Sharing of carbapenemase-encoding plasmids between Enterobacteriaceae in UK sewage uncovered by MinION sequencing

Abstract

Dissemination of carbapenem resistance among pathogenic Gram-negative bacteria is a looming medical emergency. Efficient spread of resistance within and between bacterial species is facilitated by mobile genetic elements. We hypothesized that wastewater contributes to the dissemination of carbapenemase-producing Enterobacteriaceae (CPE), and studied this through a cross-sectional observational study of wastewater in the East of England. We isolated clinically relevant species of CPE in untreated and treated wastewater, confirming that waste treatment does not prevent release of CPE into the environment. We observed that CPE-positive plants were restricted to those in direct receipt of hospital waste, suggesting that hospital effluent may play a role in disseminating carbapenem resistance. We postulated that plasmids carrying carbapenemase genes were exchanged between bacterial hosts in sewage, and used short-read (Illumina) and long-read (MinION) technologies to characterize plasmids encoding resistance to antimicrobials and heavy metals. We demonstrated that different CPE species (Enterobacter kobei and Raoultella ornithinolytica) isolated from wastewater from the same treatment plant shared two plasmids of 63 and 280 kb. The former plasmid conferred resistance to carbapenems (bla_OXA-48), and the latter to numerous drug classes and heavy metals. We also report the complete genome sequence for Enterobacter kobei. Small, portable sequencing instruments such as the MinION have the potential to improve the quality of information gathered on antimicrobial resistance in the environment.

Keywords: ESBL- producing Enterobacteriaceae; MinION; sequencing; wastewater.

Fig. 1.

Map of wastewater treatment plants in the East of England tested for CPE. Black dots, negative for CPE; red dots, positive for CPE. Plants positive for CPE are numbered 1–4. Sewer refers to sampling at the CUH facility. Hospitals situated upstream of the study wastewater treatment plants (approximate median distance of 5.3 km; range 3.3–9.6 km) are denoted by H.

Fig. 2.

CPE isolates referred from diagnostic microbiology laboratories in the East of England to the AMRHAI Reference Unit between 2008 and 2015. One representative of the same CPE per month per hospital is shown. One sample received by PHE in 2006 was excluded as date of isolation was unknown. See Table S1 for a complete list of all isolates.

Fig. 3.

Comparison of whole-genome assemblies of Enterobacter kobei and R. ornithinolytica isolates using the Artemis comparison tool. The grey and dark blue vertical blocks represent blast hits between the isolates in the same orientation or in the inverted orientation, respectively. Only hits longer than 1 kb are shown. Contigs are highlighted in alternating orange and brown colours. The MDR plasmid is highlighted in light blue, and the bla_OXA-48 plasmid is highlighted in purple.

Fig. 4.

Comparison of shared Enterobacter kobei and R. ornithinolytica plasmids. (a) bla_OXA-48 pOXA-48a-like plasmid. (b) Multidrug-resistance plasmid. Plasmid maps of the shared plasmids are shown, with genes of interest annotated. The grey and blue blocks represent blast hits between the isolates in the same orientation and inverted orientation, respectively. The colour intensity is proportional to the per cent identity of the match, within the specific region. Gene colour code indicates function: dark blue, heavy-metal resistance; light blue, conjugational transfer; dark pink, antibiotic resistance; light pink, IS elements and transposases; yellow, replication, maintenance, partitioning genes; light green, other (hypothetical proteins, host metabolism, regulators and pseudogenes).