Underrepresented high diversity of class 1 integrons in the environment uncovered by PacBio sequencing using a new primer

Class 1 integrons (CL1s) are one of the major contributors to the horizontal transfer of antibiotic resistance genes (ARGs). However, our knowledge of CL1 in the environment is still very limited due to the limitations of the current PCR primers and the sequencing methods adopted. This study developed a new primer coupled with PacBio sequencing to investigate the underrepresented diversity of CL1s in a mixed environmental sample (i.e. activated sludge from wastewater treatment plant and pig feces from animal farm). The new primer successfully uncovered 20 extra ARGs subtypes and 57% (422/739) more unique integron array structures than the previous primers. Compared to the whole genome database, CL1s revealed in the environment in this study were of much greater diversity, having 93% (900/967) novel array structures. Antibiotic resistance is the predominant function (78.3% genes) carried by CL1, and a vast majority (98.6% genes) of them confer resistance to aminoglycoside, beta-lactam, trimethoprim, or chloramphenicol. Additionally, 78.5% unique CL1 arrays carried more than one ARGs, and 25.9% of them carried ARGs of clinical relevance with high transferability potential posing threat to the general public. Our results indicated the importance of CL1s in the spread of ARGs. Overall, combining PacBio sequencing with the new primer designed in this study largely broadened our knowledge of CL1s in the environment and their significance in the environmental proliferation of ARGs.