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Clostridioides difficile is an important human pathogen, for which there are very limited treatment options, primarily the glycopeptide antibiotic vancomycin. In recent years, vancomycin resistance has emerged as a serious problem in several gram-positive pathogens, but high-level resistance has yet to be reported for C. difficile, although it is not known if this is due to constraints upon resistance evolution in this species. Here, we show that resistance to vancomycin can evolve rapidly under ramping selection but is accompanied by fitness costs and pleiotropic trade-offs, including sporulation defects that would be expected to severely impact transmission. We identified 2 distinct pathways to resistance, both of which are predicted to result in changes to the muropeptide terminal D-Ala-D-Ala that is the primary target of vancomycin. One of these pathways involves a previously uncharacterised D,D-carboxypeptidase, expression of which is controlled by a dedicated two-component signal transduction system. Our findings suggest that while C. difficile is capable of evolving high-level vancomycin resistance, this outcome may be limited clinically due to pleiotropic effects on key pathogenicity traits. Moreover, our data identify potential mutational routes to resistance that should be considered in genomic surveillance.
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Antibacterianos , Clostridioides difficile , Resistência a Vancomicina , Vancomicina , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/genética , Clostridioides difficile/patogenicidade , Resistência a Vancomicina/genética , Vancomicina/farmacologia , Antibacterianos/farmacologia , Aptidão Genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Transdução de Sinais , Mutação , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/genéticaRESUMO
At the end of 2022 into early 2023, the UK Health Security Agency reported unusually high levels of scarlet fever and invasive disease caused by Streptococcus pyogenes (StrepA or group A Streptococcus). During this time, we collected and genome-sequenced 341 non-invasive throat and skin S. pyogenes isolates identified during routine clinical diagnostic testing in Sheffield, a large UK city. We compared the data with that obtained from a similar collection of 165 isolates from 2016 to 2017. Numbers of throat-associated isolates collected peaked in early December 2022, reflecting the national scarlet fever upsurge, while skin infections peaked later in December. The most common emm-types in 2022-2023 were emm1 (28.7â%), emm12 (24.9â%) and emm22 (7.7â%) in throat and emm1 (22â%), emm12 (10â%), emm76 (18â%) and emm49 (7â%) in skin. While all emm1 isolates were the M1UK lineage, the comparison with 2016-2017 revealed diverse lineages in other emm-types, including emm12, and emergent lineages within other types including a new acapsular emm75 lineage, demonstrating that the upsurge was not completely driven by a single genotype. The analysis of the capsule locus predicted that only 51â% of throat isolates would produce capsule compared with 78% of skin isolates. Ninety per cent of throat isolates were also predicted to have high NADase and streptolysin O (SLO) expression, based on the promoter sequence, compared with only 56% of skin isolates. Our study has highlighted the value in analysis of non-invasive isolates to characterize tissue tropisms, as well as changing strain diversity and emerging genomic features which may have implications for spillover into invasive disease and future S. pyogenes upsurges.
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Infecções Estreptocócicas , Streptococcus pyogenes , Streptococcus pyogenes/genética , Streptococcus pyogenes/classificação , Streptococcus pyogenes/isolamento & purificação , Humanos , Reino Unido , Infecções Estreptocócicas/microbiologia , Proteínas da Membrana Bacteriana Externa/genética , Antígenos de Bactérias/genética , Faringe/microbiologia , Escarlatina/microbiologia , Escarlatina/epidemiologia , Proteínas de Transporte/genética , Estreptolisinas/genética , Sequenciamento Completo do Genoma/métodos , Proteínas de Bactérias/genética , Filogenia , Criança , Adulto , NAD+ Nucleosidase/genética , NAD+ Nucleosidase/metabolismo , Pele/microbiologia , Pré-Escolar , MasculinoRESUMO
There is growing awareness of the unique etiology, biology, and clinical presentation of invasive lobular breast cancer (ILC), but additional research is needed to ensure translation of findings into management and treatment guidelines. We conducted a survey with input from breast cancer physicians, laboratory-based researchers, and patients to analyze the current understanding of ILC, and identify consensus research questions. 1774 participants from 66 countries respondents self-identified as clinicians (N = 413), researchers (N = 376), and breast cancer patients and advocates (N = 1120), with some belonging to more than one category. The majority of physicians reported being very/extremely (41%) to moderately (42%) confident in describing the differences between ILC and invasive breast cancer of no special type (NST). Knowledge of histology was seen as important (73%) and as affecting treatment decisions (51%), and most agreed that refining treatment guidelines would be valuable (76%). 85% of clinicians have never powered a clinical trial to allow subset analysis for histological subtypes, but the majority would consider it, and would participate in an ILC clinical trials consortium. The majority of laboratory researchers, reported being and very/extremely (48%) to moderately (29%) confident in describing differences between ILC and NST. They reported that ILCs are inadequately presented in large genomic data sets, and that ILC models are insufficient. The majority have adequate access to tissue or blood from patients with ILC. The majority of patients and advocates (52%) thought that their health care providers did not sufficiently explain the unique features of ILC. They identified improvement of ILC screening/early detection, and identification of better imaging tools as top research priorities. In contrast, both researchers and clinicians identified understanding of endocrine resistance and identifying novel drugs that can be tested in clinical trials as top research priority. In summary, we have gathered information from an international community of physicians, researchers, and patients/advocates that we expect will lay the foundation for a community-informed collaborative research agenda, with the goal of improving management and personalizing treatment for patients with ILC.
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BACKGROUND: Streptococcus pyogenes-related skin infections are increasingly implicated in the development of rheumatic heart disease (RHD) in lower-resourced settings, where they are often associated with scabies. The true prevalence of S. pyogenes-related pyoderma may be underestimated by bacterial culture. METHODS: A multiplex qPCR for S. pyogenes, Staphylococcus aureus and Sarcoptes scabiei was applied to 250 pyoderma swabs from a cross-sectional study of children <5 years in The Gambia. Direct PCR-based emm-typing was used to supplement previous whole genome sequencing (WGS) of cultured isolates. RESULTS: Pyoderma lesions with S. pyogenes increased from 51% (127/250) using culture to 80% (199/250) with qPCR. Compared to qPCR, the sensitivity of culture was 95.4% for S. pyogenes (95% CI 77.2-99.9) in samples with S. pyogenes alone (22/250, 9%), but 59.9% (95% CI 52.3-67.2) for samples with S. aureus co-infection (177/250, 71%). Direct PCR-based emm-typing was successful in 50% (46/92) of cases, identifying 27 emm-types, including six not identified by WGS (total 52 emm-types). CONCLUSIONS: Bacterial culture significantly underestimates the burden of S. pyogenes in pyoderma, particularly when co-infected with S. aureus. Molecular methods should be used to enhance the detection of S. pyogenes in surveillance studies and clinical trials of preventative measures in RHD-endemic settings.
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BACKGROUND: Streptococcus pyogenes causes more than 500 000 deaths per year globally, which occur disproportionately in low-income and middle-income countries. The roles of S pyogenes skin and pharyngeal carriage in transmission are unclear. We aimed to investigate the clinical epidemiology and household transmission dynamics of both S pyogenes asymptomatic carriage and infection in a high-burden setting. METHODS: We did a 1-year prospective, longitudinal, household cohort study, recruiting healthy participants from households in Sukuta, The Gambia. Households were eligible if they comprised at least three members, including one child younger than 18 years, and were excluded if more than half of household members declined to participate. Households were identified by random GPS coordinates derived from census data. At monthly visits, pharyngeal and normal skin swabs were collected for S pyogenes culture, and sociodemographic data were recorded by interview. Incident pharyngitis and pyoderma infections were captured. Cultured isolates underwent emm genotyping. The primary outcome measures were incidence of S pyogenes carriage and disease. Additional outcomes were prevalence of S pyogenes skin and pharyngeal carriage, S pyogenes skin and pharyngeal clearance time, S pyogenes emm type, risk factors for carriage and disease events, household secondary attack rate, and emm-linked household transmission events. The study is registered on ClinicalTrials.gov, NCT05117528. FINDINGS: Between July 27, 2021, and Sept 28, 2022, 442 participants were enrolled from 44 households. The median age was 15 years (IQR 6-28) and 233 (53%) were female. We identified 17 pharyngitis and 99 pyoderma events and 49 pharyngeal and 39 skin S pyogenes carriage acquisition events. Mean monthly prevalence was 1·4% (95% CI 1·1-1·9) for S pyogenes pharyngeal carriage and 1·2% (0·9-1·6) for S pyogenes skin carriage. Incidence was 120 per 1000 person-years (95% CI 87-166) for S pyogenes pharyngeal carriage, 124 per 1000 person-years (90-170) for S pyogenes skin carriage, 51 per 1000 person-years (31-84) for S pyogenes pharyngitis, and 263 per 1000 person-years (212-327) for S pyogenes pyoderma. Pharyngeal carriage risk was higher during the rainy season (HR 5·67, 95% CI 2·19-14·69) and in larger households (per additional person: 1·03, 1·00-1·05), as was pharyngitis risk (rainy season: 3·00, 1·10-8·22; household size: 1·04, 1·02-1·07). Skin carriage risk was not affected by season or household size, but was lower in female than in male participants (0·45, 0·22-0·92) and highest in children younger than 5 years compared with adults (22·69, 3·08-167·21), with similar findings for pyoderma (female sex: 0·34, 0·19-0·61; age <5 years: 7·00, 2·78-17·64). Median clearance time after carriage acquisition was 4·0 days for both skin (IQR 3·5-7·0) and pharynx (3·5-7·3). The mean household secondary attack rate was 4·9 (95% CI 3·5-6·3) for epidemiologically linked S pyogenes events and 0·74 (0·3-1·2) for emm-linked S pyogenes events. Of the 204 carriage and disease events, emm types were available for 179 (88%). Only 18 emm-linked between-visit household transmission events were identified. Pyoderma was the most common source of S pyogenes household transmissions in 11 (61%) of 18 emm-linked transmissions. Both pharynx to skin and skin to pharynx transmission events were observed. INTERPRETATION: S pyogenes carriage and infection are common in The Gambia, particularly in children. Most events are non-household acquisitions, but skin carriage and pyoderma have an important role in S pyogenes household transmission and bidirectional transmission between skin and pharynx occurs. FUNDING: Wellcome Trust, Chadwick Trust, Fonds National de la Recherche Scientifique (Belgium), European Society for Paediatric Infectious Diseases, and Medical Research Council (UK).
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Portador Sadio , Características da Família , Faringe , Infecções Estreptocócicas , Streptococcus pyogenes , Humanos , Streptococcus pyogenes/isolamento & purificação , Gâmbia/epidemiologia , Feminino , Estudos Longitudinais , Masculino , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/transmissão , Portador Sadio/epidemiologia , Portador Sadio/microbiologia , Criança , Adulto , Adolescente , Estudos Prospectivos , Adulto Jovem , Pré-Escolar , Faringe/microbiologia , Prevalência , Incidência , Fatores de Risco , Faringite/microbiologia , Faringite/epidemiologia , Pele/microbiologia , Estudos de Coortes , Pioderma/epidemiologia , Pioderma/microbiologia , Pessoa de Meia-Idade , LactenteRESUMO
Pathogenic loss-of-function variants in BGN, an X-linked gene encoding biglycan, are associated with Meester-Loeys syndrome (MRLS), a thoracic aortic aneurysm/dissection syndrome. Since the initial publication of five probands in 2017, we have considerably expanded our MRLS cohort to a total of 18 probands (16 males and 2 females). Segregation analyses identified 36 additional BGN variant-harboring family members (9 males and 27 females). The identified BGN variants were shown to lead to loss-of-function by cDNA and Western Blot analyses of skin fibroblasts or were strongly predicted to lead to loss-of-function based on the nature of the variant. No (likely) pathogenic missense variants without additional (predicted) splice effects were identified. Interestingly, a male proband with a deletion spanning the coding sequence of BGN and the 5' untranslated region of the downstream gene (ATP2B3) presented with a more severe skeletal phenotype. This may possibly be explained by expressional activation of the downstream ATPase ATP2B3 (normally repressed in skin fibroblasts) driven by the remnant BGN promotor. This study highlights that aneurysms and dissections in MRLS extend beyond the thoracic aorta, affecting the entire arterial tree, and cardiovascular symptoms may coincide with non-specific connective tissue features. Furthermore, the clinical presentation is more severe and penetrant in males compared to females. Extensive analysis at RNA, cDNA, and/or protein level is recommended to prove a loss-of-function effect before determining the pathogenicity of identified BGN missense and non-canonical splice variants. In conclusion, distinct mechanisms may underlie the wide phenotypic spectrum of MRLS patients carrying loss-of-function variants in BGN.
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Invasive lobular breast cancer (ILC) differs from invasive breast cancer of no special type in many ways. Evidence on treatment efficacy for ILC is, however, lacking. We studied the degree of documentation and representation of ILC in phase III/IV clinical trials for novel breast cancer treatments. Trials were identified on Pubmed and clinicaltrials.gov. Inclusion/exclusion criteria were reviewed for requirements on histological subtype and tumor measurability. Documentation of ILC was assessed and ILC inclusion rate, central pathology and subgroup analyses were evaluated. Inclusion restrictions concerning tumor measurability were found in 39/93 manuscripts. Inclusion rates for ILC were documented in 13/93 manuscripts and varied between 2.0 and 26.0%. No central pathology for ILC was reported and 3/13 manuscripts had ILC sub-analyses. ILC is largely disregarded in most trials with poor representation and documentation. The current inclusion criteria using RECIST v1.1, fall short in recognizing the unique non-measurable metastatic infiltration of ILC.
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INTRODUCTION: Thebaine is an alkaloid in poppy seeds that is neurotoxic to animals. Data on its clinical effects and toxicokinetics in people are minimal. In 2022, poppy seeds high in thebaine entered the Australian food market, and people consuming tea made from these poppy seeds developed poisoning. METHODS: Three patients who drank poppy seed tea and developed neuromuscular toxicity consented for thebaine to be quantitated in serial blood samples. Blood samples were analyzed by liquid chromatography with high-resolution mass spectrometry. RESULTS: Case 1: A man in his 60s presented with drowsiness, vomiting, malaise and myoclonus. He developed metabolic acidosis with hyperlactataemia, acute kidney injury requiring haemodialysis, convulsions, rhabdomyolysis, and was in the hospital for 18 days. The admission thebaine blood concentration was 2.1 mg/L, and the apparent elimination half-life was 14.8 h. Case 2: A man in his 30s presented with myoclonus, rigidity, vomiting, and dizziness. He developed metabolic acidosis with hyperlactataemia, acute kidney injury, and myalgias. The admission thebaine blood concentration was 4.1 mg/L, and the apparent elimination half-life was 11.6 h. Case 3: A man in his 30s presented with myoclonus, rigidity, clonus, diaphoresis, and abdominal pain. The admission thebaine blood concentration was 2.2 mg/L, and the apparent elimination half-life was 8.3 h. DISCUSSION: Neuromuscular toxicity, metabolic acidosis with hyperlactataemia, acute kidney injury, and gastrointestinal symptoms were prominent clinical features in these patients after drinking poppy seed tea. Effects persisted for days, and all survived, despite thebaine concentrations far exceeding those in published forensic reports, although human data are sparse. Compared to rats, the thebaine apparent elimination half-life is much longer in humans who develop symptoms at lower concentrations. CONCLUSIONS: Despite relatively high thebaine blood concentrations and moderate to severe poisoning, outcomes were favourable with early presentations. It is possible that acute kidney injury prolongs the apparent elimination half-life of thebaine.
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Acidose , Injúria Renal Aguda , Mioclonia , Papaver , Masculino , Humanos , Animais , Ratos , Tebaína/análise , Morfina , Papaver/química , Toxicocinética , Austrália , Sementes/química , Chá , Injúria Renal Aguda/induzido quimicamente , Vômito/induzido quimicamenteRESUMO
INTRODUCTION: Poppy seed tea is used for its opioid effects and contains multiple opium alkaloids, including morphine, codeine, papaverine, and thebaine. Animal studies indicate thebaine has strychnine-like properties, but there is limited literature describing human thebaine poisoning. We describe a cluster of acute thebaine poisoning in people ingesting tea made using poppy seeds with high thebaine content that entered the Australian food supply chain. METHODS: This is an observational study of patients poisoned after drinking poppy seed tea. Cases were identified by three prospective toxicovigilance systems: the Emerging Drug Network of Australia collaboration, the New South Wales Prescription, Recreational and Illicit Substance Evaluation program, and the Emerging Drugs Network of Australia Victoria study. We report characteristics of clinical toxicity in cases with reported ingestion of poppy seed tea and analytical confirmation of thebaine exposure. RESULTS: Forty cases presenting with multi-system toxicity following poppy seed tea ingestion were identified across seven Australian states/territories from November 2022 to January 2023. Blood testing in 23 cases confirmed high thebaine concentrations. All 23 were male (median age 35, range 16-71 years). All patients experienced muscle spasms. Rigidity was described in nine, convulsions in six, while rhabdomyolysis, acute kidney injury, and metabolic acidosis occurred in five patients. There were two cardiac arrests. The thebaine median admission blood concentration was 1.6 mg/L, with a range of 0.1-5.6 mg/L, and was the dominant opium alkaloid in all samples. Convulsions, acute kidney injury, metabolic acidosis, and cardiac arrest were associated with increasing median thebaine concentrations. Four patients were managed in the Intensive Care Unit, with two receiving continuous kidney replacement therapy (one also received intermittent haemodialysis) for kidney injury. There was one death. CONCLUSIONS: Thebaine toxicity, like strychnine poisoning, resulted in neuromuscular excitation characterized by muscle spasm, rigidity, and convulsions. Severe toxicity, including acute kidney injury, metabolic acidosis, and cardiac arrest, appears dose-dependent.
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Acidose , Injúria Renal Aguda , Parada Cardíaca , Papaver , Animais , Humanos , Masculino , Adolescente , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Feminino , Tebaína/análise , Ópio , Estudos Prospectivos , Estricnina , Morfina , Codeína , Sementes/química , Convulsões , Chá , VitóriaRESUMO
Epithelial colonization is a critical first step in bacterial pathogenesis. Staphylococcus aureus can utilize several host factors to associate with cells, including α5ß1 integrin and heparan sulfate proteoglycans, such as the syndecans. Here, we demonstrate that a partner protein of both integrins and syndecans, the host membrane adapter protein tetraspanin CD9, is essential for syndecan-mediated staphylococcal adhesion. Fibronectin is also essential in this process, while integrins are only critical for post-adhesion entry into human epithelial cells. Treatment of epithelial cells with CD9-derived peptide or heparin caused significant reductions in staphylococcal adherence, dependent on both CD9 and syndecan-1. Exogenous fibronectin caused a CD9-dependent increase in staphylococcal adhesion, whereas blockade of ß1 integrins did not affect adhesion but did reduce the subsequent internalization of adhered bacteria. CD9 disruption or deletion increased ß1 integrin-mediated internalization, suggesting that CD9 coordinates sequential staphylococcal adhesion and internalization. CD9 controls staphylococcal adhesion through syndecan-1, using a mechanism that likely requires CD9-mediated syndecan organization to correctly display fibronectin at the host cell surface. We propose that CD9-derived peptides or heparin analogs could be developed as anti-adhesion treatments to inhibit the initial stages of staphylococcal pathogenesis. IMPORTANCE Staphylococcus aureus infection is a significant cause of disease and morbidity. Staphylococci utilize multiple adhesion pathways to associate with epithelial cells, including interactions with proteoglycans or ß1 integrins through a fibronectin bridge. Interference with another host protein, tetraspanin CD9, halves staphylococcal adherence to epithelial cells, although CD9 does not interact directly with bacteria. Here, we define the role of CD9 in staphylococcal adherence and uptake, observing that CD9 coordinates syndecan-1, fibronectin, and ß1 integrins to allow efficient staphylococcal infection. Two treatments that disrupt this action are effective and may provide an alternative to antibiotics. We provide insights into the mechanisms that underlie staphylococcal infection of host cells, linking two known adhesion pathways together through CD9 for the first time.
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Infecções Estafilocócicas , Sindecana-1 , Humanos , Sindecana-1/genética , Fibronectinas/metabolismo , Adesão Celular , Integrinas , Proteínas de Membrana , Integrina beta1/metabolismo , Heparina , Tetraspaninas , Tetraspanina 29RESUMO
Antibody drug conjugates, a class of biotherapeutic proteins, have been extensively developed in recent years, resulting in new approvals and improved standard of care for cancer patients. Among the numerous strategies of conjugating cytotoxic payloads to monoclonal antibodies, insertion of a cysteine residue achieves a tightly controlled, site-specific drug to antibody ratio. Tailored analytical tools are required to direct the development of processes capable of manufacturing novel antibody scaffolds with the desired product quality. Here, we describe the development of a 12 min, mass-spectrometry-based method capable of monitoring four distinct quality attributes simultaneously: variations in the thiol state of the inserted cysteines, N-linked glycosylation, reduction of interchain disulfide bonds, and polypeptide fragmentation. This method provides new insight into the properties of the antibody intermediate and associated manufacturing processes. Oxidized thiol states are formed within the bioreactor, of which a variant containing an additional disulfide bond was produced and remained relatively constant throughout the fed-batch process; reduced thiol variants were introduced upon harvest. Nearly 20 percent of N-linked glycans contained sialic acid, substantially higher than anticipated for wildtype IgG1. Lastly, previously unreported polypeptide fragmentation sites were identified in the C239i constant domain, and the relationship between fragmentation and glycoform were explored. This work illustrates the utility of applying a high-throughput liquid chromatography-mass spectrometry multi-attribute monitoring method to support the development of engineered antibody scaffolds.
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Anticorpos Monoclonais , Imunoconjugados , Humanos , Anticorpos Monoclonais/química , Cromatografia Líquida/métodos , Imunoconjugados/química , Cisteína/química , Compostos de Sulfidrila , Dissulfetos/químicaRESUMO
BACKGROUND: Immunity to Streptococcus pyogenes in high burden settings is poorly understood. We explored S. pyogenes nasopharyngeal colonization after intranasal live attenuated influenza vaccine (LAIV) among Gambian children aged 24-59 months, and resulting serological response to 7 antigens. METHODS: A post hoc analysis was performed in 320 children randomized to receive LAIV at baseline (LAIV group) or not (control). S. pyogenes colonization was determined by quantitative polymerase chain reaction (qPCR) on nasopharyngeal swabs from baseline (day 0), day 7, and day 21. Anti-streptococcal IgG was quantified, including a subset with paired serum before/after S. pyogenes acquisition. RESULTS: The point prevalence of S. pyogenes colonization was 7%-13%. In children negative at day 0, S. pyogenes was detected at day 7 or 21 in 18% of LAIV group and 11% of control group participants (P = .12). The odds ratio (OR) for colonization over time was significantly increased in the LAIV group (day 21 vs day 0 OR, 3.18; P = .003) but not in the control group (OR, 0.86; P = .79). The highest IgG increases following asymptomatic colonization were seen for M1 and SpyCEP proteins. CONCLUSIONS: Asymptomatic S. pyogenes colonization appears modestly increased by LAIV, and may be immunologically significant. LAIV could be used to study influenza-S. pyogenes interactions. Clinical Trials Registration. NCT02972957.
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Vacinas contra Influenza , Influenza Humana , Humanos , Criança , Gâmbia/epidemiologia , Streptococcus pyogenes , Influenza Humana/epidemiologia , Influenza Humana/prevenção & controle , Vacinas Atenuadas , Imunoglobulina GRESUMO
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.
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Proteômica , Streptococcus pyogenes , Streptococcus pyogenes/genética , Filogenia , Antígenos de Bactérias/genética , InglaterraRESUMO
Life-threatening bacterial infections in women after childbirth, known as puerperal sepsis, resulted in classical epidemics and remain a global health problem. While outbreaks of puerperal sepsis have been ascribed to Streptococcus pyogenes, little is known about disease mechanisms. Here, we show that the bacterial R28 protein, which is epidemiologically associated with outbreaks of puerperal sepsis, specifically targets the human receptor CEACAM1. This interaction triggers events that would favor the development of puerperal sepsis, including adhesion to cervical cells, suppression of epithelial wound repair and subversion of innate immune responses. High-resolution structural analysis showed that an R28 domain with IgI3-like fold binds to the N-terminal domain of CEACAM1. Together, these findings demonstrate that a single adhesin-receptor interaction can drive the pathogenesis of bacterial sepsis and provide molecular insights into the pathogenesis of one of the most important infectious diseases in medical history.
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Infecção Puerperal , Sepse , Infecções Estreptocócicas , Feminino , Humanos , Gravidez , Adesinas Bacterianas/genética , Proteínas de Bactérias/genética , Infecção Puerperal/epidemiologia , Infecção Puerperal/microbiologia , Sepse/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenesRESUMO
Staphylococcus aureus is an opportunistic bacterial pathogen that often results in difficult-to-treat infections. One mechanism used by S. aureus to enhance survival during infection is the stringent response. This is a stress survival pathway that utilizes the nucleotides (p)ppGpp to reallocate bacterial resources, shutting down growth until conditions improve. Small colony variants (SCVs) of S. aureus are frequently associated with chronic infections, and this phenotype has previously been linked to a hyperactive stringent response. Here, we examine the role of (p)ppGpp in the long-term survival of S. aureus under nutrient-restricted conditions. When starved, a (p)ppGpp-null S. aureus mutant strain ((p)ppGpp0) initially had decreased viability. However, after 3 days we observed the presence and dominance of a population of small colonies. Similar to SCVs, these small colony isolates (p0-SCIs) had reduced growth but remained hemolytic and sensitive to gentamicin, phenotypes that have been tied to SCVs previously. Genomic analysis of the p0-SCIs revealed mutations arising within gmk, encoding an enzyme in the GTP synthesis pathway. We show that a (p)ppGpp0 strain has elevated levels of GTP, and that the mutations in the p0-SCIs all lower Gmk enzyme activity and consequently cellular GTP levels. We further show that in the absence of (p)ppGpp, cell viability can be rescued using the GuaA inhibitor decoyinine, which artificially lowers the intracellular GTP concentration. Our study highlights the role of (p)ppGpp in GTP homeostasis and underscores the importance of nucleotide signaling for long-term survival of S. aureus in nutrient-limiting conditions, such as those encountered during infections. IMPORTANCE Staphylococcus aureus is a human pathogen that upon invasion of a host encounters stresses, such as nutritional restriction. The bacteria respond by switching on a signaling cascade controlled by the nucleotides (p)ppGpp. These nucleotides function to shut down bacterial growth until conditions improve. Therefore, (p)ppGpp are important for bacterial survival and have been implicated in promoting chronic infections. Here, we investigate the importance of (p)ppGpp for long-term survival of bacteria in nutrient-limiting conditions similar to those in a human host. We discovered that in the absence of (p)ppGpp, bacterial viability decreases due to dysregulation of GTP homeostasis. However, the (p)ppGpp-null bacteria were able to compensate by introducing mutations in the GTP synthesis pathway that led to a reduction in GTP build-up and a rescue of viability. This study therefore highlights the importance of (p)ppGpp for the regulation of GTP levels and for long-term survival of S. aureus in restricted environments.
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The genetic etiologies of more than half of rare diseases remain unknown. Standardized genome sequencing and phenotyping of large patient cohorts provide an opportunity for discovering the unknown etiologies, but this depends on efficient and powerful analytical methods. We built a compact database, the 'Rareservoir', containing the rare variant genotypes and phenotypes of 77,539 participants sequenced by the 100,000 Genomes Project. We then used the Bayesian genetic association method BeviMed to infer associations between genes and each of 269 rare disease classes assigned by clinicians to the participants. We identified 241 known and 19 previously unidentified associations. We validated associations with ERG, PMEPA1 and GPR156 by searching for pedigrees in other cohorts and using bioinformatic and experimental approaches. We provide evidence that (1) loss-of-function variants in the Erythroblast Transformation Specific (ETS)-family transcription factor encoding gene ERG lead to primary lymphoedema, (2) truncating variants in the last exon of transforming growth factor-ß regulator PMEPA1 result in Loeys-Dietz syndrome and (3) loss-of-function variants in GPR156 give rise to recessive congenital hearing impairment. The Rareservoir provides a lightweight, flexible and portable system for synthesizing the genetic and phenotypic data required to study rare disease cohorts with tens of thousands of participants.
Assuntos
Estudo de Associação Genômica Ampla , Doenças Raras , Humanos , Doenças Raras/genética , Teorema de Bayes , Genótipo , Estudo de Associação Genômica Ampla/métodos , Fenótipo , Proteínas de MembranaRESUMO
Streptococcus pyogenes is a leading cause of human morbidity and mortality, especially in resource-limited settings. The development of a vaccine against S. pyogenes is a global health priority to reduce the burden of postinfection rheumatic heart disease. To support this, molecular characterization of circulating S. pyogenes isolates is needed. We performed whole-genome analyses of S. pyogenes isolates from skin and soft tissue infections in Sukuta, The Gambia, a low-income country (LIC) in West Africa where there is a high burden of such infections. To act as a comparator to these LIC isolates, skin infection isolates from Sheffield, United Kingdom (a high-income country [HIC]), were also sequenced. The LIC isolates from The Gambia were genetically more diverse (46 emm types in 107 isolates) than the HIC isolates from Sheffield (23 emm types in 142 isolates), with only 7 overlapping emm types. Other molecular markers were shared, including a high prevalence of the skin infection-associated emm pattern D and the variable fibronectin-collagen-T antigen (FCT) types FCT-3 and FCT-4. Fewer of the Gambian LIC isolates carried prophage-associated superantigens (64%) and DNases (26%) than did the Sheffield HIC isolates (99% and 95%, respectively). We also identified streptococcin genes unique to 36% of the Gambian LIC isolates and a higher prevalence (48%) of glucuronic acid utilization pathway genes in the Gambian LIC isolates than in the Sheffield HIC isolates (26%). Comparison to a wider collection of HIC and LIC isolate genomes supported our findings of differing emm diversity and prevalence of bacterial factors. Our study provides insight into the genetics of LIC isolates and how they compare to HIC isolates. IMPORTANCE The global burden of rheumatic heart disease (RHD) has triggered a World Health Organization response to drive forward development of a vaccine against the causative human pathogen Streptococcus pyogenes. This burden stems primarily from low- and middle-income settings where there are high levels of S. pyogenes skin and soft tissue infections, which can lead to RHD. Our study provides much needed whole-genome-based molecular characterization of isolates causing skin infections in Sukuta, The Gambia, a low-income country (LIC) in West Africa where infection and RHD rates are high. Although we identified a greater level of diversity in these LIC isolates than in isolates from Sheffield, United Kingdom (a high-income country), there were some shared features. There were also some features that differed by geographical region, warranting further investigation into their contribution to infection. Our study has also contributed data essential for the development of a vaccine that would target geographically relevant strains.
Assuntos
Cardiopatia Reumática , Infecções dos Tecidos Moles , Infecções Estreptocócicas , Humanos , Streptococcus pyogenes/genética , Infecções dos Tecidos Moles/epidemiologia , Infecções Estreptocócicas/microbiologia , Antígenos de Bactérias , GenômicaRESUMO
Colorectal symptoms are common but only infrequently represent serious pathology, including colorectal cancer (CRC). A large number of invasive tests are presently performed for reassurance. We investigated the feasibility of urinary volatile organic compound (VOC) testing as a potential triage tool in patients fast-tracked for assessment for possible CRC. A prospective, multi-center, observational feasibility study was performed across three sites. Patients referred to NHS fast-track pathways for potential CRC provided a urine sample that underwent Gas Chromatography-Mass Spectrometry (GC-MS), Field Asymmetric Ion Mobility Spectrometry (FAIMS), and Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) analysis. Patients underwent colonoscopy and/or CT colonography and were grouped as either CRC, adenomatous polyp(s), or controls to explore the diagnostic accuracy of VOC output data supported by an artificial neural network (ANN) model. 558 patients participated with 23 (4%) CRC diagnosed. 59% of colonoscopies and 86% of CT colonographies showed no abnormalities. Urinary VOC testing was feasible, acceptable to patients, and applicable within the clinical fast track pathway. GC-MS showed the highest clinical utility for CRC and polyp detection vs. controls (sensitivity = 0.878, specificity = 0.882, AUROC = 0.896) but it is labour intensive. Urinary VOC testing and analysis are feasible within NHS fast-track CRC pathways. Clinically meaningful differences between patients with cancer, polyps, or no pathology were identified suggesting VOC analysis may have future utility as a triage tool.