A low-affinity penicillin-binding protein 2x variant is required for heteroresistance in Streptococcus pneumoniae.

Heteroresistance to penicillin in Streptococcus pneumoniae is the ability of subpopulations to grow at a higher antibiotic concentration than expected from the MIC. This may render conventional resistance testing unreliable and lead to therapeutic failure. We investigated the role of the primary ß-lactam resistance determinants, penicillin-binding protein 2b (PBP2b) and PBP2x, and the secondary resistance determinant PBP1a in heteroresistance to penicillin. Transformants containing PBP genes from the heteroresistant strain Spain(23F) 2349 in the nonheteroresistant strain R6 background were tested for heteroresistance by population analysis profiling (PAP). We found that pbp2x, but not pbp2b or pbp1a alone, conferred heteroresistance to R6. However, a change of pbp2x expression was not observed, and therefore, expression does not correlate with an increased proportion of resistant subpopulations. In addition, the influence of the CiaRH system, mediating PBP-independent ß-lactam resistance, was assessed by PAP on ciaR disruption mutants but revealed no heteroresistant phenotype. We also showed that the highly resistant subpopulations (HOM*) of transformants containing low-affinity pbp2x undergo an increase in resistance upon selection on penicillin plates that partially reverts after passaging on selection-free medium. Shotgun proteomic analysis showed an upregulation of phosphate ABC transporter subunit proteins encoded by pstS, phoU, pstB, and pstC in these highly resistant subpopulations. In conclusion, the presence of low-affinity pbp2x enables certain pneumococcal colonies to survive in the presence of ß-lactams. Upregulation of phosphate ABC transporter genes may represent a reversible adaptation to antibiotic stress.

Heteroresistance to fosfomycin is predominant in Streptococcus pneumoniae and depends on the murA1 gene.

Fosfomycin targets the first step of peptidoglycan biosynthesis in Streptococcus pneumoniae catalyzed by UDP-N-acetylglucosamine enolpyruvyltransferase (MurA1). We investigated whether heteroresistance to fosfomycin occurs in S. pneumoniae. We found that of 11 strains tested, all but 1 (Hungary(19A)) displayed heteroresistance and that deletion of murA1 abolished heteroresistance. Hungary(19A) differs from the other strains by a single amino acid substitution in MurA1 (Ala(364)Thr). To test whether this substitution is responsible for the lack of heteroresistance, it was introduced into strain D39. The heteroresistance phenotype of strain D39 was not changed. Furthermore, no relevant structural differences between the MurA1 crystal structures of heteroresistant strain D39 and nonheteroresistant strain Hungary(19A) were found. Our results reveal that heteroresistance to fosfomycin is the predominant phenotype of S. pneumoniae and that MurA1 is required for heteroresistance to fosfomycin but is not the only factor involved. The findings provide a caveat for any future use of fosfomycin in the treatment of pneumococcal infections.

Budesonide is effective in adolescent and adult patients with active eosinophilic esophagitis.

BACKGROUND & AIMS: Eosinophilic esophagitis (EoE) is a chronic inflammatory disease of the esophagus characterized by dense tissue eosinophilia; it is refractory to proton pump inhibitor therapy. EoE affects all age groups but most frequently individuals between 20 and 50 years of age. Topical corticosteroids are effective in pediatric patients with EoE, but no controlled studies of corticosteroids have been reported in adult patients. METHODS: We performed a randomized, double-blind, placebo-controlled trial to evaluate the effect of oral budesonide (1 mg twice daily for 15 days) in adolescent and adult patients with active EoE. Pretreatment and posttreatment disease activity was assessed clinically, endoscopically, and histologically. The primary end point was reduced mean numbers of eosinophils in the esophageal epithelium (number per high-power field [hpf] = esophageal eosinophil load). Esophageal biopsy and blood samples were analyzed using immunofluorescence and immunoassays, respectively, for biomarkers of inflammation and treatment response. RESULTS: A 15-day course of therapy significantly decreased the number of eosinophils in the esophageal epithelium in patients given budesonide (from 68.2 to 5.5 eosinophils/hpf; P < .0001) but not in the placebo group (from 62.3 to 56.5 eosinophils/hpf; P = .48). Dysphagia scores significantly improved among patients given budesonide compared with those given placebo (5.61 vs 2.22; P < .0001). White exudates and red furrows were reversed in patients given budesonide, based on endoscopy examination. Budesonide, but not placebo, also reduced apoptosis of epithelial cells and molecular remodeling events in the esophagus; no serious adverse events were observed. CONCLUSIONS: A 15-day course of treatment with budesonide is well tolerated and highly effective in inducing a histologic and clinical remission in adolescent and adult patients with active EoE.

Global phylogenomic analysis of nonencapsulated Streptococcus pneumoniae reveals a deep-branching classic lineage that is distinct from multiple sporadic lineages.

The surrounding capsule of Streptococcus pneumoniae has been identified as a major virulence factor and is targeted by pneumococcal conjugate vaccines (PCV). However, nonencapsulated S. pneumoniae (non-Ec-Sp) have also been isolated globally, mainly in carriage studies. It is unknown if non-Ec-Sp evolve sporadically, if they have high antibiotic nonsusceptiblity rates and a unique, specific gene content. Here, whole-genome sequencing of 131 non-Ec-Sp isolates sourced from 17 different locations around the world was performed. Results revealed a deep-branching classic lineage that is distinct from multiple sporadic lineages. The sporadic lineages clustered with a previously sequenced, global collection of encapsulated S. pneumoniae (Ec-Sp) isolates while the classic lineage is comprised mainly of the frequently identified multilocus sequences types (STs) ST344 (n = 39) and ST448 (n = 40). All ST344 and nine ST448 isolates had high nonsusceptiblity rates to ß-lactams and other antimicrobials. Analysis of the accessory genome reveals that the classic non-Ec-Sp contained an increased number of mobile elements, than Ec-Sp and sporadic non-Ec-Sp. Performing adherence assays to human epithelial cells for selected classic and sporadic non-Ec-Sp revealed that the presence of a integrative conjugative element (ICE) results in increased adherence to human epithelial cells (P = 0.005). In contrast, sporadic non-Ec-Sp lacking the ICE had greater growth in vitro possibly resulting in improved fitness. In conclusion, non-Ec-Sp isolates from the classic lineage have evolved separately. They have spread globally, are well adapted to nasopharyngeal carriage and are able to coexist with Ec-Sp. Due to continued use of PCV, non-Ec-Sp may become more prevalent.