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

Staphylococcus aureus biofilms are poorly responsive to antibiotics. Underlying reasons include a matrix effect preventing drug access to embedded bacteria, or the presence of dormant bacteria with reduced growth rate. Using 18 clinical isolates previously characterized for their moxifloxacin-resistant and moxifloxacin-persister character in stationary-phase culture, we studied their biofilm production and matrix composition and the anti-biofilm activity of moxifloxacin. Biofilms were grown in microtiter plates and their abundance quantified by crystal violet staining and colony counting; their content in polysaccharides, extracellular DNA and proteins was measured. Moxifloxacin activity was assessed after 24 h of incubation with a broad range of concentrations to establish full concentration-response curves. All clinical isolates produced more biofilm biomass than the reference strain ATCC 25923, the difference being more important for those with high relative persister fractions to moxifloxacin, most of which being also resistant. High biofilm producers expressed icaA to higher levels, enriching the matrix in polysaccharides. Moxifloxacin was less potent against biofilms from clinical isolates than from ATCC 25923, especially against moxifloxacin-resistant isolates with high persister fractions, which was ascribed to a lower concentration of moxifloxacin in these biofilms. Time-kill curves in biofilms revealed the presence of a moxifloxacin-tolerant subpopulation, with low multiplication capacity, whatever the persister character of the isolate. Thus, moxifloxacin activity depends on its local concentration in biofilm, which is reduced in most isolates with high-relative persister fractions due to matrix effects, and insufficient to kill resistant isolates due to their high MIC.

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.

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.

High prevalence of colonisation with carbapenem-resistant Enterobacteriaceae among patients admitted to Vietnamese hospitals: Risk factors and burden of disease.

BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE) is an increasing problem worldwide, but particularly problematic in low- and middle-income countries (LMIC) due to limitations of resources for surveillance of CRE and infection prevention and control (IPC). METHODS: A point prevalence survey (PPS) with screening for colonisation with CRE was conducted on 2233 patients admitted to neonatal, paediatric and adult care at 12 Vietnamese hospitals located in northern, central and southern Vietnam during 2017 and 2018. CRE colonisation was determined by culturing of faecal specimens on selective agar for CRE. Risk factors for CRE colonisation were evaluated. A CRE admission and discharge screening sub-study was conducted among one of the most vulnerable patient groups; infants treated at an 80-bed Neonatal ICU from March throughout June 2017 to assess CRE acquisition, hospital-acquired infection (HAI) and treatment outcome. RESULTS: A total of 1165 (52%) patients were colonised with CRE, most commonly Klebsiella pneumoniae (n = 805), Escherichia coli (n = 682) and Enterobacter spp. (n = 61). Duration of hospital stay, HAI and treatment with a carbapenem were independent risk factors for CRE colonisation. The PPS showed that the prevalence of CRE colonisation increased on average 4.2% per day and mean CRE colonisation rates increased from 13% on the day of admission to 89% at day 15 of hospital stay. At the NICU, CRE colonisation increased from 32% at admission to 87% at discharge, mortality was significantly associated (OR 5·5, P < 0·01) with CRE colonisation and HAI on admission. CONCLUSION: These data indicate that there is an epidemic spread of CRE in Vietnamese hospitals with rapid transmission to hospitalised patients.