Harvesting and amplifying gene cassettes confers cross-resistance to critically important antibiotics.

Amikacin and piperacillin/tazobactam are frequent antibiotic choices to treat bloodstream infection, which is commonly fatal and most often caused by bacteria from the family Enterobacterales. Here we show that two gene cassettes located side-by-side in and ancestral integron similar to In37 have been "harvested" by insertion sequence IS26 as a transposon that is widely disseminated among the Enterobacterales. This transposon encodes the enzymes AAC(6')-Ib-cr and OXA-1, reported, respectively, as amikacin and piperacillin/tazobactam resistance mechanisms. However, by studying bloodstream infection isolates from 769 patients from three hospitals serving a population of 1.2 million people in South West England, we show that increased enzyme production due to mutation in an IS26/In37-derived hybrid promoter or, more commonly, increased transposon copy number is required to simultaneously remove these two key therapeutic options; in many cases leaving only the last-resort antibiotic, meropenem. These findings may help improve the accuracy of predicting piperacillin/tazobactam treatment failure, allowing stratification of patients to receive meropenem or piperacillin/tazobactam, which may improve outcome and slow the emergence of meropenem resistance.

Improving nitrofurantoin resistance prediction in Escherichia coli from whole-genome sequence by integrating NfsA/B enzyme assays.

Nitrofurantoin resistance in Escherichia coli is primarily caused by mutations damaging two enzymes, NfsA and NfsB. Studies based on small isolate collections with defined nitrofurantoin MICs have found significant random genetic drift in nfsA and nfsB, making it extremely difficult to predict nitrofurantoin resistance from whole-genome sequence (WGS) where both genes are not obviously disrupted by nonsense or frameshift mutations or insertional inactivation. Here, we report a WGS survey of 200 oqxAB-negative E. coli from community urine samples, of which 34 were nitrofurantoin resistant. We characterized individual non-synonymous mutations seen in nfsA and nfsB among this collection using complementation cloning and NfsA/B enzyme assays in cell extracts. We definitively identified R203C, H11Y, W212R, A112E, and A112T in NfsA and R121C, Q142H, F84S, P163H, W46R, K57E, and V191G in NfsB as amino acid substitutions that reduce enzyme activity sufficiently to cause resistance. In contrast, E58D, I117T, K141E, L157F, A172S, G187D, and A188V in NfsA and G66D, M75I, V93A, and A174E in NfsB are functionally silent in this context. We identified that 9/166 (5.4%) nitrofurantoin-susceptible isolates were "pre-resistant," defined as having loss of function mutations in nfsA or nfsB. Finally, using NfsA/B enzyme assays and proteomics, we demonstrated that 9/34 (26.5%) ribE wild-type nitrofurantoin-resistant isolates also carried functionally wild-type nfsB or nfsB/nfsA. In these cases, NfsA/B activity was reduced through downregulated gene expression. Our biological understanding of nitrofurantoin resistance is greatly improved by this analysis but is still insufficient to allow its reliable prediction from WGS data.

Hound: a novel tool for automated mapping of genotype to phenotype in bacterial genomes assembled de novo.

Increasing evidence suggests that microbial species have a strong within species genetic heterogeneity. This can be problematic for the analysis of prokaryote genomes, which commonly relies on a reference genome to guide the assembly process. Differences between reference and sample genomes will therefore introduce errors in final assembly, jeopardizing the detection from structural variations to point mutations-critical for genomic surveillance of antibiotic resistance. Here we present Hound, a pipeline that integrates publicly available tools to assemble prokaryote genomes de novo, detect user-given genes by similarity to report mutations found in the coding sequence, promoter, as well as relative gene copy number within the assembly. Importantly, Hound can use the query sequence as a guide to merge contigs, and reconstruct genes that were fragmented by the assembler. To showcase Hound, we screened through 5032 bacterial whole-genome sequences isolated from farmed animals and human infections, using the amino acid sequence encoded by blaTEM-1, to detect and predict resistance to amoxicillin/clavulanate which is driven by over-expression of this gene. We believe this tool can facilitate the analysis of prokaryote species that currently lack a reference genome, and can be scaled either up to build automated systems for genomic surveillance or down to integrate into antibiotic susceptibility point-of-care diagnostics.

Klebsiella pneumoniae Mutants Resistant to Ceftazidime-Avibactam Plus Aztreonam, Imipenem-Relebactam, Meropenem-Vaborbactam, and Cefepime-Taniborbactam.

We show that a previously described Klebsiella pneumoniae variant that is resistant to ceftazidime-avibactam plus meropenem-vaborbactam, has a ramR plus ompK36 mutation, and produces the V239G variant KPC-3 (V240G per the standard numbering system) exhibits resistance to ceftazidime-avibactam plus aztreonam and imipenem-relebactam but not cefepime-taniborbactam. The V239G variant does not generate collateral ß-lactam susceptibility like many KPC-3 variants associated with ceftazidime-avibactam resistance. Additional mutation of ompK35 and production of the OXA-48-like carbapenemase OXA-232 were required to confer cefepime-taniborbactam resistance.

OmpF Downregulation Mediated by Sigma E or OmpR Activation Confers Cefalexin Resistance in Escherichia coli in the Absence of Acquired ß-Lactamases.

Cefalexin is a widely used first-generation cephalosporin, and resistance in Escherichia coli is caused by extended-spectrum (e.g., CTX-M) and AmpC ß-lactamase production and therefore frequently coincides with third-generation cephalosporin resistance. However, we have recently identified large numbers of E. coli isolates from human infections, and from cattle, where cefalexin resistance is not ß-lactamase mediated. Here, we show, by studying laboratory-selected mutants, clinical isolates, and isolates from cattle, that OmpF porin disruption or downregulation is a major cause of cefalexin resistance in E. coli. Importantly, we identify multiple regulatory mutations that cause OmpF downregulation. In addition to mutation of ompR, already known to downregulate OmpF and OmpC porin production, we find that rseA mutation, which strongly activates the sigma E regulon, greatly increases DegP production, which degrades OmpF, OmpC, and OmpA. Furthermore, we reveal that mutations affecting lipopolysaccharide structure, exemplified by the loss of GmhB, essential for lipopolysaccharide heptosylation, also modestly activate DegP production, resulting in OmpF degradation. Remarkably, given the critical importance attached to such systems for normal E. coli physiology, we find evidence for DegP-mediated OmpF downregulation and gmhB and rseA loss-of-function mutation in E. coli isolates derived from human infections. Finally, we show that these regulatory mutations enhance the ability of group 1 CTX-M ß-lactamase to confer reduced carbapenem susceptibility, particularly those mutations that cause OmpC in addition to OmpF downregulation.

Over-Expression of Hypochlorite Inducible Major Facilitator Superfamily (MFS) Pumps Reduces Antimicrobial Drug Susceptibility by Increasing the Production of MexXY Mediated by ArmZ in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a well-known cause of nosocomial infection, is frequently antibiotic resistant and this complicates treatment. Links between oxidative stress responses inducing antibiotic resistance through over-production of RND-type efflux pumps have been reported in P. aeruginosa, but this has not previously been associated with MFS-type efflux pumps. Two MFS efflux pumps encoded by mfs1 and mfs2 were selected for study because they were found to be sodium hypochlorite (NaOCl) inducible. Antibiotic susceptibility testing was used to define the importance of these MFS pumps in antibiotic resistance and proteomics was used to characterize the resistance mechanisms involved. The results revealed that mfs1 is NaOCl inducible whereas mfs2 is NaOCl, N-Ethylmaleimide and t-butyl hydroperoxide inducible. Deletion of mfs1 or mfs2 did not affect antibiotic or paraquat susceptibility. However, over-production of Mfs1 and Mfs2 reduced susceptibility to aminoglycosides, quinolones, and paraquat. Proteomics, gene expression analysis and targeted mutagenesis showed that over-production of the MexXY RND-type efflux pump in a manner dependent upon armZ, but not amgRS, is the cause of reduced antibiotic susceptibility upon over-production of Mfs1 and Mfs2. mexXY operon expression analysis in strains carrying various lengths of mfs1 and mfs2 revealed that at least three transmembrane domains are necessary for mexXY over-expression and decreased antibiotic susceptibility. Over-expression of the MFS-type efflux pump gene tetA(C) did not give the same effect. Changes in paraquat susceptibility were independent of mexXY and armZ suggesting that it is a substrate of Mfs1 and Mfs2. Altogether, this is the first evidence of cascade effects where the over-production of an MFS pump causes over-production of an RND pump, in this case MexXY via increased armZ expression.