The PitA protein contributes to colistin susceptibility in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of problematic infections in individuals with predisposing conditions. Infections can be treated with colistin but some isolates are resistant to this antibiotic. To better understand the genetic basis of resistance, we experimentally evolved 19 independent resistant mutants from the susceptible laboratory strain PAO1. Whole genome sequencing identified mutations in multiple genes including phoQ and pmrB that have previously been associated with resistance, pitA that encodes a phosphate transporter, and carB and eno that encode enzymes of metabolism. Individual mutations were engineered into the genome of strain PAO1. Mutations in pitA, pmrB and phoQ increased the minimum inhibitory concentration (MIC) for colistin 8-fold, making the bacteria resistant. Engineered pitA/phoQ and pitA/pmrB double mutants had higher MICs than single mutants, demonstrating additive effects on colistin susceptibility. Single carB and eno mutations did not increase the MIC suggesting that their effect is dependent on the presence of other mutations. Many of the resistant mutants had increased susceptibility to ß-lactams and lower growth rates than the parental strain demonstrating that colistin resistance can impose a fitness cost. Two hundred and fourteen P. aeruginosa isolates from a range of sources were tested and 18 (7.8%) were colistin resistant. Sequence variants in genes identified by experimental evolution were present in the 18 resistant isolates and may contribute to resistance. Overall our results identify pitA mutations as novel contributors to colistin resistance and demonstrate that resistance can reduce fitness of the bacteria.

Mutations in the gdpP gene are a clinically relevant mechanism for ß-lactam resistance in meticillin-resistant Staphylococcus aureus lacking mec determinants.

In Staphylococcus aureus, resistance to ß-lactamase stable ß-lactam antibiotics is mediated by the penicillinbinding protein 2a, encoded by mecA or by its homologues mecB or mecC. However, a substantial number of meticillin-resistant isolates lack known mec genes and, thus, are called meticillin resistant lacking mec (MRLM). This study aims to identify the genetic mechanisms underlying the MRLM phenotype. A total of 141 MRLM isolates and 142 meticillin-susceptible controls were included in this study. Oxacillin and cefoxitin minimum inhibitory concentrations were determined by broth microdilution and the presence of mec genes was excluded by PCR. Comparative genomics and a genome-wide association study (GWAS) approach were applied to identify genetic polymorphisms associated with the MRLM phenotype. The potential impact of such mutations on the expression of PBP4, as well as on cell morphology and biofilm formation, was investigated. GWAS revealed that mutations in gdpP were significantly associated with the MRLM phenotype. GdpP is a phosphodiesterase enzyme involved in the degradation of the second messenger cyclic-di-AMP in S. aureus. A total of 131 MRLM isolates carried truncations, insertions or deletions as well as amino acid substitutions, mainly located in the functional DHH-domain of GdpP. We experimentally verified the contribution of these gdpP mutations to the MRLM phenotype by heterologous complementation experiments. The mutations in gdpP had no effect on transcription levels of pbp4; however, cell sizes of MRLM strains were reduced. The impact on biofilm formation was highly strain dependent. We report mutations in gdpP as a clinically relevant mechanism for ß-lactam resistance in MRLM isolates. This observation is of particular clinical relevance, since MRLM are easily misclassified as MSSA (meticillin-susceptible S. aureus), which may lead to unnoticed spread of ß-lactam-resistant isolates and subsequent treatment failure.