High throughput sequencing reveals the abundance and diversity of antibiotic-resistant bacteria in aquaculture wastewaters, Shandong, China

Abstract

An innovative investigation was undertaken into the abundance and diversity of high antibiotic-resistant bacteria in aquaculture waters in Shandong Province, China, through cumulation incubation, PCR amplification of 16S rDNA, and high-throughput sequencing. The results showed that Vibrio, Bacillus, Vagococcus, Acinetobacter, Shewanella, Psychrobacter, Lactococcus, Enterococcus, Marinimonus and Myroids were abundant in the aquaculture waters, whereas other phylum including Actinobacteria, Deinococcus-Thermus, Omnitrophica and Nitrospirae had relatively lower abundance. Our studies revealed the presence of different bacteria in different locations in the aquaculture waters, most of which were resistant to multiple antibiotics. That is, the same microbial species from the same aquaculture wastewater can resist different antibiotics. Altogether, a considerable portion of the microbial community were found to be multi-drug resistant. It is essential that the spread of the antibiotic-resistant bacteria is controlled so that the distribution of antibiotic resistance genes to other environments is avoided.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-021-02656-4.

Keywords: Antibiotic-resistant bacteria; Aquaculture waters; High-throughput sequencing; Multi-drug resistant.

Fig. 1

VENN graph. A1 sample A incubated with 8 µg/mL ciprofloxacin A2 sample A incubated with 8 µg/mL amikacin sulfate A3 sample A incubated with 8 µg/mL polymyxin E A4 sample A with 8 µg/mL tetracycline, and so on as B1, B2, B3, B4 and C1, C2, C3, C4. The petals contain two numbers. The upper number represents the quantity of OTUs that each sample contains and the number in the brackets represents the quantity of unique OTUs that the sample contains. The number in the white centre circle represents the core OTU number

Fig. 2

The horizontal difference matrix of species plus OTU. A1 sample A incubated with 8 µg/mL ciprofloxacin A2 sample A incubated with 8 µg/mL amikacin sulfate A3 sample A incubated with 8 µg/mL polymyxin E A4 sample A with 8 µg/mL tetracycline, and so on as B1, B2, B3, B4 and C1, C2, C3, C4

Fig. 3

Abundance of different genus in different samples. A1 sample A incubated with 8 µg/mL ciprofloxacin A2 sample A incubated with 8 µg/mL amikacin sulfate A3 sample A incubated with 8 µg/mL polymyxin E A4 sample A with 8 µg/mL tetracycline, and so on as B1, B2, B3, B4 and C1, C2, C3, C4

Fig. 4

Star graph of the top 10 genera of abundance. A1 sample A incubated with 8 µg/mL ciprofloxacin A2 sample A incubated with 8 µg/mL amikacin sulfate A3 sample A incubated with 8 µg/mL polymyxin E A4 sample A with 8 µg/mL tetracycline, and so on as B1, B2, B3, B4 and C1, C2, C3, C4

Fig. 5

Taxonomic cladogram obtained from LEfSe analysis of 16S rRNA sequences. Linkages with LDA of 2.0 or higher are shown in the cladogram. The black circles represent phylum, class, order, family, genus, and species, respectively from inner to outer, while red, green and purple circles represent sample A, B and C, respectively

Fig. 6

Taxa enriched in moderately zone are shown in the three groups of sample A, B and C with LDA values higher than 3.0

Fig. 7

Heatmap showing the 50 different microbial composition at genus level between the samples. Color-coded bar plot showing the microbial species relative abundance across different samples. A1 sample A incubated with 8 µg/mL ciprofloxacin A2 sample A incubated with 8 µg/mL amikacin sulfate A3 sample A incubated with 8 µg/mL polymyxin E A4 sample A with 8 µg/mL tetracycline, and so on as B1, B2, B3, B4 and C1, C2, C3, C4

Fig. 8

Plasmid analysis of antibiotic-resistant isolates. B the plasmid was digested by BamH I, and E the plasmid was digested by EcoR I