A Study on Genetic Variability of Pathogenic Aeromonas hydrophila Strains and the Varied Responses of the Strains Towards Phyto-extracts

INTRODUCTION

Aeromonas hydrophila and Pseudomonas are common in freshwater and can survive and multiply in that environment provided there is enough organic matter and suitable growth temperature. Most bacteria in this group are not usually capable of multiplying or causing disease below 10-12°C, although some strains are pathogenic at temperatures as low as 5°C. They are capable of producing a variety of diseases, which predominate during summer months when water temperatures and organic loadings are high. Skin infections may appear resulting in bright red blotches around the vent, back and sides. Haemorrhages may appear in the internal organs and in advanced stages, the kidney appears liquefied (Hazen et al., 1978; Kaper et al., 1980; Van der Kooij, 1988). Aeromonas infections are probably the most common bacterial disease diagnosed in cultured warmwater fish. Usually, mortality rates are low (10% or less) and losses may occur over a period of time (2-3 weeks or longer). In these instances, some factor; usually stress, has caused the fish to become more susceptible to the bacteria. Common sources of stress are poor water quality, overcrowding, or rough handling. (Mathewson and Dupont, 1992; Larsen and Jensen, 1977). It was considered as a opportunistic pathogen in the past, but recent surveys have emphasized its emergence an primary pathogen, particularly in compromised hosts or in wound infections (Davis et al., 1978; Fraire, 1978, Salyers and Whitt, 1994).

Several diseased conditions such as tail rot, fin rot and haemorrhagic septicemia has been associated with A. hydrophila infection (Miyazaki and Kaige, 1985). Apart from infecting fish, it also causes food borne diseases in humans (Palumbo et al., 1989). Because of its high adaptability in different environments, it would seem that genetic and phenotypic diversity of A. hydrophila is a natural phenomenon.

As use of antibiotic to control microbial pathogens such as Aeromonas leads to multidrug resistance, antibiotic residues in environment, transmission of antibiotic in the food chain leads to several problems. So it is inevitable to probe for alternative methods of controlling pathogens (Rahim et al., 1984; Bonjar and Nik, 2004). One such alternative is the use of herbal medicinal plant extracts as feed supplement which not only enhance immunity but also increase the size of fishes. Hence, in this study antimicrobial activity of three selected medicinal plants Ocimum sanctum, Adathoda vasica and Calendula officinalis were used against Aeromonas hydrophila. The present study was also intended to establish a correlation between the antibiotic susceptibility patterns and genetic diversity of Aeromonas hydrophila strains using DNA-PCR (RAPD) analysis.

MATERIALS AND METHODS

Isolation and identification of A. hydrophila: Diseased common freshwater fish Cyprinus carpio were covered in plastic wrap and transported from the fish farm in an ice chamber to the laboratory. Four strains A. hydrophila were isolated from swab specimens of superficial skin ulcers. A. hydrophila was cultured on tryptone soya agar (Himedia) and harvested in tryptone soya broth (Himedia). The inoculated broth was incubated in shaker at 200 rpm for 12 h at 27°C and centrifuged at 10000 rpm for 20 min at 4°C. Supernatant was discarded and the bacterial pellet was used for further analysis.

Extract preparation: Leaves of Adathoda vasica, Ocimum sanctum and Calendula officinalis were collected from various areas. Collected leaves were dried in shade and ground into fine powder and stored in containers. The 125 g of powdered leaf was successfully extracted with cold butanol, using Soxhlet apparatus. Fractions were completely dried by evaporation at room temperature and were stored in sterile container.

Antibacterial assay: Petri plates containing 20 mL of tryptone soya agar medium were seeded with a 24 h old culture of the bacterial strain. The leaf extracts and fractions were dissolved in Dimethyl sulphoxide (DMSO) and filtered by using sartorius syringe filter (pore size of 0.22 mm). 40, 50 and 75 μL, of leaf extracts were impregnated into the sterile 6 mm diameter discs. Discs were dried in room temperature and dispensed on the solidified tryptone soya agar inoculated with test microorganisms. Incubation was made at 37°C for 24 h. Assessment of antibacterial activity was based on matching the diameter of the inhibition zone formed around the discs with interpretive criteria on Kirby-Bauer chart (NCCLS, 2002).

Co-culture of A. hydrophila strains with plant extract: The 0.25 and 0.50 mL of the plant extracts Adathoda vasica and Ocimum sanctum were added to the broth culture flasks of A. hydrophila and the growth of bacteria was assessed by checking the optical density, daily, for a period of seven days.

Biochemical tests: The bacterial strains used in this study were identified using standard morphological, physiological and biochemical tests (Holt et al., 1994).

Antibiotic susceptibility: Susceptibility of A. hydrophila to different standard antibiotics was tested by agar diffusion method using discs purchased from Himedia.

Extraction of genomic DNA for PCR-RAPD analysis: Bacterial samples were well ground and mixed with cTAb extraction buffer. This homogenate was incubated with 150 μg mL^-1 of proteinase K at 50°C for 4-12 h. It was then extracted with equal volume of phenol:chloroform (1: 1). It was then centrifuged at 10,000 rpm at room temperature for 5 min. The upper aqueous phase was collected. To this equal volume of chloroform:isoamyl alcohol (24: 1) was added and mixed by gentle shaking. Contents were centrifuged for 5 min at room temperature. The upper aqueous phase was collected. DNA was precipitated with cold absolute ethanol. The contents were centrifuged at 5000 rpm for few minutes and the pellet was then dissolved in 400 μL of 1 N NaCl . To this 2 μL of RNAase was added and incubated at 37°C for 30 min. To this 1 mL of cold absolute ethanol was added and kept at -20°C for 30 min. The sample was centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was discarded, the pellet was washed with 70% ethanol and the centrifugation was repeated. The pellet was collected; the ethanol content was evaporated and dissolved in 1X TE buffer.

Amplification of DNA using random primer: PCR amplification was performed as described by Muyzer et al. (1993). Twenty RAPD primers (Kit A1-A20) were obtained from IDT (New Delhi) and were tested with DNA samples of Aeromonas.

Data analysis: The data analysis was performed using the Diversity database software (Bio-Rad) and similarities among isolates were estimated by means of dice co-efficient. The program calculated all the Pearson Correlation Coefficients between pairs of variables, transformed these coefficients into distances and made a clustering using Unweighted Pair Group Method with Arithmetic Mean (UPGMA). Dendrograms were produced based on the UPGMA clustering (Garcia-Vallve et al., 1999).

RESULTS

Biochemical and physiological characterization: Four strains of A. hydrophila were isolated and characterized using standard biochemical tests. All the four strains were gram negative, oxidase positive and fermenting glucose. But there was slight variation in the degree of fermentation. All the four strains could utilize sucrose and arabinose. Aeromonas strain 1-3 were able to utilize sorbitol, strain 2 and 3 could utilize xylase and only strain III was able to utilize lactose (Table 1).

Antibiotic sensitivity test: The antibiotic concentrations per disc were (in micrograms) as follows: Amphicillin 30; Amikacin 30; Cefuroxime 30; Chloramphenical 30; Ciprofloxacin 5; Ceffriaxone ; Cotrimaxazole ; Gentamycin 10; Levofloxacin 5; Imipenem 10; Nalidixic acid 30; Norfloxacin 10; Novobiocin 30; Ofloxacin 5; tetracycline 30; Trimethoprim 5. All the four strains showed varied susceptibility to standard antibiotics. Among 16 antibiotics tested Aeromonas-I was susceptible to 7 antibiotics and resistant to eight antibiotics. Strain 2 was highly multidrug resistant and showed resistance against 10 antibiotics. Strain 3 was susceptible to 11antibiotics and strain 4 showed resistance against 8 antibiotics. Thus except strain 3 other strains were highly multidrug resistant strains. All the four strains were susceptible to chloramphenical and Ceffriaxone. Resistance was observed against Novobiocin and Nalidixic acid in all the four strains (Table 2).

Disc diffusion method: Antibiotic sensitivity pattern assessed for all bacterial isolates revealed that except for Novobiocin and Nalidixic acid the bacterial isolates were sensitive to all other antibiotics. Their zones of inhibition were compared with standard zones as per Kirby Bauer Chart and the degree of inhibitions by the two antibiotics could serve as a benchmark for the antimicrobial activity of plant extracts.

Antimicrobial activity of butanolic extracts (40, 50 and 75 μg disc^-1) of Adathoda vasica against Aeromonas strains is presented in Fig. 2. There was not much variation between A. hydrophila-II and Aeromonas IV in the inhibitory activity. Maximum inhibitory activity was observed against Aeromonas-IV at 75 μL concentration. Minimum inhibitory zone were observed against A. hydrophila-I in all the three concentrations.

Butanolic extracts (40, 50 and 75 μg disc^-1) of Ocimum sanctum showed maximum activity against A. hydrophila-II and III with a zone of 18 and 17 mm dm^-1. Inhibitory zone observed against A. hydrophila-I and Aeromonas-IV were similar in all the three concentrations (Fig. 3).

Inhibitory zones could be observed against the microbes using butanolic extracts of Calendula officinalis. It showed significant inhibitory effect on all the four strains of Aeromonas, in which the maximum zone of inhibition was observed against Aeromonas hydrophila-II. (21 mm at 75 μL concentrations).

Table 1:	Identification of Aeromonas hydrophilla by biochemical characterization

+: Positive, -: Negative, V: Variable, A/A: Acid/Acid, A/Ak: Acid/Alkaline

Table 2:	Antibiotic susceptibilities of A.hydrophila strains

R: Resistance, S: Susceptibility, MS: Mild susceptibility

Minimum inhibitory zone was observed against Aeromonas-IV (10 mm at 40 μL). Similar levels of inhibitory zones were observed for Aeromonas I and III (Fig. 4).

Butanolic leaf extract of all the three plants Ocimum sanctum, Adathoda vasica and Calendula officinalis thus showed antimicrobial activity against the strains of Aeromonas.

Co-culture technique: Inhibitory effect of plant extracts on Aeromonas strains in broth culture both the concentrations (0.25 and 0.50/200 mL broth) of Adathoda vasica showed inhibitory activity against Aeromonas hydrophila-I at the later stage of culturing i.e., beyond the 5th day. 0.50 mL A. vasica group showed higher inhibition when compared with 0.25 mL concentration of A. vasica (Fig. 5).


Fig. 1:	PCR pattern of genetic variability


Fig. 2:	Antimicrobial activity of butanolic extract of Adathoda vasica


Fig. 3:	Antimicrobial activity of butanolic extract of Ocimum sanctum

Ocimum sanctum also showed, similar level of inhibitory action in both 0.25 and 0.50 mL concentrations. 0.50 mL concentration of Ocimum sanctum inhibited the growth of A. hydrophila in 5 days of culture.

These results indicated that two plant extracts inhibited the growth of A. hydrophila-I in similar manner. Extracts of Adathoda vasica and Ocimum sanctum had no apparent effect on A. hydrophila-II as evidenced by the progressive O.D values observed with the progression of the experiment. This indicates that A. hydrophila-II strain could not be inhibited by the two plants extracts (Fig. 6).

PCR-RAPD studies: The RAPD procedure was used in the present study because of its simplicity and speed in identifying the genetic polymorphisms within the species level. DNA samples of Aeromonas were screened by using the arbitrary 10-mer primers.

From the 20 random primers tested the primer A 03 gave reproducible, consistent and gave scorable fragments. Following is the sequence of the primer A 03 which gave results 5”AGT CAG CCAC 3’.

The pattern of RAPD profile for Aeromonas in this study revealed characteristics of genetic variability of each population. PCR pattern of genetic variability is shown in Fig. 1a.

Similarity indices and dendrogram were computed and presented in Fig. 1b and c. Amplified fragments ranged from 840 to 154 bp.


Fig. 4:	Antimicrobial activity of butanolic extract of Calendula officinalis


Fig. 5:	Effect of plant extracts on Aeromonas hydrophila-I

Species specific fragments were identified at 702 bp (Fig. 1a). An interesting finding of this study was that 100% similarity did not exist among the four populations studied (Fig. 1b). The similarity indices and the dendrogram constructed by UPGMA, clearly supported PCR pattern of genetic variability in the populations. A maximum similarity index was exhibited among isolates 1 and 3 Aeromonas followed by isolates 1 and 2 and between 2 and 3. The UPGMA based dendrogram analysis grouped isolates 1 and 3. The first cluster was further sub grouped with isolate 2 and then with isolate 4. As Aeromonas species isolates were grouped under different clusters, in general one could assume that distinct genetic variations existed in these microbial populations.


Fig. 6:	Effect of plant extracts on Aeromonas hydrophila-II

DISCUSSION

Because of the side effects and the development of resistance by microbes against conventional antibiotics, much attention has been paid recently to medicinal plants, as a source of safe compounds to be used as drugs for human beings and also in aquaculture environments. Observation by Samy and Ignacimuthu (2000) on the folklore medicines of Western Ghats (South India) has highlighted the potential of plant based products in tackling many of the modern day diseases. Screening of 165 medicinal plants by Pereira et al. (2004) revealed that medicinal property in plants are not universal and hence specific compounds have to be tested against each bacterial strain, so that suitable plant source may be identified and scientific validation of those compounds may be carried out. While testing against Pseudomonas and Aeromonads, they could identify only 13 species of plants coming under 12 families which had the relevant antimicrobial potential.

In the present study, based on their traditional medicinal value, three plants Adathoda vasica, Ocimum sanctum and Calendula officinalis were selected and tested against A. hydrophila strains an opportunistic pathogen affecting cultivable fish species. Antimicrobial activity of plant extracts could be established effectively in the present study (Graph 1-3). Anti Aeromonas effect of Begonia malabarica leaf extract could be established by Ramesh et al. (2002). Disease resistance and immunostimulatory effect could be observed in A. hydrophila challenged Oreochromis mosambicus administered with leaf extract of Solanum trilobatum (Divyagnaneswari et al., 2007). Resistance against A. hydrophila could also be observed in the leaf extracts of many plants like Eclipta alba, Achyranthes aspera, Zingiber officinalis (Rao et al., 2006; Christybapita et al., 2007). Enhancement of disease resistance could be observed achieved by using the leaf extract of Ocimum sanctum against A. hydrophila in Orechromis mosambicus by Logambal et al. (2000). All the above works reveal the susceptibility of A. hydrophila towards medicinal plant extracts, as observed in the present study. Although bacterial infection can be controlled by antibiotic treatment, the use of antibiotics leads to environmental hazards and the development of antibiotic resistant genes in the bacteria. Several strains of microbes have no effective therapeutic measures at all (Itami et al., 1998). In this context search for nutraceuticals gain much importance in improving disease resistance in animals (Gerin, 1999). From the preceding reports, it can be inferred that all the works were pertaining to a single strain of the bacterium in contention and as one has taken into account their differences in function and resistance development based on their genetic variability, which should be at high frequency taking into considerably their low generation time and higher rate of mutations. Hence in the present study genetic variability was given due importance while studying their susceptibility to plant extracts as well as antibiotics.

Susceptibility of A. hydrophila to different antibiotics was estimated using disc diffusion method (Fig. 1-3). Among sixteen antibiotics used, susceptibility pattern of the four strains of A. hydrophila, varied from the observation by earlier workers like McNicol et al. (1980), Fass and Barnishan (1981) and Fainstein et al. (1982). The four strains of A. hydrophila exhibited susceptibility to chloramphenicol; three strains exhibited resistance to tetracycline and two strains exhibited resistance towards trimethoprim, which was in accordance with the result observed with McNicol et al. (1980). In contrast to the result obtained by Rahim et al. (1984), these four strains of A. hydrophila showed complete resistance for novobiocin and nalidixic acid. It was observed by many workers that almost all strains of A. hydrophila are resistant to ampicillin, hence it was recommended for the selective medium in 30 mcg mL^-1 concentration for isolation of A. hydrophila (Rogol et al., 1979). Recovery of multidrug resistant strains in cultured fish and water was reported by Hayashi et al. (1982) and McNicol et al. (1980). Three strains of multidrug resistant Pseudomonas spp. were isolated from Channas gachua, which exhibited resistance against eleven standard antibiotics and sensitivity towards chloramphenical and gentamycin susceptibility of A. hydrophila to three plant extract was also strain dependent (Fig. 1-3).

Genetic variations between bacterial species and genomic polymorphism between bacterial isolates can be identified by the variations in sizes and number of fragments. Randomly amplified polymorphic DNA (RAPD) is the simplest and easily reproducible DNA fingerprinting method (Williams et al., 1990). RAPD and PCR are established methods for generating DNA fingerprints and find discrimination among strains of A. hydrophila (Szczuka and Kaznowski, 2004, 2007; Aguilera-Arreola et al., 2005). Genetic heterogeneity among the strains of A. salmonicida isolated from different species of fish was reported by Garcia et al. (2000). The scattered RAPD profiles observed in the present study resembles to an extent the genetic variations observed in A. hydrophila isolated from Rainbow trout by Miyata et al. (1995) and Lee et al. (2000). The results of the present study confirm the fact, while testing antimicrobial compounds, due importance should be given to the variety of strains available further studies of their genomic identity.

CONCLUSION

From the present study it was concluded that all the four strains of Aeromonas was susceptible to butanolic extracts of three medicinal plants namely Calendula officinalis, Adathoda vasica and Ocimum sanctum; eventhough they show genetic variation and varied responses towards standard antibiotics.

ACKNOWLEDGMENT

Authors are grateful to Ministry of Earth Sciences, Ocean Atmospheric Science Technology Cell (MOES-OASTC), UGC, Government of India for providing financial support.