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Among the many oral streptococci, Streptococcus pneumoniae (Spn) stands out for the capacity of encapsulated strains to cause invasive infection. Spread beyond upper airways, however, is a biological dead end for the organism, raising the question of the benefits of expending energy to coat its surface in a thick layer of capsular polysaccharide (CPS). In this study, we compare mutants of two serotypes expressing different amounts of CPS and test these in murine models of colonization, invasion infection and transmission. Our analysis of the effect of CPS amount shows that Spn expresses a capsule of sufficient thickness to shield its surface from the deposition of complement and binding of antibody to underlying epitopes. While effective shielding is permissive for invasive infection, its primary contribution to the organism appears to be in the dynamics of colonization. A thicker capsule increases bacterial retention in the nasopharynx, the first event in colonization, and also impedes IL-17-dependent clearance during late colonization. Enhanced colonization is associated with increased opportunity for host-to-host transmission. Additionally, we document substantial differences in CPS amount among clinical isolates of three common serotypes. Together, our findings show that CPS amount is highly variable among Spn and could be an independent determinant affecting host interactions.
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Infecções Pneumocócicas , Streptococcus pneumoniae , Animais , Camundongos , Streptococcus pneumoniae/metabolismo , Streptococcus , Polissacarídeos/metabolismo , Nasofaringe/microbiologia , Nariz , Infecções Pneumocócicas/microbiologia , Cápsulas Bacterianas/genéticaRESUMO
Staphylococcus aureus (S. aureus) is a serious global pathogen that causes a diverse range of invasive diseases. S. aureus utilizes a family of pore-forming toxins, known as bi-component leukocidins, to evade the host immune response and promote infection. Among these is LukAB (leukocidin A/leukocidin B), a toxin that assembles into an octameric ß-barrel pore in the target cell membrane, resulting in host cell death. The established cellular receptor for LukAB is CD11b of the Mac-1 complex. Here, we show that hydrogen voltage-gated channel 1 is also required for the cytotoxicity of all major LukAB variants. We demonstrate that while each receptor is sufficient to recruit LukAB to the plasma membrane, both receptors are required for maximal lytic activity. Why LukAB requires two receptors, and how each of these receptors contributes to pore-formation remains unknown. To begin to resolve this, we performed an alanine scanning mutagenesis screen to identify mutations that allow LukAB to maintain cytotoxicity without CD11b. We discovered 30 mutations primarily localized in the stem domains of LukA and LukB that enable LukAB to exhibit full cytotoxicity in the absence of CD11b. Using crosslinking, electron microscopy, and hydroxyl radical protein footprinting, we show these mutations increase the solvent accessibility of the stem domain, priming LukAB for oligomerization. Together, our data support a model in which CD11b binding unlatches the membrane penetrating stem domains of LukAB, and this change in flexibility promotes toxin oligomerization.
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Proteínas de Bactérias , Leucocidinas , Staphylococcus aureus , Toxinas Biológicas , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Leucocidinas/genética , Leucocidinas/metabolismo , Leucocidinas/toxicidade , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Toxinas Biológicas/metabolismo , Mutação , Ligação Proteica/genética , Domínios Proteicos , Linhagem Celular , Células CHO , Cricetulus , AnimaisRESUMO
Staphylococcus aureus is a gram-positive pathogen that poses a major health concern, in part due to its large array of virulence factors that allow infection and evasion of the immune system. One of these virulence factors is the bicomponent pore-forming leukocidin LukAB. The regulation of lukAB expression is not completely understood, especially in the presence of immune cells such as human polymorphonuclear neutrophils (hPMNs). Here, we screened for transcriptional regulators of lukAB during the infection of primary hPMNs. We uncovered that PerR, a peroxide sensor, is vital for hPMN-mediated induction of lukAB and that PerR upregulates cytotoxicity during the infection of hPMNs. Exposure of S. aureus to hydrogen peroxide (H2O2) alone also results in increased lukAB promoter activity, a phenotype dependent on PerR. Collectively, our data suggest that S. aureus uses PerR to sense the H2O2 produced by hPMNs to stimulate the expression of lukAB, allowing the bacteria to withstand these critical innate immune cells.IMPORTANCEStaphylococcus aureus utilizes a diverse set of virulence factors, such as leukocidins, to subvert human neutrophils, but how these toxins are regulated is incompletely defined. Here, we identified the peroxide-sensitive repressor, PerR, as a required protein involved in the induction of lukAB in the presence of primary human neutrophils, a phenotype directly linked to the ability of PerR to sense H2O2. Thus, we show that S. aureus coordinates sensing and resistance to oxidative stress with toxin production to promote pathogen survival.
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Infecções Estafilocócicas , Staphylococcus aureus , Humanos , Neutrófilos , Peróxido de Hidrogênio/toxicidade , Peróxido de Hidrogênio/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Proteínas de Bactérias/metabolismo , Leucocidinas , Infecções Estafilocócicas/microbiologiaRESUMO
OBJECTIVE: Whereas genetic susceptibility for systemic lupus erythematosus (SLE) has been well explored, the triggers for clinical disease flares remain elusive. To investigate relationships between microbiota community resilience and disease activity, we performed the first longitudinal analyses of lupus gut-microbiota communities. METHODS: In an observational study, taxononomic analyses, including multivariate analysis of ß-diversity, assessed time-dependent alterations in faecal communities from patients and healthy controls. From gut blooms, strains were isolated, with genomes and associated glycans analysed. RESULTS: Multivariate analyses documented that, unlike healthy controls, significant temporal community-wide ecological microbiota instability was common in SLE patients, and transient intestinal growth spikes of several pathogenic species were documented. Expansions of only the anaerobic commensal, Ruminococcus (blautia) gnavus (RG) occurred at times of high-disease activity, and were detected in almost half of patients during lupus nephritis (LN) disease flares. Whole genome sequence analysis of RG strains isolated during these flares documented 34 genes postulated to aid adaptation and expansion within a host with an inflammatory condition. Yet, the most specific feature of strains found during lupus flares was the common expression of a novel type of cell membrane-associated lipoglycan. These lipoglycans share conserved structural features documented by mass spectroscopy, and highly immunogenic repetitive antigenic-determinants, recognised by high-level serum IgG2 antibodies, that spontaneously arose, concurrent with RG blooms and lupus flares. CONCLUSIONS: Our findings rationalise how blooms of the RG pathobiont may be common drivers of clinical flares of often remitting-relapsing lupus disease, and highlight the potential pathogenic properties of specific strains isolated from active LN patients.
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Microbioma Gastrointestinal , Lúpus Eritematoso Sistêmico , Nefrite Lúpica , Microbiota , Humanos , Microbioma Gastrointestinal/genética , Exacerbação dos Sintomas , Fezes , Nefrite Lúpica/genéticaRESUMO
Identifying resistance to antiretroviral drugs is crucial for ensuring the successful treatment of patients infected with viruses such as human immunodeficiency virus (HIV) or hepatitis C virus (HCV). In contrast to Sanger sequencing, next-generation sequencing (NGS) can detect resistance mutations in minority populations. Thus, genotypic resistance testing based on NGS data can offer novel, treatment-relevant insights. Since existing web services for analyzing resistance in NGS samples are subject to long processing times and follow strictly rules-based approaches, we developed geno2pheno[ngs-freq], a web service for rapidly identifying drug resistance in HIV-1 and HCV samples. By relying on frequency files that provide the read counts of nucleotides or codons along a viral genome, the time-intensive step of processing raw NGS data is eliminated. Once a frequency file has been uploaded, consensus sequences are generated for a set of user-defined prevalence cutoffs, such that the constructed sequences contain only those nucleotides whose codon prevalence exceeds a given cutoff. After locally aligning the sequences to a set of references, resistance is predicted using the well-established approaches of geno2pheno[resistance] and geno2pheno[hcv]. geno2pheno[ngs-freq] can assist clinical decision making by enabling users to explore resistance in viral populations with different abundances and is freely available at http://ngs.geno2pheno.org.
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Farmacorresistência Viral/genética , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Software , Genoma Viral/genética , Genótipo , Infecções por HIV/genética , Infecções por HIV/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MutaçãoRESUMO
Resistance to colistin, a polypeptide drug used as an agent of last resort for the treatment of infections caused by multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacteria, including carbapenem-resistant Enterobacteriaceae (CRE), severely limits treatment options and may even transform an XDR organism into one that is pan-resistant. We investigated the synergistic activity of colistin in combination with 19 antibiotics against a collection of 20 colistin-resistant Enterobacteriaceae isolates, 15 of which were also CRE. All combinations were tested against all strains using an inkjet printer-assisted digital dispensing checkerboard array, and the activities of those that demonstrated synergy by this method were evaluated against a single isolate in a time-kill synergy study. Eighteen of 19 combinations demonstrated synergy against two or more isolates, and the 4 most highly synergistic combinations (colistin combined with linezolid, rifampin, azithromycin, and fusidic acid) were synergistic against ≥90% of strains. Sixteen of 18 combinations (88.9%) that were synergistic in the checkerboard array were also synergistic in a time-kill study. Our findings demonstrate that colistin in combination with a range of antibiotics, particularly protein and RNA synthesis inhibitors, exhibits synergy against colistin-resistant strains, suggesting that colistin may exert a subinhibitory permeabilizing effect on the Gram-negative bacterial outer membrane even in isolates that are resistant to it. These findings suggest that colistin combination therapy may have promise as a treatment approach for patients infected with colistin-resistant XDR Gram-negative pathogens.
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Antibacterianos/farmacologia , Colistina/farmacologia , Enterobacteriaceae/efeitos dos fármacos , Azitromicina/farmacologia , Farmacorresistência Bacteriana Múltipla , Sinergismo Farmacológico , Ácido Fusídico/farmacologia , Linezolida/farmacologia , Testes de Sensibilidade Microbiana , Rifampina/farmacologiaRESUMO
In Mycobacterium tuberculosis proteins that are post-translationally modified with Pup, a prokaryotic ubiquitin-like protein, can be degraded by proteasomes. While pupylation is reversible, mechanisms regulating substrate specificity have not been identified. Here, we identify the first depupylation regulators: CoaX, a pseudokinase, and pantothenate, an essential, central metabolite. In a Δ coaX mutant, pantothenate synthesis enzymes were more abundant, including PanB, a substrate of the Pup-proteasome system. Media supplementation with pantothenate decreased PanB levels in a coaX and Pup-proteasome-dependent manner. In vitro , CoaX accelerated depupylation of Pupâ¼PanB, while addition of pantothenate inhibited this reaction. Collectively, we propose CoaX contributes to proteasomal degradation of PanB by modulating depupylation of Pupâ¼PanB in response to pantothenate levels. One Sentence Summary: A pseudo-pantothenate kinase regulates proteasomal degradation of a pantothenate synthesis enzyme in M. tuberculosis .
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We recently described the evolution of a community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 variant responsible for an outbreak of skin and soft tissue infections. Acquisition of a mosaic version of the Φ11 prophage (mΦ11) that increases skin abscess size was an early step in CA-MRSA adaptation that primed the successful spread of the clone. The present report shows how prophage mΦ11 exerts its effect on virulence for skin infection without encoding a known toxin or fitness genes. Abscess size and skin inflammation were associated with DNA methylase activity of an mΦ11-encoded adenine methyltransferase (designated pamA). pamA increased expression of fibronectin-binding protein A (fnbA; FnBPA), and inactivation of fnbA eliminated the effect of pamA on abscess virulence without affecting strains lacking pamA. Thus, fnbA is a pamA-specific virulence factor. Mechanistically, pamA was shown to promote biofilm formation in vivo in skin abscesses, a phenotype linked to FnBPA's role in biofilm formation. Collectively, these data reveal a novel mechanism-epigenetic regulation of staphylococcal gene expression-by which phage can regulate virulence to drive adaptive leaps by S. aureus.
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Depletion of microbiota increases susceptibility to gastrointestinal colonization and subsequent infection by opportunistic pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). How the absence of gut microbiota impacts the evolution of MRSA is unknown. The present report used germ-free mice to investigate the evolutionary dynamics of MRSA in the absence of gut microbiota. Through genomic analyses and competition assays, we found that MRSA adapts to the microbiota-free gut through sequential genetic mutations and structural changes that enhance fitness. Initially, these adaptations increase carbohydrate transport; subsequently, evolutionary pathways largely diverge to enhance either arginine metabolism or cell wall biosynthesis. Increased fitness in arginine pathway mutants depended on arginine catabolic genes, especially nos and arcC, which promote microaerobic respiration and ATP generation, respectively. Thus, arginine adaptation likely improves redox balance and energy production in the oxygen-limited gut environment. Findings were supported by human gut metagenomic analyses, which suggest the influence of arginine metabolism on colonization. Surprisingly, these adaptive genetic changes often reduced MRSA's antimicrobial resistance and virulence. Furthermore, resistance mutation, typically associated with decreased virulence, also reduced colonization fitness, indicating evolutionary trade-offs among these traits. The presence of normal microbiota inhibited these adaptations, preserving MRSA's wild-type characteristics that effectively balance virulence, resistance, and colonization fitness. The results highlight the protective role of gut microbiota in preserving a balance of key MRSA traits for long-term ecological success in commensal populations, underscoring the potential consequences on MRSA's survival and fitness during and after host hospitalization and antimicrobial treatment.
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The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H2O2, a crucial host defense against S. aureus. We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr increased both respiration and fermentation but decreased ATP levels and growth, suggesting that Δagr cells assume a hyperactive metabolic state in response to reduced metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of Δagr strains to lethal H2O2 doses. Increased survival of wild-type agr cells during H2O2 exposure required sodA, which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected Δagr cells from killing by H2O2. Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived "memory" of agr-mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient (Nox2-/-) mice. These results demonstrate the importance of protection that anticipates impending ROS-mediated immune attack. The ubiquity of quorum sensing suggests that it protects many bacterial species from oxidative damage.
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The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H2O2, a crucial host defense against S. aureus. We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr resulted in decreased ATP levels and growth, despite increased rates of respiration or fermentation at appropriate oxygen tensions, suggesting that Δagr cells undergo a shift towards a hyperactive metabolic state in response to diminished metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of Δagr strains to lethal H2O2 doses. Increased survival of wild-type agr cells during H2O2 exposure required sodA, which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected Δagr cells from killing by H2O2. Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived 'memory' of agr-mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient (Cybb-/-) mice. These results demonstrate the importance of protection that anticipates impending ROS-mediated immune attack. The ubiquity of quorum sensing suggests that it protects many bacterial species from oxidative damage.
Assuntos
Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica , Peróxido de Hidrogênio , Estresse Oxidativo , Percepção de Quorum , Staphylococcus aureus , Transativadores , Staphylococcus aureus/genética , Staphylococcus aureus/fisiologia , Staphylococcus aureus/metabolismo , Percepção de Quorum/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Animais , Transativadores/metabolismo , Transativadores/genética , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Camundongos , Infecções Estafilocócicas/microbiologia , Viabilidade Microbiana , Espécies Reativas de Oxigênio/metabolismo , Deleção de GenesRESUMO
Severe COVID-19 has been associated with coinfections with bacterial and fungal pathogens. Notably, patients with COVID-19 who develop Staphylococcus aureus bacteremia exhibit higher rates of mortality than those infected with either pathogen alone. To understand this clinical scenario, we collected and examined S. aureus blood and respiratory isolates from a hospital in New York City during the early phase of the pandemic from both SARS-CoV-2+ and SARS-CoV-2- patients. Whole genome sequencing of these S. aureus isolates revealed broad phylogenetic diversity in both patient groups, suggesting that SARS-CoV-2 coinfection was not associated with a particular S. aureus lineage. Phenotypic characterization of the contemporary collection of S. aureus isolates from SARS-CoV-2+ and SARS-CoV-2- patients revealed no notable differences in several virulence traits examined. However, we noted a trend toward overrepresentation of S. aureus bloodstream strains with low cytotoxicity in the SARS-CoV-2+ group. We observed that patients coinfected with SARS-CoV-2 and S. aureus were more likely to die during the acute phase of infection when the coinfecting S. aureus strain exhibited high or low cytotoxicity. To further investigate the relationship between SARS-CoV-2 and S. aureus infections, we developed a murine coinfection model. These studies revealed that infection with SARS-CoV-2 renders mice susceptible to subsequent superinfection with low cytotoxicity S. aureus. Thus, SARS-CoV-2 infection sensitizes the host to coinfections, including S. aureus isolates with low intrinsic virulence. IMPORTANCE: The COVID-19 pandemic has had an enormous impact on healthcare across the globe. Patients who were severely infected with SARS-CoV-2, the virus causing COVID-19, sometimes became infected with other pathogens, which is termed coinfection. If the coinfecting pathogen is the bacterium Staphylococcus aureus, there is an increased risk of patient death. We collected S. aureus strains that coinfected patients with SARS-CoV-2 to study the disease outcome caused by the interaction of these two important pathogens. We found that both in patients and in mice, coinfection with an S. aureus strain lacking toxicity resulted in more severe disease during the early phase of infection, compared with infection with either pathogen alone. Thus, SARS-CoV-2 infection can directly increase the severity of S. aureus infection.
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COVID-19 , Coinfecção , SARS-CoV-2 , Infecções Estafilocócicas , Staphylococcus aureus , COVID-19/complicações , COVID-19/microbiologia , Coinfecção/microbiologia , Coinfecção/virologia , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Infecções Estafilocócicas/microbiologia , Humanos , Animais , Camundongos , SARS-CoV-2/genética , Filogenia , Feminino , Cidade de Nova Iorque/epidemiologia , Masculino , Virulência , Pessoa de Meia-Idade , Sequenciamento Completo do Genoma , Bacteriemia/microbiologia , Modelos Animais de Doenças , IdosoRESUMO
Considering human immunodeficiency virus type 2 (HIV-2) phenotypic data and experience from HIV type 1 and from the follow-up of HIV-2-infected patients, a panel of European experts voted on a rule set for interpretation of mutations in HIV-2 protease, reverse transcriptase, and integrase and an automated tool for HIV-2 drug resistance analyses freely available on the Internet (http://www.hiv-grade.de).
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Infecções por HIV/virologia , HIV-2/classificação , Mutação , Fármacos Anti-HIV/farmacologia , Conferências de Consenso como Assunto , Farmacorresistência Viral , Europa (Continente) , Integrase de HIV/genética , Protease de HIV/genética , Transcriptase Reversa do HIV/genética , HIV-2/efeitos dos fármacos , HIV-2/enzimologia , HIV-2/genética , Humanos , Internet , Interface Usuário-ComputadorRESUMO
Mycobacterium tuberculosis is a major human pathogen and the causative agent of tuberculosis disease. M. tuberculosis is able to persist in the face of host-derived antimicrobial molecules nitric oxide (NO) and copper (Cu). However, M. tuberculosis with defective proteasome activity is highly sensitive to NO and Cu, making the proteasome an attractive target for drug development. Previous work linked NO susceptibility with the accumulation of para-hydroxybenzaldehyde (pHBA) in M. tuberculosis mutants with defective proteasomal degradation. In this study, we found that pHBA accumulation was also responsible for Cu sensitivity in these strains. We showed that exogenous addition of pHBA to wild-type M. tuberculosis cultures sensitized bacteria to Cu to a degree similar to that of a proteasomal degradation mutant. We determined that pHBA reduced the production and function of critical Cu resistance proteins of the regulated in copper repressor (RicR) regulon. Furthermore, we extended these Cu-sensitizing effects to an aldehyde that M. tuberculosis may face within the macrophage. Collectively, this study is the first to mechanistically propose how aldehydes can render M. tuberculosis susceptible to an existing host defense and could support a broader role for aldehydes in controlling M. tuberculosis infections. IMPORTANCE M. tuberculosis is a leading cause of death by a single infectious agent, causing 1.5 million deaths annually. An effective vaccine for M. tuberculosis infections is currently lacking, and prior infection does not typically provide robust immunity to subsequent infections. Nonetheless, immunocompetent humans can control M. tuberculosis infections for decades. For these reasons, a clear understanding of how mammalian immunity inhibits mycobacterial growth is warranted. In this study, we show aldehydes can increase M. tuberculosis susceptibility to copper, an established antibacterial metal used by immune cells to control M. tuberculosis and other microbes. Given that activated macrophages produce increased amounts of aldehydes during infection, we propose host-derived aldehydes may help control bacterial infections, making aldehydes a previously unappreciated antimicrobial defense.
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Mycobacterium tuberculosis , Tuberculose , Animais , Humanos , Mycobacterium tuberculosis/metabolismo , Cobre/farmacologia , Cobre/metabolismo , Aldeídos/metabolismo , Aldeídos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Tuberculose/microbiologia , Antibacterianos/farmacologia , Mamíferos/metabolismoRESUMO
Loss of antimicrobial proteins such as REG3 family members compromises the integrity of the intestinal barrier. Here, we demonstrate that overproduction of REG3 proteins can also be detrimental by reducing a protective species in the microbiota. Patients with inflammatory bowel disease (IBD) experiencing flares displayed heightened levels of secreted REG3 proteins that mediated depletion of Enterococcus faecium (Efm) from the gut microbiota. Efm inoculation of mice ameliorated intestinal inflammation through activation of the innate immune receptor NOD2, which was associated with the bacterial DL-endopeptidase SagA that generates NOD2-stimulating muropeptides. NOD2 activation in myeloid cells induced interleukin-1ß (IL-1ß) secretion to increase the proportion of IL-22-producing CD4+ T helper cells and innate lymphoid cells that promote tissue repair. Finally, Efm was unable to protect mice carrying a NOD2 gene variant commonly found in IBD patients. Our findings demonstrate that inflammation self-perpetuates by causing aberrant antimicrobial activity that disrupts symbiotic relationships with gut microbes.
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Anti-Infecciosos , Enterococcus faecium , Doenças Inflamatórias Intestinais , Animais , Camundongos , Imunidade Inata , Linfócitos , InflamaçãoRESUMO
Nosocomial infections caused by multidrug-resistant (MDR) Enterobacter cloacae complex (ECC) pathogens are on the rise. However, the virulence strategies employed by these pathogens remain elusive. Here, we study the interaction of ECC clinical isolates with human serum to define how this pathogen evades the antimicrobial action of complement, one of the first lines of host-mediated immune defense. We identified a small number of serum-sensitive strains, including Enterobacter hormaechei strain NR3055, which we exploited for the in vitro selection of serum-resistant clones. Comparative genomics between the serum-sensitive NR3055 strain and the isolated serum-resistant clones revealed a premature stop codon in the wzy gene of the capsular polysaccharide biosynthesis locus of NR3055. The complementation of wzy conferred serum resistance to NR3055, prevented the deposition of complement proteins on the bacterial surface, inhibited phagocytosis by human neutrophils, and rendered the bacteria virulent in a mouse model of peritonitis. Mice exposed to a nonlethal dose of encapsulated NR3055 were protected from subsequent lethal infections by encapsulated NR3055, whereas mice that were previously exposed to unencapsulated NR3055 succumbed to infection. Thus, capsule is a key immune evasion determinant for E. hormaechei, and it is a potential target for prophylactics and therapeutics to combat these increasingly MDR human pathogens. IMPORTANCE Infections caused by antimicrobial resistant bacteria are of increasing concern, especially those due to carbapenem-resistant Enterobacteriaceae pathogens. Included in this group are species of the Enterobacter cloacae complex, regarding which there is a paucity of knowledge on the infection biology of the pathogens, despite their clinical relevance. In this study, we combine techniques in comparative genomics, bacterial genetics, and diverse models of infection to establish capsule as an important mechanism of Enterobacter pathogens to resist the antibacterial activity of serum, a first line of host defense against bacterial infections. We also show that immune memory targeting the Enterobacter capsule protects against lethal infection. The further characterization of Enterobacter infection biology and the immune response to infection are needed for the development of therapies and preventative interventions targeting these highly antibiotic resistant pathogens.
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Enterobacter , Infecções por Enterobacteriaceae , Humanos , Camundongos , Animais , Virulência , Enterobacter/genética , Antibacterianos/farmacologia , Polissacarídeos , Testes de Sensibilidade Microbiana , Infecções por Enterobacteriaceae/microbiologiaRESUMO
BACKGROUND: Genotypic drug resistance testing provides essential information for guiding treatment in HIV-infected patients. It may either be used for identifying patients with transmitted drug resistance or to clarify reasons for treatment failure and to check for remaining treatment options. While different approaches for the interpretation of HIV sequence information are already available, no other available rules-based systems specifically have looked into the effects of combinations of drugs. HIV-GRADE (Genotypischer Resistenz Algorithmus Deutschland) was planned as a countrywide approach to establish standardized drug resistance interpretation in Germany and also to introduce rules for estimating the influence of mutations on drug combinations. The rules for HIV-GRADE are taken from the literature, clinical follow-up data and from a bioinformatics-driven interpretation system (geno2pheno([resistance])). HIV-GRADE presents the option of seeing the rules and results of other drug resistance algorithms for a given sequence simultaneously. METHODS: The HIV-GRADE rules-based interpretation system was developed by the members of the HIV-GRADE registered society. For continuous updates, this expert committee meets twice a year to analyze data from various sources. Besides data from clinical studies and the centers involved, published correlations for mutations with drug resistance and genotype-phenotype correlation data information from the bioinformatic models of geno2pheno are used to generate the rules for the HIV-GRADE interpretation system. A freely available online tool was developed on the basis of the Stanford HIVdb rules interpretation tool using the algorithm specification interface. Clinical validation of the interpretation system was performed on the data of treatment episodes consisting of sequence information, antiretroviral treatment and viral load, before and 3 months after treatment change. Data were analyzed using multiple linear regression. RESULTS: As the developed online tool allows easy comparison of different drug resistance interpretation systems, coefficients of determination (R(2)) were compared for the freely available rules-based systems. HIV-GRADE (R(2) = 0.40), Stanford HIVdb (R(2) = 0.40), REGA algorithm (R(2) = 0.36) and ANRS (R(2) = 0.35) had a very similar performance using this multiple linear regression model. CONCLUSION: The performance of HIV-GRADE is comparable to alternative rules-based interpretation systems. While there is still room for improvement, HIV-GRADE has been made publicly available to allow access to our approach regarding the interpretation of resistance against single drugs and drug combinations.
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Algoritmos , Fármacos Anti-HIV/farmacologia , Biologia Computacional/métodos , HIV-1/efeitos dos fármacos , HIV-1/genética , Testes de Sensibilidade Microbiana/métodos , Tipagem Molecular/métodos , Alemanha , Infecções por HIV/virologia , Humanos , InternetRESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common causes of hospital-acquired pneumonia. To better manage patients with MRSA pneumonia, we require a greater understanding of the host-pathogen interactions during infection. MRSA research focuses on highly virulent and cytotoxic strains, which demonstrate robust phenotypes in animal models of infection. However, nosocomial infections are often caused by hospital-acquired MRSA (HA-MRSA) isolates that exhibit low cytotoxicity and few or no phenotypes in mice, thereby confounding mechanistic studies of pathogenesis. Consequently, virulence pathways utilized by HA-MRSA in nosocomial pneumonia are largely unknown. Here, we report that conditioning mice with broad-spectrum antibiotics lowers the barrier to pneumonia, thereby transforming otherwise avirulent HA-MRSA isolates into lethal pathogens. HA-MRSA isolates are avirulent in gnotobiotic mice, mimicking results in conventional animals. Thus, the observed enhanced susceptibility to infection in antibiotic-treated mice is not due to depletion of the microbiota. More generally, we found that antibiotic conditioning leads to increased susceptibility to infection by diverse antimicrobial-resistant (AMR) pathogens of low virulence. Treatment with antibiotics leads to dehydration and malnutrition, suggesting a potential role for these clinically relevant and reducible hospital complications in susceptibility to pathogens. In sum, the model described here mitigates the impact of low virulence in immunocompetent mice, providing a convenient model to gain fundamental insight into the pathogenesis of nosocomial pathogens. IMPORTANCE Antimicrobial-resistant (AMR) pathogens are responsible for over 2.8 million infections and over 35,000 deaths per year in the United States. To study these microbes, animal models that are susceptible to these pathogens are required. However, many of these pathogens exhibit low virulence in conventional mice, which has negatively impacted mechanistic studies. Here, we show that mice treated with antibiotics in their drinking water become exquisitely susceptible to low-virulence AMR pathogens. Surprisingly, the increased susceptibility was independent of the impact of antibiotics on the microbiome and seems to be due to an unintended consequence of antibiotic treatment: weight loss due to dehydration and caloric restriction. Unlike other models used to sensitize mice to low-virulence pathogens, our model does not reduce phagocyte numbers. Thus, here, we describe an immunocompetent mouse model to facilitate the identification of novel targets and accelerate the development of preventives and therapeutics to combat infections by AMR pathogens.
Assuntos
Infecções Comunitárias Adquiridas , Infecção Hospitalar , Staphylococcus aureus Resistente à Meticilina , Microbiota , Infecções Estafilocócicas , Animais , Antibacterianos/farmacologia , Desidratação , Modelos Animais de Doenças , Staphylococcus aureus Resistente à Meticilina/genética , Camundongos , Testes de Sensibilidade MicrobianaRESUMO
Background: The epidemiology of nosocomial bloodstream infections (NBSIs) in patients with coronavirus disease 2019 (COVID-19) is poorly understood, due in part to substantial disease heterogeneity resulting from multiple potential pathogens. Methods: We identified risk factors for NBSIs and examined the association between NBSIs and mortality in a retrospective cohort of patients hospitalized with COVID-19 in 2 New York City hospitals during the height of the pandemic. We adjusted for the potential effects of factors likely to confound that association, including age, race, illness severity upon admission, and underlying health status. Results: Between January 1 and October 1, 2020, 1403 patients had a positive blood culture, and 79 and 101 met the stringent criteria for NBSI among non-COVID-19 and COVID-19 patients, respectively. NBSIs occurred almost exclusively among patients who were severely ill with COVID-19 at hospital admission. NBSIs were associated with elevated mortality, even after adjusting for baseline differences in COVID-19 illness (55% cases vs 45% controls; Pâ =â .13). Mortality was concentrated in patients with early-onset pneumonia caused by S. aureus and gram-negative bacteria. Less virulent Candida (49%) and Enterococcus (12%) species were the predominant cause of NBSI in the latter stages of hospitalization, after antibiotic treatment and COVID-19 treatments that attenuate immune response. Most Enterococcus and Candida infections did not have an identifiable source and were not associated with common risk factors for infection by these organisms. Conclusions: Pathogen species and mortality exhibited temporal differences. Early recognition of risk factors among COVID-19 patients could potentially decrease NBSI-associated mortality through early COVID-19 and antimicrobial treatment.
RESUMO
Soil-transmitted intestinal worms known as helminths colonize over 1.5 billion people worldwide. Although helminth colonization has been associated with altered composition of the gut microbiota, such as increases in Clostridia, individual species have not been isolated and characterized. Here, we isolate and sequence the genome of 13 Clostridia from the Orang Asli, an indigenous population in Malaysia with a high prevalence of helminth infections. Metagenomic analysis of 650 fecal samples from urban and rural Malaysians confirm the prevalence of species corresponding to these isolates and reveal a specific association between Peptostreptococcaceae family members and helminth colonization. Remarkably, Peptostreptococcaceae isolated from the Orang Asli display superior capacity to promote the life cycle of whipworm species, including hatching of eggs from Trichuris muris and Trichuris trichiura. These findings support a model in which helminths select for gut colonization of microbes that support their life cycle.