Serotype distribution and antimicrobial susceptibility of Streptococcus pneumoniae isolates from a Phase III community-acquired bacterial pneumonia (CABP) trial.

OBJECTIVES: To report Streptococcus pneumoniae serotyping and susceptibility data from a recent clinical trial (ML-3341-306) comparing delafloxacin with moxifloxacin in the treatment of adults with community-acquired bacterial pneumonia (CABP). METHODS: Serotyping and susceptibility testing were conducted on 142 baseline S. pneumoniae isolates recovered from subjects participating in a CABP clinical trial. RESULTS: Overall, 113/142 (79.6%) isolates were vaccine serotypes. 76.8% (109/142) of serotyped isolates were PPSV23 serotypes and 59.9% (85/142) of isolates were PCV13 serotypes. 15.5% (22/142) of serotyped isolates were serotypes not covered by either vaccine; 4.9% (7/142) of tested isolates were non-typeable. The most common serotypes were serotypes 3 (19.0%; 27/142), 19F (9.9%; 14/142) and 23F (7.0%; 10/142). All of the 142 isolates were susceptible to delafloxacin and moxifloxacin, 76.1% were susceptible to azithromycin and 71.8% were susceptible to penicillin. Multidrug resistance was found among 19A (4/5; 80%), 6A (1/4; 25%), 6B (1/4; 25%), 14 (1/4; 25%), 19F (1/14; 7.1%), and 23F serotypes (2/10; 20%), and among non-typeable S. pneumoniae isolates (1/7; 14.3%). CONCLUSIONS: S. pneumoniae vaccine-targeted serotypes were the main cause of CABP in this Phase 3 CABP study. Fluoroquinolones including delafloxacin remain a good treatment option for CABP in adults caused by S. pneumoniae.

Delafloxacin In Vitro Broth Microdilution and Disk Diffusion Antimicrobial Susceptibility Testing Guidelines: Susceptibility Breakpoint Criteria and Quality Control Ranges for an Expanded-Spectrum Anionic Fluoroquinolone.

Delafloxacin, a recently approved anionic fluoroquinolone, was tested within an international resistance surveillance program. The in vitro susceptibilities of 7,914 indicated pathogens causing acute bacterial skin and skin structure infections (ABSSSI) were determined using Clinical and Laboratory Standards Institute (CLSI) broth microdilution MIC testing methods. The U.S. Food and Drug Administration (FDA) susceptibility testing breakpoints and quality control ranges for routine broth microdilution and disk diffusion methods were confirmed. The delafloxacin MIC50/90 (% susceptibility) results were as follows: Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), 0.008/0.25 µg/ml (92.8%); Staphylococcus lugdunensis, 0.016/0.03 µg/ml (99.3%); Streptococcus pyogenes, 0.016/0.03 µg/ml (100.0%); Streptococcus anginosus group, 0.008/0.016 µg/ml (100.0%); Enterococcus faecalis, 0.12/1 µg/ml (66.2%); and Enterobacteriaceae, 0.12/4 µg/ml (69.5%). The FDA clinical breakpoints were used to assess intermethod test agreement between delafloxacin MIC and disk diffusion methods for the indicated pathogens. The intermethod susceptibility test categorical agreement for delafloxacin was acceptable, with only 0.4% very major, false-susceptible errors among S. aureus strains. Across all FDA-indicated species, the selected breakpoints produced only 0.0 to 1.7% rates of serious (very major and major errors) intermethod error. Quality control ranges for these standardized delafloxacin susceptibility test methods were calculated from three multilaboratory (12 total sites) studies for six control organisms. In conclusion, the application of FDA MIC breakpoints for delafloxacin against contemporary (2014 to 2016) isolates of ABSSSI pathogens provides additional support for the use of delafloxacin in the treatment of adults with ABSSSI. Delafloxacin MIC and disk diffusion susceptibility testing methods have been standardized for clinical application, achieving high intermethod categorical agreement.

Effects of phosphoprotein on collagen fibril formation in vitro.

Transmission electron microscopy showed that when either dentinal phosphoprotein or calcium-treated phosphoprotein or phosvitin were introduced during type I collagen fibrillogenesis the fibrils formed were significantly wider and the cross-banding was more distinct than in the absence of phosphoprotein. The collagen fibril width also increased with increasing concentrations of these molecules. When either bovine serum albumin (BSA) or dephosphorylated dentine phosphoprotein were used, no differences in the fibril characteristics were seen when compared to the controls that contained no phosphoprotein or BSA. When these dialysed matrices were placed into mineralizing solutions, no mineral was observed in any of the samples.

Comparison of phosphoprotein isolated from mature and immature human tooth roots.

Mature (average patient age = 29.5 yr, closed apical foramen) and immature (average patient age = 17.5 yr, open apical foramen) root shards were placed in dialysis tubing and demineralized to completion using either 10% disodium EDTA plus protease inhibitors or 0.6 N HCl. The demineralized shards were re-extracted (five times) with 0.05 M tris-HCl, 1.0 M NaCl and then collagenase digested. No major differences were observed in chromatograms of extracts, re-extracts or collagenase digests from root shards demineralized in either way. In contrast, chromatograms of immature and mature roots showed qualitative differences. Chromatograms of mature roots demineralized in either way showed broader protein peaks and less organic phosphorus than those from immature tooth roots. A distinct band amid degraded phosphoprotein (150 K) was found in SDS-PAGE gels (7.5%) from EDTA-extracted immature tooth roots but not from mature tooth roots. Electroelution of this band revealed a typical phosphoprotein amino-acid profile containing increased aspartic acid and serine residues. Comparison of the total phosphoprotein and amino acid composition of extracts, re-extracts and collagenase digests revealed that phosphoprotein, serine and to a lesser extent aspartic acid were recovered in greater quantities from immature roots than mature tooth roots. These data suggest that the degree of maturation is crucial to the isolation of an intact phosphoprotein and provides additional evidence that human dentine phosphoprotein undergoes amino acid compositional changes during maturation.

Effects of phosphoprotein moieties on the remineralization of human root caries.

The noncollagenous proteins, especially phosphoprotein, have been shown to modulate biomineralization. The objective of this study was to investigate the remineralization potential of human tooth root organic matrices which did or did not contain soluble non-collagenous proteins including phosphoprotein. Human tooth roots were completely demineralized using conditions that either removed or did not remove soluble phosphoprotein and were then subjected to remineralization conditions. Removal of soluble phosphoprotein resulted in remineralization while no remineralization occurred in tooth roots that still contained soluble phosphoprotein. Transmission electron microscopy and microradiography demonstrated that demineralized cementum did not remineralize under any of the conditions used in this study. Collagenase digestion of demineralized and salt-reextracted tooth root organic matrices revealed that a nonsoluble phosphoprotein was present in the matrices. Amino acid analysis and SDS-PAGE showed that this nonsoluble phosphoprotein was similar in composition to the soluble phosphoprotein. This work suggests that the removal of soluble, noncollagenous proteins, especially phosphoprotein from root caries lesions, may enhance their remineralization potential.

Phosphoprotein extraction from the dentine/cementum complex of human tooth roots.

Root shards were placed in dialysis tubing and demineralized to completion in either 10% disodium EDTA, pH 7.4, 0.6 M HCl, 0.1 M HCl, 0.5 M acetic or 75 mM-25 mM lactic-acetic acids. The demineralized shards were then re-extracted with 0.05 M tris-HCl, 1.0 M NaCl. DEAE chromatography revealed that the major peak of the 0.6 M CHl and EDTA extracts contained organic phosphorus, whereas much less organic phosphorus was found in the major peak of the 0.1 M HCl extract. Analysis of the re-extracts gave a pattern opposite to that obtained from the initial extractions. Measurements of protein and organic phosphorus released during extraction and re-extraction confirmed these results. Staining of SDS-PAGE gels for phosphoprotein with Stains-All resulted in a blue smear in fractions containing organic phosphorus. Thus the extraction of phosphoproteins from human tooth roots differed depending upon the demineralizing conditions. This ability to remove phosphoprotein differentially will allow further investigation of the role of phosphoprotein in mineralization and remineralization.