Molecular characterization and pathogenicity of a ...

Aquaculture 471 (2017) 157–162

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Molecular characterization and pathogenicity of a virulent Acinetobacter baumannii associated with mortality of farmed Indian Major Carp Labeo rohita (Hamilton 1822) B.K. Behera ⁎, P. Paria, A. Das, S. Bhowmick, A.K. Sahoo, B.K. Das ICAR-Central Inland Fisheries Research Institute, Barrackpore, Kolkata 700120, West Bengal, India

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Article history: Received 5 December 2016 Received in revised form 18 January 2017 Accepted 19 January 2017 Available online 20 January 2017 Keywords: Labeo rohita 16S rRNA gene Acinetobacter baumannii Histology Molecular phylogeny

a b s t r a c t Indian Major Carp Labeo rohita (Ham.) is the most preferred cultivable fish species under freshwater aquaculture in India. The present study was conducted to characterize the bacteria at molecular level and to understand its pathogenicity associated with mortality of farmed rohu, Labeo rohita. Diseased fish samples were collected from cultured ponds of Nadia, West Bengal, India for the isolation of the pathogen. The 16S rRNA gene sequence of the bacteria revealed that the isolate was 100% identical with Acinetobacter baumannii (NCBI Accession Number KT156752). Intraperitoneal injection with the isolate at the level of 2.1 × 108 cells/ml in fish causes mortality. The challenged fish had loss of mucus and reddish lesion near the pectoral fin, however there was no sign in the gill. The histology of experimentally challenged Labeo rohita showed hemorrhages and shrunken glomeruli with densely basophilic nuclei in kidney whereas degenerated hepatic tissue and increased hepatocyte vacuolation in liver. This study underlines the first time involvement of Acinetobacter baumannii in the disease outbreak of Labeo rohita. The pathology and pathogenesis studies of this emerging pathogen in cultured carps would help in management of the outbreak of disease in aquaculture. © 2017 Elsevier B.V. All rights reserved.

1. Introduction The genus Acinetobacter comprises of Gram-negative, strictly aerobic, non-fermentative, rod-shaped bacteria (Nemec et al., 2010; Falagas et al., 2006). Some Acinetobacter sp. like A. baumannii, A. lwoffii, A. johnsonii and A. calcoaceticus were identified as emerging opportunistic pathogens in the fish farm of rainbow trout and common carp in Poland (Kozińska et al., 2014). Reddy and Mastan, 2013 identified Acinetobacter schindleri involved in red eye infection in Pangasius fingerlings in India. Only in the last decade, association of Acinetobacter baumannii in the infection of various fish species like Siniperca chuatsi, Barbus pentazona hexazona, Ictalurus punctatus, Channa striata, and Oncorhynchus mykiss (Gu et al., 1997; Rauta et al., 2011; Musa et al., 2008; Xia et al., 2008), have been reported by many workers from different parts of the world (Malaysia, China, Turkey, India and Poland) which indicate that this bacteria is able to grow in wide range of environment and infect wide range of fish species. Acinetobacter sp. posses many virulence factors like cell surface hydrophobicity,

⁎ Corresponding author. E-mail address: [email protected] (B.K. Behera).

http://dx.doi.org/10.1016/j.aquaculture.2017.01.018 0044-8486/© 2017 Elsevier B.V. All rights reserved.

enzymes, toxic slime polysaccharides, verotoxins, siderophores and outer membrane proteins which enable the bacteria to infect human (Bergogne-Berezin and Towner, 1996; Dorsey et al., 2004; Zimbler et al., 2013). Out of these species A. baumannii accounts for 80% of the reported infections in human. Experimental infection with this bacteria reveled that it is able to infect wide range of animal species including rodent, non rodent, non mammals and fishes (Rauta et al., 2011; Xia et al., 2008; McConnell et al., 2013). Indian Major Carp (IMC), rohu is one of the most preferred aquaculture fish species among the carps in the country and command a higher market price. However with increase in aquaculture production of the rohu, outbreak of infectious diseases acts as significant setback for successful aquaculture production and trade in India (Yunxia et al., 2001). Identification of the diseases and their causative agents will help to develop strategies to control and prevent the incidence of diseases in farmed Labeo rohita, thus reducing loss to farmers. The aim of the study was to investigate disease outbreaks in cultured L. rohita and identifying the etiological agents based on morphological, biochemical characteristics and 16S rRNA gene sequence analysis. Experimental challenge was conducted through intraperitoneal injection of the pure culture of bacterium, isolated from the diseased fish to confirm the pathogenicity.

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2. Materials and methods

ensure coherency of the clusters formed. Bootstrapping was performed (10,000 replications) using the MEGA 6 program.

2.1. Sampling 2.5. Experimental challenge Diseased samples of L. rohita were collected from Nadia (N 23°07′ 0.828″; E 088°41′0.152″) West Bengal, India, for the isolation of pathogenic Acinetobacter baumannii. The moribund fishes with reddish lesions near the pectoral fin were used for bacterial isolation. Fishes were anaesthetized by using an overdose of clove oil (Merck, Germany) (50 μl/l). The fishes were cleaned with alcohol and then dissected. Samples of liver and kidney were taken aseptically and plated directly on Tryptic Soya Agar (TSA, Hi-media). The plates were incubated at 37 °C for 24 h. Single colony isolates were selected and re-streaked on fresh TSA plates to obtain the pure culture. The pure culture (NBL13) was grown in Tryptic Soya Broth (TSB) and maintained as glycerol stock at −20 °C. 2.2. Biochemical characterization of bacterial isolate The isolated strain was primarily characterized by Gram-staining, ONPG (β-galactosidase), lysine utilization, urease, nitrate reduction, Voges Proskauer's (VP), malonate utilization, esculin hydrolysis, arabinose, xylose, adonitol, rhamnose, cellobiose, melibiose, saccharose, raffinose, trehalose, glucose, lactose oxidase, ornithine utilization, phenylalanine deamination, H2S production, citrate utilization, methyl red and indole were performed (KB-003, HiMedia). Additional 41 biochemical tests were carried out using an automated bacterial identification system (VITEK 2 compact, BioMerieux, France). 2.3. DNA isolation and 16S rRNA gene amplification Bacterial genomic DNA was extracted from isolated bacteria by Sarkosyl method (Sambrook and Russel, 2001). The DNA concentration was quantified using UV visible spectrophotometer. The PCR amplification of the 16S rRNA gene was performed using 16S rRNA specific primers with the thermal cycler Gene Amp PCR system 9700 (Applied Biosystems, Foster City, CA). The primers chosen for amplification of 16S rRNA gene were UFF2 5′-GTTGATCATGGCTCAG-3′ as forward primer and URF2 5′-GGTTCACTTGTTACGACTT-3′ as reverse primer (Kumar et al., 2014). The final volume of the PCR reaction mixture (50 μl) was composed of 100 ng of isolated genomic DNA, 1 U Taq DNA polymerase (Sigma, USA), 5 μl of 10× PCR buffer, 1 μl of 50 mM MgCl2, 1 μl of 10 mM dNTPs (Sigma, USA), and 1 μl of 10 pmol of each primer (Sigma, USA) using the thermal cycler. The thermal profile consisted of initial denaturation for 2 min at 95 °C, 35 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 45 s and extension at 72 °C for 1.30 min, final extension for 10 min at 72 °C. PCR product was visualized on 1.8% agarose gel containing ethidium bromide. 2.4. Molecular identification of the bacterium The PCR product was sequenced in both directions using an ABI 3730xl capillary sequencer (Applied Biosystems, Foster City, CA) to check the validity of the sequence data (Behera et al., 2014). The forward and reverse sequences of the isolate NBL13 were aligned using the software Bio Edit version 7.0.0. The sequence of forward strand was proofread using the sequence of complementary strand. The16S rRNA gene sequence of the isolate NBL13 was around 1414 bp which was then compared with sequences available in NCBI GenBank using the NCBI–BLAST program facility (http://www.ncbi.nlm.nih.gov/ BLAST). The 16S rRNA gene sequence of the Acinetobacter baumannii was aligned with 16S rRNA gene sequences of most known species of Acinetobacter retrieved from NCBI GenBank using MEGA 6 (Tamura et al., 2013). The 16S rRNA gene sequence of Psychrobacter immobilis was considered as an outgroup. Phylogenetic analysis was carried out by applying the neighbour-joining and maximum-likelihood algorithms to

A total of 70 healthy Labeo rohita (mean weight; 23 ± 5 g) were collected from local carp hatchery and were acclimatized under laboratory conditions for 15 days by providing commercially available fish diet twice daily @ 2% of their body weight. The fishes were randomly assigned to seven experimental tanks (one tank as control and other six tanks for experimental challenge) for challenge study. A total number of 10 fishes were kept in each 200 l tank. 2.6. LD50 determination The experimental challenge study was conducted to determine the cumulative mortality and the lowest bacterial dose for 50% mortality (LD50) in L. rohita. The bacteria were sub-cultured in 10 ml of Brain Heart Infusion (BHI) broth and the suspension was static-incubated at 37 °C for 24 h. After the incubation, the culture was centrifuged for 5 min at 5000 rpm. The supernatant was discarded and pellet was washed two times with normal saline (NS) water and finally resuspended in 10 ml NS water. 1 ml of cell suspension was suitably diluted up to 10−6 in NS and the number of cells/ml of suspension was determined by spread plate method after incubation at 37 °C for 24 h. The fishes were injected with 0.2 ml of bacterial suspension intraperitoneally with final concentration of 2.1 × 105, 2.1 × 106, 2.1 × 107, 2.1 × 108, 2.1 × 109 and 2.1 × 1010 CFU ml−1, respectively. The control fishes were injected with 0.2 ml of saline water. The experiment was carried out in triplicates. Fish mortality was recorded in every 24 h interval for 7 days. The injected bacterial isolate as a pathogen was re-isolated from the blood and liver of moribund fish to satisfy Koch's postulates. Following Reed and Muench (1938), LD50 was calculated using the cumulative mortalities.

Table 1 Comparative biochemical analysis of NBL13 isolated from Labeo rohita with published biochemical data of Acinetobacter baumannii. Biochemical test

NBL13

ChEa

A. baumanniib

ONPG (β-galactosidase) Lysine Ornithine Urease Phenylalanine deamination Nitrate H2S Citrate V·P Methyl red Indole Oxidase Malonate Esculin hydrolase Anabinose Xylose Adonitol Rhamnose Cellobiose Mellibiose Saccharose Raffinose Trehalose Glucose Lactose Sorbitol Mannitol Sucrose

− − − + − − − + − − − + + − + + − − + + − − − + − − − −

na − − − − + − +

na − − − − na − − na na − − na na + − na na na na na − na na na − − na

“+”: positive; “−”: negative. a Rauta et al. (2011). b Gu et al. (1997) and Xia et al. (2008).

− − na − + + + na + na na − + na − + −

B.K. Behera et al. / Aquaculture 471 (2017) 157–162 Table 2 Additional biochemical characteristics of Acinetobacter baumannii (NBL13) strain as assessed by using VITEK 2 compact (BioMerieux, France). Biochemical test

(PyrA)

L-Arabitol (IARL) Beta-galactosidase (BGAL) Beta-N-acetyl-glucosaminidase (BNAG) Glutamyl arylamidase pNA (AGLTp) Gamma-glutamyl transferase (GGT) Fermentation/glucose (OFF) Beta-glucosidase (BGLU) D-Maltose (dMAL) D-Mannitol (dMAN) D-Mannose (dMNE) Beta-xylosidase (BXYL) Beta-alanine arylamidase pNA (BAlap) L-Proline arylamidase (ProA) Lipase (LIP) Palatinose (PLE) Tyrosine arylamidase (TyrA) D-Tagatose

(dTAG)

D-Trehalose

(dTRE) 5-Keto D-gluconate (5KG) L-Lactate alkalinisation (ILATk) Alpha-glucosidase (AGLU) Succinate alkalinisation (SUCT) Beta-N-acetyl-galactosaminidase (NAGA) Alpha-galactosidase (AGAL) Phosphatase (PHOS) Glycine arylamidase (GlyA) Lysine decarboxylase (LDC) Orinithine decarboxylase (ODEC) L-Histidine assimilation (IHISa) Coumarate (CMT) Beta-glucoronidase (BGUR) O/129 resistance (O129R) Glu–Gly–Arg–arylamidase (GGAA) L-Malate assimilation (IMLTa) Ellman (ELLM) L-Lactate

assimilation (ILATa)

“+”: positive; “−”: negative.

2.7. Histopathology

− − − − − − − −

The internal organs like kidney and liver from the infected fish samples were collected after recording the external clinical signs and fixed in 10% Neutral Buffered Formalin (NBF) for histopathological studies. The preserved tissues were cut into 1–2 mm size and kept in 10% NBF for overnight. Then the tissues were dehydrated with a series of different concentrations of alcohol and cleared in xylene and embedded into paraffin following the impregnation technique (Leica EG 1140H, Germany). The paraffin-embedded tissues were sectioned at 5-mm thickness using a microtome and stained with haematoxylin and eosin (Luna, 1968). Pathological changes manifested in the tissue sections were noted down and microphotographs were taken (Olympus CX31, Japan), wherever necessary.

+ − − −

3. Results

Result

Ala–Phe–Pro–arylamidase (APPA) L-Pyrrolydonyl–arylamidase

159

− − −

− − + − − − + − + − − − − − − + + − − − + − +

3.1. Biochemical characterization and molecular identification of the bacterium The biochemical test revealed that the Acinetobacter baumannii isolate was Gram negative, rods. The other tests revealed that it was positive for citrate, urease, malonate, arabinose, cellobiose, mellibiose, glucose, oxidase, whereas it was showing negative for ONPG, lysine, ornithine, phenylalanine deamination, nitrate, H2S, methyl red, indole, VP, esculin hydrolase, adonitol, rhamnose, saccharose, raffinose, trehalose, lactose, sorbitol, sucrose, and mannitol (Table 1). Additional biochemical test showed that the bacterium was positive for D mannose, tyrosine acrylamidase, L-lactate alkalinisation, succinate alkalinisation, L-histidine assimilation, L-malate assimilation, L-lactate assimilation (Table 2). The PCR amplified 16S rRNA gene from the isolated bacterium (GenBank Accession Number: KT156752) was sequenced and analyzed by using BLAST program. The results revealed that the 16S rRNA gene sequence of the isolated bacterium (NBL13) has 100% homology with Acinetobacter baumannii. The molecular sequence analysis and biochemical analysis confirmed that the isolated bacterial sample was Acinetobacter baumannii.

Fig. 1. Phylogenetic tree analysis of Acinetobacter sp. based on 16S rRNA nucleotide sequences. Phylogenetic tree was generated using neighbour-joining method by the MEGA 6 software. The numbers next to the branches indicate percentage values for 10,000 bootstrap replicates. Bootstrap values are shown at the nodes. The isolate NBL13 identified in this study are indicated by the shaded triangle.

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glomeruli with densely basophilic nuclei (Fig. 3B) in kidney. The liver section exhibited degenerated hepatic tissue (Fig. 4A) and increased hepatocyte vacuolation (Fig. 4B). 4. Discussion

Fig. 2. Cumulative mortality curves for the determination of LD50 values in Indian Major Carp Labeo rohita challenged with Acinetobacter baumannii by intra-peritoneal injection at different concentrations.

3.2. Phylogenetic analysis The phylogenetic tree was constructed using the neighbour-joining algorithm revealed the relationships of isolated bacterium NBL13 with all other Acinetobacter species. The 16S rRNA sequences of different Acinetobacter sp. were acquired from NCBI GenBank. Isolate NBL13 was clustered with Acinetobacter baumannii. This cluster was supported by high bootstrap value (Fig. 1). The same clusters were also observed in trees constructed using the maximum-likelihood algorithm. 3.3. Cumulative mortality and determination of LD50 Cumulative mortality rate of Labeo rohita after post infection with Acinetobacter baumannii are shown in Fig. 2. Mortality was not observed after 7 days post injection in the control fishes which were injected with NS. Fishes which were challenged by intraperitoneal injection of the isolated bacterium had shown the similar clinical symptoms. The bacterium was re-isolated from the kidney; liver and blood were found to be same as that of the injected isolate NBL13. LD50 value of Acinetobacter baumannii is 1.5 × 105 CFU per fish calculated by following Reed and Muench (1938). 3.4. Histopathology The clinical symptoms of the infected fishes were observed as loss of mucus and reddish lesion near the pectoral fin, however there was no sign in the gill. The histopathological examination of the experimentally challenged Labeo rohita showed hemorrhages (Fig. 3A) and shrunken

Diseases are the major constraints for sustainable development of carp aquaculture in India. In the present study, Acinetobacter baumannii was isolated from the diseased L. rohita from the culture ponds. Isolation of this bacterium from ulcerated fishes showed its association in contributing the pathogenesis of ulcerative lesions in the diseased fishes. The occurrence of disease in fish due to infection of A. baumannii has been reported in different fish species like Cyprinus carpio, Oncorhynchus mykiss, Channa striata and Siniperca chuatsi (Gu et al., 1997; Rauta et al., 2011; Musa et al., 2008; Xia et al., 2008). The biochemical tests together with molecular identification were carried out for identification of the isolate NBL13. Most of the biochemical test results were found similar with biochemical result of Acinetobacter baumannii, reported by the earlier workers (Rauta et al., 2011; Xia et al., 2008). The present isolate showed slight variation in some of the biochemical test like citrate, trehalose, nitrate and xylose. The isolate NBL13 was able to utilize citrate, xylose and was unable to utilize adinitol, trehalose and mannitol. Rauta et al., 2011 isolated Acinetobacter baumannii from diseased fish species Channa striata and that isolate was able to utilize citrate, mannitol, xylose and trehalose. Whereas Acinetobacter baumannii isolated from diseased channel catfish (Ictalurus punctatus) in China was unable to utilize citrate and mannitol as a carbon source (Xia et al., 2008). The infection in mandarin fish Siniperca chuatsi with this bacterium was first time reported by Gu et al., 1997. He had identified the bacterium based on the biochemical test. Molecular evolutionary analysis of 14 different Acinetobacter species, reveled that 16S rRNA gene sequence of NBL13 has 100% homology with Acinetobacter baumannii (gi 645320408 and gi EO760624) and they clustered within a sister group in the phylogenetic tree with highest bootstrap value. Xia et al., 2008 had identified infection associated with Acinetobacter baumannii in channel catfish Ictalurus punctatus using biochemical test and 16S rRNA gene sequence analysis. The level and dose of pathogenicity of the bacteria in several carp species were also found to be variable (Sarkar and Rashid, 2012; Ali et al., 2014; Sahoo et al., 2008). Therefore, for the first time, the optimum dose of bacterial inoculation and pathogenicity was determined in L. rohita. Rey et al., 2009 observed the determination of LD50 in study organism before the experimental challenge is advantageous for successful experiment and induction of clinical signs and symptoms. In the present study, LD50 value of 1.5 × 105 CFU per fish was standardized for A. baumannii in Labeo rohita. LD50 value of A. baumannii was standardized for in vivo experiments that would serve as a baseline data for future immune response studies.

Fig. 3. A–B. Photomicrograph of kidney of Acinetobacter baumannii challenged Labeo rohita showing [A]: hemorrhages (H) (H & E staining; 60×); [B]: Shrunken glomeruli with densely basophilic nuclei (SG) (H & E staining; 60×).


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Fig. 4. A–B. Photomicrograph of Liver of Acinetobacter baumannii challenged Labeo rohita showing [A]: degenerated hepatic tissue (DH) (H & E staining; 60×); [B]: increased hepatocyte vacuolation (HV) (H & E staining; 60×).

The systemic pathological alterations were seen in the kidney of experimentally challenged Labeo rohita. The kidney of the diseased fish showed hemorrhages and shrunken glomeruli with densely basophilic nuclei and increased space within the Bowman's capsule (Fig. 3A–B). Abraham et al., 2015 observed similar types of histological changes in kidney of African catfish Clarias gariepinus infected by Edwardsiella tarda. The degenerated hepatic tissue and increased hepatocyte vacuolation was observed in the liver section of L. rohita challenged with A. baumannii (Fig. 4A–B). Feist et al., 2015 studied the liver tissue of infected fishes (Hoplostethus atlanticus, Aphanopus carbo, Phycis blennoides, Coryphaenoides rupestris) collected from Northeast Atlantic Ocean and found the individual hepatocytes displayed a significant variation in appearance within H & E stained sections, with the trabecular arrangement of hepatocytes. Ramkumar et al., 2014 have observed hepatic necrosis and irregular cytoplasmic vacculation with converging sinusoids in the experimentally challenged Labeo rohita with Providencia vermicola. The findings of the experimental challenge in rohu fish underlined the pathogenicity potential of Acinetobacter baumannii. The fish mortality was confirmed through the bacterial transmission intraperitoneally to fish during infection studies. This is the first time report of Acinetobacter baumannii involvement in the disease outbreak of Indian Major Carp Labeo rohita. Since Acinetobacter baumannii was found pathogenic against fish species, therefore better management practices in aquaculture is needed to prevent the outbreak of this emerging disease. In addition to that, more studies are required to understand its virulence factors responsible for infection in the fish and the prevalence rate of this bacterium in aquaculture system. Conflict of interest The authors have declared no conflict of interest. Acknowledgments This work was funded by the National Fisheries Development Board (NFDB) (G/Nat. Surveillance/2013 dated 16.08.2013), Hyderabad under “National Surveillance Program for Aquatic Animal Diseases (NSPAAD)” Project. The authors are thankful to Dr. Joykrushna Jena, Deputy Director General (Fishery Science), Indian Council of Agricultural Research, New Delhi for his support and guidance. The authors would like to express their sincere gratitude to Prof. T. J Abraham, Dept of Aquatic Animal Health, Faculty of Fishery Sciences, West Bengal University of Animal and Fishery Sciences, India for providing the equipment for biochemical analysis. The authors are also thankful to Mr. Asim Kumar Jana, Mr. Hirak Jyoti Chakraborty and Mr. Sudip Pakrashi for helping in sampling and laboratory assistance.

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