Wastewater (WW) is considered a source of antibiotic-resistant bacteria with clinical relevance and may, thus, be important for their dissemination into the environment, especially in countries with poor WW treatment. To obtain an overview of the occurrence and characteristics of carbapenem-resistant Gram-negative bacteria (CR-GNB) in WW of Belgrade, we investigated samples from the four main sewer outlets prior to effluent into international rivers, the Sava and the Danube. Thirty-four CR-GNB isolates were selected for antimicrobial susceptibility testing (AST) and whole-genome sequencing (WGS). AST revealed that all isolates were multidrug-resistant. WGS showed that they belonged to eight different species and 25 different sequence types (STs), seven of which were new. ST101 K. pneumoniae (bla_CTX-M-15/bla_OXA-48) with novel plasmid p101_srb was the most frequent isolate, detected at nearly all the sampling sites. The most frequent resistance genes to aminoglycosides, quinolones, trimethroprim-sulfamethoxazole, tetracycline and fosfomycin were aac(6′)-Ib-cr (55.9%), oqxA (32.3%), dfrA14 (47.1%), sul1 (52.9%), tet(A) (23.5%) and fosA (50%), respectively. Acquired resistance to colistin via chromosomal-mediated mechanisms was detected in K. pneumoniae (mutations in mgrB and basRS) and P. aeruginosa (mutation in basRS), while a plasmid-mediated mechanism was confirmed in the E. cloacae complex (mcr-9.1 gene). The highest number of virulence genes (>300) was recorded in P. aeruginosa isolates. Further research is needed to systematically track the occurrence and distribution of these bacteria so as to mitigate their threat.

For bacterial genome sequencing, libraries from different strains are usually multiplexed in a single run. Normalized libraries are most often pooled in equal volumes, as recommended by next-generation sequencing platform manufacturers. This equal-volume strategy is well suited for multiplexing isolates from the same species. However, for runs involving multiple microbial species, an equimolar library pooling is more adapted because of the variation in bacterial genome size. To demonstrate its utility in clinical microbiology, we compared both equal-volume and equimolar strategies using a menu comprising 13 bacterial species involved in healthcare-associated infections. We show that equimolar pooling limits the retesting risk due to insufficient coverage depth, particularly when interspecies genome size difference is more than 2-fold. The use of this alternative strategy for multiplexing pathogenic bacteria should lead to more cost effective whole-genome sequencing applications in clinical microbiology.