SummaryBackgroundSince 2014, England has seen increased scarlet fever activity unprecedented in modern times. In 2016, England's scarlet fever seasonal rise coincided with an unexpected elevation in invasive Streptococcus pyogenes infections. We describe the molecular epidemiological investigation of these events.MethodsWe analysed changes in S pyogenes emm genotypes, and notifications of scarlet fever and invasive disease in 2014–16 using regional (northwest London) and national (England and Wales) data. Genomes of 135 non-invasive and 552 invasive emm1 isolates from 2009–16 were analysed and compared with 2800 global emm1 sequences. Transcript and protein expression of streptococcal pyrogenic exotoxin A (SpeA; also known as scarlet fever or erythrogenic toxin A) in sequenced, non-invasive emm1 isolates was quantified by real-time PCR and western blot analyses.FindingsCoincident with national increases in scarlet fever and invasive disease notifications, emm1 S pyogenes upper respiratory tract isolates increased significantly in northwest London in the March to May period, from five (5%) of 96 isolates in 2014, to 28 (19%) of 147 isolates in 2015 (p=0·0021 vs 2014 values), to 47 (33%) of 144 in 2016 (p=0·0080 vs 2015 values). Similarly, invasive emm1 isolates collected nationally in the same period increased from 183 (31%) of 587 in 2015 to 267 (42%) of 637 in 2016 (p<0·0001). Sequences of emm1 isolates from 2009–16 showed emergence of a new emm1 lineage (designated M1UK)—with overlap of pharyngitis, scarlet fever, and invasive M1UK strains—which could be genotypically distinguished from pandemic emm1 isolates (M1global) by 27 single-nucleotide polymorphisms. Median SpeA protein concentration in supernatant was nine-times higher among M1UK isolates (190·2 ng/mL [IQR 168·9–200·4]; n=10) than M1global isolates (20·9 ng/mL [0·0–27·3]; n=10; p<0·0001). M1UK expanded nationally to represent 252 (84%) of all 299 emm1 genomes in 2016. Phylogenetic analysis of published datasets identified single M1UK isolates in Denmark and the USA.InterpretationA dominant new emm1 S pyogenes lineage characterised by increased SpeA production has emerged during increased S pyogenes activity in England. The expanded reservoir of M1UK and recognised invasive potential of emm1 S pyogenes provide plausible explanation for the increased incidence of invasive disease, and rationale for global surveillance.FundingUK Medical Research Council, UK National Institute for Health Research, Wellcome Trust, Rosetrees Trust, Stoneygate Trust.

Microbes communicate with each other by using quorum sensing (QS) systems and modulate their collective ‘behavior’ for in-host colonization and virulence, biofilm formation, and environmental adaptation. The recent increase in genome data availability reveals the presence of several putative QS sensing circuits in microbial pathogens, but many of these have not been functionally characterized yet, despite their possible utility as drug targets. To increase the repertoire of functionally characterized QS systems in bacteria, we studied Rgg144/Shp144 and Rgg939/Shp939, two putative QS systems in the important human pathogen Streptococcus pneumoniae. We find that both of these QS circuits are induced by short hydrophobic peptides (Shp) upon sensing sugars found in the respiratory tract, such as galactose and mannose. Microarray analyses using cultures grown on mannose and galactose revealed that the expression of a large number of genes is controlled by these QS systems, especially those encoding for essential physiological functions and virulence-related genes such as the capsular locus. Moreover, the array data revealed evidence for cross-talk between these systems. Finally, these Rgg systems play a key role in colonization and virulence, as deletion mutants of these QS systems are attenuated in the mouse models of colonization and pneumonia.

Antimicrobial resistance in enteric or urinary Escherichia coli is a risk factor for invasive E. coli infections. Due to widespread trimethoprim resistance amongst urinary E. coli and increased bacteraemia incidence, a national recommendation to prescribe nitrofurantoin for uncomplicated urinary tract infection was made in 2014. Nitrofurantoin resistance is reported in <6% urinary E. coli isolates in the UK, however, mechanisms underpinning nitrofurantoin resistance in these isolates remain unknown. This study aimed to identify the genetic basis of nitrofurantoin resistance in urinary E. coli isolates collected from north west London and then elucidate resistance-associated genetic alterations in available UK E. coli genomes. As a result, an algorithm was developed to predict nitrofurantoin susceptibility. Deleterious mutations and gene-inactivating insertion sequences in chromosomal nitroreductase genes nfsA and/or nfsB were identified in genomes of nine confirmed nitrofurantoin-resistant urinary E. coli isolates and additional 11 E. coli isolates that were highlighted by the prediction algorithm and subsequently validated to be nitrofurantoin-resistant. Eight categories of allelic changes in nfsA, nfsB, and the associated gene ribE were detected in 12412 E. coli genomes from the UK. Evolutionary analysis of these three genes revealed homoplasic mutations and explained the previously reported order of stepwise mutations. The mobile gene complex oqxAB, which is associated with reduced nitrofurantoin susceptibility, was identified in only one of the 12412 genomes. In conclusion, mutations and insertion sequences in nfsA and nfsB were leading causes of nitrofurantoin resistance in UK E. coli . As nitrofurantoin exposure increases in human populations, the prevalence of nitrofurantoin resistance in carriage E. coli isolates and those from urinary and bloodstream infections should be monitored.

Background Since 2014, England has seen increased scarlet fever activity unprecedented in modern times. In 2016, England's scarlet fever seasonal rise coincided with an unexpected elevation in invasive Streptococcus pyogenes infections. We describe the molecular epidemiological investigation of these events. Methods We analysed changes in S pyogenes emm genotypes, and notifications of scarlet fever and invasive disease in 2014-16 using regional (northwest London) and national (England and Wales) data. Genomes of 135 non-invasive and 552 invasive emm1 isolates from 2009-16 were analysed and compared with 2800 global emm1 sequences. Transcript and protein expression of streptococcal pyrogenic exotoxin A (SpeA; also known as scarlet fever or erythrogenic toxin A) in sequenced, non-invasive emm1 isolates was quantified by real-time PCR and western blot analyses. Findings Coincident with national increases in scarlet fever and invasive disease notifications, emm1 S pyogenes upper respiratory tract isolates increased significantly in northwest London in the March to May period, from five (5%) of 96 isolates in 2014, to 28 (19%) of 147 isolates in 2015 (p=0•0021 vs 2014 values), to 47 (33%) of 144 in 2016 (p=0•0080 vs 2015 values). Similarly, invasive emm1 isolates collected nationally in the same period increased from 183 (31%) of 587 in 2015 to 267 (42%) of 637 in 2016 (p<0•0001). Sequences of emm1 isolates from 2009-16 showed emergence of a new emm1 lineage (designated M1 UK)-with overlap of pharyngitis, scarlet fever, and invasive M1 UK strains-which could be genotypically distinguished from pandemic emm1 isolates (M1 global) by 27 single-nucleotide polymorphisms. Median SpeA protein concentration in supernatant was nine-times higher among M1 UK isolates (190•2 ng/mL [IQR 168•9-200•4]; n=10) than M1 global isolates (20•9 ng/mL [0•0-27•3]; n=10; p<0•0001). M1 UK expanded nationally to represent 252 (84%) of all 299 emm1 genomes in 2016. Phylogenetic analysis of published datasets identified single M1 UK isolates in Denmark and the USA. Interpretation A dominant new emm1 S pyogenes lineage characterised by increased SpeA production has emerged during increased S pyogenes activity in England. The expanded reservoir of M1 UK and recognised invasive potential of emm1 S pyogenes provide plausible explanation for the increased incidence of invasive disease, and rationale for global surveillance.

Emm1 Streptococcus pyogenes is a successful, globally-distributed epidemic clone that is regarded as inherently invasive. An emm1 sublineage, M1UK, that expresses increased SpeA toxin, was associated with increased scarlet fever and invasive infections in England in 2015/2016. Defined by 27 SNPs in the core genome, M1UK is now dominant in England. To more fully characterise M1UK, we undertook comparative transcriptomic and proteomic analyses of M1UK and contemporary non-M1UK emm1 strains (M1global). Just seven genes were differentially expressed by M1UK compared with contemporary M1global strains. In addition to speA, five genes in the operon that includes glycerol dehydrogenase were upregulated in M1UK (gldA, mipB/talC, pflD, and pts system IIC and IIB components), while aquaporin (glpF2) was downregulated. M1UK strains have a stop codon in gldA. Deletion of the gldA gene in M1global abrogated glycerol dehydrogenase activity, and recapitulated upregulation of gene expression within the operon that includes gldA, consistent with a feedback effect. Phylogenetic analysis identified two intermediate emm1 sublineages in England comprising 13/27 (M1_13SNPs) and 23/27 SNPs (M1_23SNPs) respectively, that had failed to expand in the population. Proteomic analysis of these four major phylogenetic emm1 groups highlighted sublineage-specific changes in carbohydrate metabolism, protein synthesis and protein processing; upregulation of SpeA was not observed in chemically-defined medium. In rich broth however, transcription and secretion of SpeA was upregulated ~10-fold in both M1_23SNPs and M1UK sublineages, compared with M1_13SNPs and M1global. We conclude that stepwise accumulation of SNPs led to the emergence of M1UK. While increased expression of SpeA is a key indicator of M1UK and undoubtedly important, M1UK strains have outcompeted M1_23SNPs and other emm types that produce similar or more superantigen toxin. We speculate that an accumulation of adaptive SNPs has contributed to a wider fitness advantage in M1UK on an inherently successful emm1 streptococcal background.

Increasing reports of invasive Streptococcus pyogenes infections mandate surveillance for toxigenic lineage M1 UK . An allele-specific PCR was developed to distinguish M1 UK from other emm 1 strains. The M1 UK lineage represented 91% of invasive emm 1 isolates in England in 2020. Allele-specific PCR will permit surveillance for M1 UK without need for genome sequencing.

Streptococcus pyogenes genotype emm1 is a successful, globally distributed epidemic clone that is regarded as inherently virulent. An emm1 sublineage, M1UK, that produces increased levels of SpeA toxin was associated with increased scarlet fever and invasive infections in England in 2015/2016. Defined by 27 SNPs in the core genome, M1UK is now dominant in England. To more fully characterize M1UK, we undertook comparative transcriptomic and proteomic analyses of M1UK and contemporary non-M1UK emm1 strains (M1global). Just seven genes were differentially expressed by M1UK compared with contemporary M1global strains. In addition to speA, five genes in the operon that includes glycerol dehydrogenase were upregulated in M1UK (gldA, mipB/talC, pflD, and phosphotransferase system IIC and IIB components), while aquaporin (glpF2) was downregulated. M1UK strains have a stop codon in gldA. Deletion of gldA in M1global abrogated glycerol dehydrogenase activity, and recapitulated upregulation of gene expression within the operon that includes gldA, consistent with a feedback effect. Phylogenetic analysis identified two intermediate emm1 sublineages in England comprising 13/27 (M113SNPs) and 23/27 SNPs (M123SNPs), respectively, that had failed to expand in the population. Proteomic analysis of invasive strains from the four phylogenetic emm1 groups highlighted sublineage-specific changes in carbohydrate metabolism, protein synthesis and protein processing; upregulation of SpeA was not observed in chemically defined medium. In rich broth, however, expression of SpeA was upregulated ~10-fold in both M123SNPs and M1UK sublineages, compared with M113SNPs and M1global. We conclude that stepwise accumulation of SNPs led to the emergence of M1UK. While increased expression of SpeA is a key indicator of M1UK and undoubtedly important, M1UK strains have outcompeted M123SNPs and other emm types that produce similar or more superantigen toxin. We speculate that an accumulation of adaptive SNPs has contributed to a wider fitness advantage in M1UK on an inherently successful emm1 streptococcal background.

For the generation of energy, the important human pathogen Streptococcus pneumoniae relies on host-derived sugars, including ␤-glucoside analogs. The catabolism of these nutrients involves the action of 6-phospho-␤-glucosidase to convert them into usable monosaccharaides. In this study, we characterized a 6-phospho-␤-glucosidase (BglA3) encoded by SPD_0247. We found that this enzyme has a cell membrane localization and is active only against a phosphorylated substrate. A mutated pneumococcal ⌬SPD0247 strain had reduced 6-phospho-glucosidase activity and was attenuated in growth on cellobiose and hyaluronic acid compared to the growth of wild-type D39. ⌬SPD0247-infected mice survived significantly longer than the wild-type-infected cohort, and the colony counts of the mutant were lower than those of the wild type in the lungs. The expression of SPD_0247 in S. pneumoniae harvested from infected tissues was significantly increased relative to its expression in vitro on glucose. Additionally, ⌬SPD0247 is severely impaired in its attachment to an abiotic surface. These results indicate the importance of ␤-glucoside metabolism in pneumococcal survival and virulence. Streptococcus pneumoniae is a frequent occupant of the human nasopharynx, where it resides without causing symptoms (1). Conversely, the bacterium also is a major human pathogen, being a leading cause of bacterial pneumonia, otitis media, meningitis, and septicemia (1). The increasing trend of antibiotic resistance and the shortcomings of existing vaccines mean that a better understanding of the pathogenesis of pneumococcal diseases is required.Almost one-third of the transporters in the pneumococcal genome are dedicated to sugars (2), stressing the important role that these carbon sources play in the ability of pneumococci to survive in the host. The pneumococcus has been shown to ferment 32 different sugars in vitro, including various hexoses, ␣-and ␤-galactosides, and glucosides, as well as polysaccharides (3, 4). However, the concentrations of readily available simple carbohydrates in the respiratory tract are low (5); thus, the pneumococcus relies on complex host glycans for growth in vivo (4). Central to this is the ability to sequentially deglycosylate host glycoproteins (6).Mammalian extracellular matrix is rich in glycosaminoglycans (GAGs; e.g., hyaluronic acid), which contain ␤-linked disaccharide repeating units (7,8). The degradation of GAGs leads to the generation of structural analogues of cellobiose or N,N=-diacetylchitobiose [(GlcNAc) 2 ] and other ␤-linked disaccharides (9). Disaccharides such as these can be transported into the bacterial cell through phosphoenolpyruvate-dependent phosphotransferase systems (PTS), which transport and phosphorylate sugars simultaneously (10). The phosphorylated disaccharides then are hydrolyzed by cytoplasmic phospho-␤-glucosidases (EC 3.2.1.86) or phospho-␤-galactosidases (EC 3.2.1.85) that usually do not have hydrolytic activity toward nonphosphorylated substrates. The resulting glucose and glucose 6-pho...