gastrointestinal tract of fish and water bodies and has ability to grow at low temperature. A. hydrophila is a diet tolerated pathogen that is accountable for severe zoonotic diseases [8][9][10] . Seasonal incidents also cause more stress in fish farms. In particular during Monsoon period when fish breeding season is at peak in Punjab region, the high temperature, high levels of ammonia and nitrites, less amount of dissolved oxygen (DO), presence of high amount of carbon dioxide (CO 2 ), organic pollution and malnutrition enable the A. hydrophila to develop in aquaculture quite rapidly. Moreover, heavy infestation with parasites, presence of injured fish and spawning activity creates a stressed environment for fish while favorable environment for A. hydrophila to grow 11,12 .
In a variety of freshwater species, the existence and pathogenicity of A. hydrophila has previously been reported in different fish species particularly Carassius auratus 13 , Cyprinus carpio 13 , Anabas testudineus 14 , Clarias gariepinus 15 , Oncorhynchus mykiss 16 and Potamotrygon motoro 17 . It has been recently reported that A. hydrophila is the key bacterial pathogen that causes diseases in freshwater fish species 18 . The pathogen has recently emerged in United States where it has been responsible for extensive farm losses in Catfish aquaculture 19 .
The fish diseases caused by A. hydrophila are responsible for major economical losses of local fish farmers in Pakistan. Diseases in inland aquaculture are currently being treated by antibiotic products. To our knowledge, disease prevention mechanisms in terms of local or imported vaccine products have not been explored for fish culture systems in the country to date. Under these circumstances, it will be worthwhile to characterize the local bacterial strains that may be considered as potential threat to fish farming industry and pave the way forward for its preventive measures in terms of autogenous vaccines.
The premier objective of present work is to perform phenotypic identification, molecular characterization and phylogenetic analysis of local strains of A. hydrophila in cultured carps of Pakistan. The resulting bacterial isolates are to be used for subsequent remedial actions against A. hydrophila strains for prevention of disease in farmed fish species of Pakistan.

Ethics declarations
• All methods in this work were carried out in accordance with "National Biosafety Guidelines" of Pakistan Environmental Protection Agency, Ministry of Environment, Government of Pakistan (http://www.environment.gov.pk/ images/guidelines/BiosftyGlines2005.pdf).
• This study was carried out in compliance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines https://arriveguidelines.org/arrive-guidelines.

Isolation of bacteria
For isolation of bacteria Nutrient Agar, Nutrient Broth and MacConkey Agar media were used with standard compositions. Sterilization was done by autoclaving media at 121ºC under 15 lbs and glassware in hot air oven at 180ºC for 10 minutes. Pouring of autoclaved media was done for preparation of MacConkey agar plates. For the sterilization check, the plates were incubated at 37ºC for 24h. Body surfaces of fish samples were first cleaned with 70% alcohol and then dissected under sterile laboratory conditions. Smears were taken aseptically using sterile loop from kidney, liver and spleen and streaked to MacConkey agar plates. After streaking the plates were incubated at 37°C for 24 hours following the procedure described in 20 and 16 .

Phenotypic characterization of isolates
The identifications of bacterial isolates were verified by colony morphology, microscopy and biochemical characterization. The isolated strains were tested for ten different phenotypic tests. These tests were performed in a conventional format as previously described, and appropriate positive and negative controls were included for each test 21 . Morphological characterization of isolates included size and shape of colony and Gram's reaction along with motility test 22 . Catalase test, Urease test, Voges-Proskauer (VP) test and five (5) different carbohydrate fermentation tests were also conducted for bio-chemical characterization of A. hydrophila 23 . Observations on carbohydrate metabolism reaction included production of acid and gas in Sucrose and Glycerol tests; and production of acid in L-arabinose, D-mannitol, and salicin tests. For biochemical tests, bioMérieux ® API-20E microbiological kit was used 24 .

Genetic analysis of isolates
The DNA extraction was done from a representative isolate of the biochemical results using GeneAll ® ExGene TM DNA purification kit following manufacturer's protocol 25 . The cells were harvested by centrifugation. The supernatant was discarded

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and then re-suspended to 20 µl of proteinase K solution (20 mg/ml) and 200 µl of CL buffer. The mixture was heated in water bath at 56ºC for 15 minutes. Then concisely spun down the tube to eliminate drops from the lid. The tube was filled with 200 µl BL buffer. Then placed in the water bath for 10 minutes at 70ºC and the mixture was concisely spin down the tube to eliminate drops from the lid. Absolute ethanol (200 µl) was added in tube, mixed by vortex and was spin to eliminate the drops. The mixture was carefully moved to SV column then centrifuged at 8000 rpm for 1 minute. After that 600 µl BW buffer was added and centrifuged at 8000 rpm for 1 minute. Mixture was transferred into new SV column. After adding 700 µl TW buffer mixture was centrifuged at 8000 rpm for 1 minute and supernatant was removed. Then SV column was transferred into collection tube. The residual wash buffer was eliminated by centrifuging for 1 minute at 13000 rpm. SV column was placed in 1.5 ml of micro centrifuge tube and 200 µl of AE buffer was added. As a last step, tubes were incubated and centrifuged for 1 minute at 13000 rpm. The genomic DNA of A. hydrophila was amplified by using universal primer purchased from Gene Link TM NY, USA. For 16S ribotyping, universal primer with sense 5'GGG AGT GCC TTC GGG AAT CAG A'3 and antisense 5'TCA CCG CAA CAT TCT GAT TTG'3 with product size of 356 bp was used. For PCR, reaction mixture of 25 µl was comprised of followings: Master mix (12.5 µl), DNA sample (2 µl), forward primer (1 µl), reverse primer (1 µl) and nucleus free water (8.5 µl). The composition of the master mixture was 0.05 U/µL Taq DNA polymerase, reaction buffer, 4 mM MgCl 2 and 0.4 mM of each dNTP (dATP, dCTP, dGTP and dTTP). PCR amplification was done by using BIO-RAD ® T100 Thermal Cycler TM for 50 cycles with each cycle's conditions as follows: 5 minutes for initial denaturation at 95ºC, 0.5 minute for denaturation at 95ºC, 0.5 minute for annealing at 56.9ºC, 0.5 minutes for elongation at 72ºC and finally 7 minutes for elongation at 72ºC. After the completion of 50 cycles the yield was observed by gel electrophoresis.
For electrophoresis, the buffer was prepared as per manufacturer's instruction. Gel was prepared by adding 100 ml of TAE buffer in 1 gram of agarose. The solution was heated in microwave oven until it became transparent and 5 µl ethidium bromide was added. Agarose solution was then poured carefully in electrophoresis tray with suitable comb. After 30 to 40 minutes the gel solidified the comb was removed and tray was placed in electrophoresis tank filled with electrophoresis buffer. The 2 µl of loading dye was mixed with 5 µl of DNA sample. By using micropipette the samples were loaded on gel. After that 2 µl of DNA ladder was loaded in first well at the right and last well at left side of the gel. After power on, the gel was run and DNA moved towards the anode. The voltage was applied at 120 V/cm for 35 minutes. The gel tray was further removed and placed on a Tran's illuminator. The DNA bands were visualized under Bio-Rad ® UV transilluminator. The amplified PCR products were sequenced directly from MacroGen ® , South Korea.
The initial and final portions of the sequences were manually trimmed in BioEdit sequence alignment editor software. The resulting high-quality fragments of the sequences were exported in FASTA format for comparison with GenBank database using online BLAST (by NCBI, USA) optimized for highly similar sequences (megablast). The query coverage and percent identity values ≥ 98% were considered for specific identification. The three (03) sequences obtained in the present study were deposited at NCBI GenBank under nucleotide accession numbers MT249820, MT249821 and MT249822. The supplementary information in terms of fish species, place of origin, year and season of collection, and size of amplified PCR products was also provided.
The phylogenetic diagrams were constructed for verification of sequencing data using highly similar reference sequences and out groups from NCBI Genebank along with three sequences of present study. All the sequences were taken in FASTA format for subsequent pair-wise and multiple alignment using the ClustalW alignment tool 26 . The aligned sequences were then subject to phylogenetic analysis. As a result of the preliminary analysis, a neighbor-joining phylogenetic diagram was constructed using the Kimura 2-parameter model, a gamma-shape parameter with 5 categories and nearest-neighbor-interchange tree inference options. Bootstrap analysis with 1000 replications was used for assessment of stability in internal nodes.

Antibiotic sensitivity
Antibiogram of the of isolates against antibiotics was studied by standard disc diffusion assay method [27][28][29] . The sterilized media was poured into petri plates. By using sterile swabs the isolates were spread on the plates. Antibiotic disc was placed on the surface of the nutrient agar for the sensitivity test. The plates were incubated at 37ºC for 24 hours. Nine antibiotics were used is this study are: Penicillin, Colistin sulphate, Oxytetracycline, Novobiocin, Ciprofloxacin, Gentamicin, Trimethoprim, Tetracycline and Nitrofurantoin. After 24 hours the zones of inhibition were measured 30 .

Results
The fish was analyzed in terms of total length, fork length and weight. The morphometric data and. The clinical signs of naturally infected Labeo rohita showed symptoms like abdominal swelling, scales extrusions, hemorrhagic septicemia, necrotizing ulcer on body, exophthalmia and sero-hemorrhagic fluids from the vent as seen from Figure 1

Phenotypic characterization
Out of 90 samples, 67 (74%) were identified as A. hydrophila through phenotypic characterization. A. hydrophila colonies appeared in pale yellowish color, round shaped with rounded end on MacConkey agar medium when incubated at 37°C for 24 hours (Figure 2(a)).
For the microscopic study, the bacterial cultures were examined by Gram's staining method. After staining they were observed as rod shape, single or paired and infrequently as short chains non-spore forming gram-negative bacillus (Figure 2(b)). All the isolates were motile in distilled water as well as peptone water on slide, which indicates the positive result in distilled water motility test. Biochemical test results for three (03) isolates are provided in Table 1, which show that bacterial isolates are uniformly positive for catalase and Voges proskauer (VP) tests whereas negative reaction was observed for Urease test. Out of ten (10) biochemical tests, five (05) glucose fermentation tests were performed in which isolates utilized Sucrose, D-mannitol, Glycerol and Salicin. However L-arabinose did not ferment. On the basis of biochemical reactions and glucose fermentation, the isolates were identified as A. hydrophila. The results are in close correspondence with work of Halima et al. 33 , Furmanek-Blaszk 34 and Nawaz et al. 35 .

Genetic analysis
Molecular characterization revealed that all the isolates were A. hydrophila based on amplification by real-time polymerase chain reaction (PCR) technique. The genomic DNA of bacterial isolates were extracted as per protocols described by GeneAll ® for ExGene TM DNA purification kit. Universal 16S rRNA gene has been amplified from genomic DNA of all A. hydrophila isolates. The amplification primers listed in Table 2 were used for the forward and reverse sequencing. The target genes amplified in this study along with primer pairs, sequences, amplified product size and references are also provided in Table 2. The DNA were examined by electrophoresis on 1% agarose gels with 100bp ladder for estimating the bands. The DNA bands were documented and pictured in Gel documentation system (Biorad ® Gel Doc XR system). The samples provided were 4/12   Figure 3.
To validate the Aeromonas characterization, the chosen strains were identified by 16S rRNA PCR pattern analysis on 1% agarose gel electrophoresis. The product was selected for subsequent sequencing from MacroGen ® , South Korea. The 16S rRNA sequences of the bacterial strains acquired in this study were aligned with bacterial nucleotide sequence data available at GenBank database using online BLAST program (Basic Local Alignment Search Tool) by National Center for Biotechnology Information (NCBI), USA. The obtained sequences highly resembled with nucleotide of the same bacterial species as listed in Table 3.
The consensus nucleotide sequences were aligned with the help of ClustalW bioinformatics software 38 . The genetic distance of aligned sequences were obtained using "Kimura's Two-parameter Model" 39 . Phylogenetic analysis was performed for isolated strains with reference strains using neighbour-joining method, and bootstrap values were calculated with 1000 replicates. Evolutionary trees were constructed with the help of MEGA v. 6.06 software by Kumar et al. 40 . The phylogenetic tree of Figure 4 shows close resemblance of our isolate QOL788 to A. hydrophila strains on GenBank database after performing BLAST.
The phylogenetic tree of Figure 5 depicts a consolidated genetic relationship of isolated strains among each other as well as with reference strains. The trees also show genetic heterogeneity and distance within the species due to different sources of collection. However all three isolates were confirmed as A. hydrophila.

Antibiotic sensitivity
The results of the antibiotic resistance tests of each isolate on nine (09) antibiotics are presented in Table 4. Resistance test results of A. hydrophila against some antibiotics were marked by the formation of clear zones around the antibiotic dishes. A. hydrophila isolates were resistant to Penicillin and Novobiocin and sensitive to Colistin sulphate, Oxytetracycline, Ciprofloxacin, Gentamicin, Trimethoprim, Tetracycline and Nitrofurantoin. Figure 6 shows the measurement of inhibition zones of different antibiotics for QOL787 sample (Sample 1).

Discussion
Fish is a significant source of protein in daily food and plays a vital role in agricultural economy. Farmed fish species especially Labeo rohita, is suffering from infectious diseases caused by microbial pathogens 12 . Predominantly these pathogens have bacterial origin and among those, Aeromonas species like A. hydrophila causes majority of the diseases in different fish species. The gram-negative A. hydrophila is pervasive in freshwater ponds causing fish mortality and economic losses to farmers mainly in semi-intensive culture system in Pakistan 1 .
In present study three (03) isolates of A. hydrophila were recovered from infected Labeo rohita samples collected from different fish farms in Punjab, Pakistan. Post mortem findings of the infected fish were hemorrhages at the base of fin and edge of head, ulcerative skin lesions on body and tail erosion which are more or less similar with the findings of Mathur et al. 41 and Rahman et al. 42 . Congested liver and internal organs were also observed in the infected fishes by Dahdouh et al. 43    et al. 44 . Minor variations might be due to different fish species and seasonal variations that influence the disease incidence. For bacterial identification, biochemical reactions are very important. These reactions help bacteria to provide energy by oxidation of organic substances or by fermentation. Based on ten bio-chemical reactions conducted in this study, the bacteria were identified as A. hydrophila which conform to previous biochemical studies in India 21,23 and Egypt 45 . Our study extends the work of Shahzad et al. 22 towards molecular characterization using 16S rRNA universal gene. The 16S rRNA gene sequencing is an accurate and objective method for identification of microorganisms in the clinical laboratory 46,47 .
Species of the genus Aeromonas exhibit very high levels of overall 16S rRNA gene sequence similarity in nucleotides. Species such as A. hydrophila, A. dhakensis and A. caviae exhibit very few differences. Through phylogenetic analysis, it is found that local isolate MT249820 is most closely related to A. hydrophila strains (Accession no. AB901365) which was reported earlier in 2014 from Tamilnadu, India. Our strains are very closely related to Aeromonas dhakensis strain (Accession no. MT193203) which is more recently discovered strain from Tamilnadu, India. The results of present study confirm that A. hydrophila strains are geographically more related to those in India 48, 49 , Bangladesh 33, 50 , China 51, 52 and South Korea 17 .
The results of sensitivity tests to a wide number of antibiotics could be used for diagnosis of MAS and other infectious diseases in different outbreak and epidemiological conditions in aquaculture. Current results showed that all A. hydrophila isolates were sensitive to the selected antibiotics of Colistin sulphate, Oxytetracycline, Ciprofloxacin, Gentamicin, Trimethoprim, Tetracycline and Nitrofurantoin. Shahzad et al. 22 from Pakistan and Kusdarwati et al. 53 from Indonesia obtained similar results on the chosen antibiotics. Resistance of A. hydrophila isolates against amoxicillin and penicillin has previously been reported from fish affected with bacterial diseases in India 54 , United Arab Emirates 55 , Bangladesh 56 , Egypt 57 and South Korea 17 .   Table 4. Antibiotic sensitivity tests of A. hydrophila strains (QOL787 -QOL789) S = sensitive, R = resistant Subsequently from this study, fish farmers will be benefited for controlling Abdominal dropsy and other infectious diseases caused by A. hydrophila by the administration of specific therapeutants. In particular, the A. hydrophila isolates of present study will be used for future research towards disease prevention against local strains of A. hydrophila in aquaculture of Pakistan.