Insights into a Novel blaKPC-2- Encoding IncP-6 Plasmid Reveal Carbapenem-Resistance Circulation in Several Enterobacteriaceae Species from Wastewater and a Hospital Source in Spain

Abstract

Untreated wastewater, particularly from hospitals and other healthcare facilities, is considered to be a reservoir for multidrug-resistant bacteria. However, its role in the spread of antibiotic resistances in the human population remains poorly investigated. We used whole genome sequencing to analyze 25 KPC-2-producing Enterobacteriaceae isolates from sewage water collected during a 3-year period and three clinical Citrobacter freundii isolates from a tertiary hospital in the same collection area in Spain. We detected a common, recently described, IncP-6 plasmid carrying the gene bla_KPC-2 in 21 isolates from both sources. The plasmid was present in diverse environmental bacterial species of opportunistic pathogens such as C. freundii, Enterobacter cloacae, Klebsiella oxytoca, and Raoultella ornithinolytica. The 40,186 bp IncP-6 plasmid encoded 52 coding sequences and was composed of three uniquely combined regions that were derived from other plasmids recently reported in different countries of South America. The region harboring the carbapenem resistance gene (14 kb) contained a Tn3 transposon disrupted by an ISApu-flanked element and the core sequence composed by ISKpn6/bla_KPC-2/Δbla_TEM-1/ISKpn27. We document here the presence of a novel promiscuous bla_KPC-2 plasmid circulating in environmental bacteria in wastewater and human populations.

Keywords: Enterobacteriaceae; IncP-6 plasmid; KPC2 carbapenemase; Spain; wastewater and hospital source.

FIGURE 1

Genetic Map of the p121SC21-KPC2. The backbone of the plasmid contains one region responsible for mobilization and another for replication and maintenance (both indicated in dark green). The gene bla_KPC-2 (fucsia arrow) is located upstream of an interrupted bla_TEM-1 gene and flanked by ISKpn6 and ISKpn27. It is adjacent to a Tn3 transposon with an ISApu-mediated interrupted transposase.

FIGURE 2

Structure comparison of the p121SC21-KPC2 with p12065 and pCOL-1. Genes (open reading frames) and their corresponding transcriptional orientations are indicated by broad horizontal arrows. The orange- and green-shaded areas show identical regions among the structures compared, in the same orientation or inverted, respectively.

FIGURE 3

Genetic bla_KPC-2 environments and their distributions. Four different genetic environments of the gene bla_KPC-2 were present in plasmids of different incompatibility groups of IncP-6, IncX3, IncN, and IncU. The locus adjacent to the Tn3-tnpR was variable. The predominant genetic environment, IncP-6 type, contained a disrupted Tn3 transposase by insertion of an ISApu1/ISApu2-element and was encountered in 21 isolates belonging to several species during the whole period and in isolates of both clinical and wastewater sources.