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There is substantial genomic heterogeneity among Staphylococcus aureus isolates of children with acute hematogenous osteomyelitis (AHO) but transcriptional behavior of clinically differentiated strains has not been previously described. This study evaluates transcriptional activity of S. aureus isolates of children with AHO that may regulate metabolism, biosynthesis, or virulence during bacterial growth and pathogenesis. In vitro growth kinetics were compared between three S. aureus clinical isolates from children with AHO who had mild, moderate, and severe illness. Total RNA sequencing was performed for each isolate at six separate time points throughout the logarithmic phase of growth. The NASA RNA-Sequencing Consensus Pipeline was used to identify differentially expressed genes allowing for 54 comparisons between the three isolates during growth. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathways were used to evaluate transcriptional variation in metabolism, biosynthesis pathways and virulence potential of the isolates. The S. aureus isolates demonstrated differing growth kinetics under standardized conditions with the mild isolate having higher optical densities with earlier and higher peak rates of growth than that of the other isolates (p<0.001). Enrichment pathway analysis established distinct transcriptional signatures according to both sampling time and clinical severity. Moderate and severe isolates demonstrated pathways of bacterial invasion, S. aureus infection, quorum sensing and two component systems. In comparison, the mild strain favored biosynthesis and metabolism. These findings suggest that transcriptional regulation during the growth of S. aureus may impact the pathogenetic mechanisms involved in the progression of severity of illness in childhood osteomyelitis. The clinical isolates studied demonstrated a tradeoff between growth and virulence. Further investigation is needed to evaluate these transcriptional pathways in an animal model or during active clinical infections of children with AHO.
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Osteomielite , Infecções Estafilocócicas , Animais , Staphylococcus aureus , Transcriptoma , Osteomielite/microbiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Infecções Estafilocócicas/microbiologiaRESUMO
BACKGROUND: Pseudomonas aeruginosa infection is the leading cause of death among patients with cystic fibrosis (CF) and a common cause of difficult-to-treat hospital-acquired infections. P. aeruginosa uses several mechanisms to resist different antibiotic classes and an individual CF patient can harbour multiple resistance phenotypes. OBJECTIVES: To determine the rates and distribution of polyclonal heteroresistance (PHR) in P. aeruginosa by random, prospective evaluation of respiratory cultures from CF patients at a large referral centre over a 1â year period. METHODS: We obtained 28 unique sputum samples from 19 CF patients and took multiple isolates from each, even when morphologically similar, yielding 280 unique isolates. We performed antimicrobial susceptibility testing (AST) on all isolates and calculated PHR on the basis of variability in AST in a given sample. We then performed whole-genome sequencing on 134 isolates and used a machine-learning association model to interrogate phenotypic PHR from genomic data. RESULTS: PHR was identified in most sampled patients (nâ=â15/19; 79%). Importantly, resistant phenotypes were not detected by routine AST in 26% of patients (nâ=â5/19). The machine-learning model, using the extended sampling, identified at least one genetic variant associated with phenotypic resistance in 94.3% of isolates (nâ=â1392/1476). CONCLUSION: PHR is common among P. aeruginosa in the CF lung. While traditional microbiological methods often fail to detect resistant subpopulations, extended sampling of isolates and conventional AST identified PHR in most patients. A machine-learning tool successfully identified at least one resistance variant in almost all resistant isolates by leveraging this extended sampling and conventional AST.
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Fibrose Cística , Infecções por Pseudomonas , Humanos , Pseudomonas aeruginosa/genética , Fibrose Cística/microbiologia , Infecções por Pseudomonas/microbiologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Sistema Respiratório/microbiologia , Testes de Sensibilidade MicrobianaRESUMO
OBJECTIVE: To examine the association between uropathogens and pyuria in children <24 months of age. STUDY DESIGN: A retrospective study of children <24 months of age evaluated in the emergency department for suspected urinary tract infection (UTI) with paired urinalysis and urine culture during a 6-year period. Bagged urine specimens or urine culture growing mixed/multiple urogenital organisms were excluded. Analysis was limited to children with positive urine culture as defined by the American Academy of Pediatrics clinical practice guideline culture thresholds. RESULTS: Of 30 462 children, 1916 had microscopic urinalysis and positive urine culture. Urine was obtained by transurethral in-and-out catheterization in 98.3% of cases. Pyuria (≥5 white blood cells per high-powered field) and positive leukocyte esterase (small or more) on the urine dipstick were present in 1690 (88.2%) and 1692 (88.3%) of the children respectively. Children with non-Escherichia coli species were less likely to exhibit microscopic pyuria than children with E coli (OR 0.24, 95% CI 0.17-0.34) with more pronounced effect on Enterococcus and Klebsiella (OR 0.08, 95% CI 0.03-0.18 and OR 0.18, 95% CI 0.11-0.27 respectively). Similarly, positive leukocyte esterase was less frequently seen in non-E coli uropathogens compared with E coli. CONCLUSIONS: Pyuria and leukocyte esterase are not sensitive markers to identify non-E coli UTI in young children. More sensitive screening biomarkers are needed to identify UTI with these uropathogens.
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Piúria , Infecções Urinárias , Biomarcadores , Criança , Pré-Escolar , Escherichia coli , Humanos , Estudos Retrospectivos , Urinálise , Infecções Urinárias/complicações , Infecções Urinárias/diagnósticoRESUMO
Here we report a case of a 14-week-old girl with a history of intrauterine drug exposure and hypoxic ischemic encephalopathy secondary to cardiac arrest requiring prolonged resuscitation at birth presented with irritability and a bulging anterior fontanelle. After neurosurgical resection, pathologic examination showed fungal hyphae, and Epicoccum nigrum was detected by fungal PCR and sequencing. To our knowledge, this is the first reported case of a central nervous system infection due to Epicoccum nigrum.
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BACKGROUND: The COVID-19 pandemic has resulted in shortages of diagnostic tests, personal protective equipment, hospital beds, and other critical resources. OBJECTIVE: We sought to improve the management of scarce resources by leveraging electronic health record (EHR) functionality, computerized provider order entry, clinical decision support (CDS), and data analytics. METHODS: Due to the complex eligibility criteria for COVID-19 tests and the EHR implementation-related challenges of ordering these tests, care providers have faced obstacles in selecting the appropriate test modality. As test choice is dependent upon specific patient criteria, we built a decision tree within the EHR to automate the test selection process by using a branching series of questions that linked clinical criteria to the appropriate SARS-CoV-2 test and triggered an EHR flag for patients who met our institutional persons under investigation criteria. RESULTS: The percentage of tests that had to be canceled and reordered due to errors in selecting the correct testing modality was 3.8% (23/608) before CDS implementation and 1% (262/26,643) after CDS implementation (P<.001). Patients for whom multiple tests were ordered during a 24-hour period accounted for 0.8% (5/608) and 0.3% (76/26,643) of pre- and post-CDS implementation orders, respectively (P=.03). Nasopharyngeal molecular assay results were positive in 3.4% (826/24,170) of patients who were classified as asymptomatic and 10.9% (1421/13,074) of symptomatic patients (P<.001). Positive tests were more frequent among asymptomatic patients with a history of exposure to COVID-19 (36/283, 12.7%) than among asymptomatic patients without such a history (790/23,887, 3.3%; P<.001). CONCLUSIONS: The leveraging of EHRs and our CDS algorithm resulted in a decreased incidence of order entry errors and the appropriate flagging of persons under investigation. These interventions optimized reagent and personal protective equipment usage. Data regarding symptoms and COVID-19 exposure status that were collected by using the decision tree correlated with the likelihood of positive test results, suggesting that clinicians appropriately used the questions in the decision tree algorithm.
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OBJECTIVES: Accuracy of pyuria for urinary tract infection (UTI) varies with urine concentration. Our objective of this study was to determine the optimal white blood cell (WBC) cutoff for UTI in young children at different urine concentrations as measured by urine specific gravity. METHODS: Retrospective cross-sectional study of children <24 months of age evaluated in the emergency department for suspected UTI with paired urinalysis and urine culture during a 6-year period. The primary outcome was positive urine culture result as described in the American Academy of Pediatrics clinical practice guideline culture thresholds. Test characteristics for microscopic pyuria cut points and positive leukocyte esterase (LE) were calculated across 3 urine specific gravity groups: low <1.011, moderate 1.011 to 1.020, and high >1.020. RESULTS: Of the total 24 171 patients analyzed, urine culture result was positive in 2003 (8.3%). Urine was obtained by transurethral in-and-out catheterization in 97.9%. Optimal WBC cutoffs per high-power field (HPF) were 3 (positive likelihood ratio [LR+] 10.5; negative likelihood ratio [LR-] 0.12) at low, 6 (LR+ 12; LR- 0.14) at moderate, and 8 (LR+ 11.1; LR- 0.35) at high urine concentrations. Likelihood ratios for small positive LE from low to high urine concentrations (LR+ 25.2, LR- 0.12; LR+ 33.1, LR- 0.15; LR+ 37.6, LR- 0.41) remained excellent. CONCLUSIONS: Optimal pyuria cut point in predicting positive urine culture results changes with urine concentration in young children. Pyuria thresholds of 3 WBCs per HPF at low urine concentrations whereas 8 WBCs per HPF at high urine concentrations have optimal predictive value for UTI. Positive LE is a strong predictor of UTI regardless of urine concentration.
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Piúria/diagnóstico , Piúria/urina , Urinálise/métodos , Pré-Escolar , Estudos Transversais , Feminino , Humanos , Lactente , Recém-Nascido , Leucócitos/metabolismo , Masculino , Piúria/terapia , Estudos Retrospectivos , Urinálise/normas , Infecções Urinárias/diagnóstico , Infecções Urinárias/terapia , Infecções Urinárias/urinaRESUMO
BACKGROUND: Metagenomic next generation sequencing (mNGS) is becoming increasingly available for pathogen detection directly from clinical specimens. These tests use target-independent, shotgun sequencing to detect potentially unlimited organisms. The promise of this methodology to aid infection diagnosis is demonstrated through early case reports and clinical studies. However, the optimal role of mNGS in clinical microbiology remains uncertain. CONTENT: We reviewed studies reporting clinical use of mNGS for pathogen detection from various specimen types, including cerebrospinal fluid, plasma, lower respiratory specimens, and others. Published clinical study data were critically evaluated and summarized to identify promising clinical indications for mNGS-based testing, to assess the clinical impact of mNGS for each indication, and to recognize test limitations. Based on these clinical studies, early testing recommendations are made to guide clinical utilization of mNGS for pathogen detection. Finally, current barriers to routine clinical laboratory implementation of mNGS tests are highlighted. SUMMARY: The promise of direct-from-specimen mNGS to enable challenging infection diagnoses has been demonstrated through early clinical studies of patients with meningitis or encephalitis, invasive fungal infections, community acquired pneumonia, and other clinical indications. However, the proportion of patient cases with positive clinical impact due to mNGS testing is low in published studies and the cost of testing is high, emphasizing the importance of improving our understanding of 'when to test' and for which patients mNGS testing is appropriate.
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Líquidos Corporais/microbiologia , Líquidos Corporais/parasitologia , Sequenciamento de Nucleotídeos em Larga Escala/normas , Metagenômica/normas , Alveolados/genética , Bactérias/genética , Infecções Bacterianas/diagnóstico , Fungos/genética , Humanos , Micoses/diagnóstico , Infecções por Protozoários/diagnósticoRESUMO
Cystic fibrosis (CF) is a genetic disease that causes patients to accumulate thick, dehydrated mucus in the lung and develop chronic, polymicrobial infections due to reduced mucociliary clearance. These chronic polymicrobial infections and subsequent decline in lung function are significant factors in the morbidity and mortality of CF. Pseudomonas aeruginosa and Streptococcus spp. are among the most prevalent organisms in the CF lung; the presence of P. aeruginosa correlates with lung function decline, and the Streptococcus milleri group (SMG), a subgroup of the viridans streptococci, is associated with exacerbations in patients with CF. Here we characterized the interspecies interactions that occur between these two genera. We demonstrated that multiple P. aeruginosa laboratory strains and clinical CF isolates promote the growth of multiple SMG strains and oral streptococci in an in vitro coculture system. We investigated the mechanism by which P. aeruginosa enhances growth of streptococci by screening for mutants of P. aeruginosa PA14 that are unable to enhance Streptococcus growth, and we identified the P. aeruginosapqsL::TnM mutant, which failed to promote growth of Streptococcus constellatus and S. sanguinis Characterization of the P. aeruginosa ΔpqsL mutant revealed that this strain cannot promote Streptococcus growth. Our genetic data and growth studies support a model whereby the P. aeruginosa ΔpqsL mutant overproduces siderophores and thus likely outcompetes Streptococcus sanguinis for limited iron. We propose a model whereby competition for iron represents one important means of interaction between P. aeruginosa and Streptococcus spp.IMPORTANCE Cystic fibrosis (CF) lung infections are increasingly recognized for their polymicrobial nature. These polymicrobial infections may alter the biology of the organisms involved in CF-related infections, leading to changes in growth, virulence, and/or antibiotic tolerance, and could thereby affect patient health and response to treatment. In this study, we demonstrate interactions between P. aeruginosa and streptococci using a coculture model and show that one interaction between these microbes is likely competition for iron. Thus, these data indicate that one CF pathogen may influence the growth of another, and they add to our limited knowledge of polymicrobial interactions in the CF airway.
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Proteínas de Bactérias/metabolismo , Interações Microbianas , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Sideróforos/metabolismo , Streptococcus milleri (Grupo)/efeitos dos fármacos , Streptococcus milleri (Grupo)/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Deleção de Genes , Testes Genéticos , Ferro/metabolismo , Mutagênese Insercional , Pseudomonas aeruginosa/genéticaRESUMO
Here, we report an approach to detect diverse bacterial and fungal taxa in complex samples by direct analysis of community RNA in one step using NanoString probe sets. We designed rRNA-targeting probe sets to detect 42 bacterial and fungal genera or species common in cystic fibrosis (CF) sputum and demonstrated the taxon specificity of these probes, as well as a linear response over more than 3 logs of input RNA. Culture-based analyses correlated qualitatively with relative abundance data on bacterial and fungal taxa obtained by NanoString, and the analysis of serial samples demonstrated the use of this method to simultaneously detect bacteria and fungi and to detect microbes at low abundance without an amplification step. Compared at the genus level, the relative abundances of bacterial taxa detected by analysis of RNA correlated with the relative abundances of the same taxa as measured by sequencing of the V4V5 region of the 16S rRNA gene amplified from community DNA from the same sample. We propose that this method may complement other methods designed to understand dynamic microbial communities, may provide information on bacteria and fungi in the same sample with a single assay, and with further development, may provide quick and easily interpreted diagnostic information on diverse bacteria and fungi at the genus or species level.IMPORTANCE Here we demonstrate the use of an RNA-based analysis of specific taxa of interest, including bacteria and fungi, within microbial communities. This multiplex method may be useful as a means to identify samples with specific combinations of taxa and to gain information on how specific populations vary over time and space or in response to perturbation. A rapid means to measure bacterial and fungal populations may aid in the study of host response to changes in microbial communities.
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Bactérias/classificação , Biota , Fibrose Cística/microbiologia , Fungos/classificação , RNA Bacteriano/genética , RNA Fúngico/genética , Escarro/microbiologia , Bactérias/genética , Bactérias/isolamento & purificação , Fungos/genética , Fungos/isolamento & purificação , Humanos , Sondas de Oligonucleotídeos/genética , RNA Bacteriano/análise , RNA Fúngico/análise , RNA Ribossômico/análise , RNA Ribossômico/genética , Análise de Sequência de DNARESUMO
We report the rare finding of Strongyloides stercoralis rhabditiform larvae in biliary fluid, here associated with cholecystitis and near total portal vein thrombosis. The role of S. stercoralis leading to atypical clinical presentations and difficulty diagnosing strongyloidiasis in such patients with appropriate geographic exposure is discussed.
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UNLABELLED: The airways of patients with cystic fibrosis are colonized with diverse bacterial communities that change dynamically during pediatric years and early adulthood. Staphylococcus aureus is the most prevalent pathogen during early childhood, but during late teens and early adulthood, a shift in microbial composition occurs leading to Pseudomonas aeruginosa community predominance in â¼50% of adults. We developed a robust dual-bacterial in vitro coculture system of P. aeruginosa and S. aureus on monolayers of human bronchial epithelial cells homozygous for the ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) mutation to better model the mechanisms of this interaction. We show that P. aeruginosa drives the S. aureus expression profile from that of aerobic respiration to fermentation. This shift is dependent on the production of both 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) and siderophores by P. aeruginosa. Furthermore, S. aureus-produced lactate is a carbon source that P. aeruginosa preferentially consumes over medium-supplied glucose. We find that initially S. aureus and P. aeruginosa coexist; however, over extended coculture P. aeruginosa reduces S. aureus viability, also in an HQNO- and P. aeruginosa siderophore-dependent manner. Interestingly, S. aureus small-colony-variant (SCV) genetic mutant strains, which have defects in their electron transport chain, experience reduced killing by P. aeruginosa compared to their wild-type parent strains; thus, SCVs may provide a mechanism for persistence of S. aureus in the presence of P. aeruginosa. We propose that the mechanism of P. aeruginosa-mediated killing of S. aureus is multifactorial, requiring HQNO and P. aeruginosa siderophores as well as additional genetic, environmental, and nutritional factors. IMPORTANCE: In individuals with cystic fibrosis, Staphylococcus aureus is the primary respiratory pathogen during childhood. During adulthood, Pseudomonas aeruginosa predominates and correlates with worse patient outcome. The mechanism(s) by which P. aeruginosa outcompetes or kills S. aureus is not well understood. We describe an in vitro dual-bacterial species coculture system on cystic fibrosis-derived airway cells, which models interactions relevant to patients with cystic fibrosis. Further, we show that molecules produced by P. aeruginosa additively induce a transition of S. aureus metabolism from aerobic respiration to fermentation and eventually lead to loss of S. aureus viability. Elucidating the molecular mechanisms of P. aeruginosa community predominance can provide new therapeutic targets and approaches to impede this microbial community transition and subsequent patient worsening.
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Técnicas Bacteriológicas , Técnicas de Cocultura , Fibrose Cística/microbiologia , Viabilidade Microbiana , Pseudomonas aeruginosa/fisiologia , Staphylococcus aureus/metabolismo , Aerobiose , Meios de Cultura/química , Glucose/química , Glucose/metabolismo , Hidroxiquinolinas/farmacologia , Mutação , Sideróforos/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genéticaRESUMO
BACKGROUND: Human-associated microbial communities include fungi, but we understand little about which fungal species are present, their relative and absolute abundances, and how antimicrobial therapy impacts fungal communities. The disease cystic fibrosis (CF) often involves chronic airway colonization by bacteria and fungi, and these infections cause irreversible lung damage. Fungi are detected more frequently in CF sputum samples upon initiation of antimicrobial therapy, and several studies have implicated the detection of fungi in sputum with worse outcomes. Thus, a more complete understanding of fungi in CF is required. RESULTS: We characterized the fungi and bacteria in expectorated sputa from six CF subjects. Samples were collected upon admission for systemic antibacterial therapy and upon the completion of treatment and analyzed using a pyrosequencing-based analysis of fungal internal transcribed spacer 1 (ITS1) and bacterial 16S rDNA sequences. A mixture of Candida species and Malassezia dominated the mycobiome in all samples (74%-99% of fungal reads). There was not a striking trend correlating fungal and bacterial richness, and richness showed a decline after antibiotic therapy particularly for the bacteria. The fungal communities within a sputum sample resembled other samples from that subject despite the aggressive antibacterial therapy. Quantitative PCR analysis of fungal 18S rDNA sequences to assess fungal burden showed variation in fungal density in sputum before and after antibacterial therapy but no consistent directional trend. Analysis of Candida ITS1 sequences amplified from sputum or pure culture-derived genomic DNA from individual Candida species found little (<0.5%) or no variation in ITS1 sequences within or between strains, thereby validating this locus for the purpose of Candida species identification. We also report the enhancement of the publically available Visualization and Analysis of Microbial Population Structures (VAMPS) tool for the analysis of fungal communities in clinical samples. CONCLUSIONS: Fungi are present in CF respiratory sputum. In CF, the use of intravenous antibiotic therapy often does not profoundly impact bacterial community structure, and we observed a similar stability in fungal species composition. Further studies are required to predict the effects of antibacterials on fungal burden in CF and fungal community stability in non-CF populations.
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Pseudomonas aeruginosa produces outer membrane vesicles (OMVs) that contain a number of secreted bacterial proteins, including phospholipases, alkaline phosphatase, and the CFTR inhibitory factor (Cif). Previously, Cif, an epoxide hydrolase, was shown to be regulated at the transcriptional level by epoxides, which serve as ligands of the repressor, CifR. Here, we tested whether epoxides have an effect on Cif levels in OMVs. We showed that growth of P. aeruginosa in the presence of specific epoxides but not a hydrolysis product increased Cif packaging into OMVs in a CifR-independent fashion. The outer membrane protein, OprF, was also increased under these conditions, but alkaline phosphatase activity was not significantly altered. Additionally, we demonstrated that OMV shape and density were affected by epoxide treatment, with two distinct vesicle fractions present when cells were treated with epibromohydrin (EBH), a model epoxide. Vesicles isolated from the two density fractions exhibited different protein profiles in Western blotting and silver staining. We have shown that a variety of clinically or host-relevant treatments, including antibiotics, also alter the proteins packaged in OMVs. Proteomic analysis of purified OMVs followed by an analysis of transposon mutant OMVs yielded mutants with altered vesicle packaging. Finally, epithelial cell cytotoxicity was reduced in the vesicles formed in the presence of EBH, suggesting that this epoxide alters the function of the OMVs. Our data support a model whereby clinically or host-relevant signals mediate differential packaging of virulence factors in OMVs, which results in functional consequences for host-pathogen interactions.
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Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Pseudomonas aeruginosa/metabolismo , Fatores de Virulência/metabolismo , Fosfatase Alcalina , Proteínas de Bactérias/genética , Compostos de Epóxi/metabolismo , Fosfolipases/metabolismo , Transporte Proteico , Pseudomonas aeruginosa/genética , Estresse Fisiológico , Fatores de Virulência/genéticaRESUMO
Streptococcus pneumoniae (pneumococci) adhere to human nasopharyngeal (NP) epithelial cells as a first step in colonization and adherence of pneumococci to lung epithelia may be required to establish pneumonia. We sought to determine if PcpA can serve as an adhesin to human NP (D562) and lung (A549) epithelial cells and whether PcpA mediated adherence can be inhibited by human anti-PcpA antibodies. A PcpA isogenic mutant was constructed in a pneumococcal TIGR4 background. When the mutant and wild type strains were compared for their adherence to D562 and A549 cell lines, a reduction in adherence by the mutant was observed (p = 0.0001 for both cell types). PcpA was ectopically expressed on the surface of minimally-adherent heterologous host Escherichia coli resulting in augmented adherence to D562 (p = 0.002) and A549 (p = 0.015) cells. Total IgG was purified from a pool of 6 human sera having high IgG titers of anti-pneumococcal proteins. The purified IgG reduced TIGR4 adherence to D562 cells but we determined that this effect was largely due to bacterial cell aggregation as determined by flow cytometry and confocal microscopy. Fab fragments were prepared from pooled IgG sera. Inhibition of TIGR4 adherence to D562 cells was observed using the Fab fragments without causing bacterial aggregation (p = 0.0001). Depletion of PcpA-specific Fab fragments resulted in an increase in adherence of TIGR4 to D562 cells (p = 0.028). We conclude that PcpA can mediate adherence of pneumococci to human NP and lung epithelial cells and PcpA mediated adherence can be inhibited by human anti-PcpA antibodies.