Detection of multidrug resistance Aeromonas hydrophila in farm raised fresh water prawns

This study was undertaken for isolation, identification and determination of antibiogram profile of Aeromonas hydrophila in farm raised fresh water prawn (Macrobrachium rosenbergii) on five commercial ghers. Fresh water prawns (n=25) were collected from five ghers located at Satkhira, Bagerhat and Khulna districts of Bangladesh. Brain (n=25), muscle (n=25) and intestine (n=25) samples were collected aseptically from fresh water prawn and inoculated into alkaline peptone (APW) water for enrichment at 37°C for 8 h. Enriched cultured was streaked into Thiosulfate Citrate Bile Salt Sucrose (TCBS) agar to isolate bacteria. Bacteria were identified by cultural, Gram staining, biochemical properties and polymerase chain reaction (PCR) assay. The antibiogram profiles of bacteria were investigated against 5 commonly used antibiotics (Gentamicin, Cefalexin, Ampicillin, Azithromycin and Ciprofloxacin) by disc diffusion method. Thirteen A. hydrophila isolates were identified and the prevalence of the A. hydrophila in fresh water prawn was 17.33%. All 13 (100%) isolates were sensitive to Ciprofloxacin, Gentamicin, Azithromycin and resistant to Ampicillin and Cefalexin. The results of this study indicate that farm raised fresh water prawn harbor multidrug resistant A. hydrophila which might causes public health problem if enter into human food chain.


INTRODUCTION
Fresh water prawn (Macrobrachium rosenbergii) is a popular food item and the second largest exportable commodity in Bangladesh.Fresh water prawn is cultured in the ghers (modified rice field) of southern region of Bangladesh like Satkhira, Bagerhat, Khulna, and Cox's Bazar.Prawn is one kind of shellfish.Bacteria, virus and parasites are common to cause infection in shellfish (FDA, 2011).Ten bacterial genera are known to cause diseases in shellfish.Feldhusen (2000) reported three groups of pathogenic bacteria in shellfish namely indigenous bacteria (bacteria commonly found in fresh and marine water including Vibrio spp., Aeromonas hydrophila, Listeria monocytogenes and Clostridium botulinum), non indigenous bacteria (enteric bacteria of fecal origin including Salmonella spp.), and bacterial contamination during processing.Shellfish infected with A. hydrophila are unsuitable for export because these bacteria can cause food borne infection in humans (Li and Saghaian, 2011).These bacterial infections cause tail rot, fin rot, and hemorrahagic septicemia both in fresh water and marine fishes (Aoki, 1999).
Gram-negative, facultative anaerobic A. hydrophila is normal bacterial flora of fish and responsible for diseases in fish at the time of stress (Peters et al., 1988).In humans, it has been associated with gastroenteritis and localized wound infection (Nemetz and Shotts, 1993).Waters get contaminated with antimicrobial agents from human and animal wastes which leads to the emergence of multidrug resistance (MDR) bacterial flora in the aquatic environment (Morita et al., 1994).
The multidrug resistance was reported in the genus of Aeromonas (Albert et al., 2000;Palu et al., 2006).Few reports are available on the isolation of A. hydrophila from fresh water fish and prawn sold at market in Bangladesh (Rahim et al., 1984;Rahim and Aziz, 1994).However, no study has been conducted so far on the detection of A. hydrophila in farm raised fresh water prawn at ghers in the Southern districts of Bangladesh.The objectives of this study were (i) Isolation and identification of A. hydrophila from farm raised fresh water prawn, and (ii) determination of antibiogram profile of A. hydrophila against five commonly used antibiotics.Processing of samples: Brain (n=25), muscle (n=25) and intestinal (n=25) specimens of prawns were aseptically collected.Muscle samples (0.5 g) were mixed with 4.5 mL alkaline peptone water (APW) and grinded by pastle and morter to prepare a 10% tissue suspension.Intestinal samples were cut by scissors into several small pieces and homogenized in PBS to prepare a 10% tissue suspension.

Enrichment of samples:
A loopful of brain fluid was inoculated into 4.5 mL of APW and incubated at 37 for 8 h.Suspension of muscle (0.5 mL) and intestine (0.5 mL) were separately inoculated into test tubes containing 4.5 mL APW and incubated at 37 for 8 h.

Isolation of bacteria:
One loopful of enrichment culture of brain, muscle and intestine was separately streaked duplicate onto Thiosulfate citrate bile salts sucrose (TCBS) agar (Himedia, India) and incubated aerobically at 37 for 24 h.Single colony grown onto the TCBS agar was further sub cultured onto TCBS agar until pure cultures were obtained.
Identification of bacteria: Identification of bacteria was conducted by observing cultural characteristics and colony morphology on the TCBS agar and growth of bacteria into nutrient broth containing 0% and 6% sodium chloride (NaCl

RESULTS AND DISCUSSION
Multidrug-resistant Aeromonas strains have been emerged as serious public health pathogens which can cause gastroenteritis, septicemia and skin infections in humans (Igbinosa et al., 2012).A. hydrophila is naturally found in the aquatic environment (Austin and Adam, 1996).It produces various diseases in fishes.These bacteria enter into human body through ingestion of contaminated food and water and open wounds.A. hydrophila have been found in various fish species (Daskalov, 2006;Deng et al., 2009).
In the present study, isolation of Aeromonas spp was done on TCBS agar since it is used for the growth of both Vibrio spp.and Aeromonas spp.(Kaysner and Depaola, 2004).The most frequently isolated species of Vibrio in fresh water are V. cholera and V. mimicus (Fouz et al., 2002).In TCBS agar V. cholera produce yellow colour colony and V. mimicus produce green colour colony (Kaysner and Depaola, 2004).Aeromonas spp. on the other hand also produce yellow colour colony on TCBS agar.In this study, DNA extracted from yellow colour colony grown on TCBS agar successfully amplified 276-bp fragment of lipase gene confirmed prawn bacterial isolates belonged to Aeromonas spp.(Delamare et al., 2012) (Figure 1).In PCR assay positive control was not available for use.Islam et al., 2013;Kaysner and Depaola, 2004).In this study, bacteria isolated from farm raised fresh water prawn were Gram negative, rod shaped, lactose fermenter and VP test positive which are characteristics for A. hydrophila.Another differential diagnosis carried out to differentiate Vibrio spp.from Aeromonas spp.by their growth characteristics in nutrient broth containing 0% and 6% NaCl (Kaysner and Depaola, 2004).In the present study, prawn bacterial isolates grew in nutrient broth containing both 0% and 6% NaCl which were important growth characteristics of A. hydrophila.Several Aeromonas spp.were reported in fresh water fish and shellfish.Ashiru et al. (2011) reported A. hydrophila, A. caviae and A. sobria both in Tilapia and Catfish.These three species of Areromonas can be differentiated by lactose and sucrose fermentation tests.
A. hydrophila can ferment lactose and sucrose.On the contrary, A. caviae and A. sobria cannot ferment lactose and sucrose (Ashiru et al., 2011).In this study, bacterial isolates of prawns fermented lactose and sucrose confirming their identity as A. hydrophila.Results of sugar fermentation and biochemical tests of A. hydrophila isolates from fresh water prawn are summarized in the Table 2.
In present study, 13 A. hydrophila were isolated and identified from the farm raised fresh water prawns.The prevalence of A. hydrophila in fresh water farm raised prawns in this study was 17.33%.Rahim and Aziz (1994) recorded 31% prevalence of A. hydrophila in fresh water prawn which were collected from fish market of Dhaka, Bangladesh.In shrimp 13.89% prevalence of A. hydrophila was reported in Iran by Khamesipour et al. (2014).Vivekanandhan et al. (2005) reported 17.62% prevalence of A. hydrophila in prawns in India.In the current study, brain, muscle and intestine samples were screened for Aeromonas spp.since these samples were also analyzed by other investigators (Ashiru et al., 2011;Jayasinghe et al., 2008).This study detected the presence of A. hydrophila in brain, muscle and intestine samples of prawns.The prevalence of A. hydrophila was the highest in intestine (28%) followed by muscle (16%) and brain (8%).The overall prevalence of A. hydrophila in fresh water prawn was 17.33% (Table 3).Vivekanandhan et al. (2005) also recorded the highest prevalence of A. hydrophila in the intestine (38.43%) as compared to body surface (32.46%) and gill (29.10%).
Prevalence of A. hydrophila was 26.67% in Satkhira gher, 20% in Dumuria gher, 13.33% in Assasuni gher, and 13.33% in Bagerhat gher and 13.33% in Rampal gher (Figure 2).   A. hydrophila are sensitive to ciprofloxacin and gentamicin (Ko et al., 2003;Truong et al., 2008;Overman, 1980) and resistant to ampicillin (Geiss and Freij, 1989;Overman, 1980).In this study, all A. hydrophila were found sensitive to ciprofloxacin, gentamicin and azithromycin and resistant to ampicillin and cefalexin (Figure 3).Antibiotic resistance pattern among bacterial strains may be varied which is linked to place of origin of the strains (Ko et al., 1996).In the present study, A. hydrophila isolated from prawns in the Dumuria Gher of Khulna were sensitive to all five antibiotics (Table 4).

CONCLUSION
Data of this study suggest that A. hydrophila are prevalent in fresh water farm raised prawn in the ghers of the southern part of the Bangladesh.Data of antibiogram study suggest that farm raised fresh water prawn harbors multidrug resistant A. hydrophila which may cause public health hazard if enter into the food chain.

Figure 2 .
Figure 2. Prevalence of A. hydrophila in fresh water prawn in five commercial ghers located at Satkhira, Khulna and Bagerhat districts of Bangladesh

Table 1 .
Number of samples collected from each study area

Study area (District) Location of gher (Upazila) No. of samples collected
). Gram's staining method, motility test, sugar fermentation and biochemical tests (oxidase test, catalase test, citrate test, indole test and MR-VP test) were performed to identify bacteria.
Antibiotic sensitivity test: Antibiogram profile of 13 A. hydrophila isolates was done against five different antibiotic such as: Gentamicin, Azithromycin, Ciprofloxacin, Ampicillin and cefalexin (Himedia, India).The antibiotics sensitivity testing was carried out according to instructions of the Clinical and Laboratory Standard Institute(CLSI, 2011).

Table 2 .
Summary of sugar fermentation and biochemical test results for Aeromonas hydrophila.

Table 3 .
Prevalence of Aeromonas hydrophila in brain, muscle and intestine samples of fresh water prawn.

Table 4 .
Summary of Antibiogram profile of Aeromonas hydrophila against five commonly used antibiotics.