RESUMO
Pseudomonas aeruginosa causes chronic lung infection in cystic fibrosis (CF), resulting in structural lung damage and progressive pulmonary decline. P. aeruginosa in the CF lung undergoes numerous changes, adapting to host-specific airway pressures while establishing chronic infection. P. aeruginosa undergoes lipid A structural modification during CF chronic infection that is not seen in any other disease state. Lipid A, the membrane anchor of LPS (i.e., endotoxin), comprises the majority of the outer membrane of Gram-negative bacteria and is a potent Toll-like receptor 4 (TLR4) agonist. The structure of P. aeruginosa lipid A is intimately linked with its recognition by TLR4 and subsequent immune response. Prior work has identified P. aeruginosa strains with altered lipid A structures that arise during chronic CF lung infection; however, the impact of the P. aeruginosa lipid A structure on airway disease has not been investigated. Here, we show that P. aeruginosa lipid A lacks PagL-mediated deacylation during human airway infection using a direct-from-sample mass spectrometry approach on human BAL fluid. This structure triggers increased proinflammatory cytokine production by primary human macrophages. Furthermore, alterations in lipid A 2-hydroxylation impact cytokine response in a site-specific manner, independent of CF transmembrane conductance regulator function. It is interesting that there is a CF-specific reduction in IL-8 secretion within the epithelial-cell compartment that only occurs in CF bronchial epithelial cells when infected with CF-adapted P. aeruginosa that lacks PagL-mediated lipid A deacylation. Taken together, we show that P. aeruginosa alters its lipid A structure during acute lung infection and that this lipid A structure induces stronger signaling through TLR4.
Assuntos
Fibrose Cística , Lipídeo A , Infecções por Pseudomonas , Pseudomonas aeruginosa , Pseudomonas aeruginosa/imunologia , Humanos , Lipídeo A/metabolismo , Lipídeo A/imunologia , Fibrose Cística/microbiologia , Fibrose Cística/imunologia , Fibrose Cística/metabolismo , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/imunologia , Citocinas/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Líquido da Lavagem Broncoalveolar/imunologia , Pulmão/microbiologia , Pulmão/imunologia , Pulmão/metabolismoRESUMO
The coordination of single or multiple microorganisms are required for the manufacture of traditional fermented foods, improving the flavour and nutrition of the food materials. However, both the additional economic benefits and safety concerns have been raised by microbiotas in fermented products. Among the fermented products, Lactobacillus and Saccharomyces cerevisiae are one of the stable microbiotas, suggesting their interaction is mediated by coexistence-relevant mechanisms and prevent to be excluded by other microbial species. Thus, aiming to guide the manufacture of fermented foods, this review will focus on interactions of coexistence-relevant mechanisms between Lactobacillus and S. cerevisiae, including metabolites communications, aggregation, and polymicrobial biofilm. Also, the molecular regulatory network of the coexistence-relevant mechanisms is discussed according to omics researches.
Assuntos
Lactobacillus/fisiologia , Saccharomyces cerevisiae/fisiologia , Alimentos Fermentados/microbiologia , Microbiologia de Alimentos , Lactobacillus/genética , Interações Microbianas , Saccharomyces cerevisiae/genéticaRESUMO
Cronobacter sakazakii is an emerging opportunistic foodborne pathogen causing rare but severe infections in neonates. Furthermore, the formation of biofilm allows C. sakazakii to persist in different environments. We have demonstrated that the mutator phenotype ascribed to deficiency of the pmrA gene results in more biomass in the first 24 h but less during the post maturation stage (7-14 d) compared with BAA 894. The present study aimed to investigate the regulatory mechanism modulating biofilm formation due to pmrA mutation. The transcriptomic analyses of BAA 894 and s-3 were performed by RNA-sequencing on planktonic and biofilm cells collected at different time points. According to the results, when comparing biofilm to planktonic cells, expression of genes encoding outer membrane proteins, lysozyme, etc. were up-regulated, with LysR family transcriptional regulators, periplasmic proteins, etc. down-regulated. During biofilm formation, cellulose synthase operon genes, flagella-related genes, etc. played essential roles in different stages. Remarkably, pmrA varies the expression of a number of genes related to motility, biofilm formation, and antimicrobial resistance, including srfB, virK, mviM encoding virulence factor, flgF, fliN, etc. encoding flagellar assembly, and marA, ramA, etc. encoding AraC family transcriptional regulators in C. sakazakii. This study provides valuable insights into transcriptional regulation of C. sakazakii pmrA mutant during biofilm formation.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes , Cronobacter sakazakii/genética , Plâncton/genética , Transcriptoma , Proteínas de Bactérias/genética , Cronobacter sakazakii/crescimento & desenvolvimento , Cronobacter sakazakii/fisiologia , Regulação Bacteriana da Expressão Gênica , Plâncton/crescimento & desenvolvimento , Plâncton/fisiologia , Transcrição Gênica , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
The opportunistic pathogen Pseudomonas aeruginosa is responsible for much of the morbidity and mortality associated with cystic fibrosis (CF), a condition that predisposes patients to chronic lung infections. P. aeruginosa lung infections are difficult to treat because P. aeruginosa adapts to the CF lung, can develop multidrug resistance, and can form biofilms. Despite the clinical significance of P. aeruginosa, modeling P. aeruginosa infections in CF has been challenging. Here, we characterize Scnn1b-transgenic (Tg) BALB/c mice as P. aeruginosa lung infection models. Scnn1b-Tg mice overexpress the epithelial Na+ channel (ENaC) in their lungs, driving increased sodium absorption that causes lung pathology similar to CF. We intranasally infected Scnn1b-Tg mice and wild-type littermates with the laboratory P. aeruginosa strain PAO1 and CF clinical isolates and then assessed differences in bacterial clearance, cytokine responses, and histological features up to 12 days postinfection. Scnn1b-Tg mice carried higher bacterial burdens when infected with biofilm-grown rather than planktonic PAO1; Scnn1b-Tg mice also cleared infections more slowly than their wild-type littermates. Infection with PAO1 elicited significant increases in proinflammatory and Th17-linked cytokines on day 3. Scnn1b-Tg mice infected with nonmucoid early CF isolates maintained bacterial burdens and mounted immune responses similar to those of PAO1-infected Scnn1b-Tg mice. In contrast, Scnn1b-Tg mice infected with a mucoid CF isolate carried high bacterial burdens, produced significantly more interleukin 1ß (IL-1ß), IL-13, IL-17, IL-22, and KC, and showed severe immune cell infiltration into the bronchioles. Taken together, these results show the promise of Scnn1b-Tg mice as models of early P. aeruginosa colonization in the CF lung.
Assuntos
Fibrose Cística/genética , Modelos Animais de Doenças , Canais Epiteliais de Sódio/genética , Infecções Oportunistas/genética , Infecções por Pseudomonas/genética , Pseudomonas aeruginosa/imunologia , Animais , Carga Bacteriana , Biofilmes/crescimento & desenvolvimento , Fibrose Cística/imunologia , Fibrose Cística/microbiologia , Fibrose Cística/patologia , Canais Epiteliais de Sódio/imunologia , Feminino , Regulação da Expressão Gênica , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-13/genética , Interleucina-13/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Interleucinas/genética , Interleucinas/imunologia , Transporte de Íons , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Infecções Oportunistas/imunologia , Infecções Oportunistas/microbiologia , Infecções Oportunistas/patologia , Plâncton/crescimento & desenvolvimento , Plâncton/imunologia , Plâncton/patogenicidade , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Sódio/metabolismo , Interleucina 22RESUMO
Prosthetic joint infections are difficult to diagnose and treat due to biofilm formation by the causative pathogens. Pathogen identification relies on microbial culture that requires days to weeks, and in the case of chronic biofilm infections, lacks sensitivity. Diagnosis of infection is often delayed past the point of effective treatment such that only the removal of the implant is curative. Early diagnosis of an infection based on antibody detection might lead to less invasive, early interventions. Our study examined antibody-based assays against the Staphylococcus aureus biofilm-upregulated antigens SAOCOL0486 (a lipoprotein), glucosaminidase (a domain of SACOL1062), and SACOL0688 (the manganese transporter MntC) for detection of chronic S. aureus infection. We evaluated these antigens by enzyme-linked immunosorbent assay (ELISA) using sera from naive rabbits and rabbits with S. aureus-mediated osteomyelitis, and then we validated a proof of concept for the lateral flow assay (LFA). The SACOL0688 LFA demonstrated 100% specificity and 100% sensitivity. We demonstrated the clinical diagnostic utility of the SACOL0688 antigen using synovial fluid (SF) from humans with orthopedic implant infections. Elevated antibody levels to SACOL0688 in clinical SF specimens correlated with 91% sensitivity and 100% specificity for the diagnosis of S. aureus infection by ELISA. We found measuring antibodies levels to SACOL0688 in SF using ELISA or LFA provides a tool for the sensitive and specific diagnosis of S. aureus prosthetic joint infection. Development of the LFA diagnostic modality is a desirable, cost-effective option, potentially providing rapid readout in minutes for chronic biofilm infections.
Assuntos
Osteomielite , Infecções Estafilocócicas , Animais , Antígenos de Bactérias , Biofilmes , Coelhos , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureusRESUMO
Periprosthetic joint infection (PJI) occurring after artificial joint replacement is a major clinical issue requiring multiple surgeries and antibiotic interventions. Staphylococcus aureus is the bacterium most commonly responsible for PJI. Recent in vitro research has shown that staphylococcal strains rapidly form aggregates in the presence of synovial fluid (SF). We hypothesize that these aggregates provide early protection to bacteria entering the wound site, allowing them time to attach to the implant surface, leading to biofilm formation. Thus, understanding the attachment kinetics of these aggregates is critical in understanding their adhesion to various biomaterial surfaces. In this study, the number, size, and surface area coverage of aggregates as well as of single cells of S. aureus were quantified under various conditions on different orthopedic materials relevant to orthopedic surgery: stainless steel (316L), titanium (Ti), hydroxyapatite (HA), and polyethylene (PE). It was observed that, regardless of the material type, SF-induced aggregation resulted in reduced aggregate surface attachment and greater aggregate size than the single-cell populations under various shear stresses. Additionally, the surface area coverage of bacterial aggregates on PE was relatively high compared to that on other materials, which could potentially be due to the rougher surface of PE. Furthermore, increasing shear stress to 78 mPa decreased aggregate attachment to Ti and HA while increasing the aggregates' average size. Therefore, this study demonstrates that SF induced inhibition of aggregate attachment to all materials, suggesting that biofilm formation is initiated by lodging of aggregates on the surface features of implants and host tissues.IMPORTANCE Periprosthetic joint infection occurring after artificial joint replacement is a major clinical issue that require repeated surgeries and antibiotic interventions. Unfortunately, 26% of patients die within 5 years of developing these infections. Staphylococcus aureus is the bacterium most commonly responsible for this problem and can form biofilms to provide protection from antibiotics as well as the immune system. Although biofilms are evident on the infected implants, it is unclear how these are attached to the surface in the first place. Recent in vitro investigations have shown that staphylococcal strains rapidly form aggregates in the presence of synovial fluid and provide protection to bacteria, thus allowing them time to attach to the implant surface, leading to biofilm formation. In this study, we investigated the attachment kinetics of Staphylococcus aureus aggregates on different orthopedic materials. The information presented in this article will be useful in surgical management and implant design.
Assuntos
Equipamentos Ortopédicos/microbiologia , Resistência ao Cisalhamento , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia , Estresse Mecânico , Líquido Sinovial/microbiologiaRESUMO
Streptococcus agalactiae is considered as a leading case of bacterial infection among neonates. Although relative protection strategies have been performed in many high-income countries, resulting in a massive reduction in the occurrences of early-onset GBS disease, the late-onset disease has not affected. Here, the whole genome of S. agalactiae Guangzhou-SAG036 was sequenced by the Pacific Biosciences Sequel using the P4-C2 chemistry and the continuous long reads were used for de novo assembly using HGAP. Besides, genes prediction and multiply annotation were performed by comparing it with diverse databases. The whole genome has a length of 2,206,504 bp and contains 2162 predicted genes with an average G + C content of 35.85%. Based on the whole genome sequence, 2 large prophages, 20 virulence factors genes, and 8 antibiotic resistant genes were identified. MLST analysis revealed S. agalactiae Guangzhou-SAG036 was identified as ST-17. The virulence factors genes were identified with different functions including adherence, antiphagocytosis, spreading factor, immune evasion, invasion, toxin. Besides, the antibiotic-resistant genes may provide S. agalactiae with resistance to multi-drugs including erythromycin, streptomycin, azithromycin, spiramycin, ampicillin, kanamycin, cationic peptides, and tetracycline. Therefore, the infection of S. agalactiae Guangzhou-SAG036 ST-17 strain maybe caused by the complex virulence factors and multi-drugs resistance. These results contribute to further understand GBS epidemiology and surveillance targets.
Assuntos
Infecções Estreptocócicas , Streptococcus agalactiae , Eritromicina/farmacologia , Humanos , Recém-Nascido , Tipagem de Sequências Multilocus , Streptococcus agalactiae/genética , Fatores de Virulência/genéticaRESUMO
BACKGROUND: Lactobacillus brevis is a major contaminant of spoiled beer. And it was able to enter VBNC state and cause false negative detection, which poses a major challenge to the brewing industry. METHODS: The genomic DNA of L. brevis BM-LB13908 was extracted and purified to form a sequencing library that meets the quality requirements and was sequenced. The sequencing results were then screened and assembled to obtain the entire genome sequence of L. brevis. Predicted genes were annotated by GO database, KEGG pathway database and COG functional classification system. RESULTS: The final assembly yielded 275 scaffolds of a total length of 2 840 080 bp with a G + C content of 53.35%. There were 2357, 701, 1519 predicted genes with corresponding GO functional, COG functional, and KEGG biological pathway annotations, respectively. The genome of L. brevis BM-LB13908 contains hop resistance gene horA and multiple genes related to the formation of VBNC state. CONCLUSIONS: This report describes the draft genome sequence of L. brevis BM-LB13908, a spoilage strain isolated from finished beer sample. This study may support further study on L. brevis and other beer spoilage bacteria, and prevent and control beer spoilage caused by microorganisms.
Assuntos
Levilactobacillus brevis , Bactérias , Cerveja , Microbiologia de Alimentos , Genômica , Levilactobacillus brevis/genéticaRESUMO
Staphylococcus aureus is a causative agent of chronic biofilm-associated infections that are recalcitrant to resolution by the immune system or antibiotics. To combat these infections, an antistaphylococcal, biofilm-specific quadrivalent vaccine against an osteomyelitis model in rabbits has previously been developed and shown to be effective at eliminating biofilm-embedded bacterial populations. However, the addition of antibiotics was required to eradicate remaining planktonic populations. In this study, a planktonic upregulated antigen was combined with the quadrivalent vaccine to remove the need for antibiotic therapy. Immunization with this pentavalent vaccine followed by intraperitoneal challenge of BALB/c mice with S. aureus resulted in 16.7% and 91.7% mortality in pentavalent vaccine and control groups, respectively (P < 0.001). Complete bacterial elimination was found in 66.7% of the pentavalent cohort, while only 8.3% of the control animals cleared the infection (P < 0.05). Further protective efficacy was observed in immunized rabbits following intramedullary challenge with S. aureus, where 62.5% of the pentavalent cohort completely cleared the infection, versus none of the control animals (P < 0.05). Passive immunization of BALB/c mice with serum IgG against the vaccine antigens prior to intraperitoneal challenge with S. aureus prevented mortality in 100% of mice and eliminated bacteria in 33.3% of the challenged mice. These results demonstrate that targeting both the planktonic and biofilm stages with the pentavalent vaccine or the IgG elicited by immunization can effectively protect against S. aureus infection.
Assuntos
Antígenos de Bactérias/imunologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/prevenção & controle , Vacinas Antiestafilocócicas/imunologia , Staphylococcus aureus/imunologia , Animais , Anticorpos Antibacterianos/administração & dosagem , Anticorpos Antibacterianos/imunologia , Modelos Animais de Doenças , Imunização Passiva , Imunoglobulina G/administração & dosagem , Imunoglobulina G/imunologia , Camundongos Endogâmicos BALB C , Coelhos , Vacinas Antiestafilocócicas/administração & dosagem , Análise de Sobrevida , Resultado do TratamentoRESUMO
Pseudomonas aeruginosa can survive in a myriad of environments, partially due to modifications of its lipid A, the membrane anchor of lipopolysaccharide. We previously demonstrated that divergent late acyltransferase paralogs, HtrB1 and HtrB2, add acyloxyacyl laurate to lipid A 2- and 2'-acyl chains, respectively. The genome of P. aeruginosa also has genes which encode two dioxygenase enzymes, LpxO1 and LpxO2, that individually hydroxylate a specific secondary laurate. LpxO1 acts on the 2'-acyloxyacyl laurate (added by HtrB2), whereas LpxO2 acts on the 2-acyloxyacyl laurate (added by HtrB1) in a site-specific manner. Furthermore, while both enzyme pairs are evolutionarily linked, phylogenomic analysis suggests the LpxO1/HtrB2 enzyme pair as being of ancestral origin, present throughout the Pseudomonas lineage, whereas the LpxO2/HtrB1 enzyme pair likely arose via horizontal gene transfer and has been retained in P. aeruginosa over time. Using a murine pulmonary infection model, we showed that both LpxO1 and LpxO2 enzymes are functional in vivo, as direct analysis of in vivo lipid A structure from bronchoalveolar lavage fluid revealed 2-hydroxylated lipid A. Gene expression analysis reveals increased lpxO2 but unchanged lpxO1 expression in vivo, suggesting differential regulation of these enzymes during infection. We also demonstrate that loss-of-function mutations arise in lpxO1 and lpxO2 during chronic lung infection in people with cystic fibrosis (CF), indicating a potential role for pathogenesis and airway adaptation. Collectively, our study characterizes lipid A 2-hydroxylation during P. aeruginosa airway infection that is regulated by two distinct lipid A dioxygenase enzymes.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen that causes severe infection in hospitalized and chronically ill individuals. During infection, P. aeruginosa undergoes adaptive changes to evade host defenses and therapeutic interventions, increasing mortality and morbidity. Lipid A structural alteration is one such change that P. aeruginosa isolates undergo during chronic lung infection in CF. Investigating genetic drivers of this lipid A structural variation is crucial in understanding P. aeruginosa adaptation during infection. Here, we describe two lipid A dioxygenases with acyl-chain site specificity, each with different evolutionary origins. Further, we show that loss of function in these enzymes occurs in CF clinical isolates, suggesting a potential pathoadaptive phenotype. Studying these bacterial adaptations provides insight into selection pressures of the CF airway on P. aeruginosa phenotypes that persist during chronic infection. Understanding these adaptive changes may ultimately provide clinicians better control over bacterial populations during chronic infection.
Assuntos
Fibrose Cística , Dioxigenases , Infecções por Pseudomonas , Humanos , Animais , Camundongos , Pseudomonas aeruginosa/metabolismo , Lipídeo A/metabolismo , Infecção Persistente , Lauratos/metabolismo , Hidroxilação , Fibrose Cística/microbiologia , Infecções por Pseudomonas/microbiologia , Dioxigenases/metabolismoRESUMO
The anterior nares of humans are the major reservoir for Staphylococcus aureus colonization. Approximately 20% of the healthy human population is persistently and 80% is intermittently colonized with S. aureus in the nasal cavity. Previous studies have shown a strong causal connection between S. aureus nasal carriage and increased risk of nosocomial infection, as well as increased carriage due to immune dysfunction. However, the immune responses that permit persistence or mediate clearance of S. aureus on the nasal mucosa are fundamentally undefined. In this study, we developed a carriage model in C57BL/6J mice and showed that clearance begins 14 days postinoculation. In contrast, SCID mice that have a deficient adaptive immune response are unable to eliminate S. aureus even after 28 days postinoculation. Furthermore, decolonization was found to be T cell mediated but B cell independent by evaluating carriage clearance in T-cell receptor ß/δ (TCR-ß/δ) knockout (KO) and IgH-µ KO mice, respectively. Upregulation of the cytokines interleukin 1ß (IL-1ß), KC (also termed CXC ligand 1 [CXCL1]), and IL-17A occurred following inoculation with intranasal S. aureus. IL-17A production was crucial for clearance, since IL-17A-deficient mice were unable to effectively eliminate S. aureus carriage. Subsequently, cell differential counts were evaluated from nasal lavage fluid obtained from wild-type and IL-17A-deficient colonized mice. These counts displayed IL-17A-dependent neutrophil migration. Antibody-mediated depletion of neutrophils in colonized mice caused reduced clearance compared to that in isotype-treated controls. Our data suggest that the Th17-associated immune response is required for nasal decolonization. This response is T cell dependent and mediated via IL-17A production and neutrophil influx. Th17-associated immune responses may be targeted for strategies to mitigate distal infections originating from persistent S. aureus carriage in humans.
Assuntos
Interleucina-17/metabolismo , Neutrófilos/fisiologia , Nariz/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/fisiologia , Linfócitos T/fisiologia , Animais , Linfócitos B , Portador Sadio/imunologia , Portador Sadio/microbiologia , Regulação Bacteriana da Expressão Gênica , Humanos , Interleucina-17/genética , Interleucina-23/genética , Interleucina-23/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nariz/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologiaRESUMO
This work aimed to study the effects of dietary lipid composition and content on cecal and fecal microbiota of mice fed the following diets for 8 weeks: palm olein (PO)-based low-fat diet, PO-based high-fat diet, palm stearin (PS)-based low-fat diet, and PS-based high-fat diet. Increasing the dietary PS level favored the growth of Firmicutes over Bacteroidetes in the cecum and feces. In addition, it significantly elevated the total lipid (p < 0.01) and bile acid content (p < 0.01) in feces, resulting in the enrichment of fat-degrading and bile-acid tolerant genera within the families Ruminococcaceae and Lachnospiraceae. Although increasing the PO intake also caused obesity in mice, it did not affect the microbial structure. When fat intake is constant, only at a high-fat level can PS (vs PO) induce the above-mentioned microbial shifts. These results highlighted the combined roles of lipid quality and quantity on the gut microbiota.
Assuntos
Gorduras na Dieta , Microbiota , Camundongos , Animais , Camundongos Endogâmicos C57BL , Gorduras na Dieta/farmacologia , Ceco , Dieta Hiperlipídica/efeitos adversos , Fezes , Ácidos e Sais Biliares , Óleo de PalmeiraRESUMO
Many microbes of concern to human health remain without vaccines. We have developed a whole-microbe inactivation technology that enables us to rapidly inactivate large quantities of a pathogen while retaining epitopes that were destroyed by previous inactivation methods. The method that we call UVC-MDP inactivation can be used to make whole-cell vaccines with increased potency. We and others are exploring the possibility of using improved irradiation-inactivation technologies to develop whole-cell vaccines for numerous antibiotic-resistant microbes. Here, we apply UVC-MDP to produce candidate MRSA vaccines which we test in a stringent tibia implant model of infection challenged with a virulent MSRA strain. We report high levels of clearance in the model and observe a pattern of protection that correlates with the immunogen protein profile used for vaccination.
RESUMO
BACKGROUND: Next-generation DNA sequencing (NGS) detects bacteria-specific DNA corresponding to the 16S ribosomal RNA gene and can identify bacterial presence with greater accuracy than traditional culture methods. The clinical relevance of these findings is unknown. The purpose of the present study was to compare the results from bacterial culture and NGS in order to characterize the potential use of NGS in orthopaedic trauma patients. METHODS: A prospective cohort study was performed at a single academic, level-I trauma center. Three patient groups were enrolled: (1) patients undergoing surgical treatment of acute closed fractures (presumed to have no bacteria), (2) patients undergoing implant removal at the site of a healed fracture without infection, and (3) patients undergoing a first procedure for the treatment of a fracture nonunion who might or might not have subclinical infection. Surgical site tissue was sent for culture and NGS. The proportions of culture and NGS positivity were compared among the groups. The agreement between culture and NGS results was assessed with use of the Cohen kappa statistic. RESULTS: Bacterial cultures were positive in 9 of 111 surgical sites (110 patients), whereas NGS was positive in 27 of 111 surgical sites (110 patients). Significantly more cases were positive on NGS as compared with culture (24% vs. 8.1%; p = 0.001), primarily in the acute closed fracture group. No difference was found in terms of the percent positivity of NGS when comparing the acute closed fracture, implant removal, and nonunion groups. With respect to bacterial identification, culture and NGS agreed in 73% of cases (κ = 0.051; 95% confidence interval, -0.12 to 0.22) indicating only slight agreement compared with expected chance agreement of 50%. CONCLUSIONS: NGS identified bacterial presence more frequently than culture, but with only slight agreement between culture and NGS. It is possible that the increased frequency of bacterial detection with molecular methods is reflective of biofilm presence on metal or colonization with nonpathogenic bacteria, as culture methods have selection pressure posed by restrictive, artificial growth conditions and there are low metabolic activity and replication rates of bacteria in biofilms. Our data suggest that NGS should not currently substitute for or complement conventional culture in orthopaedic trauma cases with low suspicion of infection. LEVEL OF EVIDENCE: Diagnostic Level II. See Instructions for Authors for a complete description of levels of evidence.
Assuntos
Fraturas Fechadas , Ortopedia , Bactérias/genética , DNA Bacteriano/genética , Humanos , Estudos Prospectivos , Análise de Sequência de DNARESUMO
Staphylococcus aureus has reemerged as an important human pathogen in recent decades. Although many infections caused by this microbial species persist through a biofilm mode of growth, little is known about how the host's adaptive immune system responds to these biofilm infections. In this study, S. aureus cells adhered to pins in culture and were subsequently inserted into the tibiae of C57BL/6 mice, with an infecting dose of 2 × 105 CFU. This model was utilized to determine local cytokine levels, antibody (Ab) function, and T cell populations at multiple time points throughout infection. Like human hosts, S. aureus implant infection was chronic and remained localized in 100% of C57BL/6 mice at a consistent level of approximately 10(7) CFU/gram bone tissue after day 7. This infection persisted locally for >49 days and was recalcitrant to clearance by the host immune response and antimicrobial therapy. Local inflammatory cytokines of the Th1 (interleukin-2 [IL-2], IL-12 p70, tumor necrosis factor alpha [TNF-α], and IL-1ß) and Th17 (IL-6 and IL-17) responses were upregulated throughout the infection, except IL-12 p70, which dwindled late in the infection. In addition, Th1 Ab subtypes against a biofilm antigen (SA0486) were upregulated early in the infection, while Th2 Abs and anti-inflammatory regulatory T cells (Tregs) were not upregulated until later. These results indicate that early Th1 and Th17 inflammatory responses and downregulated Th2 and Treg responses occur during the development of a chronic biofilm implant infection. This unrestrained inflammatory response may cause tissue damage, thereby enabling S. aureus to attach and thrive in a biofilm mode of growth.
Assuntos
Biofilmes/crescimento & desenvolvimento , Pinos Ortopédicos/microbiologia , Infecções Relacionadas à Prótese/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/fisiologia , Animais , Doença Crônica , Citocinas/imunologia , Citometria de Fluxo , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologiaRESUMO
Staphylococcus aureus is a common cause of prosthetic implant infections, which can become chronic due to the ability of S. aureus to grow as a biofilm. Little is known about adaptive immune responses to these infections in vivo. We hypothesized that S. aureus elicits inflammatory Th1/Th17 responses, associated with biofilm formation, instead of protective Th2/Treg responses. We used an adapted mouse model of biofilm-mediated prosthetic implant infection to determine chronic infection rates, Treg cell frequencies, and local cytokine levels in Th1-biased C57BL/6 and Th2-biased BALB/c mice. All C57BL/6 mice developed chronic S. aureus implant infection at all time points tested. However, over 75% of BALB/c mice spontaneously cleared the infection without adjunctive therapy and demonstrated higher levels of Th2 cytokines and anti-inflammatory Treg cells. When chronic infection rates in mice deficient in the Th2 cytokine interleukin-4 (IL-4) via STAT6 mutation in a BALB/c background were assessed, the mice were unable to clear the S. aureus implant infection. Additionally, BALB/c mice depleted of Treg cells via an anti-CD25 monoclonal antibody (MAb) were also unable to clear the infection. In contrast, the C57BL/6 mice that were susceptible to infection were able to eliminate S. aureus biofilm populations on infected intramedullary pins once the Th1 and Th17 responses were diminished by MAb treatment with anti-IL-12 p40. Together, these results indicate that Th2/Treg responses are mechanisms of protection against chronic S. aureus implant infection, as opposed to Th1/Th17 responses, which may play a role in the development of chronic infection.
Assuntos
Biofilmes/crescimento & desenvolvimento , Inflamação/prevenção & controle , Staphylococcus aureus Resistente à Meticilina/fisiologia , Infecções Estafilocócicas/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Doença Crônica , Imunidade Inata , Subunidade p40 da Interleucina-12/imunologia , Interleucina-6/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Próteses e Implantes/microbiologia , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismo , Infecções Estafilocócicas/microbiologia , Linfócitos T Reguladores/imunologia , Células Th2/fisiologiaRESUMO
OBJECTIVES: To evaluate the efficacy of intraoperative tobramycin powder in preventing surgical site infection (SSI) and implant colonization with Enterobacter cloacae in a rabbit fixation model. Gram-negative rods, particularly Enterobacter species, comprise an increasing percentage of SSI at our institution. METHODS: Eighteen New Zealand White rabbits underwent surgical fixation of the left tibia with implantation of a plate and screws. The surgical site and implant were inoculated with 1 × 107 CFUs E. cloacae. The selected E. cloacae isolate was resistant to tobramycin and capable of forming biofilms. Nine rabbits received 125 mg tobramycin powder directly into the surgical site, overlying the implant. The control group was untreated. Fourteen days postinfection, the tibiae and implants were explanted. Radiographs were taken with and without the implants in place. One tibia from each group was examined after hematoxylin and eosin staining. The remaining tibiae and implants were morselized or sonicated, respectively, and plated on agar to determine infection burden. Data were analyzed with Fisher exact tests and Mann-Whitney U tests. RESULTS: No bone infection or implant colonization occurred in the tobramycin-treated group. In the control group, 7 of 8 rabbits developed bone infections (P = 0.001), and 4 of 8 implants were colonized (P = 0.07). No gross disruption of the normal bone architecture was observed in either group. CONCLUSIONS: Intraoperative tobramycin powder applied at the time of contamination prevented bone infection with E. cloacae in this rabbit fixation model. The results are encouraging because the E. cloacae isolate was tobramycin-resistant, demonstrating the utility of intraoperative powdered antibiotics.
Assuntos
Infecção da Ferida Cirúrgica , Tobramicina , Animais , Antibacterianos/uso terapêutico , Enterobacter cloacae , Pós , Coelhos , Infecção da Ferida Cirúrgica/tratamento farmacológico , Infecção da Ferida Cirúrgica/prevenção & controleRESUMO
Bacterial biofilms are responsible for a variety of serious human infections and are notoriously difficult to treat due to their recalcitrance to antibiotics. Further work is necessary to elicit a full understanding of the mechanism of this antibiotic tolerance. The arginine deiminase (ADI) pathway is responsible for bacterial pH maintenance and is highly expressed during biofilm growth in multiple bacterial species. Using the group A Streptococcus (GAS) as a model human pathogen, the ADI pathway was demonstrated to contribute to biofilm growth. The inability of antibiotics to reduce GAS populations when in a biofilm was demonstrated by in vitro studies and a novel animal model of nasopharyngeal infection. However, disruption of the ADI pathway returned GAS biofilms to planktonic levels of antibiotic sensitivity, suggesting the ADI pathway is influential in biofilm-related antibiotic treatment failure and provides a new strategic target for the treatment of biofilm infections in GAS and potentially numerous other bacterial species.IMPORTANCE Biofilm-mediated bacterial infections are a major threat to human health because of their recalcitrance to antibiotic treatment. Through the study of Streptococcus pyogenes, a significant human pathogen that is known to form antibiotic-tolerant biofilms, we demonstrated the role that a bacterial pathway known for responding to acid stress plays in biofilm growth and antibiotic tolerance. This not only provides some insight into antibiotic treatment failure in S. pyogenes infections but also, given the widespread nature of this pathway, provides a potentially broad target for antibiofilm therapies. This discovery has the potential to impact the treatment of many different types of recalcitrant biofilm infections.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Hidrolases/metabolismo , Streptococcus pyogenes/efeitos dos fármacos , Animais , Biofilmes/efeitos dos fármacos , Feminino , Regulação Bacteriana da Expressão Gênica , Humanos , Masculino , Redes e Vias Metabólicas , Camundongos Endogâmicos C57BL , Streptococcus pyogenes/enzimologiaRESUMO
The impacts of lipid physical state and content on lipid digestion behavior were investigated using 4 and 20% palm olein-in-water emulsions (4% PO and 20% PO) and 4 and 20% palm stearin-in-water emulsions (4% PS and 20% PS). The changes of lipid physical state, particle size, and microstructure during gastrointestinal digestion; the free fatty acid (FFA) released in the intestinal phase; and the fatty acid composition of micellar phases were investigated. After gastric digestion, all emulsions underwent flocculation and coalescence, with 20% PS showing the most extensive aggregation. During intestinal digestion, the FFA release rate and level decreased as the lipid content increased from 4 to 20%, with 4% PO presenting the highest digestion rate and extent. Besides, the solid fat in 4% PS and 20% PS decreased and increased the maximum lipid digestibility, respectively. These results highlighted the combined roles of lipid physical state and content in modulating dietary lipid digestion.
Assuntos
Mucosa Intestinal/metabolismo , Óleo de Palmeira/metabolismo , Digestão , Emulsões/química , Emulsões/metabolismo , Ácidos Graxos não Esterificados/química , Ácidos Graxos não Esterificados/metabolismo , Humanos , Óleo de Palmeira/química , Tamanho da Partícula , Água/químicaRESUMO
This study aimed to evaluate the Staphylococcus aureus biofilm formation and Nε-carboxymethyl-lysine generation ability under food heat processing conditions including pH (5.0-9.0), temperature (25 °C, 31 °C, 37 °C, 42 °C and 65 °C), NaCl concentration (10%, 15% and 20%, w/v) and glucose concentration (0.5%, 1%, 2%, 3%, 5%, 10%, w/v). S. aureus biofilm genetic character was obtained by PCR detecting atl, ica operon, sasG and agr. Biofilm biomass and metabolic activity were quantified with crystal violet and methyl thiazolyl tetrazolium staining methods. S. aureus biofilm was sensitive to food heat processing conditions with 37 °C, pH 7.0, 2% glucose concentration (w/v) and 10% NaCl concentration (w/v) were favorable conditions. Besides, free and bound Nε-carboxymethyl-lysine level in weak, moderate and strong biofilm were detected by optimized high performance liquid chromatography tandem mass spectrometry. Nε-carboxymethyl-lysine level in S. aureus biofilm possessed a significant gap between strong, moderate and weak biofilm strains. This investigation revealed the biological and chemical hazard of Staphylococcus aureus biofilm to food processing environment.