Drug resistant eskapee pathogens in clinical isolates and hospital effluent.

Abstract

Background: The ESKAPEE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp, and Escherichia coli) is a group of Gram-negative and -positive pathogens that exhibit antimicrobial resistance (AMR) to commonly used antibiotics. Aim: This study compared clinical ESKAPEE isolates from patients and hospital effluent in terms of antibiotic resistance patterns, antibiotic resistance genes, mobile genetic elements (MGEs) and phylogenomic relationships. Methodology: Samples were collected and pooled from the final effluent point of a regional hospital in the uMgungundlovu district, Kwa-Zulu Natal, South Africa. Clinical isolates were also collected from the same hospital. Selective culture media was used for isolation and identification. Antimicrobial susceptibility testing (AST) was performed using the VITEK® 2 system. DNA was extracted using the GenElute extraction kits prior to whole genome sequencing. The resistome, mobilome and phylogenetic lineages of sequenced isolates were assessed using bioinformatics analysis. ResFinder, PlasmidFinder, INTEGRALL and PGAP & ISFinder were used to annotate and identify resistance genes, plasmids, integrons and insertion sequences and transposons, respectively. MLST was used to identify sequence types, BV-BRC was used to construct the phylogenetic trees, and iTOL was used to view, edit and annotate the generated phylogenetic trees. Results: A total of 112 presumptive ESKAPEE constituted the sample of which 42 were clinical isolates and 70 were isolates from hospital effluent. Of these, 36 isolates consisting of 16 K. pneumoniae, 9 E. faecium, 7 Enterobacter hormaechei, 3 E. coli and 1 P. aeruginosa were positively identified as ESKAPEE pathogens by WGS. The effluent E. faecium isolates were totally resistant to six of the antibiotics tested (tetracycline, doxycycline, erythromycin, azithromycin, fosfomycin and levofloxacin). They also harboured antibiotic resistance genes (ARGs) that confer resistance to aminoglycosides (aac(6')-Ii, aac(6')-aph(2''), aph(2'')-Ia), macrolides (msr(C)), tetracycline (tet(M), tet(L)), and trimethoprim (dfrG), none of which were carried on any MGEs. In the K. pneumoniae isolates, ARGs conferring resistance to ß-lactam antibiotics were the most common among the clinical isolates while effluent K. pneumoniae carried markedly fewer ARGs. Aminoglycoside resistance genes (aph(6)-Id, aph(3'')-Ib, aac(6')-Ib-cr aadA16), quinolone (qnrB1, qnrB6, qnrS1, OqxA and oqxB), ß-lactam (blaSHV group, blaCTX-M-15, and blaTEM group) and trimethoprim-sulfamethoxazole (sul1, sul2, and dfrA27, drfA14) were common among the clinical K. pneumoniae isolates. ARGs were associated with diverse MGEs, particularly in the clinical isolates and a common plasmid replicon IncFIB(K) was identified in both clinical and effluent isolates. MLST showed no relation between the K. pneumoniae clinical and effluent isolates. The clinical P. aeruginosa isolate harboured ß-lactam [blaOXA-50, blaPAO], aminoglycoside (aph(3')-Iib), and fluoroquinolone (crpP) ARGs that were not associated with MGEs. The isolate had a sequence type ST275, which showed no relation with other isolates it was compared with. The majority of clinical E. hormaechei isolates showed total resistance to aminoglycosides, ß-lactams and tetracycline antibiotics. The E. hormaechei isolates, except one, harboured [blaOXA-1, blaCTX-M-15, blaACT-5 and blaTEM-1B], dfrA14, tet(A), [aph(6)-Id, aph(3'')-Ib, aac(3)-Iia, aac(6')-Ib-cr] and sul2 ARGs conferring resistance to ß-lactams, trimethoprim, tetracycline, aminoglycoside, and sulfamethoxazole, respectively. Clinical and effluent isolates of E. coli displayed similar resistance patterns. ARGs conferring resistance to ß-lactam (blaCTXM-15 and blaOXA-1), aminoglycosides (aph(6)-Id, aph(3'')-Ib, aac(6')-Ib-cr, aadA5 and aac(3)-IId), trimethoprim-sulfamethoxazole (dfrA14, dfrA17, sul1 and sul2), and tetracycline (tet(A) and tet(B)) were observed among the clinical isolates; while the effluent isolate harboured ß-lactam (blaCTXM-15, blaOXA-1, blaTEM-1B and blaOXA-10), aminoglycosides (aac(3)-Iid, aac(3)-IIa, aac(6')-Ib-cr, aph(6)-Id, aph(3'')-Ib), trimethoprim-sulfamethoxazole (dfrA14, dfrA23, sul1 and sul2), and tetracycline (tet(A)). The clinical E. coli isolates had sequence types ST69 and ST131 and the effluent isolate belonged to ST10. The clinical and effluent isolates from this study that did not cluster together were not closely related and belonged to different sequence types. Conclusion: These findings demonstrate the prevalence of ESKAPEE pathogens in hospital effluent. While the effluent did not mirror AMR in the clinical setting, presence of antibiotic-resistant bacteria (ARB) in the effluent cannot be overlooked. This study highlights the need for continuous monitoring of the effluent to track the spread of resistant bacteria from the hospital to the environment.