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ABSTRACTNext-generation sequencing (NGS) analysis has emerged as a promising molecular epidemiological method for investigating health care-associated outbreaks. Here, we used NGS to investigate a 3-year outbreak of multidrug-resistant Acinetobacter baumannii(MDRAB) at a large academic burn center. A reference genome from the index case was generated using de novoassembly of PacBio reads. Forty-six MDRAB isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and sequenced using an Illumina platform. After mapping to the index case reference genome, four samples were excluded due to low coverage, leaving 42 samples for further analysis. Multilocus sequence types (MLST) and the presence of acquired resistance genes were also determined from the sequencing data. A transmission network was inferred from genomic and epidemiological data using a Bayesian framework. Based on single-nucleotide variant (SNV) differences, this MDRAB outbreak represented three sequential outbreaks caused by distinct clones. The first and second outbreaks were caused by sequence type 2 (ST2), while the third outbreak was caused by ST79. For the second outbreak, the MLST and PFGE results were discordant. However, NGS-based SNV typing detected a recombination event and consequently enabled a more accurate phylogenetic analysis. The distribution of resistance genes varied among the three outbreaks. The first- and second-outbreak strains possessed a blaOXA-23-likegroup, while the third-outbreak strains harbored a blaOXA-40-likegroup. NGS-based analysis demonstrated the superior resolution of outbreak transmission networks for MDRAB and provided insight into the mechanisms of strain diversification between sequential outbreaks through recombination.

ABSTRACTDrug-resistant Plasmodium falciparummalaria is a major public health problem. An elevated pfmdr1gene copy number (CN) is known to decrease parasite sensitivity to the commonly used antimalarial mefloquine (MFQ). To understand the relationship between pfmdr1CN and mefloquine resistance, we evaluated pfmdr1transcript levels in three P. falciparumstrains with different CNs in the presence and absence of MFQ. Parasite strains with multiple pfmdr1gene copies exhibited higher relative transcript levels than single-copy parasites, and MFQ induced pfmdr1expression above the levels without treatment in all three strains evaluated. Concomitant morphology analyses of the sampled cultures revealed that MFQ treatment of synchronized ring-stage parasites induced a delay in parasite maturation through the intraerythrocytic cycle. pfmdr1expression peaks in the ring stage, and MFQ could be causing increased transcription by delaying parasite maturation. However, pretreatment with mefloquine did not affect the artemisinin in vitrohalf-maximal inhibitory concentration (IC50). These results suggest that MFQ-induced increases in pfmdr1expression are the direct result of the maturation delay at the ring stage but that this change in expression does not affect the antimalarial activity of artemisinin.

ABSTRACTEscherichia colisequence type 131 (ST131) predominates globally among multidrug-resistant (MDR) E. colistrains. We used whole-genome sequencing (WGS) to investigate 63 MDR E. coliisolates from 7 North Carolina community hospitals (2010 to 2015). Of these, 39 (62%) represented ST131, including 37 (95%) from the ST131-H30R subclone: 10 (27%) from its H30R1 subset and 27 (69%) from its H30Rx subset. ST131 core genomes differed by a median of 15 (range, 0 to 490) single-nucleotide variants (SNVs) overall versus only 7 within H30R1 (range, 3 to 12 SNVs) and 11 within H30Rx (range, 0 to 21). The four isolates with identical core genomes were all H30Rx. Epidemiological and clinical characteristics did not vary significantly by strain type, but many patients with MDR E. colior H30Rx infection were critically ill and had poor outcomes. H30Rx isolates characteristically exhibited fluoroquinolone resistance and CTX-M-15 production, had a high prevalence of trimethoprim-sulfamethoxazole resistance (89%), sul1(89%), and dfrA17(85%), and were enriched for specific virulence traits, and all qualified as extraintestinal pathogenic E. coli. The high overall prevalence of CTX-M-15 appeared to be possibly attributable to its association with the ST131-H30Rx subclone and IncF[F2:A1:B−] plasmids. Some phylogenetically clustered non-ST131 MDR E. coliisolates also had distinctive serotypes/fimHtypes, fluoroquinolone mutations, CTX-M variants, and IncF types. Thus, WGS analysis of our community hospital source MDR E. coliisolates suggested ongoing circulation and differentiation of E. coliST131 subclones, with clonal segregation of CTX-M variants, other resistance genes, Inc-type plasmids, and virulence genes.

ABSTRACTKlebsiella pneumoniaecarbapenemase (KPC)-producing Enterobacter cloacaehas been recently recognized in the United States. Whole-genome sequencing (WGS) has become a useful tool for analysis of outbreaks and for determining transmission networks of multidrug-resistant organisms in health care settings, including carbapenem-resistant Enterobacteriaceae(CRE). We experienced a prolonged outbreak of CRE E. cloacaeand K. pneumoniaeover a 3-year period at a large academic burn center despite rigorous infection control measures. To understand the molecular mechanisms that sustained this outbreak, we investigated the CRE outbreak isolates by using WGS. Twenty-two clinical isolates of CRE, including E. cloacae(n= 15) and K. pneumoniae(n= 7), were sequenced and analyzed genetically. WGS revealed that this outbreak, which seemed epidemiologically unlinked, was in fact genetically linked over a prolonged period. Multiple mechanisms were found to account for the ongoing outbreak of KPC-3-producing E. cloacaeand K. pneumoniae. This outbreak was primarily maintained by a clonal expansion of E. cloacaesequence type 114 (ST114) with distribution of multiple resistance determinants. Plasmid and transposon analyses suggested that the majority of blaKPC-3was transmitted via an identical Tn4401b element on part of a common plasmid. WGS analysis demonstrated complex transmission dynamics within the burn center at levels of the strain and/or plasmid in association with a transposon, highlighting the versatility of KPC-producing Enterobacteriaceaein their ability to utilize multiple modes to resistance gene propagation.