Targeting of PBP1 by β-lactams determines recA/SOS response activation in heterogeneous MRSA clinical strains.

The SOS response, a conserved regulatory network in bacteria that is induced in response to DNA damage, has been shown to be associated with the emergence of resistance to antibiotics. Previously, we demonstrated that heterogeneous (HeR) MRSA strains, when exposed to sub-inhibitory concentrations of oxacillin, were able to express a homogeneous high level of resistance (HoR). Moreover, we showed that oxacillin appeared to be the triggering factor of a β-lactam-mediated SOS response through lexA/recA regulators, responsible for an increased mutation rate and selection of a HoR derivative. In this work, we demonstrated, by selectively exposing to β-lactam and non-β-lactam cell wall inhibitors, that PBP1 plays a critical role in SOS-mediated recA activation and HeR-HoR selection. Functional analysis of PBP1 using an inducible PBP1-specific antisense construct showed that PBP1 depletion abolished both β-lactam-induced recA expression/activation and increased mutation rates during HeR/HoR selection. Furthermore, based on the observation that HeR/HoR selection is accompanied by compensatory increases in the expression of PBP1,-2, -2a, and -4, our study provides evidence that a combination of agents simultaneously targeting PBP1 and either PBP2 or PBP2a showed both in-vitro and in-vivo efficacy, thereby representing a therapeutic option for the treatment of highly resistant HoR-MRSA strains. The information gathered from these studies contributes to our understanding of β-lactam-mediated HeR/HoR selection and provides new insights, based on β-lactam synergistic combinations, that mitigate drug resistance for the treatment of MRSA infections

Minimal inhibitory concentration (MIC) of SA13011-HeR <i>recA</i>-lacZ (LMR21) to antimicrobial agents used in the study.

<p>Minimal inhibitory concentration (MIC) of SA13011-HeR <i>recA</i>-lacZ (LMR21) to antimicrobial agents used in the study.</p

Treatment effect using <i>Galleria mellonella</i>.

<p>Groups of larvae (10/group) were inoculated with either 10 µl of PBS (uninfected control group; A) or bacterial suspension containing 1.5×10⁶ CFU/ml of 13011-HoR (B–D) into the last proleg and incubated for 2 h at 37C. After this, 10 µl of IMP (10 mg/kg), NAF (5 mg/kg; B), CTX (10 mg/kg; C), BAL (5 mg/kg; D), or the corresponding IMP/β-lactam were administered (time 0) into the right hind-most proleg, and re-incubated for 24 h at 37°C. PBS treatment was given as a control with multiple injections. The treatment was repeated after the first 24 h incubation. Worms were checked daily and recorded for any deaths for a total of 10 days. A minimum of three independent experimental runs were performed for each experiment. Survival data were plotted using the Kaplan-Meir method.</p

Time-course analysis of growth (A) and rate of DNA replication (B) was performed in SA13011-HeR −/+ OXA (0.5 µg/ml) during β-lactam-induced HeR/HoR selection.

<p>Bacteria were grown −/+ OXA (0.5 µg/ml) overnight. Cultures without OXA (HeR) were then diluted to an optical density at 600 nm (OD₆₀₀) equivalent to the culture growing with OXA (HoR) and continued to grow at 37°C with shaking. (B) Both SA13011-HeR/-HoR cultures were supplemented with the radioactive methyl-[3H]-thymine precursor (1 µCi/ml); triplicates samples were removed at different time-intervals and collected for DNA extraction, eluted and transferred to Whatman paper filters; after drying, radioactivity was measured by liquid scintillation. Values represent the means of three biological replicates ± standard error of the mean (SEM), sampled in triplicate to minimize error by inter- and intra-samples, respectively.</p

Figure 2

<p>(A) Kinetic of growth of the LMR23 (<i>pbp1</i>-antisense) in the presence and absence of IPTG (1 mM). IPTG was added to the cultures and cells were collected at the indicated time intervals for OD measurement. LMR25 (empty-vector) was used as a control. (B) Quantitation of <i>pbp</i>1, <i>recA</i>, and <i>umuC</i> mRNAs by real-time RT-PCR. RNAs were extracted from SA13011-HeR −/+ OXA, and LMR23+IPTG, −/+ OXA (LMR24), collected at the exponential phase of growth, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061083#s2" target="_blank">Materials and Methods</a>. Relative fold change values of specific mRNAs are shown on the vertical axis; 16S rRNA was used as an internal control. Values represent the means of at least three biological replicates ± standard error of the mean (SEM), sampled in triplicate to minimize error by inter- and intra-samples, respectively. *: indicates significantly higher than SA13011-HeR, <i>P</i><0.01. Specific sets of primers are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061083#pone-0061083-t001" target="_blank">Table 1</a>. MIT: mitomycin C (0.005 µg/ml), a well-known inducer of the SOS system and modulator of <i>recA</i> promoter, was used as a positive control.</p

β-galactosidase activity (Miller units) of SA13011-HeR(<i>recA</i>P::<i>lacZ</i>) (LMR21) grown in the absence or presence of (1/4 MICs values) the corresponding antibiotics.

<p>The number of Miller units is expressed in fold change. Values represent the mean ± SD of three independent replicates.</p

Analysis of antibacterial efficacy against SA13011-HoR.

<p>Synergy time kill analysis was performed using Mueller-Hinton (MH) broth with 10⁶ CFU/ml inoculums at 0, 2, 4, 6, 8, and 24 h. Antibiotics were used as specified at the following concentrations: IMP: 0.12 µg/ml; FOX: 4 µg/ml; BAL: 0.5 µg/ml, and CTX: 4 µg/ml. A minimum of three independent experimental runs were performed for each IMP/β-lactam combination.</p

Strains, plasmids, and primers used in this study.

<p>Strains, plasmids, and primers used in this study.</p

Mutation rate analysis during OXA-induced HeR/HoR selection of parents and homogenous derivatives of SA13011, LMR25, and LMR23 strains.

<p>Mutation frequency was determined using rifampicin selection and expressed as a ratio of rifampicin resistance as a fraction of viable cells, during time points at 6, 27, and 33 h of the selection process, −/+OXA 0.5 µg/ml (*).</p

VraSR Two-Component Regulatory System Contributes to mprF-Mediated Decreased Susceptibility to Daptomycin in In Vivo-Selected Clinical Strains of Methicillin-Resistant Staphylococcus aureus

Daptomycin (DAP) is a new class of cyclic lipopeptide antibiotic highly active against methicillin-resistant Staphylococcus aureus (MRSA) infections. Proposed mechanisms involve disruption of the functional integrity of the bacterial membrane in a Ca-dependent manner. In the present work, we investigated the molecular basis of DAP resistance in a group of isogenic MRSA clinical strains obtained from patients with S. aureus infections after treatment with DAP. Different point mutations were found in the mprF gene in DAP-resistant (DR) strains. Investigation of the mprF L826F mutation in DR strains was accomplished by inactivation and transcomplementation of either full-length wild-type or mutated mprF in DAP-susceptible (DS) strains, revealing that they were mechanistically linked to the DR phenotype. However, our data suggested that mprF was not the only factor determining the resistance to DAP. Differential gene expression analysis showed upregulation of the two-component regulatory system vraSR. Inactivation of vraSR resulted in increased DAP susceptibility, while complementation of vraSR mutant strains restored DAP resistance to levels comparable to those observed in the corresponding DR wild-type strain. Electron microscopy analysis showed a thicker cell wall in DR CB5012 than DS CB5011, an effect that was related to the impact of vraSR and mprF mutations in the cell wall. Moreover, overexpression of vraSR in DS strains resulted in both increased resistance to DAP and decreased resistance to oxacillin, similar to the phenotype observed in DR strains. These results support the suggestion that, in addition to mutations in mprF, vraSR contributes to DAP resistance in the present group of clinical strains