The Persister Character of Clinical Isolates of Staphylococcus aureus Contributes to Faster Evolution to Resistance and Higher Survival in THP-1 Monocytes: A Study With Moxifloxacin

Abstract

Staphylococcus aureus may cause relapsing infections. We previously showed that S. aureus SH1000 surviving intracellularly to bactericidal antibiotics are persisters. Here, we used 54 non-duplicate clinical isolates to assess links between persistence, resistance evolution, and intracellular survival, using moxifloxacin throughout as test bactericidal antibiotic. The relative persister fraction (RPF: percentage of inoculum surviving to 100× MIC moxifloxacin in stationary phase culture for each isolate relative to ATCC 25923) was determined to categorize isolates with low (≤10) or high (>10) RPF. Evolution to resistance (moxifloxacin MIC ≥ 0.5 mg/L) was triggered by serial passages at 0.5× MIC (with daily concentration readjustments). Intracellular moxifloxacin maximal efficacy (Emax) was determined by 24 h concentration-response experiments [pharmacodynamic model (Hill-Langmuir)] with infected THP-1 monocytes exposed to moxifloxacin (0.01 to 100× MIC) after phagocytosis. Division of intracellular survivors was followed by green fluorescence protein dilution (FACS). Most (30/36) moxifloxacin-susceptible isolates showed low RPF but all moxifloxacin-resistant (n = 18) isolates harbored high RPF. Evolution to resistance of susceptible isolates was faster for those with high vs. low RPF (with SOS response and topoisomerase-encoding genes overexpression). Intracellularly, moxifloxacin Emax was decreased (less negative) for isolates with high vs. low RPF, independently from resistance. Moxifloxacin intracellular survivors were non-dividing. The data demonstrate and quantitate persisters in clinical isolates of S. aureus, and show that this phenotype accelerates resistance evolution and is associated with intracellular survival in spite of high antibiotic concentrations. Isolates with high RPF may represent a possible cause of treatment failure not directly related to resistance in patients receiving active antibiotics.

Activity of Moxifloxacin Against Biofilms Formed by Clinical Isolates of Staphylococcus aureus Differing by Their Resistant or Persister Character to Fluoroquinolones

“…The methicillin-susceptible S. aureus (MSSA) ATCC 25923 and the methicillin-resistant S. aureus (MRSA) ATCC 33591 were used as references. Two strains expressing GFP under the control of a tetracycline-inducible promoter (RN4220-palc and 1214-palc, Nguyen et al, 2020b ), were used in specific experiments. In addition, 18 clinical, non-isogenic, isolates of S. aureus collected from patients with persistent or recurrent infections at the Bach Mai hospital, Hanoi, Vietnam, were included in the study.…”

“…These isolates were previously characterized ( Supplementary Table 1 ) by molecular spa and agr typing; their MRSA character was established by measuring cefoxitine MIC and detecting mecA and mecC by PCR ( Nguyen et al, 2020a ). The MIC of moxifloxacin was previously determined ( Nguyen et al, 2020b ) by broth microdilution following CLSI recommendations ( Clinical and Laboratory Standards Institute, 2020 ) and susceptibility breakpoints were those established by EUCAST; 1 the relative persister fraction was previously established ( Nguyen et al, 2020b ) by measuring the number of CFU after 5 h incubation of a stationary phase culture with moxifloxacin at 100 times its MIC, according to a published procedure. In this method, the persister character of each isolate was evaluated by its persister fraction relative to that of a reference ( De Groote et al, 2009 ), here ATCC 25923, to allow for a more accurate comparison of persister fractions determined in independent experiments.…”

“…In the present study, we used a collection of S. aureus assembled in a tertiary hospital in Hanoi, Vietnam ( Nguyen et al, 2020a ) from samples of patients suffering from persistent or recurrent infections (defined as infections that did not resolved after 5 days of treatment with an antibiotic to which the initial isolate was susceptible, or infections that reactivated a few days after improvement of the patient’s conditions and interruption of the antibiotic treatment, respectively). All isolates were thereafter characterized in Belgium with respect to their resistance profile to commonly used antibiotics ( Nguyen et al, 2020a ) and to their persister character [as determined against stationary phase cultures exposed to moxifloxacin as reporter antibiotic ( Nguyen et al, 2020b )]. Importantly, persistence and resistance were closely related in this collection, with all resistant isolates showing high relative persister fractions (RPF) to moxifloxacin, while only a small proportion of susceptible isolates were high persisters.…”

“…Importantly, persistence and resistance were closely related in this collection, with all resistant isolates showing high relative persister fractions (RPF) to moxifloxacin, while only a small proportion of susceptible isolates were high persisters. In addition, susceptible high persisters also acquire resistance to moxifloxacin more rapidly than susceptible isolates with low RPF ( Nguyen et al, 2020b ). This indicates that persistence may promote evolution toward resistance, as previously documented for Escherichia coli ( Barrett et al, 2019 ; Windels et al, 2019 ), and therefore links to some extent these two properties.…”

“…We selected an equivalent number of non-clonal moxifloxacin-susceptible and -resistant isolates in this collection (all belonging to Agr group I; Nguyen et al, 2020a ), taking also into account their relative persister fraction to moxifloxacin ( Nguyen et al, 2020b ). We then determined their capacity to form biofilm as well as the global composition of the biofilm matrix.…”