Dissecting molecular evolution of class 1 integron gene cassettes and identifying their bacterial hosts in suburban creeks via epicPCR

Objectives

Our study aimed to sequence class 1 integrons in uncultured environmental bacterial cells in freshwater from suburban creeks and uncover the taxonomy of their bacterial hosts. We also aimed to characterize integron gene cassettes with altered DNA sequences relative to those from databases or literature and identify key signatures of their molecular evolution.

Methods

We applied a single-cell fusion PCR-based technique—emulsion, paired isolation and concatenation PCR (epicPCR)—to link class 1 integron gene cassette arrays to the phylogenetic markers of their bacterial hosts. The levels of streptomycin resistance conferred by the WT and altered aadA5 and aadA11 gene cassettes that encode aminoglycoside (3″) adenylyltransferases were experimentally quantified in an Escherichia coli host.

Results

Class 1 integron gene cassette arrays were detected in Alphaproteobacteria and Gammaproteobacteria hosts. A subset of three gene cassettes displayed signatures of molecular evolution, namely the gain of a regulatory 5′-untranslated region (5′-UTR), the loss of attC recombination sites between adjacent gene cassettes, and the invasion of a 5′-UTR by an IS element. Notably, our experimental testing of a novel variant of the aadA11 gene cassette demonstrated that gaining the observed 5′-UTR contributed to a 3-fold increase in the MIC of streptomycin relative to the ancestral reference gene cassette in E. coli.

Conclusions

Dissecting the observed signatures of molecular evolution of class 1 integrons allowed us to explain their effects on antibiotic resistance phenotypes, while identifying their bacterial hosts enabled us to make better inferences on the likely origins of novel gene cassettes and IS that invade known gene cassettes.

Methods
Approximately 200 mL freshwater samples were collected from Kikkiya Creek (-33.776, 151.117) and the Macquarie University Lake section of Mars Creek (-33.772, 151.115) in three independent biological replicates in suburban metropolitan Sydney, NSW, Australia (Figure 1A). Water samples were immediately transported back to the lab, filtered with 40 µm EASYstrainer cell strainers (Greiner AG, Germany) and centrifuged at 3,220 g for 15 minutes at 4˚C. Pellets were resuspended in 300 µL 0.01M phosphate buffered saline (PBS) solution (pH 7.4) and stored as 25% glycerol stocks for cryopreservation. Estimates of bacterial cell density were obtained by fluorescence microscopy using previously described procedures (1).

epicPCR to determine the identity of bacterial hosts of class 1 integrons in suburban creeks was performed in three technical replicates for each of the three biological replicates using previously established procedures with minor modifications (1). Approximately 150,000 cells were resuspended in 75 µL PCR mix containing the following reagents at the final concentrations indicated in the parentheses: GC buffer (1x), dNTP mix (0.4 mM), Phusion High-Fidelity DNA polymerase (0.05 U/µL) (Thermo Scientific, United States), bovine serum albumin (1 µg/µL) (Promega, United States), Lucigen Ready-Lyse lysozyme (500 U/µL) (LGC Biosearch Technologies, United States), as well as three primers R926 (2 µM), intI1_outer (1 µM), and R519-qacE bridge primer (0.04 µM) (Table S1, Supplementary Materials). The PCR was mixed at 4 ms-1 speed for 45 s in 425 µL ABIL oil using a FastPrep-24 bead beating system (MP Biomedicals, United States), and the resulting emulsion was aliquoted into 8 portions that were subject to the following conditions: 37˚C for 10 min (lysozyme lysis); 98˚C for 5 minutes (initial denaturation); 38 cycles of 98°C for 10 s (denaturation), 55°C for 30 s (annealing), and 72°C for 2 min (extension); and finally, 72°C for 5 min. DNA in the aqueous phase was extracted, purified, and size-selected to deplete <300 bp fragments.

In the second stage of epicPCR, DNA templates from the previous step were PCR amplified in eight aliquots of 12.5 µL reactions containing GC buffer (1x), dNTP mix (0.4 mM), Phusion High-Fidelity DNA polymerase (0.04 U/µL), AP27_short primer (0.8 µM), intI1_nested primer (0.4 µM), forward and reverse blocking primers (0.32 µM each) under the following conditions: 98˚C for 30 s; 23 cycles of 98°C for 10 s, 55°C for 30 s and 72°C for 2 min; followed by a final step of 72°C for 5 min. 80 µL of the recombined PCR product was added to 48 µL of Sera-Mag Select magnetic beads (Cytiva, United States) to deplete epicPCR products containing cassette-less integrons, and the remaining DNA size selection procedures were carried out according to the manufacturer’s instructions. In the third stage of epicPCR, the PCR products were further PCR amplified in eight aliquots of 12.5 µL reactions containing GC buffer (1x), dNTP mix (0.4 mM), Phusion High-Fidelity DNA polymerase (0.04 U/µL), AP27_tail primer (0.4 µM), intI1_nested primer (0.4 µM), forward and reverse blocking primers (0.32 µM each) under the following conditions: 98˚C for 30 s; 12 cycles of 98°C for 10 s, 55°C for 30 s and 72°C for 2 min. The recombined epicPCR products were visualised on 1% agarose gel by electrophoresis. 80 µL epicPCR product was added to 40 µL of Sera-Mag Select magnetic beads in the final DNA size selection step. The purified epicPCR amplicons were sequenced using the same Oxford Nanopore procedures described previously (1).

References

Qi, Q.; Ghaly, T. M.; Penesyan, A.; Rajabal, V.; Stacey, J. A.; Tetu, S. G.; Gillings, M. R. Uncovering bacterial hosts of class 1 integrons in an urban coastal aquatic environment with a single-cell fusion-polymerase chain reaction technology. Environ Sci Technol 2023, 57 (12), 4870-4879. DOI: 10.1021/acs.est.2c09739