Antibiotic and Metal Resistance offered by Aeromonas species isolated from Edible Fishes in Chennai, Tamil Nadu: A Field Investigation

 

S. Veena¹, P. Keerthana², A. Jayabalaji², M. Yasasve², L. A. Vishal²*

¹Centre for Nano Science and Technology, A. C. Tech Campus, Anna University, Chennai, Tamil Nadu, India

²Department of Biotechnology, Sree Sastha Institute of Engineering and Technology (Affiliated to Anna University), Chennai, Tamil Nadu, India

 

ABSTRACT:

Aeromonas species are common contaminants of fish and seafood. This study presents key findings of the Aeromonas species that were isolated from the organs of fishes collected from Kasimedu region in Chennai, Tamil Nadu, India. In this study, bacterial isolates from intestines and gills of three different edible fish varieties - Sardinella longiceps, Lutjanus gibbus and Pseudocaranx dentex were isolated. The isolates were identified to belong to gram negative Aeromonas species through biochemical tests. All the isolates were tested for antibiotic sensitivity for ten different antibiotics which includes Ampicillin, Chloramphenicol, Erythromycin, Gentamycin, Penicillin-G, Streptomycin, Tetracycline, Fluoroquinolone, Cephalosporin and Carbapenam. Metal resistance was checked for five metals namely Copper (Cu), Chromium (Cr), Lead (Pb), Manganese (Mn) and Nickel (Ni). All the strains exhibited Multiple Antibiotic Resistances (MAR) to at least two antibiotics. The Aeromonas strains showed sensitivity to Erythromycin and Gentamycin. The MAR index for the three strains was calculated as 0.52, 0.47 and 0.28 respectively. All the three MAR indices values were seen to be greater than 0.2 which indicates that the isolate has originated from a high-risk source of contamination. Thus, the results obtained clearly show that the fish samples collected is highly contaminated with antibiotic and metal resistant Aeromonas bacterial species.

 

 

 

INTRODUCTION:

The aquaculture industry in India produces 12.60 million metric tons of fish products annually and constitutes about 6.3% towards the global fish production. Indian fisheries rank as the third-largest in overall fish production and 50 different types of fish-shellfish products are being exported to 75 countries around the world.¹ Fishes are an affordable animal source of protein and are of great importance due to its higher nutritive value in the Indian export scenario. The quality of the fish is being monitored at regular intervals by the Food Safety and Standards of India (FSSAI) during export and consumption.

 

During consumption of raw or undercooked infected fish may cause various infections and intoxications in humans.² The contamination could be a result of a toxin, pathogen or microorganism with drug resistances (microbial quality of fish.³ Excessive use of antibiotics in aquaculture leads to drug resistance of pathogens and reduces the effectiveness of the antibiotic.

 

When fishes are harvested from water, microbes can contaminate the gill surfaces and intestinal tract.⁵ The pathogens and microorganisms present in fish at times lead to fish spoilage and one such species Aeromonas are widespread in natural habitats such as soil, fresh and brackish water, sewage and waste water.^6-7 Earlier studies reveal that Aeromonas species possess virulence factors that cause severe diarrheal infections and gastrointestinal illness in humans after consumption. The majority of Aeromonas species related for causing human-gastroenteritis include; A. veronii biovar sobria (HG-8/10), A. hydrophila (HG-1), and A. caviae (HG-4), though A. veronii biovar veronii (HG-8/10), A. trota (HG-13), and A. jandaei (HG-9) occur rarely.⁸ The most prevalent form of human Aeromonas hydrophila infection is a cholera-like disease characterized by acute secretory dysentery, watery stools and mild fever.⁹ Gastroenteritis caused by A. sobria was characterized by rice watery stools, abdominal pain and vomiting.¹⁰

 

An earlier study has reported the presence of 40% post-harvest contamination of Aeromonas species in the ready-to-eat (RTE) fishes during handling, transportation, and selling through fishermen and fish vendors.¹¹ The main virulence factors that are responsible for pathogenicity in Aeromonas species include hemolysins, cytotoxins, enterotoxins, proteases, exotoxins and structural features of the micro-organism. As the water sources are constantly exposed to contaminants through effluents and run off, mutations that lead to antibiotic resistances are developed and reported.¹²

 

This is the first field-research study which presents key findings of the Aeromonas species that were isolated from the organs of fishes collected from a local fish market in Chennai, Tamil Nadu, India. Furthermore, the antibiotic and metal resistances offered by these organisms were evaluated.

 

MATERIALS AND METHODS:

Collection of Fishes:

The fish samples were collected from a local fish market at Kasimedu back waters Chennai, Tamil Nadu, India. The samples were collected in sterile polyethylene bags and brought to the laboratory in an ice chest. Body surface, gill and intestinal content of fish were aseptically swabbed, inoculated and sterilized in peptone water. These flasks were incubated at 37^oC for 24 hours in a shaker incubator. Three edible _{fish samples namely Sardinella} longiceps (S1), Lutjanus gibbus (S2) and Pseudocaranx dentex (S3) were collected and used for _{further studies.}

 

Isolation of Aeromonas Species:

The enriched cultures were streaked on starch ampicillin agar and incubated at 37^oC for 24 hours. They were sub cultured on fresh ampicillin agar plates and stored for further process. The Aeromonas that were isolated from Sardinella longiceps, Lutjanus gibbus and Pseudocaranx dentex, were labeled as S1, S2 and S3 respectively. Identification of bacteria at genus level is done by carrying out different staining techniques and biochemical tests.¹³

 

Antibiotic Resistance Study:

Disc diffusion method for antibiotic susceptibility test was conducted.¹⁴ The strains were tested against the following antibiotic discs (Hi-Media): Ampicillin (10µg), Chloramphenicol (30µg), Erythromycin (15µg), Gentamycin (10µg), Penicillin-G (10µg), Streptomycin (10µg), Tetracycline (30µg), Fluoroquinolone (10µg), Cephalosporin (10µg) and Carbapenam (10µg). For checking the antibiotic sensitivity, Mueller Hinton agar was prepared and poured into petri plates. These plates were then spread plated with 0.1ml of overnight bacterial cultures (S1, S2 and S3) and left to stand for one minute. The antibiotics discs were placed in the centre of the petri plate and incubated at 37^oC for 24 hours. After incubation, the plates were taken out from the incubator and the diameter of the zone of inhibition around each disc was measured. The clear zone diameter results were then compared to the standard inhibition zone of each antibiotic according to CLSI 2015 (Clinical Laboratory Standards Institute) and descriptively analyzed and classified as resistant, intermediate and sensitive.

 

Multiple antibiotic resistance (MAR) index was determined for each isolate by using the formula MAR = A/B, where “A” represents the number of antibiotics to which the test isolate depicted resistance and “B” represents the total number of antibiotics to which the test isolate has been evaluated for susceptibility.¹⁵

 

The minimum inhibitory concentrations (MIC) of antibiotic resistance for the sensitive Aeromonas strains were estimated by broth dilution technique.¹⁶ The culture tubes were seeded with respective Aeromonas bacterial cultures isolated from the edible fishes and different concentration of antibiotics was added and incubated at 37°C for 24 hours. The lowest concentration of antibiotic inhibiting the growth of the organism and which did not permit any visible growth of the inoculated test organism in broth culture was regarded as the minimum MIC in each case. Escherichia coli (ATCC 25922) was used as the positive control and a negative control (sterilized culture broth only) was included in tests.

 

Metal Tolerance Study:

Metal tolerance through Minimum inhibitory concentration (MIC) of Chromium, Copper, Lead, Manganese and Nickel were determined for the isolates. _Different concentrations of each metal (K₂Cr₂O₇, CuSO₄.5H₂O, Pb(CH₃COO)₂, ^MnCl₂ ^{and NiSO}₄) ranging from 50ppm to 500ppm was prepared and mixed with nutrient broth. Nutrient broth with different concentrations of metals was inoculated in conical flasks with the isolated bacterial cultures S1, S2 and S3. After 24 hours of incubation at 37^oC, the optical density (OD) was measured at 540nm in an UV-Vis Spectrophotometer (make and model). OD of initial media and OD after incubating the bacterial culture for 24hrs was compare and the MIC was calculated.¹⁷

 

RESULTS:

The study reports the dangerous effects on humans consuming the contaminated edible fishes of Chennai region. Sardinella longiceps and Lutjannus gibbus are among the top 10 species exported by India and Pseudocaranx dentex is one of the most utilized local delicacies due to its taste and availability.¹⁸ Therefore, there is a need to assess the microbial safety among the three species. Following the biochemical tests, the Aeromonas strains isolated by our study were found to be of Aeromonas hydrophilia origin. These were isolated and sub-cultured on fresh ampicillin agar plates separately.

 

Isolation of Aeromonas species:

Appearances of golden yellow colour were considered as Aeromonas species.¹⁹ Earlier studies on isolation and identification of Aeromonas species was performed using direct plating method (DP) on Pseudomonas Aeromonas selective agar (GSP) with ampicillin added. Aeromonas species were initially identified by a clear halo around each yellow colour colony.²⁰ A. hydrophila bacterial isolation showed a good growth on Thiosulfate-citrate-bile salts-sucrose agar (TCBS) medium and isolates produced yellow colour colonies due to sucrose fermentation, with diameter of colonies ranged from 2-3 mm.²¹

 

Antibiotic Resistance:

Disc diffusion method for antibiotic susceptibility testing was carried out in triplicates to assess the resistance towards different antibiotics. The antibiotic sensitivity of the three strains isolated was checked for ten different antibiotics (Ampicillin, Chloramphenicol, Erythromycin, Gentamycin, Penicillin-G, Streptomycin, Tetracycline, Fluoroquinolone, Cephalosporin and Carbapenam). The discs were infused with the antibiotics and placed on the Mueller Hinton agar plates.

 

Ten antibiotics were exposed to the isolated bacteria and it is clear from the results that all the three strains are resistant to Ampicillin, Tetracycline, Fluoroquinolone, Cephalosporin and Carbapenam. The strains are seen to be highly sensitive to the antibiotic Gentamycin with very high values of diameter of zone of inhibition (Table 1). The strain from Sardinella longiceps fish showed resistance to six antibiotics with a MAR index of 0.42, Lutjanus gibbus strain had a MAR index of 0.57 and displayed resistance to seven antibiotics. Among the three strains, the S2 had the highest antibiotic resistance. The strain isolated from Pseudocaranx dentex showed resistance to only five antibiotics with a MAR of 0.28. MAR index equal to or less than 0.2 indicates that the collected fish were not often or never exposed to the tested antibiotics; on the other case, the fish may have a high risk of exposure to the tested antibiotics if the MAR index is more than 0.2. The minimum inhibitory concentrations (MIC) of antibiotic resistance for the sensitive Aeromonas strains were estimated and the lowest concentration of the antibiotic that will inhibit the visible growth of the sensitive Aeromonas strains was found to be 4μg/ml for Gentamycin.

 

Table 1 Antibiotic Resistance Test for different fish samples

S. No	Antibiotic Used	Sardinella longiceps (S1)		Lutjanus gibbus (S2)		Pseudocaranx dentex (S3)
		Resistant/ Sensitive	Zone of inhibition (mm)	Resistant/ Sensitive	Zone of Inhibition (mm)	Resistant/ Sensitive	Zone of inhibition (mm)
1	Ampicillin	R	-	R	-	R	-
2	Chloramphenicol	S	11.7±1.52	R	-	S	10±2.08
3	Erythromycin	S	7.5±1.15	S	6±2	S	11±1.52
4	Gentamycin	S	23±1	S	21±1.52	S	23±1.15
5	Penicillin-G	R	-	S	7±1	S	7±1.15
6	Streptomycin	S	4.7±1.12	R	-	S	7.6±1.52
7	Tetracycline	R	-	R	-	R	-
8	Fluoroquinolone	R	-	R	-	R	-
9	Cephalosporin	R	-	R	-	R	-
10	Carbapenam	R	-	R	-	R	-
	MAR Index	0.42		0.57		0.28

 

Metal Tolerance Study:

Metal tolerance of Chromium, Copper, Lead, Manganese and Nickel were determined for the isolates. The OD after 24 hours was compared with the OD at zero minute (Table 2). The increase in OD signifies that the bacteria have grown at that particular concentration. The MIC (ppm) values of five different metals are listed in Table 3. Copper and chromium trace components are crucial for homeostasis in the animal immune system. In addition, heavy metals also tend to eventually influence the Aeromonas metal tolerances and antibiotic resistance. Isolated bacteria from different animal and environmental sources have been discovered to be resistant to certain heavy metals through particular genetic mechanisms.²²

 

Table 2 Tolerance to metals at different concentration

S. No	Concentration (PPM)	OD at 540nm
		Chromium		Copper		Lead		Nickel		Manganese
		OD at 0hr	OD after 24hr	OD at 0hr	OD after 24hr	OD at 0hr	OD after 24hr	OD at 0hr	OD after 24hr	OD at 0hr	OD after 24 hr
1 2 3 4 5 6 7	50 100 150 200 300 400 500	0.009 0.012 0.033 0.066 0.094 0.214 0.410	0.12 0.013 0.035 0.068 0.098 0.212 0.421	0.003 0.009 0.013 0.036 0.053 0.077 0.097	0.07 0.12 0.013 0.038 0.059 0.080 0.10	0.003 0.007 0.011 0.017 0.029 0.041 0.056	0.061 0.094 0.172 0.212 0.024 0.039 0.058	0.002 0.009 0.016 0.09 0.043 0.066 0.081	0.016 0.021 0.033 0.028 0.042 0.069 0.080	0.001 0.008 0.014 0.036 0.057 0.069 0.084	0.06 0.13 0.29 0.67 0.83 0.067 0.082

 

Table 3 Minimum Inhibitory Concentration (MIC) of metals

S. No	Metal	MIC (ppm)
1 2 3 4 5	Chromium Copper Lead Manganese Nickel	100 150 300 400 200

 

DISCUSSION:

Fish occupies an important part in the diet of majority population residing at Chennai, Tamil Nadu. It is available as the most favourite and least expensive form of animal protein. The per capita annual consumption of fish is estimated to be 5kg/yr for the whole population and it is 8kg for the non-vegetarian population of India as against the world average of 16kg.²³ The antibiotic and metal resistance in bacterial population is clearly an environmental phenomenon of natural selection for survival. Bacterial resistance for antibiotics and metals may be due to the presence of R-Plasmid.²⁴ The use of antibiotics is an important factor in amplifying the level of resistance in a given reservoir. MAR among Aeromonas hydrophilia has been reported from many parts of the world. Under these circumstances, it is imperative to understand the emerging antibiotic resistances among the Aeromonas hydrophilia population of the region.²⁵

 

It was seen that all the three MAR indices values were greater than 0.2. This implies that the strains are from a high-risk source of contamination. Microbial flora of fish is affected by the degree of contamination of water in which they live.²⁶ Fish is generally stored and transported in refrigerated conditions and this aid in the reduction of the microbial load in the fish material. However, Aeromonas species can grow and produce toxins even in refrigerated conditions showing that refrigeration cannot be an effective method to control the pathogen. This is a point of concern for the consumers.²⁷

 

The increase of antibiotic resistance in Aeromonas bacteria owing to mechanisms such as cross or co-regulation resistance to heavy metals has been recorded to determine the pathways in these bacteria.²⁸ Heavy metals such as cadmium (Cd), cobalt (Co), copper (Cu) and zinc (Zn) account for the major metal pollutants of aquatic environments. Hence, there is an elevated probability level that these heavy metals will be absorbed by marine bacteria and thus accumulated in food chains.²⁹

 

CONCLUSION:

The results obtained clearly show that the fish samples collected is highly contaminated with antibiotic and metal resistant Aeromonas bacterial species. This study indicates the significant risk to public health due to the Aeromonas species virulence potential in edible fish. The public should therefore be made aware of the intrinsic hazard that may arise during the handling of fresh fish or the consumption of improperly cooked fish. Furthermore, characterization of Aeromonas in coastal foods will be performed and rapid diagnostic methods can also be developed in the future for the detection of Aeromonas infections.

 

ACKNOWLEDGMENT:

The work was supported by Tamilnadu State Council for Science and Technology, Chennai-600025.

 

CONFLICT OF INTEREST:

No conflict of interest between the authors associated with this work.

 

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Received on 23.11.2019           Modified on 07.01.2020

Accepted on 21.02.2020         © RJPT All right reserved