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.

Molecular Architecture of Early Dissemination and Massive Second Wave of the SARS-CoV-2 Virus in a Major Metropolitan Area.

We sequenced the genomes of 5,085 SARS-CoV-2 strains causing two COVID-19 disease waves in metropolitan Houston, Texas, an ethnically diverse region with seven million residents. The genomes were from viruses recovered in the earliest recognized phase of the pandemic in Houston, and an ongoing massive second wave of infections. The virus was originally introduced into Houston many times independently. Virtually all strains in the second wave have a Gly614 amino acid replacement in the spike protein, a polymorphism that has been linked to increased transmission and infectivity. Patients infected with the Gly614 variant strains had significantly higher virus loads in the nasopharynx on initial diagnosis. We found little evidence of a significant relationship between virus genotypes and altered virulence, stressing the linkage between disease severity, underlying medical conditions, and host genetics. Some regions of the spike protein - the primary target of global vaccine efforts - are replete with amino acid replacements, perhaps indicating the action of selection. We exploited the genomic data to generate defined single amino acid replacements in the receptor binding domain of spike protein that, importantly, produced decreased recognition by the neutralizing monoclonal antibody CR30022. Our study is the first analysis of the molecular architecture of SARS-CoV-2 in two infection waves in a major metropolitan region. The findings will help us to understand the origin, composition, and trajectory of future infection waves, and the potential effect of the host immune response and therapeutic maneuvers on SARS-CoV-2 evolution.

Molecular Architecture of Early Dissemination and Massive Second Wave of the SARS-CoV-2 Virus in a Major Metropolitan Area.

We sequenced the genomes of 5,085 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains causing two coronavirus disease 2019 (COVID-19) disease waves in metropolitan Houston, TX, an ethnically diverse region with 7 million residents. The genomes were from viruses recovered in the earliest recognized phase of the pandemic in Houston and from viruses recovered in an ongoing massive second wave of infections. The virus was originally introduced into Houston many times independently. Virtually all strains in the second wave have a Gly614 amino acid replacement in the spike protein, a polymorphism that has been linked to increased transmission and infectivity. Patients infected with the Gly614 variant strains had significantly higher virus loads in the nasopharynx on initial diagnosis. We found little evidence of a significant relationship between virus genotype and altered virulence, stressing the linkage between disease severity, underlying medical conditions, and host genetics. Some regions of the spike protein-the primary target of global vaccine efforts-are replete with amino acid replacements, perhaps indicating the action of selection. We exploited the genomic data to generate defined single amino acid replacements in the receptor binding domain of spike protein that, importantly, produced decreased recognition by the neutralizing monoclonal antibody CR3022. Our report represents the first analysis of the molecular architecture of SARS-CoV-2 in two infection waves in a major metropolitan region. The findings will help us to understand the origin, composition, and trajectory of future infection waves and the potential effect of the host immune response and therapeutic maneuvers on SARS-CoV-2 evolution.IMPORTANCE There is concern about second and subsequent waves of COVID-19 caused by the SARS-CoV-2 coronavirus occurring in communities globally that had an initial disease wave. Metropolitan Houston, TX, with a population of 7 million, is experiencing a massive second disease wave that began in late May 2020. To understand SARS-CoV-2 molecular population genomic architecture and evolution and the relationship between virus genotypes and patient features, we sequenced the genomes of 5,085 SARS-CoV-2 strains from these two waves. Our report provides the first molecular characterization of SARS-CoV-2 strains causing two distinct COVID-19 disease waves.

Development and Evaluation of a Novel Protein-Based Assay for Specific Detection of KPC ß-Lactamases from Klebsiella pneumoniae Clinical Isolates.

Carbapenemases confer resistance to nearly all ß-lactam antibiotics. The extensive spread of carbapenemase-producing multidrug-resistant bacteria contributes significantly to hospital-acquired infections. We have developed a novel protein-based binding assay that identifies KPC ß-lactamases from clinical isolates. We used the protein-protein interaction between KPCs and a soluble ß-lactamase inhibitory protein (BLIP) variant, BLIPK74T/W112D, which specifically inhibits KPCs but not other ß-lactamases. In this assay, BLIPK74T/W112D was allowed to form complexes with KPC-2 in bacterial cell lysates and then extracted using His tag binding resins. We demonstrated the presence of KPC-2 by monitoring the hydrolysis of a colorimetric ß-lactam substrate. Also, to further increase the accuracy of the method, a BLIPK74T/W112D-mediated inhibition assay was developed. The binding and inhibition assays were validated by testing 127 Klebsiella pneumoniae clinical isolates with known genome sequences for the presence of KPC. Our assays identified a total of 32 strains as KPC-2 producers, a result in 100% concordance with genome sequencing predictions. To further simplify the assay and decrease the time to obtain results, the BLIPK74T/W112D protein was tested in combination with the widely used Carba-NP assay. For this purpose, the genome-sequenced K. pneumoniae strains were tested for the presence of carbapenemases with the Carba-NP test with and without the addition of BLIPK74T/W122D The test accurately identified carbapenemase-producing strains and the addition of BLIPK74T/W112D allowed a further determination that the strains contain KPC carbapenemase. Thus, the BLIPK74T/W112D protein is an effective sensor to specifically detect KPC ß-lactamases produced by clinical isolates.IMPORTANCE Infections caused by carbapenem-resistant Enterobacteriaceae are associated with high therapeutic failure and mortality rates. Thus, it is critical to rapidly identify clinical isolates expressing KPC ß-lactamases to facilitate administration of the correct antibiotic treatment and initiate infection control strategies. To address this problem, we developed a protein-based, KPC-specific binding assay in combination with a cell lysate inhibition assay that provided a 100% identification rate of KPC from clinical isolates of known genomic sequence. In addition, this protein sensor was adapted to the Carba-NP assay to provide a rapid strategy to detect KPC-producing isolates that will facilitate informed treatment of critically ill patients.

Determination of optimal loading and maintenance doses for continuous infusion of vancomycin in critically ill patients: Population pharmacokinetic modelling and simulations for improved dosing schemes.

OBJECTIVES: Despite extensive clinical use, limited data are available on optimal loading and maintenance doses of vancomycin in critically ill patients. This study aimed to develop a rational approach for optimised dosage of vancomycin given in a continuous infusion in critically ill patients. METHODS: Vancomycin pharmacokinetic (PK) data (total serum concentrations) were obtained from 55 intensive care unit (ICU) patients (Bach Mai Hospital, Hanoi, Vietnam) receiving a 20 mg/kg loading dose followed by continuous infusion stratified by creatinine clearance (CLCr). Population PK modelling and Monte Carlo simulations were performed using a nonlinear mixed-effects modelling (NONMEM) program for a target of 20-30 mg/L to optimise efficacy and minimise nephrotoxicity. RESULTS: A two-compartment model with first-order elimination best fitted the PK data with central and peripheral volumes of distribution of 1.01 and 2.39 L/kg, respectively (allometric scaling to a 70 kg standard subject). The population total clearance of 3.63 L/h was only explained by renal function in the covariate and final model. The simulations showed that a 25-mg/kg loading dose infused over 90 minutes was optimal to reach the target range. The optimal maintenance dose for low renal function (CLCr < 45 mL/min) was 1000-1500 mg/day. For augmented renal clearance (CLCr > 130 mL/min) the dose should be up to 3500 mg/day or even 4500 mg/day to achieve adequate exposure. These simulated maintenance doses were larger than previously proposed for non-ICU patients. CONCLUSION: Large loading and maintenance doses of vancomycin are generally needed in critically ill patients. Because of high interindividual variability in vancomycin PK, drug monitoring may still be necessary.

Draft Genome Sequence of Candida auris Strain LOM, a Human Clinical Isolate from Greater Metropolitan Houston, Texas.

Candida auris is an emerging pathogen of considerable public health importance. We present the draft genome sequence of a strain recently cultured from the urine of a patient hospitalized in the greater Houston metropolitan region. Two combined Oxford Nanopore sequencing runs provided sufficient data to rapidly generate a draft genome.

Integrated analysis of population genomics, transcriptomics and virulence provides novel insights into Streptococcus pyogenes pathogenesis.

Streptococcus pyogenes causes 700 million human infections annually worldwide, yet, despite a century of intensive effort, there is no licensed vaccine against this bacterium. Although a number of large-scale genomic studies of bacterial pathogens have been published, the relationships among the genome, transcriptome, and virulence in large bacterial populations remain poorly understood. We sequenced the genomes of 2,101 emm28 S. pyogenes invasive strains, from which we selected 492 phylogenetically diverse strains for transcriptome analysis and 50 strains for virulence assessment. Data integration provided a novel understanding of the virulence mechanisms of this model organism. Genome-wide association study, expression quantitative trait loci analysis, machine learning, and isogenic mutant strains identified and confirmed a one-nucleotide indel in an intergenic region that significantly alters global transcript profiles and ultimately virulence. The integrative strategy that we used is generally applicable to any microbe and may lead to new therapeutics for many human pathogens.