Biology subjects

Sriskandan, S.

Publications and source records attributed to Sriskandan, S..

18 recordsLinked to original sources

bioRxiv2026

Diversity of penicillin-binding protein 2x in Streptococcus pyogenes from England and Wales, 2001 to 2015

Streptococcus pyogenes remains clinically susceptible to {beta}-lactam antibiotics, its first-line treatment. Yet recent reports from the US, Iceland, and Japan have identified isolates with PBP2x (a primary {beta}-lactam target) substitutions that reduced antibiotic susceptibility. To determine whether these PBP2x substitutions occurred in S. pyogenes from England and Wales, we re-analysed 2,970 invasive genomes from 2001 to 2015. We found 42 PBP2x sequence mutants in 34% (996/2,970) of the genomes, revealing an unexpected diversity in this antibiotic target. Nine emm12 isolates carried a novel double substitution, PBP2x G600A_P601H, that co-occurred with macrolide resistance genes. Experimentally, these isolates showed 2- to 4-fold elevated minimum inhibitory concentrations for four {beta}-lactam antibiotics, alongside erythromycin resistance. Other PBP2x substitutions, found across seven emm types without reduced antibiotic susceptibility, appeared driven by emm lineage expansion rather than antibiotic pressure. These findings establish a baseline for pbp2x variation, reinforcing the case for sustained genomic surveillance globally.

microbiology↗

bioRxiv2026

M1 protein expression determines niche-specific spread of Streptococcus pyogenes

Invasive infections with Streptococcus pyogenes account for significant mortality. Cell surface M protein is believed to be essential for S. pyogenes virulence, with critical roles in dissemination and immune evasion. In contrast, the role of secreted M protein in infection is less clear. Here, we assessed the role of M protein in streptococcal colonisation, persistence, and invasion in respiratory and soft tissue murine infection models, and report that while cell-surface M protein is required to establish nasopharyngeal infection, it does not drive invasive infection. Cell-surface M protein was important for binding of major host matrix proteins and growth in immune human blood, but soluble M protein was the predominant determinant of lymphatic survival, tissue spread, and systemic infection in mice. A mutant emm1 strain which only expresses secreted M protein exhibited enhanced survival in the lymph node niche and overall competitive advantage over the wildtype parent strain in co-infections. Absence of M protein at the bacterial cell surface led to enhanced hyaluronan binding, providing explanation for homing to the draining lymph node. Together, these findings highlight potential nuance in the role of M protein that may impact vaccine approaches targeting M protein. Author SummaryS. pyogenes is a significant cause of illness, both mild and lethal, worldwide. Despite this, there are currently no approved vaccines. Many vaccine candidates are based on the M protein, a surface expressed anti-phagocytic factor that is thought to be critical for the ability of S. pyogenes to evade the host immune system. The unexpected selection of an M1 protein mutant strain following in vivo soft tissue infection raised many questions for us. If M protein is meant to be the critical factor in avoiding immune killing, how was this strain able to persist? One explanation could be that, although the strain was negative for M1 protein on the bacterial surface, it still secreted a truncated version of the protein with potential inflammatory effects. Importantly, the strain was selected in the draining lymph node pointing to a niche-specific advantage. We showed that cell surface-associated M1 protein, although required to establish nasopharyngeal infection, was not required in soft tissue infection, and that expression of a soluble, truncated form of M1 protein gave a survival advantage over the wild-type strain expressing cell wall-associated M1 protein. Furthermore, the absence of cell surface M protein led to enhanced hyaluronan binding providing explanation for lymphatic niche homing.

microbiology↗

bioRxiv2025

Gene flow between environmental and enteric bacteria with environmental coselection for metal and antibiotic resistance genes: A case study

BackgroundThe global threat of antimicrobial resistance (AMR) is driven by multiple factors, including inadequate sanitation and the suboptimal use of antibiotics in healthcare. The environment is recognised as an important and underappreciated reservoir for AMR. Pollutants can exert selective pressure that co-selects for resistant phenotypes, while faecal contamination introduces enteric bacteria capable of acquiring resistance elements that provide a survival advantage. AimsThis study aimed to characterize multidrug-resistant (MDR) bacteria in sediment and water samples collected from an industrial site and an adjacent canal in 2023 and 2024. Selective agar plating was used to isolate MDR bacteria, which were subsequently subjected to whole-genome sequencing and analysed for the presence of antibiotic resistance genes (ARGs), metal resistance genes (MRGs), and plasmid content. ResultsForty-two MDR isolates were retrieved and sequenced over two seasons. Most were Gram negative and most originated from the industrial location; no MDR bacteria were found in a control location. Species included both environmental and Enterobacteriaceae, indicative of faecal contamination by wastewater, Of the 40 bacteria that possessed at least one ARG, 87.5% (35/40) also carried at least one or more MRG. ARG identified on mobile genetic elements included CTXM-15, bla-NDM1, bla-VIM1, other carbapenemases, and 16sRMTases. MRG encoded resistance to copper, silver, tellurium, arsenic, and mercury, while plasmid assemblies showed evidence of genetic exchange within the environmental ecosystem. Levels of mercury, vanadium, zinc and copper in sediments exceeded international guideline values. ConclusionIndustrial locations harbour a spectrum of pollutants that contribute to the development and spread of AMR. In this case study, heavy metals appear to act as a potentially major selective pressure driving the evolution of multidrug resistance through co-selection. The observed diversity of mobilizable ARGs and MRGs in the environment highlights the potential for such toxic environments to serve as incubators for the emergence of novel, high-risk bacterial clones.

microbiology↗

bioRxiv2025

Lymphatic dissemination drives systemic invasion by diverse clinically relevant extracellular bacterial pathogens

Extracellular bacterial pathogens cause an estimated 6 million deaths annually and drive antimicrobial resistance. Systemic bacterial infection is typically attributed to direct blood vessel invasion or intracellular transit, while draining lymph nodes are generally assumed to trap and eliminate extracellular bacteria to prevent onward lymphatic spread. However, we previously demonstrated that Streptococcus pyogenes disseminates extracellularly via the lymphatic system. Here, we show that lymphatic dissemination extends across diverse clinically important extracellular bacterial pathogens, including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus agalactiae. In a murine soft tissue infection model, clinical isolates reached local draining lymph nodes, sequential distant draining lymph nodes, and systemic organs. In contrast,non-draining lymph nodes contained few or no bacteria, even during bacteraemia, consistent with lymphatic spread rather than haematogenous seeding. Dissemination efficiency varied between species and among isolates, with S. aureus largely restricted to local draining lymph nodes. Notably, capsular polysaccharide enhanced lymphatic dissemination independently of bacterial burden at the infection site. These findings identify lymphatic dissemination as a common route of invasion by extracellular bacterial pathogens, exposing sequential draining lymph nodes to viable bacteria.

immunology↗

bioRxiv2023

Aminoglycoside heteroresistance in Enterobacter cloacae is driven by the cell envelope stress response.

Enterobacter cloacae is a Gram-negative nosocomial pathogen of the ESKAPE priority group with increasing multi-drug resistance via the acquisition of resistance plasmids. However, E. cloacae can also display phenotypic antimicrobial resistance, such as heteroresistance or persistence. Here we report that E. cloacae ATCC 13047 and six strains isolated from patients with blood infections display heteroresistance or persistence to aminoglycosides. E. cloacae heteroresistance is transient, accompanied with formation of petite colonies and increased MIC against gentamicin and other aminoglycosides used in the clinic, but not other antibiotic classes. To explore the underlying mechanisms, we performed RNA sequencing of heteroresistant bacteria, which revealed global gene-expression changes and a signature of the CpxRA cell envelope stress response. Deletion of the cpxRA two-component system abrogated aminoglycoside heteroresistance and petite colony formation, pointing to its indispensable role in phenotypic resistance. The introduction of a constitutively active allele of cpxA led to high aminoglycoside MICs, consistent with cell envelope stress driving these behaviours in E. cloacae. Cell envelope stress can be caused by environmental cues, including heavy metals. Indeed, bacterial exposure to copper increased gentamicin MIC in the wild type, but not the {Delta}cpxRA mutant. Moreover, copper exposure also elevated the gentamicin MICs of bloodstream isolates, suggesting that CpxRA- and copper-dependent aminoglycoside resistance is broadly conserved in E. cloacae strains. Altogether, we establish that E. cloacae relies on transcriptional reprogramming via the envelope stress response pathway for transient resistance to a major class of frontline antibiotic. ImportanceEnterobacter cloacae is a bacterium that belongs to the ESKAPE priority group and an increasing threat worldwide due its multidrug resistance. E. cloacae can also display phenotypic resistance to antibiotics, leading to treatment failure. We report that sensitive strains of E. cloacae, including six strains isolated from patients with bloodstream infections, show heteroresistance or persistence to aminoglycoside antibiotics. These are important frontline microbicidal drugs used against Gram-negative bacterial infections, therefore understanding how resistance develops in sensitive strains is important. We show that aminoglycoside resistance is driven by the activation of the cell envelope stress response and transcriptional reprogramming via the CpxRA two-component system. Further, heterologous activation of envelope stress via copper, typically a heavy metal with antimicrobial actions, also increased aminoglycoside MICs in all tested strains of E. cloacae. Our study suggests phenotypic aminoglycoside resistance in E. cloacae could be broadly conserved and cautions against the undesirable effects of copper.

microbiology↗

bioRxiv2023

Characterization of the RofA regulon in the pandemic M1global and emergent M1UK lineages of Streptococcus pyogenes

Background & AimsThe standalone regulator RofA is a positive regulator of the pilus locus in Streptococcus pyogenes. Found in only certain emm genotypes, RofA has been reported to regulate other virulence factors, although its role in the globally dominant emm1 S. pyogenes is unclear. Given the recent emergence of a new emm1 (M1UK) toxigenic lineage that is distinguished by three non-synonymous SNPs in rofA, we characterized the rofA regulon in six emm1 strains, that are representative of the two contemporary major emm1 lineages (M1global and M1UK) using RNAseq analysis, and then determined the specific role of the M1UK-specific rofA SNPs. ResultsDeletion of rofA in three M1global strains led to altered expression of 14 genes, including six non-pilus locus genes. In M1UK strains, deletion of rofA led to altered expression of 16 genes, including 9 genes that were unique to M1UK. Only the pilus locus genes were common to the RofA regulons of both lineages, while transcriptomic changes varied between strains even within the same lineage. Although introduction of the 3 SNPs into rofA did not impact gene expression in an M1global strain, reversal of 3 SNPs in an M1UK strain led to an unexpected number of transcriptomic changes that in part recapitulated transcriptomic changes seen when deleting RofA in the same strain. Computational analysis predicted interactions with a key histidine residue in the PRD domain of RofA would differ between M1UK and M1global. SummaryRofA is a positive regulator of the pilus locus in all emm1 strains but effects on other genes are strain- and lineage-specific, with no clear, common DNA binding motif. The SNPs in rofA that characterize M1UK may impact regulation of RofA; whether they alter phosphorylation of the RofA PRD domain requires further investigation. Author summaryRofA belongs to the group of "mga-like" bacterial regulatory proteins that comprise a DNA binding domain as well as a phosphorylation domain (PRD) that is responsive to changes in sugar availability. In certain emm genotypes of Streptococcus pyogenes, rofA sits upstream of the pilus locus, to act as a positive regulator. The recent emergence of a SpeA exotoxin-producing sublineage of emm1 S. pyogenes, (M1UK) has focused attention on the role of RofA; M1UK and its associated sublineages are characterized by 3 non-synonymous SNPs in rofA, that include adjacent SNPs in the PRD domain. Here, we determine the impact of rofA deletion and the 3 rofA SNPs in both the widely disseminated M1global clone and the newly emergent M1UK clone. While production of SpeA undoubtedly contributes to infection pathogenesis, the evolution of M1UK points to a role for metabolic regulatory rewiring in success of this lineage.

microbiology↗

bioRxiv2023

Tricontinental detection of Streptococcus pyogenes M1UK: A call for wider research and active surveillance.

The Streptococcus pyogenes M1UK lineage, characterised by an intrinsic ability to express SpeA toxin and defined by 27 single nucleotide polymorphisms in the core genome, dominates the population of emm1 S. pyogenes isolates throughout Europe, Canada, South America, Japan and Oceania. While not the sole deterministic factor, enhanced SpeA expression is likely to have contributed to M1UK lineage expansion, but was not sufficient to support expansion of intermediate lineage M123SNP, that expresses SpeA at a similar level to M1UK. A single nucleotide polymorphism (SNP) in the ssrA leader sequence upstream of speA is one of a limited number of SNPs that distinguish intermediate sublineages that differ in SpeA production. It was recently shown that this SNP leads to increased ssrA terminator read-through, and consequent increased transcription of speA, which lies downstream of ssrA. In this work, introduction of the ssrA SNP into representative isolates of the widely disseminated M1global clone and the intermediate M113SNP lineage, that cannot otherwise produce readily-detectable SpeA in culture, resulted in SpeA expression, confirming the importance of the ssrA SNP to SpeA phenotype. Consistent with this, correction of the ssrA SNP in M1UK abrogated SpeA expression. However, RNAseq analysis of 8 emm1 clinical pharyngitis strains showed that presence of the SNP was not invariably linked to read-through from the ssrA leader sequence or SpeA expression. Read-through was observed in isolates that did not possess the ssrA SNP. Critical review of existing data suggests that speA mRNA transcript length may be impacted by the two-component regulator CovRS, pointing to a complex regulatory network interaction between the bacterial chromosome and phage-encoded superantigens.

microbiology↗

bioRxiv2023

IS1-related large-scale deletion of chromosomal regions harbouring oxygen-insensitive nitroreductase gene nfsB causes nitrofurantoin heteroresistance in Escherichia coli

Nitrofurantoin is a broad-spectrum first-line antimicrobial used for managing uncomplicated urinary tract infection. Loss-of-function mutations in chromosomal genes nfsA, nfsB, and ribE of Escherichia coli are known to reduce nitrofurantoin susceptibility. Here, we report monoclonal nitrofurantoin heteroresistance in E. coli and a novel genetic mechanism associated with this phenomenon. Subpopulations with reduced nitrofurantoin susceptibility in cultures of two E. coli blood strains were identified using population analysis profiling. Four colonies of each strain growing on agar with 0.5xMIC nitrofurantoin were sub-cultured in broth with 0.5xMIC nitrofurantoin (n=2) or without nitrofurantoin (n=2). Moreover, one colony of each strain growing without nitrofurantoin exposure was selected as a reference for genomic comparison. Whole-genome sequencing of all isolates were conducted on Illumina and Nanopore MinION systems. Both strains had a nitrofurantoin MICs of 64 mg/L. The proportion of cells grown at 0.5xMIC was two and 99 per million, respectively, which is distinct to that of a homogeneously susceptible or resistant isolate. All isolates grown at 0.5xMIC had 11-66 kbp deletions in chromosomal regions harbouring nfsB, and all these deletions were immediately adjacent to IS1-family insertion sequences. Although this study is limited to E. coli and nitrofurantoin, our findings suggest IS1-associated genetic deletion represents a hitherto unrecognised mechanism of heteroresistance that could compromise infection management and impact conventional antimicrobial susceptibility testing. Impact statementNitrofurantoin is widely used for treating and preventing urinary tract infection. Prevalence of nitrofurantoin resistance generally is low in E. coli. Here, we report nitrofurantoin heteroresistance in two E. coli blood strains and attribute this phenotype to IS1-associated deletion of chromosomal regions harbouring oxygen-insensitive nitroreductase gene nfsB. Our discoveries demonstrate a novel genetic mechanism of heteroresistance and suggest detecting nitrofurantoin heteroresistance in E. coli urinary isolates for improving prescribing. Data summaryWhole-genome sequencing reads and genome assemblies generated in this study have been deposited under BioProject PRJEB58678 in the European Nucleotide Archive (ENA). Accession numbers are listed in Supplementary Table 1. Previously generated Illumina whole-genome sequencing reads of parental isolates EC0026B and EC0880B are available under ENA accessions ERR3142418 and ERR3142524, respectively.

microbiology↗

bioRxiv2022

Characterisation of emergent toxigenic M1UK Streptococcus pyogenes and associated sublineages

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 (M113SNPs) and 23/27 SNPs (M123SNPs) 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 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. Data availabilityRNAseq. All new RNAseq data are uploaded to the European Nucleotide Archive under project reference PRJEB58303 Genomic data. All genomes listed are available on the European Nucleotide Archive using accession numbers as listed in the appendix, Proteomes. Proteomic data are available on FigShare 10.6084/m9.figshare.21777809 and will be uploaded to PRIDE Impact SummaryAlthough the major Streptococcus pyogenes reservoir is in children with pharyngitis and skin infections, S. pyogenes can lead to rarer, invasive infections that are rapidly progressive and associated with high mortality and morbidity. Emm1 S. pyogenes strains are the single most frequent genotype to cause invasive infections in high income countries and are established worldwide as an epidemic clone. The M1UK S. pyogenes emm1 sublineage which is defined by 27 new SNPs in the core genome, and characterised by increased scarlet fever toxin SpeA production, emerged and rose to dominance over a period of 5-6 years since initial recognition, outcompeting other emm1 strains in England. Increased dominance of emm1 among invasive infections this winter, on a background of already-increased numbers of S. pyogenes infections, points to a key shift in host-pathogen interaction. We hypothesize that a combination of pathogen fitness, virulence, and host susceptibility have coalesced to account for the excess of circulating S. pyogenes and emm1 invasive infections. In this paper we undertake a systems-based evaluation of M1UK in comparison to older non-M1UK emm1 strains, and identify a number of pathways that are altered in addition to the previously-reported increased SpeA expression. The emergence of a new sublineage within an already virulent clone requires ongoing surveillance, and more detailed investigation of the likely mechanisms leading to increased fitness. The capacity of S. pyogenes to cause outbreaks at national scale highlights a potential need to consider strain-specific public health guidance, underlining the inherent virulence of this exclusively human pathogen.

microbiology↗

bioRxiv2022

Ongoing emergence of M1UK lineage among invasive group A streptococcus isolates in 2020 and use of allele-specific PCR

BackgroundAn increasing burden of invasive group A streptococcal infections is reported in multiple countries, notably England, where scarlet fever cases are also abundant. In England, increased scarlet fever and invasive infections have been associated with emergence of a sublineage of emm1 Streptococcus pyogenes that expresses increased SpeA scarlet fever erythrogenic toxin. Wider surveillance for toxigenic Streptococcus pyogenes lineage M1UK is much needed however, to date, lineage assignment has required genome sequencing limiting surveillance to those centres with access to such facilities. MethodsTo circumvent the requirement for genome sequencing, an allele-specific PCR was developed to distinguish M1UK from other emm1 strains. Additional PCR assays were developed to distinguish M1UK from two intermediate lineages that were detected previously. The assay was evaluated using DNA from genome-sequenced upper respiratory tract emm1 S. pyogenes strains and a further set of 16 genome-sequenced invasive S. pyogenes isolates that included the two intermediate lineages. The assay was then applied to DNA from all 305 invasive emm1 isolates that had been submitted to the reference laboratory in the one pear period Jan 1-Dec 31 2020, in order to assign lineage. ResultsThe allele specific PCR was 100% accurate when compared with genome sequencing, correctly identifying M1UK, two intermediate sublineages, and other emm1 strains. The assay demonstrated the M1UK lineage to be dominant among emm1 invasive isolates in England, representing 278/305 (91%) of invasive emm1 isolates by end of 2020. ImplicationsEmm1 S. pyogenes have a prominent role in invasive infections; any emm1 lineage that demonstrates enhanced fitness within the population is of public health concern. The allele specific PCR provides a readily available method to subtype emm1 isolates and does not require access to complex sequencing facilities. The data confirm that the M1UK lineage has persisted and further expanded in England underlining the importance of wider global surveillance.

microbiology↗

bioRxiv2021

Non-conventional serine protease activity of the CXC chemokine-cleaving streptococcal enzyme, SpyCEP

The Streptococcus pyogenes cell envelope protease (SpyCEP) is vital to streptococcal pathogenesis and disease progression. Despite its strong association with invasive disease, little is known about enzymatic function beyond the ELR+ CXC chemokine substrate range. As a serine protease, SpyCEP has a catalytic triad consisting of aspartate (D151), histidine (H279), and serine (S617) residues which are all thought to be mandatory for full activity. We utilised a range of SpyCEP constructs to investigate the protein domains and catalytic residues necessary for enzyme function. We designed a high-throughput mass spectrometry assay to measure CXCL8 cleavage and applied this for the first time to study the enzyme kinetics of SpyCEP. Results revealed a remarkably low Michaelis-Menton constant (KM) of 82 nM and a turnover of 1.65 molecules per second. We found that an N-terminally-truncated SpyCEP C-terminal construct containing just the catalytic dyad of H279 and S617 was capable of cleaving CXCL8 with a similar KM of 55 nM, albeit with a reduced substrate turnover of 2.7 molecules per hour, representing a 2,200- fold reduction in activity. We conclude that the SpyCEP C-terminus plays a key role in high affinity substrate recognition and binding, but that the N-terminus is required for full catalytic activity.

microbiology↗

bioRxiv2021

Diverse Genetic Determinants of Nitrofurantoin Resistance in UK Escherichia coli

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 2018. 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 genetic determinants of nitrofurantoin resistance in a local E. coli collection and assess their prevalence in a larger dataset of E. coli genomes. Deleterious point mutations and gene-inactivating insertion sequences in both chromosomal nitroreductase genes nfsA and nfsB were identified in genomes of nine nitrofurantoin-resistant urinary E. coli isolates collected from north west London. Eight types of genetic alterations were identified when comparing sequences of nfsA, nfsB, and the associated gene ribE in 12,412 E. coli genomes collected from across the UK. Evolutionary analysis revealed homoplasic mutations and explained the order of stepwise mutations. An algorithm was developed to predict nitrofurantoin susceptibility and predictions for 20 accessible isolates were experimentally validated. Only one genome carrying oqxAB, a mobile gene complex associated with reduced nitrofurantoin susceptibility, was identified. In conclusion, mutations and insertion sequences in nfsA and nfsB are 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. ImportanceThis study expands knowledge about the genetic basis of nitrofurantoin resistance in E. coli isolates using whole-genome sequencing and genomic analysis. We report novel and previously known deleterious mutations of chromosomal genes nfsA, nfsB, and ribE as well as the interruption of nfsA and nfsB by insertion sequences, recapitulating the roles of oxygen-insensitive nitroreductases in the development of nitrofurantoin resistance in E. coli. We revealed and categorised the genotypic diversity in these three genes in a large collection of UK E. coli genomes. A scoring algorithm is provided to predict nitrofurantoin susceptibility from genotypes. Our predictions suggest that acquired nitrofurantoin resistance is not of immediate concern in the UK. However, experimental validation of predictions suggested the involvement of mechanisms other than alterations in nfsA, nfsB, or ribE in determining nitrofurantoin susceptibility, emphasising the need for monitoring nitrofurantoin resistance amongst E. coli.

microbiology↗

bioRxiv2020

Vaccine-induced, but not natural immunity, against the Streptococcal Inhibitor of Complement protects against invasive disease

Highly pathogenic emm1 Streptococcus pyogenes strains secrete the multidomain Streptococcal inhibitor of complement (SIC) that binds and inactivates components of the innate immune response. We aimed to determine if naturally occurring or vaccine-induced antibodies to SIC are protective against invasive S. pyogenes infection. Immunisation with full length SIC protected mice against systemic bacterial dissemination following intranasal or intramuscular infection with emm1 S. pyogenes. Vaccine-induced rabbit anti-SIC antibodies, but not naturally occurring human anti-SIC antibodies, enhanced bacterial clearance in an ex vivo whole blood assay. SIC vaccination of both mice and rabbits resulted in antibody recognition of all domains of SIC, whereas naturally occurring human anti-SIC antibodies recognised the proline-rich region of SIC only. We therefore propose a model whereby natural infection with S. pyogenes generates non-protective antibodies against the proline-rich region of SIC, while vaccination with full length SIC permits development of protective antibodies against all SIC domains.

microbiology↗

bioRxiv2019

The emergence of successful Streptococcus pyogenes lineages through convergent pathways of capsule loss and recombination directing high toxin expression

Gene transfer and homologous recombination in Streptococcus pyogenes has the potential to trigger the emergence of pandemic lineages, as exemplified by lineages of emm1 and emm89 that emerged in the 1980s and 2000s respectively. Although near-identical replacement gene transfer events in the nga (NADase) and slo (Streptolysin O) locus conferring high expression of these toxins underpinned the success of these lineages, extension to other emm-genotype lineages is unreported. The emergent emm89 lineage was characterised by five regions of homologous recombination additional to nga/slo, including complete loss of the hyaluronic acid capsule synthesis locus hasABC, a genetic trait replicated in two other leading emm types and recapitulated by other emm types by inactivating mutations. We hypothesised that other leading genotypes may have undergone a similar recombination events. We analysed a longitudinal dataset of genomes from 344 clinical invasive disease isolates representative of locations across England, dating from 2001 to 2011, and an international collection of S. pyogenes genomes representing 54 different genotypes, and found frequent evidence of recombination events at the nga-slo locus predicted to confer higher toxin expression. We identified multiple associations between recombination at this locus and inactivating mutations within hasA/B, suggesting convergent evolutionary pathways in successful genotypes. This included common genotypes emm28 and emm87. The combination of no or low capsule, and high expression of nga and slo, may underpin the success for many emergent S. pyogenes lineages of different genotypes, triggering new pandemics and could change the way S. pyogenes causes disease.\n\nImportanceStreptococcus pyogenes is a genetically diverse pathogen, with over 200 different genotypes defined by emm typing, but only a minority of these genotypes are responsible for majority of human infection in high income countries. Two prevalent genotypes associated with disease rose to international dominance following recombination of a toxin locus that conferred increased expression. Here, we found that recombination of this locus and promoter has occurred in other diverse genotypes, events that may allow these genotypes to expand in the population. We identified an association between the loss of hyaluronic acid capsule synthesis and high toxin expression, which we propose may be associated with an adaptive advantage. As S. pyogenes pathogenesis depends both on capsule and toxin production, new variants with altered expression may result in abrupt changes in the molecular epidemiology of this pathogen in the human population over time.

microbiology↗

bioRxiv2019

Diversity of Escherichia coli from the enteric microbiota in patients with Escherichia coli bacteraemia

ObjectiveThe increase in Escherichia coli bloodstream infections mandates better characterisation of the relationship between commensal and invasive isolates. This study established a simple approach to characterize diversity of E. coli in the gut reservoir from patients with either E. coli bacteraemia, other Gram-negative bacteraemia, or patients without bacteraemia not receiving antibiotics. Stool or rectal swabs from patients in the three groups were cultured on selective chromogenic agar. Genetic diversity of E. coli in gut microbiota was estimated by RAPD-PCR. ResultsEnteric samples from E. coli bacteraemia patients yielded a median of one E. coli RAPD pattern (range 1-4) compared with two (range 1-5) from groups without E. coli bacteraemia. Of relevance to large-scale clinical studies, observed diversity of E. coli among E. coli bacteraemia patients was not significantly altered by sample type (rectal swab or stool), nor by increasing the colonies tested beyond ten. Overall, hospitalised patients demonstrated an apparently limited diversity of E. coli in the enteric microbiota and this was further reduced in those with E. coli bacteraemia. The reduced diversity of E. coli within the gut during E. coli bacteraemia raises the possibility that dominant strains may outcompete other lineages in patients with bloodstream infection.

microbiology↗

bioRxiv2019

Elevated risk of invasive group A streptococcal disease and host genetic variation in the human leukocyte antigen locus

Invasive group A streptococcal (GAS) disease is uncommon but carries a high case-fatality rate relative to other infectious diseases. Given the ubiquity of mild GAS infections, it remains unclear why healthy individuals will occasionally develop life-threatening infections, raising the possibility of host genetic predisposition. Here, we present the results of a case-control study including 43 invasive GAS cases and 1,540 controls. Using HLA imputation and linear mixed-models, we find each copy of the HLA-DQA1*01:03 allele associates with a two-fold increased risk of disease (odds ratio 2.3, 95% confidence interval 1.3-4.4, P=0.009), an association which persists with classical HLA typing of a subset of cases and analysis with an alternative large control dataset with validated HLA data. Moreover, we propose the association is driven by the allele itself rather than the background haplotype. Overall this finding provides impetus for further investigation of the immunogenetic basis of this devastating bacterial disease.

genetics↗

bioRxiv2018

A requirement for neutrophil glycosaminoglycans in the navigation of chemokine gradients is revealed by the streptococcal protease SpyCEP

To evade the immune system, the lethal human pathogen Streptococcus pyogenes produces SpyCEP, an enzyme that cleaves the C-terminal -helix of CXCL8, resulting in markedly impaired recruitment of neutrophils to sites of invasive infection. The basis for chemokine inactivation by SpyCEP is, however, poorly understood, as the core domain of CXCL8 known to interact with CXCL8 receptors is unaffected by enzymatic cleavage.\n\nWe examined the in vitro migration of human neutrophils and observed that their ability to efficiently navigate a CXCL8 gradient was compromised following CXCL8 cleavage by SpyCEP. SpyCEP-mediated cleavage of CXCL8 also impaired CXCL8-induced migration of transfectants expressing the human chemokine receptors CXCR1 or CXCR2. Despite possessing an intact N-terminus and preserved disulphide bonds, SpyCEP-cleaved CXCL8 had impaired binding to both CXCR1 and CXCR2, pointing to a requirement for the C terminal -helix. SpyCEP-cleaved CXCL8 had similarly impaired binding to the glycosaminoglycan heparin. Enzymatic removal of neutrophil glycosaminoglycans was observed to ablate neutrophil navigation of a CXCL8 gradient, whilst navigation of an fMLP gradient remained largely intact.\n\nWe conclude therefore, that SpyCEP cleavage of CXCL8 results in chemokine inactivation due to a requirement for glycosaminoglycan binding in productive chemokine:receptor interactions. This may inform strategies to inhibit the activity of SpyCEP, but may also influence future approaches to inhibit unwanted chemokine-induced inflammation.

immunology↗

bioRxiv2018

Streptococcal superantigen-induced expansion of human tonsil T cells leads to altered T follicular helper cell phenotype, B cell death, and reduced immunoglobulin release

BackgroundStreptococcal pyrogenic exotoxin (SPE)A expression is epidemiologically linked to streptococcal tonsillo-pharyngitis and outbreaks of scarlet fever, although the mechanisms by which superantigens confer advantage Streptococcus pyogenes are unclear. S. pyogenes is an exclusively human pathogen. As the leukocyte profile of tonsil differs from peripheral blood, the impact of SPEA production on human tonsil cell function was investigated.\n\nMethodsHuman tonsil cells from routine tonsillectomy, were co-incubated with purified streptococcal superantigens or isogenic streptococcal culture supernatants, differing only in superantigen content. Tonsil cell proliferation was quantified by tritium-incorporation, and cell surface characteristics assessed by flow-cytometry. Soluble mediators were measured using ELISA and quantitative (q)RT-PCR was performed for immunoglobulin gene expression.\n\nResultsTonsil T cells proliferated in response to SPEA and demonstrated typical release of pro-inflammatory cytokines. When cultured in the absence of superantigen, tonsil preparations released large quantities of immunoglobulin over 7d. In contrast, marked B cell apoptosis and abrogation of total IgA, IgM, and IgG production occurred in the presence of SPEA and other superantigens. In SPEA-stimulated cultures, T follicular helper (TfH) cells showed a reduction in CXCR5 expression, but up-regulation of CD134 (OX40), CD278 (ICOS) and CD150 (SLAM) expression, indicative of a phenotypic change in the TfH population and associated with impaired chemotactic response to CXCL13.\n\nConclusionsSPEA and other superantigens cause dysregulated tonsil immune function, driving T cells from TFH to a proliferating phenotype, with resultant loss of B cells and immunoglobulin production, providing superantigen-producing bacteria with a likely survival advantage.

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