Characterization of microbiota composition and presence of selected antibiotic resistance genes in carriage water of ornamental fish

Abstract

International trade with ornamental fish is gradually recognized as an important source of a wide range of different antibiotic resistant bacteria. In this study we therefore characterized the prevalence of selected antibiotic resistance genes in the microbiota found in the carriage water of ornamental fish originating from 3 different continents. Real-time PCR quantification showed that the sul1 gene was present in 11 out of 100 bacteria. tet(A) was present in 6 out of 100 bacteria and strA, tet(G), sul2 and aadA were present in 1-2 copies per 100 bacteria. Class I integrons were quite common in carriage water microbiota, however, pyrosequencing showed that only 12 different antibiotic gene cassettes were present in class I integrons. The microbiota characterized by pyrosequencing of the V3/V4 variable region of 16S rRNA genes consisted of Proteobacteria (48%), Bacteroidetes (29.5%), Firmicutes (17.8%), Actinobacteria (2.1%) and Fusobacteria (1.6%). Correlation analysis between antibiotic resistance gene prevalence and microbiota composition verified by bacterial culture showed that major reservoirs of sul1 sul2, tet(A), tet(B) tet(G), cat, cml, bla, strA, aacA, aph and aadA could be found among Alpha-, Beta- and Gammaproteobacteria with representatives of Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae and Comamonadaceae being those most positively associated with the tested antibiotic resistance genes.

Figure 1. Prevalence of antibiotic resistance genes in ornamental fish carriage water.

Antibiotic resistance gene prevalence is presented with the median, 25% and 75% percentiles (box) and the whiskers indicating the minimum and maximum values recorded.

Figure 2. Amplification products after 5CS-3CS PCR of individual carriage water samples.

L – molecular weight standard in bp. Amplification resulted in PCR products ranging from 200 to 1500 bp in size.

Figure 3. Distribution of selected genes in integron structures determined by pyrosequencing and real-time PCR.

Average values calculated from data available for 48 samples were used for both real-time PCR based pie charts. Quantification of genes in integrons by pyrosequencing and real-time PCR resulted in similar results whereas the comparison of these results with the results from carriage water DNA indicated that dfrA was associated with integrons as its representation decreased when total carriage water was used as a template in real-time PCR. On the other hand, aacA and ereA must have been common in genetic elements different from integrons.

Figure 4. Composition of ornamental fish carriage water microbiota at class level.

1 – Sphingobacteria, 2 – Gammaproteobacteria, 3 – Fusobacteria, 4 – Flavobacteria, 5 – Clostridia, 6 – Betaproteobacteria, 7 – Bacteroidia, 8 – Alphaproteobacteria, 9 – Bacilli, 10 – Actinobacteria. For the full data set see table S1.

Figure 5. Heat map showing the correlation coefficients of the presence of individual families and particular antibiotic resistance genes.

Three main clusters according to their positive and negative correlation with the prevalence of antibiotic resistance genes tested in this study are indicated by red, blue and green color.

Figure 6. Antibiotic resistances in isolates belonging to families predicted as highly associated with antibiotic resistance in the microbiota of carriage water of ornamental fish.

Data shows the percentage of isolates within a given family encoding the gene indicated on the X-axis out of all isolates tested. Black columns, results based on PCR detection of gene as indicated. Grey columns, percentage of antibiotic resistance determined in the same isolates by disk diffusion assay (ampicillin resistance is shown with bla gene, the same tetracycline resistance data are shown in tet(A), tet(B) and tet(G) genes, streptomycin resistance is shown in strA, aacA, aph and aadA genes, chloramphenicol resistance is shown in cml and cat genes, sulphonamide resistance is shown in sul1 and sul2 genes and nalidixic acid resistance is shown in qnr gene. Resistance to erythromycin (ere), trimethoprim (dfr) and rifampicin (arr) was not determined by disk diffusion assay and therefore are not shown.