Caracterização de genes de resistência antimicrobiana e seu contexto genético no complexo enterobacter cloacae isolados em um hospital terciário do Estado de São Paulo

Abstract

The Enterobacter cloacae complex (ECc) are frequently involved in health care-acquired infections (HCAI). In addition to the increasing prevalence in hospitals around the world, ECc has intrinsic resistance to some classes of antibiotics, making it difficult to treat serious infections. Thus, there are few antibiotic therapy options. The spread of antibiotic resistance contributes to the selection of this group of bacteria, the main factor being the ability to mobilize several resistance genes through the mobile genetic elements. Objectives: The aim of this study was characterize resistance genes responsible to the producion of ESBL, Carbapenemases, PMQR, AME, and 16S rRNA Methyltransferases in multidrug-resistant ECc isolated from clinical samples from a tertiary hospital in the State of São Paulo; and to analyze the complete genome of strains that have already known resistance mechanisms. Methods: Seventy-four strains of ECc isolated from 2013 to 2017 were subjected to single and multiplex PCR for the detection blaSHV-5, blaSHV-12, blaCTX-M-2, blaCTX-M-15, blaCTX-M-8, blaKPC, blaIMP, blaVIM, blaNDM, blaOXA-48, blaBKC, qnrA, qnrB, qnrS, aac(6')-Ib-cr, qepA, OqxAB; AME gene acetyltransferases (AAC), nucleotidyltransferases (ANT), phosphotransferases (APH) and 16S rRNA Methyltransferases genes armA, rmtA, rmtB, rmtC, rmtD, rmtE, rmtF, rmtG and rmtH genes. Conjugation experiment was used to identification the plasmid carrying the rmtG 16S RMTases gene. The sequencing of type: Sanger (Applied Biosystems®), MiSeq (Illumina®) and MinION (Nanopore®) were performed at the ECc for confirmation of resistance gene variants, plasmid incompatibility and genetic environment. Results: Our results showed a positivity of 63% for blaCTX-M-15, 29% blaCTX-M-2, 1% blaCTX-M-8, 6% blaSHV-5 and 1% blaSHV-12. The PMQR genes found were aac(6')-Ib-cr (46%) and qnrB (14%). Among the AME genes detected, the most prevalent were ant(2")-Ia 51%, aac(6')-Ib 45%, aac(3)-IIa 34%, aph(3')-Ia 21% and aph(3')-IV 6%. Regarding the 16S rRNA Metiltransferases genes, one isolate was positive for rmtD- 2 and two isolates for the rmtG gene. Sequencing of plasmids (pEc09 and pEc13) bearing 16S rRNA methyltransferases genes showed an association with genetic mobile elements such as ISCR2, ΔISCR2 and IS26, belonging to the incompatibility group IncA/C and transposon Tn3. Among carbapenemases, the blaKPC-2 gene has been detected in most strains except ECc 61 identified as Enterobacter hormaechei subsp. xiangfangensis carrying a new variant of BKC-2, the blaBKC-2 gene was detected an IncQ1 plasmid and lack of the ISKpn23. The mcr-9 gene belonging to IncFIB and genetic context with high similarity between two insertion sequences IS903B and IS26. Conclusion: The simultaneous detection and dissemination of several antimicrobial resistance genes in the same isolate of the Enterobacter cloacae complex, point out the impact that these genes transmitted by mobile genetic elements can cause in the hospital environment.