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ABSTRACTOXA-48-like enzymes have emerged as important extended-spectrum β-lactamases/carbapenemases in Escherichia colisequence type 131 (ST131). We report the structures of the first fully sequenced blaOXA-163plasmid and of two other blaOXA-48plasmids in this lineage. blaOXA-163was located on a 71-kb IncN plasmid with other resistance genes. blaOXA-48was present on IncL/M plasmids, genetically similar to other blaOXA-48plasmid sequences, and consistent with interspecies/interlineage spread. The presence of blaOXA-48-likegenes on epidemic plasmids in ST131 is of concern.

ABSTRACTThe blaIMP-14carbapenem resistance gene has largely previously been observed in Pseudomonas aeruginosaand Acinetobacterspp. As part of global surveillance and sequencing of carbapenem-resistant Escherichia coli, we identified a sequence type 131 strain harboring blaIMP-14within a class 1 integron, itself nested within an ∼54-kb multidrug resistance region on an epidemic IncA/C2plasmid. The emergence of blaIMP-14in this context in the ST131 lineage is of potential clinical concern.

ABSTRACTThe recent widespread emergence of carbapenem resistance in Enterobacteriaceaeis a major public health concern, as carbapenems are a therapy of last resort against this family of common bacterial pathogens. Resistance genes can mobilize via various mechanisms, including conjugation and transposition; however, the importance of this mobility in short-term evolution, such as within nosocomial outbreaks, is unknown. Using a combination of short- and long-read whole-genome sequencing of 281 blaKPC-positive Enterobacteriaceaeisolates from a single hospital over 5 years, we demonstrate rapid dissemination of this carbapenem resistance gene to multiple species, strains, and plasmids. Mobility of blaKPCoccurs at multiple nested genetic levels, with transmission of blaKPCstrains between individuals, frequent transfer of blaKPCplasmids between strains/species, and frequent transposition of blaKPCtransposon Tn4401between plasmids. We also identify a common insertion site for Tn4401within various Tn2-like elements, suggesting that homologous recombination between Tn2-like elements has enhanced the spread of Tn4401between different plasmid vectors. Furthermore, while short-read sequencing has known limitations for plasmid assembly, various studies have attempted to overcome this by the use of reference-based methods. We also demonstrate that, as a consequence of the genetic mobility observed in this study, plasmid structures can be extremely dynamic, and therefore these reference-based methods, as well as traditional partial typing methods, can produce very misleading conclusions. Overall, our findings demonstrate that nonclonal resistance gene dissemination can be extremely rapid, presenting significant challenges for public health surveillance and achieving effective control of antibiotic resistance.

Carbapenem resistance in Enterobacteralesis a public health threat. Klebsiella pneumoniaecarbapenemase (encoded by alleles of the blaKPCfamily) is one of the most common transmissible carbapenem resistance mechanisms worldwide. The dissemination of blaKPChistorically has been associated with distinct K. pneumoniaelineages (clonal group 258 [CG258]), a particular plasmid family (pKpQIL), and a composite transposon (Tn4401).

The UKMYC5 plate is a 96-well microtiter plate designed by the CRyPTIC Consortium (Comprehensive Resistance Prediction for Tuberculosis: an International Consortium) to enable the measurement of MICs of 14 different antituberculosis (anti-TB) compounds for >30,000 clinical Mycobacterium tuberculosisisolates. Unlike the MYCOTB plate, on which the UKMYC5 plate is based, the UKMYC5 plate includes two new (bedaquiline and delamanid) and two repurposed (clofazimine and linezolid) compounds.

ABSTRACTThe Klebsiella pneumoniaecarbapenemase gene (blaKPC) is typically located within mobile transposon Tn4401. Enhanced KPC expression has been associated with deletions in the putative promoter region upstream of blaKPC. Illumina sequences from blaKPC-positive clinical isolates from a single institution were mapped to a Tn4401b reference sequence, which carries no deletions. The novel isoform Tn4401h (188-bp deletion [between istBand blaKPC]) was present in 14% (39/281) of clinical isolates. MICs showed that Escherichia colistrains containing plasmids with Tn4401a and Tn4401h were more resistant to meropenem (≥16 and ≥16, respectively), ertapenem (≥8 and 4, respectively), and cefepime (≥64 and 4, respectively) than E. colistrains with Tn4401b (0.5, ≤0.5, and ≤1, respectively). Quantitative real-time PCR (qRT-PCR) demonstrated that Tn4401a had a 16-fold increase and Tn4401h a 4-fold increase in blaKPCmRNA levels compared to the reference Tn4401b. A lacZreporter plasmid was used to test the activity of the promoter regions from the different variants, and the results showed that the Tn4401a and Tn4401h promoter sequences generated higher β-galactosidase activity than the corresponding Tn4401b sequence. Further dissection of the promoter region demonstrated that putative promoter P1 was not functional. The activity of the isolated P2 promoter was greatly enhanced by inclusion of the P1-P2 intervening sequence. These studies indicated that gene expression could be an important consideration in understanding resistance phenotypes predicted by genetic signatures in the context of sequencing-based rapid diagnostics.

ABSTRACTCarbapenemase genes in Enterobacteriaceaeare mostly described as being plasmid associated. However, the genetic context of carbapenemase genes is not always confirmed in epidemiological surveys, and the frequency of their chromosomal integration therefore is unknown. A previously sequenced collection of blaKPC-positive Enterobacteriaceaefrom a single U.S. institution (2007 to 2012; n= 281 isolates from 182 patients) was analyzed to identify chromosomal insertions of Tn4401, the transposon most frequently harboring blaKPC. Using a combination of short- and long-read sequencing, we confirmed five independent chromosomal integration events from 6/182 (3%) patients, corresponding to 15/281 (5%) isolates. Three patients had isolates identified by perirectal screening, and three had infections which were all successfully treated. When a single copy of blaKPCwas in the chromosome, one or both of the phenotypic carbapenemase tests were negative. All chromosomally integrated blaKPCgenes were from Klebsiellaspp., predominantly K. pneumoniaeclonal group 258 (CG258), even though these represented only a small proportion of the isolates. Integration occurred via IS15-ΔI-mediated transposition of a larger, composite region encompassing Tn4401at one locus of chromosomal integration, seen in the same strain (K. pneumoniaeST340) in two patients. In summary, we identified five independent chromosomal integrations of blaKPCin a large outbreak, demonstrating that this is not a rare event. blaKPCwas more frequently integrated into the chromosome of epidemic CG258 K. pneumoniaelineages (ST11, ST258, and ST340) and was more difficult to detect by routine phenotypic methods in this context. The presence of chromosomally integrated blaKPCwithin successful, globally disseminated K. pneumoniaestrains therefore is likely underestimated.

ABSTRACTIn this study, using the Hain GenoType MTBDRslassays (versions 1 and 2), we found that some nonsynonymous and synonymous mutations in gyrAin Mycobacterium tuberculosisresult in systematic false-resistance results to fluoroquinolones by preventing the binding of wild-type probes. Moreover, such mutations can prevent the binding of mutant probes designed for the identification of specific resistance mutations. Although these mutations are likely rare globally, they occur in approximately 7% of multidrug-resistant tuberculosis strains in some settings.

ABSTRACTCharacterization of the sequences involved in recombination of the Haemophilusplasmid p1056 with the Haemophilus influenzaechromosome produced evidence indicating site-specific recombination with chromosomal tRNALeu. attPsequences identical to those of p1056 were found in six plasmids of diverse origin, suggesting that a family of Haemophilusplasmids recombines with chromosomal tRNALeu.