16S rRNA sequence based identification of pathogenic gut microbiota of Rohu Labeo rohita (Hamilton-Buchanan 1822) and Silver carp Hypophthalmichthys molitrix (Valenciennes 1844)

Abstract

Rohu (Labeo rohita) and Silver carp (Hypophthalmichthys molitrix), both are tropical, most common, popular and commercially important fish of Bangladesh where pathogenic gastrointestinal bacteria of these fish, indicates the acceptability or quality as a whole the economic status of these fish. Current study was, therefore, carried out to investigate and compare the gastrointestinal pathogenic bacteria of these economically important fish of Bangladesh. The experimental fishes were collected from three different fish markets of Dhaka metropolitan city viz. Nobabgonj Bazar, Palashi Bazar and Anando Bazar. In the present study, the gastrointestinal tract of experimental fishes were examined with three replicates. Homogenized samples were then used for bacteriological density enumeration using serial dilution and spread plate techniques with five selective media Luria agar (LA), Thiosulphate citrate bile salt sucrose agar (TCBS), Salmonella- Shigella agar (SS), Aeromonas agar, Mannitol Salt agar (MSA), Eosin Methylene Blue agar (EMB) plate to understand Total Bacterial Count (TBC), Total Vibrio like colony count, Total Salmonella- Shigella count, Total Aeromonas count, Total Staphylococcal count, Total coliform count. Besides conventional culture techniques, Biochemical as well as molecular techniques (16S rRNA sequencing) were performed for the purpose of isolation and identification of gut microbiota. The study was also investigated the antibiotic susceptibility of 14 antibiotics on selected isolates. On an average, TBC of Rohu samples was 5.27 ± 2.01× 107 cfu/g and in Silver carp was 3.02 ± 1.42 × 107 cfu/g; total Vibrio count from Rohu and Silver carp sample was 1.58 ± 3.51 × 106 cfu/g and 2.38 ± 3.63 × 103 cfu/g, respectively; total Salmonella and Shigella was 6.94 ± 7.15 × 106cfu/g from Rohu and 1.11 ± 0.97 × 106 cfu/g from Silver carp. In Rohu, total Aeromonas was 1.31 ± 1.06 × 107 cfu/g and in Silver carp was 6.09 ± 4.61 × 106 cfu/g; total Staphylococcal count was found 1.03 ± 0.52 × 107 cfu/g in Rohu and 5.48 ± 3.98 × 106 cfu/g in Silver carp. For Rohu sample, enteric bacteria with special reference to coliform count was 1.68 ± 0.981 × 107 cfu/g and for Silver carp was 1.39 ± 2.35 × 107 cfu/g. Bacterial density (TBC, Salmonella-Shigella and Staphylococcal count) showed significant difference between species but among markets no significant difference were observed. All types of load counts exceed the ICMSF acceptable limit. 18 different (9 isolates of Rohu and 9 isolates of Silver carp) colonies were selected based on morphology for biochemical identification tests. Among 18 isolates, 3 were gram positive. From Rohu sample, 2 Aeromonas sp. and Pseudomonas sp., 1 Vibrio sp., 1 Proteus sp., 1 Staphylococcus sp., 1 Enterobacter sp. and 1 Klebsiella sp. was provisionally identified. In Silver carp, 1 Vibrio sp., 1 Salmonella sp., 1 Pseudomonas sp., 1 Escherichia sp., 1 Klebsiella sp. and 2 Aeromonas sp. and Staphylococcus sp. were provisionally identified. 10 representative isolates, named as njp1, njp2, njp3, njp4, njp5, njp6, njp7, njp8, njp9 and njp10 were selected for sequencing by 16S rRNA gene and identified as Aeromonas hydrophila subsp. dhakensis, Proteus penneri , Pseudomonas plecoglossicida, Aeromonas caviae, Enterobacter sp., Pseudomonas aeruginosa, Aeromonas sp., Citrobacter freundii, Klebsiella pneumoniae subsp. rhinoscleromatis and Klebsiella pneumoniae subsp. rhinoscleromatis, respectively. Multiple sequence alignment was performed to find out the polymorphic sites among the sequenced strains with considering 1341 bp nucleotides where 1.49% dissimilarities were observed among the identified 3 Aeromonas sp. and 3.88% in 2 Pseudomonas sp.All of the 2 group (njp9 and njp10) were absolutely similar in all positions of the sequence to each other except in 3 positions (338, 361 and 1038) were polymorphic. Phylogenetic analysis also confirmed the taxonomic relations among the identified species. Finally, 6 different group of pathogenic gastrointestinal bacteria were identified by 16S rRNA sequencing whereas biochemical assay provisionally identified 9 different group of pathogenic bacteria. The present study also revealed that, all the isolates including reference strain (E. coli DH5α) were sensitive to Ciprofloxacin and resistant to Sulphamethoxazole. Findings of this study indicate the poor quality and unhygienic condition of the marketed fish which reflects the potential reservoir of pathogenic bacteria causing fish borne disease outbreaks.