Activity of Telavancin against S. aureus isolated from cystic fibrosis patients including those with decreased susceptibility to Ceftaroline

Methicillin-resistant Staphylococcus aureus (MRSA) acquisition in cystic fibrosis (CF) patients confers a worse clinical outcome with increased rate of declined lung function. Telavancin, an approved lipoglycopeptide used to treat infections due to S. aureus has a dual mode of action causing inhibition of the peptidoglycan synthesis and membrane depolarization. CF-associated MRSA infections remain an important problem with no foreseeable decline in prevalence rates. Although telavancin is currently in clinical use for complicated skin infections and hospital-acquired pneumonia, the activity against CF-associated S. aureus infections has not been investigated. In this work, we studied the activity of telavancin against CF S. aureus strains collected from diverse geographical CF centers in the USA. We found that telavancin-MIC90 was 0.06 μg/ml, 8-fold lower than ceftaroline or daptomycin and 25-fold lower than linezolid and vancomycin. We demonstrate that telavancin at serum-free concentrations has rapid bactericidal activity with a decrease of more than 3 log10 CFU/ml during the first 4 to 6 hours of treatment, performing better in this assay than vancomycin and ceftaroline, including S. aureus resistant to ceftaroline. Telavancin resistance was infrequent (0.3%), although we found that it can occur in-vitro in both CF-and non-CF S. aureus strains by progressive passages with sub-inhibitory concentrations. Genetic analysis of telavancin in-vitro mutants showed gene polymorphisms in cell wall and virulence genes, and increased survival in a Galleria mellonella infection model. Thus, we conclude that telavancin represents a promising therapeutic option for CF infections with potent in-vitro activity and low resistance potential.

fibrosis. These strains are well known to have altered metabolism and possess multi-drug 77 resistance due to their chronic habitation and prolonged, repeated exposures to antibiotic 78 treatments in the CF lung environment (15). 79 We hypothesized that TLV may represent a valid option for the treatment of CF-MRSA and 80 MSSA infections. Therefore, the purpose of this study was to characterize by in-vitro and in-vivo 81 approaches the antimicrobial activity of TLV in S. aureus CF chronic infections strains, 82 particularly, infections by MRSA, strains isolated from diverse CF centers in the United States. 83 Lastly, we aimed to understand TLV resistance selection within a S. aureus population derived 84 from a CF-background.  ATCC 25923 TLV-R strains, containing 1.5 x 10 6 CFU/ml, as previously described (21). Inoculum 111 was administered directly to the larval hemocoel through the last left pro-leg as previously 112 described (17, 22). Every trial included a group of 10 untreated larvae as uninfected control group 113 and 10 larvae injected with PBS as a method control. Experiments were performed in at least three 114 independent trials. Injected insects were monitored over seven days at 37°C. By the day seven, 115 pupa formation was recorded in survived larvae.

164
Together these data support the potential therapeutic application of TLV for the treatment of S. 165 aureus CF infections.  Table 2). The enhanced MICs of the mutants were stable and unaltered after 174 ten passages in the absence of TLV. As shown in Table 2, there was 3 to 4 fold increase in VAN 175 MIC going from 1.5 to 6 µg/ml and an 8 to 10 fold increase in DAP MICs suggesting potential  (Table 3). Moreover, additional SNPs were found in the in-vivo derived strain MRSA MT-  (Table 3). These results may suggest that decrease 198 susceptibility to TLV is mainly associated to changes in cell wall related genes.  In absence of suitable alternative, TLV has been already used in some CF patients with 246 successful outcomes (24), supporting its potential role in the management CF-MRSA infections. 247 We are unaware of the development of TLV resistance in clinical settings. While direct resistance may be infrequent, modest increases in MICs may be seen in some isolates as the one 249 described here (TLV MIC 0.19 g/ml) and in some strains with VAN and DAP decreased 250 susceptibility. In this context, it was advantageous to gain an understanding of the ease of TLV 251 resistance when CF strains are extensively exposed to sub-therapeutic TLV in-vitro exposure.

252
After 15 days, the strains showed an increase in TLV MIC from 0.06 g/ml up to 0.25 -1 g/ml, 253 which is above the susceptible breakpoint of 0.12 µg/mL for S. aureus, followed by a 254 progressive increase up to 3 µg/ml after 40 days of exposure. These observations may imply that 255 resistance should be monitored in patient receiving repeated and/or prolonged treatment by TLV.

256
In a previous study performed by (25) one stable mutant was obtained after 43 days in multistep 257 resistance selection studies from one of the ten MRSA strains, correlating with low mutation 258 frequency. Interestingly in our study, we were able to demonstrate that the TLV resistance 259 selection was independent of the CF -S. aureus background strains considering the fact that we 260 were able to obtain a TLV-R mutant from an ATCC 25923. We determined next the main 261 genetic changes associated to TLV-R with full genome sequencing of a representative number of 262 in-vitro obtained TLV-R strains along with the in-vivo derived CF-TLV-R that showed a modest 263 increase of MIC (0.19g/ml). We found that TLV-R harbored common mutations in genes 264 associated to cell wall (e.g. murE, tagB) and cell wall surface (spa, clfB, sdrE) genes. In the TLV 265 obtained in previous studies by Song Y et al (26)most of the differential expressed genes were 266 also associated to changes in cell envelope. These findings suggest that although TLV is an agent 267 with dual mechanism (cell membrane/cell wall) the compensatory preferential mutational 268 mechanism seems to be in higher degree linked to cell wall (CW). These cell wall mutations 269 may also function in a dual manner to reduce TLVs cell membrane mechanism potency. This is 270 evidenced by prior studies correlating mutations and reduced VAN susceptibility as a result of VAN treatment (VISA strains) with collateral reduced DAP activity. Similarly, in our study the 272 derived TLV-R mutants showed cross-resistance with reduced susceptibility to both VAN (MIC 273 3-6 µg/ml) and DAP (MIC 6-8 µg/ml).

274
Another set of genes in were we found non-synonymous SNPs were related to virulence (e.g. sdrE, 275 spa, clfB). These changes appeared to suggest that TLV-R affects S. aureus virulence fitness as