High-resolution genomics identifies pneumococcal diversity and persistence of vaccine types in children with community-acquired pneumonia in the UK and Ireland.

BACKGROUND: Streptococcus pneumoniae is a global cause of community-acquired pneumonia (CAP) and invasive disease in children. The CAP-IT trial (grant No. 13/88/11; https://www.capitstudy.org.uk/ ) collected nasopharyngeal swabs from children discharged from hospitals with clinically diagnosed CAP, and found no differences in pneumococci susceptibility between higher and lower antibiotic doses and shorter and longer durations of oral amoxicillin treatment. Here, we studied in-depth the genomic epidemiology of pneumococcal (vaccine) serotypes and their antibiotic resistance profiles. METHODS: Three-hundred and ninety pneumococci cultured from 1132 nasopharyngeal swabs from 718 children were whole-genome sequenced (Illumina) and tested for susceptibility to penicillin and amoxicillin. Genome heterogeneity analysis was performed using long-read sequenced isolates (PacBio, n = 10) and publicly available sequences. RESULTS: Among 390 unique pneumococcal isolates, serotypes 15B/C, 11 A, 15 A and 23B1 were most prevalent (n = 145, 37.2%). PCV13 serotypes 3, 19A, and 19F were also identified (n = 25, 6.4%). STs associated with 19A and 19F demonstrated high genome variability, in contrast to serotype 3 (n = 13, 3.3%) that remained highly stable over a 20-year period. Non-susceptibility to penicillin (n = 61, 15.6%) and amoxicillin (n = 10, 2.6%) was low among the pneumococci analysed here and was independent of treatment dosage and duration. However, all 23B1 isolates (n = 27, 6.9%) were penicillin non-susceptible. This serotype was also identified in ST177, which is historically associated with the PCV13 serotype 19F and penicillin susceptibility, indicating a potential capsule-switch event. CONCLUSIONS: Our data suggest that amoxicillin use does not drive pneumococcal serotype prevalence among children in the UK, and prompts consideration of PCVs with additional serotype coverage that are likely to further decrease CAP in this target population. Genotype 23B1 represents the convergence of a non-vaccine genotype with penicillin non-susceptibility and might provide a persistence strategy for ST types historically associated with vaccine serotypes. This highlights the need for continued genomic surveillance

Unusual resistance patterns in macrolide-resistant Streptococcus pyogenes harbouring erm(A)

Background: We identified erm(A)-harbouring Streptococcus pyogenes that expressed three variant phenotypes: (1) low-level resistance to erythromycin (MICs 1-4 mg/L) but high azithromycin MICs in absolute terms (16-64 mg/L; n = 6); (2) same as (1) but with a high clindamycin MIC (256 mg/L; n = 1); and (3) high-level constitutive MLS (cMLS) resistance (n = 1). Here we analysed the genetic basis of these novel phenotypes. Methods: The presence of erm (A) and the absence of macrolide/ lincosamide resistance genes erm (B), mef and cfr were confirmed by PCR. erm (A), 23S rRNA, L4 and L22 genes were sequenced. Mutant erm (A) genes were cloned and electrotransformed into the macrolide-susceptible Escherichia coli AG100A. Clonality was determined by emm typing and PFGE. Effects of the identified mutations on free energy changes (\u394 G) and putative configurations of the leader sequence were studied in silico. Results: Point mutations (G98A, A137C, C140T and G205A) were observed in the erm (A) regulatory region of all eight erm (A)-harbouring S. pyogenes. Five and two isolates belonged to emm 77 and emm 89 clones, respectively, and one isolate was an emm 1. E. coli transformed with mutant erm (A) harbouring G98A, A137C or C140T mutations (phenotypes 1 and 2) did not express high-level azithromycin or clindamycin resistance. However, cMLS resistance was clearly observed in transformants with erm (A) harbouring both A137C and G205A mutations (phenotype 3). In silico analysis showed that \u394 G was minor except for the G205A mutation. Secondary structure predictions further showed that the A137C and G205A mutations together abolished the hairpin sequestering the ribosome-binding and initiation sites of the erm (A) gene, explaining the cMLS phenotype 3. Conclusions: We report point mutations in the erm (A) regulatory region leading to constitutive methylase expression and the presence of additional, as yet unidentified mechanisms mediating high-level azithromycin and clindamycin resistance in erm (A)-harbouring S. pyogenes

Impact of a short pre-enrichment on detection and bacterial loads of methicillin-resistant staphylococcus aureaus\/ from screening specimens

We compared the impacts of direct plating on a chromogenic medium and of plating after enrichment (4 h and overnight) on the detection of methicillin-resistant Staphylococcus aureus (MRSA) from 52 patient screening samples. MRSA colony counts for approximately 70% of samples after overnight pre-enrichment were >20-fold higher than the counts after the other two treatments. The qualitative differences (sample was MRSA positive/negative) between the results of the direct plating and 4-h pre-enrichment treatments were not significant, indicating no advantage of the latter; however, the number of samples positive for MRSA increased significantly after an overnight sample pre-enrichment (P < 0.005).status: publishe