Objectives To describe the prevalence of colistin heteroresistance in carbapenem-resistant Pseudomonas aeruginosa (CRPA) and evaluate the association with clinical outcomes. Methods Colistin heteroresistance was evaluated in CRPA isolates collected from patients without cystic fibrosis in Atlanta, Georgia, USA using two definitions: HR1, growth at 4 and 8 mg/L of colistin at a frequency ≥1 × 10−6 the main population; and HR2, growth at a colistin concentration ≥8× the MIC of the main population at a frequency ≥1 × 10−7. A modified population analysis profile (mPAP) technique was compared with reference PAP for detecting heteroresistance. For adults hospitalized at the time of or within 1 week of CRPA culture, multivariable logistic regression estimated the association between heteroresistance and 90 day mortality. Results Of 143 colistin-susceptible CRPA isolates, 8 (6%) met the HR1 definition and 37 (26%) met the HR2 definition. Compared with the reference PAP, mPAP had a sensitivity and specificity of 50% and 100% for HR1 and 32% and 99% for HR2. Of 82 hospitalized patients, 45 (56%) were male and the median age was 63 years (IQR 49–73). Heteroresistance was not associated with 90 day mortality using HR1 (0% in heteroresistant versus 22% in non-heteroresistant group; P = 0.6) or HR2 (12% in heteroresistant versus 24% in non-heteroresistant group; P = 0.4; adjusted OR 0.8; 95% CI 0.2–3.4). Conclusions Colistin heteroresistance was identified in up to 26% of patients with CRPA in our sample, although the prevalence varied depending on the definition. We did not observe an apparent association between colistin heteroresistance and 90 day mortality.

Vancomycin-intermediate Staphylococcus aureus (VISA) typically arises through accumulation of chromosomal mutations that alter cell-wall thickness and global regulatory pathways. Genome-based prediction of VISA requires understanding whether strain background influences patterns of mutation that lead to resistance. We used an iterative method to experimentally evolve three important methicillin-resistant S. aureus (MRSA) strain backgrounds—(CC1, CC5 and CC8 (USA300)) to generate a library of 120 laboratory selected VISA isolates. At the endpoint, isolates had vancomycin MICs ranging from 4 to 10 μg/mL. We detected mutations in more than 150 genes, but only six genes (already known to be associated with VISA from prior studies) were mutated in all three background strains (walK, prs, rpoB, rpoC, vraS, yvqF). We found evidence of interactions between loci (e.g., vraS and yvqF mutants were significantly negatively correlated) and rpoB, rpoC, vraS and yvqF were more frequently mutated in one of the backgrounds. Increasing vancomycin resistance was correlated with lower maximal growth rates (a proxy for fitness) regardless of background. However, CC5 VISA isolates had higher MICs with fewer rounds of selection and had lower fitness costs than the CC8 VISA isolates. Using multivariable regression, we found that genes differed in their contribution to overall MIC depending on the background. Overall, these results demonstrated that VISA evolved through mutations in a similar set of loci in all backgrounds, but the effect of mutation in common genes differed with regard to fitness and contribution to resistance in different strains.

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Bacteremia mortality is known to increase rapidly with time after infection, making rapid diagnostics and treatment necessary. Successful treatment depends on correct administration of antibiotics based on knowledge of strain antibiotic susceptibility. Staphylococcus aureus is a major causative agent of bacteremia that is also commonly antibiotic resistant.

Staphylococcus aureus is a major cause of bacteremia and other hospital-acquired infections. The cell-wall active antibiotic vancomycin is commonly used to treat both methicillin-resistant (MRSA) and sensitive (MSSA) infections, but vancomycin intermediate S. aureus (VISA) variants can arise through de novo mutations. Here we performed pilot experiments to develop a combined PCR/long-read sequencing-based method for detection of previously known VISA-causing mutations. We amplified 16 genes (walR, walK, rpoB, graR, graS, vraF, vraG, stpI, vraR, vraS, agrA, sarA, clpP, ccpA, prsA, and yvqF) known from prior studies to be associated with mutations responsible for VISA as 10 amplicons and sequenced amplicon pools as long-reads with Oxford Nanopore adapter ligation on Flongle flow cells. We then detected mutations by mapping reads against a parental consensus or known reference sequence and comparing called variants against a database of known VISA mutations from laboratory selection. There was high (>1000x) coverage of each amplicon in the pool, no relationship between amplicon length and coverage, and the ability to detect the causative mutation (walK 646C>G) in a VISA mutant derived from the USA300 strain (N384-3 from parental strain N384). Mixing mutant (N384-3) and parental (N384) DNA at various ratios from 0 to 1 mutant suggested a mutation detection threshold of roughly the average minor allele frequency of 6.5% at 95% confidence (two standard errors above mean mutation frequency). The study lays the groundwork for direct S. aureus antibiotic phenotype inference using rapid nanopore sequencing from clinical samples.

We describe a facile strategy to identify sites for the incorporation of noncanonical amino acids into lysostaphin—an enzyme that degrades the cell wall of Staphylococcus aureus—while retaining stapholytic activity. We used this strategy to generate active variants of lysostaphin incorporating para‐azidophenylalanine. The incorporation of this “reactive handle” enabled the orthogonal site‐specific modification of the enzyme variants with polyethylene glycol (PEG) using copper‐free click cycloaddition. PEGylated lysostaphin variants could retain their stapholytic activity, with the extent of retention depending on the site of modification and the PEG molecular weight. The site‐specific modification of lysostaphin could be useful not only for PEGylation to improve biocompatibility but also for the incorporation of the enzyme into hydrogels and other biomaterials and for studies of protein structure and dynamics. Moreover, the approach described herein could be readily applied to identify suitable sites for the incorporation of reactive handles into other proteins of interest.