Research Article

Antimicrobial Resistance of Aeromonas Species Isolated from Cultured Indian Major Carp, Labeo rohita: Possible Public Health Concern

Chethurajupalli Lavanya, Tambireddy Neeraja, P. Hari Babu, T. V. Ramana, A. Balasubramanian, Supradhnya Meshram and P. Sruthi

  • Published online: 31 Oct 2021
  • DOI : HTTPS://DOI.ORG/10.23910/1.2021.2483

  • Abstract

In India, Labeo rohita is widely cultured and consumed freshwater fish. Aeromonadsare etiological agents of major bacterial fish diseases like furunculosis, haemorrhagic septicaemia, skin ulcers, fin/tail rot and dropsy, causing significant economic losses in carp culture.Aeromonas species are widely distributed in aquatic environment which is considered as important vehicle of Aeromonas infections to fish and humans. Some of the Aeromonas spp. causes gastroenteritis, septicaemia, peritonitis, meningitis and eye infections in humans. In the present study Aeromonas species were isolated from diseased freshwater fish Labeo rohita collected from two districts viz., West Godavari and SPSR Nellore of Andhra Pradesh, India. A Total of 12 Aeromonas spp. were isolated and identified by biochemical tests. A. veronii bv. veronii (35%) was dominant when compared to other Aeromonas spp. Further, Antimicrobial resistance and multiple Antimicrobial resistance (MAR) of all Aeromonas spp. were tested against 17 antibiotics being frequently used for human diseases. The Antimicrobial resistance of all the 12 Aeromonas spp. have shown significantly high (p<0.05) resistance (100%) to ampicillin, amoxyclave and oxytetracycline except A. cavernicola when compared to other antibiotics. The MAR index of Aeromonas spp. ranged from 0.18-0.76, which indicates origination of isolated Aeromonas spp. from high risk sources of contamination. A. hydrophila, A. veronii bv. sobria, A. veronii bv. veronii, A. schubertii and A. jandaei isolated in this study were found to be pathogenic to humans also. The results revealed the pathogenic potential of Aeromonas infections in freshwater fish culture and emerging threats to public health.

Keywords :   Aeromonas species, antimicrobial resistance, biochemical characterization, Labeo rohita

  • Introduction

    Among the freshwater fishes, Indian major carps are the most widely cultivated freshwater fishes in India contributing to 80% of production (Anonymous, 2017). In India, the increase in aquaculture production particularly an expansion into semi-intensive and intensive methods of production led to an increase in fish diseases due to higher stocking densities and stress conditions that favour the occurrence and spread of infectious diseases (Das and Mishra, 2014). The occurrence of bacterial diseases in freshwater fish was considered to be a serious problem in India (Mishra et al., 2017). The most common bacteria associated with disease outbreaks in fish culture systems are Aeromonas spp. (Tomas, 2012). Aeromonas spp. are etiological agents of fish diseases like furunculosis (Reith et al., 2008), haemorrhagic septicemia, skin ulcers (Figuras et al., 2009), fin/tail rot (Cizek et al., 2010) and dropsy (Sreedharan, 2008). Aeromonas spp. are pathogenic to fish and humans, their presence in fish culture systems is of concern (Bomo et al., 2003). The genus Aeromonas is widely distributed in the aquatic environment (Popoff, 1984; Daskalov, 2006) and can easily transmit to humans posing threat to public health (Austin and Adams, 1996; Borrell et al.,1998; Janda and Abbott, 1998). The diseases caused by aeromonads in humans are generally the result of ingestion of contaminated water and aquatic food (Alavandi and Ananthan, 2003). Aeromonas species are also frequently isolated from various meat products, milk, vegetables (Stratev et al., 2016) which are considered as an important vehicle of Aeromonas infections to humans (Altwegg et al., 1991; Kirov, 1993). Some of theAeromonas species such as A. caviae, A. hydrophila, A.  veronii bv. sobria, A. veronii bv. veronii, A. trota, A. schubertii, A. jandaei causing gastroenteritis in adults (Sinha et al., 2004); A. hydrophila, A.  veronii bv. sobria, A. caviae, A. media, A. trota and A. jandaei causing gastroenteritis in children (Albert et al., 2000); A. caviae causing septicaemia (Dwivedi et al., 2008) and in rear occasions A. caviae, A. hydrophila, A. sobria causing peritonitis, meningitis and eye infections in humans especially in immune compromised patients (Kelly et al., 1993). Among the Aeromonads, A. hydrophila, A. caviae and A. veronii bv. sobria are responsible for 85% of gastrointestinal diseases (Kuijper et al., 1989; Altwegg et al.,1990;  Havelaar et al., 1992; Khun et al., 1997; Borrell et al., 1998; Stratev et al., 2012).

    Antibiotics are a major source to treat bacterial infections in aquaculture. However, overuse of antibiotics for the treatment of bacterial diseases or their incorporation in animal feeds as growth promoters in sub-therapeutic doses may lead to the development of antibiotic resistance (Vivekanadhan et al., 2002). The antibiotic resistance of Aeromonas species is considered a serious threat worldwide (El-ghareeb et al., 2019). The present study aimed to evaluate the prevalence of Aeromonas spp. isolated from majorly cultured and consumed freshwater fish, Labeo rohita from Andhra Pradesh, India and their antimicrobial resistance patterns against most commonly used antibiotics in treating human infections.

  • Materials and Methods

    2.1.  Fish Sampling

    The diseased Labeo rohita fishes were collected from fish farms of two districts viz.,West Godavari and SPSR Nellore of Andhra Pradesh, India. All the samples were aseptically brought to the laboratory of the Department of Aquatic animal health management, College of fishery science, Muthukur, Nellore district for microbiological study.

    2.2.  Isolation of Aeromonas spp.

    A total of 33 rohu fish showing clinical signs of bacterial hemorrhagic septicemia were collected and Aeromonas spp. were isolated from skin, liver, abdominal fluid and kidney. The inoculums from these organs were streaked on Rimlershotts (RS) medium and Glutamate Starch Phenol red Agar base (GSPA). On RSA plates, Aeromonas forms yellow coloured, convex and glossy colonies of 0.5-3.0 mm diameter, after 18-24 h of streaking. On GSPA medium, Aeromonas produce yellow, circular, convex and glossy colonies of 0.5-3.0 mm diameter after 48-72 h of streaking. Typical colonies were picked from the plates, sub-cultured in trypticase soy agar (TSA) slants for biochemical characterization and antibiotic resistance tests.

    2.3.  Characterization of Aeromonas spp.

    Identification of bacterial isolates was carried out by biochemical tests in the conventional method. A series of biochemical reactions as described by Martin-Carnahan and Joseph (2005) were performed to identify bacteria at the genus and species level. Taxonomic keys proposed by Abbott et al. (2003), Martinez-Murcia et al. (2008) and Beaz-Hidalgo et al. (2009 and 2010) were followed for identification of Aeromonas spp.

    2.4. Antimicrobial resistance assay

    Antibiotic resistance of  Aeromonas spp.was carried out against 17 potential antibiotics by disc diffusion method on Mullen Hinton agar.The antibiotic discs and sterile swabs were procured from HiMediaDCMTM, Mumbai, Maharashtra, India. The antibiotics used, their concentrationand the interpretation of antimicrobial resistance of bacteria to various antibiotics are shown in Table 1. Young cultures of bacteria (20 h old) from TSA slants were inoculated individually into Tryptone Soya Broth (TSB) and incubated for 10-12 h at 30±2°C. Inoculum from TSB grown culture of each bacterium was collected using sterile cotton swabs and spread onto Mullen Hinton agar (MHA) plate. Antibiotic impregnated discs were placed aseptically onto the inoculated agar plates at equal distances and sufficiently separated from each other to avoid overlapping of the zone of inhibition and at least 15 mm away from the edge. The plates were then incubated for 24 h at 30±2°C and the diameter of the zone of inhibition was measured.

    2.5.  Multiple antibiotic resistance (MAR) and  MAR index

    The resistance profiles and resistance patterns of Aeromonas spp. for 17 potential antibiotics were determined using antibiogram data (Table 1). Multiple antibiotic resistance (MAR) of each Aeromonassp. at least resistant to three antibiotics was recorded and MAR index was calculated as per Orozova et al. (2010).

    MAR Index=(Number of antibiotics to which the bacterium is resistant/The total number of antibiotics tested)

    2.6.  Statistical analysis

    Statistical analysis for antibiotic resistance was carried out using R studio software (3.6.1 version) and graphs were obtained using Microsoft Excel –2007. Pearson chi square test was used to test the significant difference in the prevalence of isolated Aeromonas spp. and their significant difference towards antimicrobial resistance.

  • Results and Discussion

    3.1.  Gross pathological signs of diseased fish

    The clinical symptoms observed in the infected fish were haemorrhages on the body surface and fin bases; reddish bulged vent with bloody fluid discharge; fluid accumulation in the intestine and visceral cavity; pale gills; fin and tail rot; discoloration of internal organs like kidney, liver, spleen and pinpoint haemorrhages on the kidney. The major gross pathological signs observed in diseased fish are presented in Plate 1.

    3.2.  Identification of Aeromonas spp.

    The isolated Aeromonas spp. were characterized by conventional biochemical tests and the results are given in  Table 2.

    Twelve Aeromonas species isolated from Labeo rohita were A. veronii bv. veronii (n=21), A. tecta (n=10), A. veronii bv. sobria (n=4), A. aquariorum (n=4),  A. popoffii (n=4), A. schubertii (n=4), A. jandaei (n=3), A. molluscorum (n=3), A. hydrophila (n=3), A. piscicola (n=2),  A. enchelia (n =1) and A. cavernicola (n=1). The letter ‘n’ represents number of each species of Aeromonas isolated. Among the 60 Aeromonas isolates of L. rohita identified, the highest prevalence (35%) was recorded for A. veroniibv. veronii, followed by 16.7% of A. tecta, 6.67% each of A. veronii bv. sobria, A. aquariorum, A. popoffii, A. schubertii and 5% A. jandaei, A. molluscorum, A. hydrophila and 3.33% of A. piscicola, 1.67% each of A. cavernicola and A. enchelia. The prevalence of Aeromonas species in rohu was given in Figure 1.

    3.3.  Antimicrobial resistance assay

    The 60 isolates of Aeromonas spp. were individually tested for antimicrobial resistance against 17 antibiotics. The percentage of antimicrobial resistance of Aeromonas spp. to the 17 antibiotics is given in Figure 2.

    The Aeromonas isolates displayed 91.67% resistance to ampicillin, amoxyclav, and oxytetracycline except for A. cavernicola; 78.77% resistance to erythromycin; 65.02% resistance to chloramphenicol; 59.72% resistance to novobiocin; 57.14% resistance to doxycycline hydrochloride; 55.87% resistance to nitrofuratoin; 52.94% resistance to oxacillin; 51.68% resistance to amikacin; 47.62% resistance to ciprofloxacin; 42.95% resistance to neomycin; 42.66% resistance to trimethoprim; 31.78% resistance to co- trimoxazole; 27.42% resistance to nalidixic acid; 24.62% resistance to enrofloxacin and 22.62% resistance to gentamycin.

    Statistical analysis by Pearson chi-square showed that the antibiotic resistance of Aeromonas isolates to ampicillin, amoxyclav and oxytetracycline was significantly high (p<0.05) in comparison to the other 14 antibiotics tested. The calculated chi-square value was 151.97 while the table value for chi-square at 16 degrees of freedom is 26.296. The results of the statistical analysis showed a significant difference in antibiotic resistance of Aeromnas isolates to various tested antibiotics.

    3.4. Multiple antibiotic resistance (MAR) index and percentage

    The Multiple antibiotic resistance patterns of 12 Aeromonas species were calculated and details of the MAR index are presented in Table 3.

    The minimum and maximum MAR index observed for each species of Aeromonas ranged between 0.18 and 0.76. The MAR index ranged between 0.29-0.58 in A. aquariorum, 0.47 in A. cavernicola, 0.76 in  A. enchelia, 0.29-0.53 in A. jandaei, 0.29 to 0.58 in A. hydrophila; 0.59-0.71 in A. molluscorum, 0.18-0.41 in A. piscicola, 0.24-0.41 in A. popoffii, 0.18-0.70 in A. schubertii, 0.29-0.52 in A. tecta, 0.24-0.58 in A. veronii bv. sobria and0.24-0.71in A. veronii bv. veronii. All the isolates of 12 Aeromonas species were found to be multiple antibiotic resistant.

    Aeromonas species are widely distributed in aquatic environments and were isolated from drinking water (Havelaar et al., 1992), squid (Baldrias and Alvero, 1999), shrimp (Thayumanavan et al., 2003; Vivekanandhan et al., 2005), mussels (Ottaviani et al., 2006), ornamental fishes (Sreedharan et al., 2012, Mohan and Unni, 2012). Aeromonas contaminated water, food and their products caused health issues to the consumers (Joseph et al., 2013). Aeromonas spp. are opportunistic, which provokes clinical signs in stressed fish or fish affected by concurrent infections (van der Marel et al., 2008). These Aeromonas species are causing hemorrhagic septicaemia, tail and fin rot, ulcer disease, or red-sore disease in a variety of freshwater and marine fish of the world (Roberts et al.,1989) and causing significant economic losses in the aquaculture industry worldwide (Maniati et al., 2005). Motile Aeromonads like A. hydrophila (Dallal and Moez Ardalan, 2004),  A. sobria (Taher et al., 2000) associated with gastroenteritis in humans. Janda and Abbott (1998) classified A. veronii bv. veronii, A. jandaei and A. schubertii as minor human pathogens. A. aquariorum was reported to cause bacteraemia and wound infections in humans (Figueras et al., 2009) and A. punctata caused gastroenteritis (Gilardi, 1967). In aquaculture and animal husbandry antibiotics are used for treating bacterial diseases and sub-therapeutic doses causing the emergence of several environmental and public health problems, such as the generation of multi-resistant bacteria (Moore et al., 2014).

    In the present investigation, Aeromonas infected rohu exhibited clinical signs like  haemorrhages on the body surface, fin bases, reddish vent, discharge of bloody fluid from the vent, pale gills, fin rot, tail rot, scale loss, reduced mucus on the body surface, distended abdomen, spleen enlargement, discoloration of internal organs like kidney and liver. More or less similar observations were also made by Mathur et al. (2005) in mrigal; Alsaphar and Al-Faragi (2012), Stratev et al. (2015) in common carp and  Sruthi et al. (2021) in catla and rohu.

    Hu et al. (2012)  isolated A. veronii (60%) as the dominant species in fish followed by A. hydrophila, A. sobria, A. media, A. caviae, A. jandaei, A. salmonicida, A. allosaccharophila, A. bivalvium. Tong Li et al. (2020) also isolated 54% of A. veronii from several freshwater fish like Carassius auratus, Cyprinus carpio, Ctenopharyngodon idella and Silurusa sotus. Further, U-taynapun et al. (2020) also recorded A. veronii bv. veronii (78%) followed by A. hydrophila (12%), A. veronii bv. sobria (6%) and A. jandaei (4%) in motile Aeromonas septicemia tilapia. Among the 12 Aeromonas isolated in the present study, the prevalence of A. veroniibiovarveronii in diseased rohu was significantly highest with 35% than A. tecta, A.veronii biovarsobria, A. aquariorum, A. popoffii, A. schubertii, A. jandaei, A. molluscorum, A. hydrophila, A. piscicola, A. cavernicola and A. enchelia and these results are agreeing with the previous findings.  The prevalence of Aeromonas spp. are more in the freshwater culture system where A. hydrophila, A. veronii bv. sobria, A. caviae were reported to be the major human pathogens and A. veronii bv. veronii, A . jandaei and A. schubertii are the minor human pathogens (Janda and Abbott, 1998). The present investigation also revealed occurrence of zoonotic Aeromonads in rohu viz., A. veronii bv. veronii to greater extent and A. veronii bv. sobria, A. schubertii and A. hydrophila to lesser extent warranting the chances of public health concern, if the fish are handled and cooked improperly. Albert (2000), Alavandi and Ananthan (2003) cautioned that high prevalence of Aeromonas spp. in the environment is considered a threat to public health through ingestion of contaminated water and fish.

    In recent years the number of antibiotic-resistant bacteria in aquaculture has increased dramatically in different parts of the world. In aquaculture and animal husbandry systems antibiotics are extensively applied for controlling bacterial diseases (Praveen et al., 2014).According to Redmayne (1989), the continued usage of medicated feeds and their application to the rapidly developing fish and shellfish farming can foster greater dissemination of virulent and resistant bacterial pathogens in the natural environment and thus potentially into the human food chain. In our study, the antimicrobial resistance pattern of 12 Aeromonas spp. isolated from cultured rohu fish from fish farms of Andhra Pradesh against 17 antibiotics revealed significantly high antibiotic resistance to three antibiotics viz., ampicillin, amoxyclav and oxytetracycline. Further, significantly low resistance (p <0.05) was shown for the remaining 14 antibiotics. Similar results were recorded by Yucel et al. (2004) isolated A. hydrophila, A. veronibv. sobria, A. caviae from fish were 100% resistant to ampicillin. Hatha et al. (2005) found Aeromonas spp. were highly resistant to oxytetracycline, amoxycillin, ampicillin, novobiocin and polymixin-B. Further, Roy et al. (2013) reported that A. veronii isolated from Fresh Water Loach,Lepidocephalycthys guntea showed resistance erythromycin, kanamycin, ciprofloxacin and gentamycin. Further, Raj et al. (2019) reported that A. veronii isolated from tilapiashowed resistant to bacitracin, nitrofurantoin, furazolidone, erythromycin, azithromycin, cefalexin, amoxycillin, cenrofloxacin, ampicillin. Further, Hassan et al. (2017) reported the resistance of A. veronii to ampicillin and other beta-lactam antibiotics such as amoxicillin. Wassif (2018) indicating that A. veroniibvsobria wasresistant to ampicillin, amoxicillin, oxytetracycline. Zdanowicz et al. (2020) reported that isolates from the water of carp ponds were mostly resistant to amoxicillin, ampicillin, clindamycin and penicillin. The present study findings were completely in concurrence with the above studies. Further, in contrast to our findings Sanyal et al. (2018) reported high antibiotic sensitivity of Aeromonas to chloramphenicol. The Aeromonas spp. isolates from a number of clinical and environmental sources were 100% sensitive to amikacin, ciprofloxacin, chloramphenicol, gentamycin (Kampfer et al., 1999), in contrast to the resistance to ciprofloxacin and gentamycin found in the present study. In contrast to the present study, Sreedharan et al. (2012) reported 100% sensitivity of Aeromonas spp. to trimethoprim isolated from freshwater ornamental fish culture systems. The variation in resistance/sensitivity of Aeromonas spp. to similar antibiotics in different studies might be due to the utilization of water in aquaculture systems from high/low risk sources.

    The multiple antibiotic resistance (MAR) index is clinically very important due to its zoonotic importance (Le et al., 2018). A high MAR index of zoonotic bacterial pathogens is a potential risk for humans by direct contact with diseased fish (Lowry and Smith, 2007). MAR among Aeromonas spp. has been reported from many parts of the world (Koet al.,1996; Mirand and Zemelman, 2002; Vivekanandhan et al., 2002; Holmstrom et al., 2003; Sinha et al., 2004). According to Krumperman (1985), the MAR index value is more than 0.2 or more is said to have originated from high risk sources of contamination where antibiotics are often used. In the present study, all the Aeromonas spp. showed a MAR index of more than 0.2, indicating that the pathogenic Aeromonas spp. isolated from rohu could have been originated from high-risk antibiotic contamination water sources. Our study is supported by Abraham (2011), who reported the association of opportunistic human bacterial pathogens and their sensitivity to broad spectrum antibiotics in cultured freshwater fishes where the MAR index was high in the bacterial flora of carps grown in sewage-fed farms than the carps grown in non sewage fed aquaculture systems of Kolkata, India. Zhou et al. (2019) studied MAR of Aeromonas spp. from 115 human samples, among which 28.7% exhibited multiple-drug resistance (MDR) patterns to 15 antimicrobial agents. 80% strains of A. hydrophila and 81.2% strains of A. dhakensis presented with MDR, while fewer MDR isolates were A. caviae (39.6%) and A. veronii (16.7%) from hospital patient samples. Isolation of antimicrobial resistance and multiple antibiotic resistant Aeromonas spp. from freshwater aquaculture systems in many countries along with our findings has shown a growing concern in the treatment of Aeromonas infections in fish and humans.

  • Conclusion

    Occurence of Aeromonas spp. in farm grown rohu and their resistance to antimicrobials with high multi-antibiotic resistant index indicates the origin of bacteria from high-risk sources and intern their threat to public health. Proper measures required in the selection of water source for aquaculture and the food fish need to be handled and cooked appropriately in order to prevent the introduction of antimicrobial resistant bacteria in fish and humans respectively.

  • Acknowledgement

    The first author is thankful to the Vice Chancellor, Prof. V. Padmanabha Reddy and the former Dean of Fishery Science, Dr. T. VenkataRamana of Sri Venkateswara Veterinary University (SVVU), Tirupati for providing stipend and the facilities provided for carrying out research work during her Ph.D. (Aquatic Animal Health) study. The chemicals, consumables and fish samples of the project entitled ‘National Surveillance Programme for Aquatic Animal Diseases (NSPAAD) as NSPAAD sub-center- 12 being operated at the Department of Aquatic Animal Health Management, College of Fishery Science, SVVU, under the principal investigatorship of the corresponding author was provided to the first author. The authors duly acknowledge NFDB, Govt. of India, Hyderabad and NBFGR (ICAR), Lucknow, India for providing funds under the NSPAAD project.

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