The Microbiome and Pediatric Transplantation.

The microbial communities that inhabit our bodies have been increasingly linked to host physiology and pathophysiology. This microbiome, through its role in colonization resistance, influences the risk of infections after transplantation, including those caused by multidrug-resistant organisms. In addition, through both direct interactions with the host immune system and via the production of metabolites that impact local and systemic immunity, the microbiome plays an important role in the establishment of immune tolerance after transplantation, and conversely, in the development of graft-versus-host disease and graft rejection. This review offers a comprehensive overview of the evidence for the role of the microbiome in hematopoietic cell and solid organ transplant complications, drivers of microbiome shift during transplantation, and the potential of microbiome-based therapies to improve pediatric transplantation outcomes.

Isolation and Characterization of a Novel Alpha-Hemolytic Streptococcus spp. from the Oral Cavity and Blood of Septicemic Periparturient Immunodeficient Mice.

MISTRG is an immunodeficient mouse strain that expresses multiple human cytokines that support hematopoietic stem cell maintenance and myelopoiesis. While establishing a breeding colony of MISTRG mice in a dedicated barrier room, 6 cases of death or disease occurred in pregnant or postpartum mice. Clinically, this manifested as hunched posture, dyspnea, and 1 case of emaciation with ataxia. Pathologic analysis of 7 mice revealed multisystemic necrosuppurative inflammation variably affecting the uterus and placenta, joints, meninges, inner and middle ears, kidneys, and small intestine. Bacteria cultured from the blood of septic mice were identified with 89% probability by the Vitek 2 identification system as Streptococcus sanguinus with atypical biochemical parameters; the API 20E/NE system fully differentiated the isolates as a novel Streptococcus species. MALDI Biotyper-based mass spectrometry also indicated that the phenotype represented a novel Streptococcus spp. Sequencing revealed that the full-length 16S rRNA gene identity was below 97% with known Streptococcus species, including the 2 closest species Streptococcus acidominimus and Streptococcus azizii. We propose the name Streptococcus murisepticum spp. nov to our novel isolates. All male mice in this colony remained healthy despite their association with diseased female mice. Overall, 19% of the colony carried the novel Streptococcus in their oral cavity, but it could not be detected in feces. The organism was sensitive to amoxicillin, which was administered via drinking water throughout pregnancy and weaning to establish a colony of pathogen-negative future breeders. The colony remained disease-free and culture-negative for Streptococcus murisepticum spp. nov after treatment with amoxicillin. We suspect that oral colonization of MISTRG mice with the novel Streptococcus species and its associated unique pathology in periparturient mice is potentially the principal cause of loss of this strain at several institutions. Therefore, screening the oral cavity for α-hemolytic streptococci followed by targeted antibiotic treatment may be necessary when establishing MISTRG and allied immunodeficient mouse strains.

Microbiota Predict Infections and Acute Graft-Versus-Host Disease After Pediatric Allogeneic Hematopoietic Stem Cell Transplantation.

BACKGROUND: Despite preventive measures, infections continue to pose significant risks to pediatric allogeneic hematopoietic cell transplantation (allo-HCT) recipients. The gut microbiota has been linked to clinical outcomes following adult allo-HCT. This study evaluated whether similar disruptions or differing microbiota patterns were associated with infection risk in pediatric allo-HCT. METHODS: In a prospective observational study, fecal samples were obtained from 74 children before conditioning and upon neutrophil recovery. Microbiome signatures identified through sequencing were examined for their associations with infections or acute graft-versus-host disease (aGVHD) in the first-year post-HCT using Cox proportional hazards analysis. RESULTS: Microbiome disruption in adults, did not predict infection risk in pediatric allo-HCT. Unique microbiota signatures were associated with different infections or aGVHD. A ratio of strict and facultative anaerobes (eg, Lachnoclostridium, Parabacteroides) prior to conditioning predicted bacteremia risk (Cox hazard ratio [HR], 3.89). A distinct ratio of oral (eg, Rothia, Veillonella) to intestinal anaerobes (eg, Anaerobutyricum, Romboutsia) at neutrophil recovery predicted likelihood of bacterial infections (Cox HR, 1.81) and viral enterocolitis (Cox HR, 1.96). CONCLUSIONS: Interactions between medical interventions, pediatric hosts, and microbial communities contribute to microbiota signatures that predict infections. Further multicenter study is necessary to validate the generalizability of these ratios as biomarkers.

Real-time quantitative PCR method for detection and quantification of Clostridioides difficile cells and spores.

A quantitative PCR method was developed for the detection of Clostridioides difficile Chaperonin-60 gene. The method had a specificity for C. difficile strains of clinical and epidemiological importance and allowed for quantification of C. difficile cells and spores.

Antibiotic prophylaxis and the gastrointestinal resistome in paediatric patients with acute lymphoblastic leukaemia: a cohort study with metagenomic sequencing analysis.

BACKGROUND: Although antibiotic prophylaxis with levofloxacin can reduce the risk of serious infection in immunocompromised patients, the potential contribution of prophylaxis to antibiotic resistance is a major drawback. We aimed to identify the effects of levofloxacin prophylaxis, given to paediatric patients with acute lymphoblastic leukaemia to prevent infections during induction chemotherapy, on antibiotic resistance in gastrointestinal microbiota after completion of induction and consolidation therapy. METHODS: This prospective, single-centre (St Jude Children's Research Hospital, Memphis, TN, USA) cohort study included children (≤18 years) receiving therapy for newly diagnosed acute lymphoblastic leukaemia and who received either primary levofloxacin prophylaxis or no antibacterial prophylaxis (aside from Pneumocystis jirovecii prophylaxis with trimethoprim-sulfamethoxazole) and provided at least two stool samples, including one after completion of induction therapy. We used metagenomic sequencing to identify bacterial genes that confer resistance to fluoroquinolones, trimethoprim-sulfamethoxazole, or other antibiotics, and to identify point mutations in bacterial topoisomerases (gyrA, parC) that confer resistance to fluoroquinolones. We then used generalised linear mixed models to compare the prevalence and relative abundance of antibiotic resistance gene groups after completion of induction and consolidation therapy between participants who had received levofloxacin and those who received no prophylaxis. FINDINGS: Between Feb 1, 2012, and April 30, 2016, 118 stool samples (32 baseline, 49 after induction, and 37 after consolidation) were collected from 49 evaluable participants; of these participants, 31 (63%) received levofloxacin prophylaxis during induction therapy and 18 (37%) received no antibacterial prophylaxis. Over the course of induction therapy, there was an overall increase in the relative abundance of trimethoprim-sulfamethoxazole resistance genes (estimated mean fold change 5·9, 95% CI 3·6-9·6; p<0·0001), which was not modified by levofloxacin prophylaxis (p=0·46). By contrast, the prevalence of topoisomerase point mutations increased over the course of induction therapy in levofloxacin recipients (mean prevalence 10·4% [95% CI 3·2-25·4] after induction therapy vs 3·7% [0·2-22·5] at baseline) but not other participants (0% vs 0%; p<0·0001). There was no significant difference between prophylaxis groups with respect to changes in aminoglycoside, ß-lactam, vancomycin, or multidrug resistance genes after completion of induction or consolidation therapy. INTERPRETATION: Analysing the gastrointestinal resistome can provide insights into the effects of antibiotics on the risk of antibiotic-resistant infections. In this study, antibiotic prophylaxis with trimethoprim-sulfamethoxazole or levofloxacin during induction therapy for acute lymphoblastic leukaemia appeared to increase the short-term and medium-term risk of colonisation with bacteria resistant to these antibiotics, but not to other drugs. More research is needed to determine the longer-term effects of antibacterial prophylaxis on colonisation with antibiotic-resistant bacteria. FUNDING: Children's Infection Defense Center at St Jude Children's Research Hospital, American Lebanese Syrian Associated Charities, and National Institutes of Health.

Respiratory Bacteria Stabilize and Promote Airborne Transmission of Influenza A Virus.

Influenza A virus (IAV) is a major pathogen of the human respiratory tract, where the virus coexists and interacts with bacterial populations comprising the respiratory tract microbiome. Synergies between IAV and respiratory bacterial pathogens promote enhanced inflammation and disease burden that exacerbate morbidity and mortality. We demonstrate that direct interactions between IAV and encapsulated bacteria commonly found in the respiratory tract promote environmental stability and infectivity of IAV. Antibiotic-mediated depletion of the respiratory bacterial flora abrogated IAV transmission in ferret models, indicating that these virus-bacterium interactions are operative for airborne transmission of IAV. Restoring IAV airborne transmission in antibiotic-treated ferrets by coinfection with Streptococcus pneumoniae confirmed a role for specific members of the bacterial respiratory community in promoting IAV transmission. These results implicate a role for the bacterial respiratory flora in promoting airborne transmission of IAV.IMPORTANCE Infection with influenza A virus (IAV), especially when complicated with a secondary bacterial infection, is a leading cause of global mortality and morbidity. Gaining a greater understanding of the transmission dynamics of IAV is important during seasonal IAV epidemics and in the event of a pandemic. Direct bacterium-virus interactions are a recently appreciated aspect of infectious disease biology. Direct interactions between IAV and specific bacterial species of the human upper respiratory tract were found to promote the stability and infectivity of IAV during desiccation stress. Viral environmental stability is an important aspect during transmission, suggesting a potential role for bacterial respiratory communities in IAV transmission. Airborne transmission of IAV was abrogated upon depletion of nasal bacterial flora with topical antibiotics. This defect could be functionally complemented by S. pneumoniae coinfection. These data suggest that bacterial coinfection may be an underappreciated aspect of IAV transmission dynamics.

Vancomycin Heteroresistance and Clinical Outcomes in Bloodstream Infections Caused by Coagulase-Negative Staphylococci.

Coagulase-negative staphylococci (CoNS) are a common etiology of serious and recurrent infections in immunocompromised patients. Although most isolates appear susceptible to vancomycin, a single strain might have a subpopulation of resistant bacteria. This phenomenon is termed heteroresistance and may adversely affect the response to treatment. A retrospective cohort study was performed of pediatric patients with leukemia treated at St. Jude Children's Research Hospital who developed CoNS central line-associated bloodstream infection (CLABSI). Available isolates were sequenced and tested for vancomycin heteroresistance by population analysis profiling. Risk factors for heteroresistance and the association of heteroresistance with treatment failure (death or relapse of infection) or poor clinical response to vancomycin therapy (treatment failure or persistent bacteremia after vancomycin initiation) were evaluated. For 65 participants with CoNS CLABSI, 62 initial isolates were evaluable, of which 24 (39%) were vancomycin heteroresistant. All heteroresistant isolates were of Staphylococcus epidermidis and comprised multiple sequence types. Participants with heteroresistant bacteria had more exposure to vancomycin prophylaxis (P = 0.026) during the 60 days prior to infection. Of the 40 participants evaluable for clinical outcomes, heteroresistance increased the risk of treatment failure (P = 0.012) and poor clinical response (P = 0.001). This effect persisted after controlling for identified confounders. These data indicate that vancomycin heteroresistance is common in CoNS isolates from CLABSIs in pediatric patients with leukemia and is associated with poor clinical outcomes. Validation of these findings in an independent cohort and evaluation of alternative antibiotic therapy in patients with heteroresistant infections have the potential to improve care for serious CoNS infections.

Astrovirus infects actively secreting goblet cells and alters the gut mucus barrier.

Astroviruses are a global cause of pediatric diarrhea, but they are largely understudied, and it is unclear how and where they replicate in the gut. Using an in vivo model, here we report that murine astrovirus preferentially infects actively secreting small intestinal goblet cells, specialized epithelial cells that maintain the mucus barrier. Consequently, virus infection alters mucus production, leading to an increase in mucus-associated bacteria and resistance to enteropathogenic E. coli colonization. These studies establish the main target cell type and region of the gut for productive murine astrovirus infection. They further define a mechanism by which an enteric virus can regulate the mucus barrier, induce functional changes to commensal microbial communities, and alter host susceptibility to pathogenic bacteria.

Astrovirus and the microbiome.

Although astroviruses are most commonly associated with acute gastrointestinal illness in humans, their ability to infect a broad range of hosts and cause a spectrum of disease makes them widespread and complex pathogens. The precise mechanisms that dictate the course of astrovirus disease have not been studied extensively but are likely driven by multifactorial host-microbe interactions. Recent insights from studies of animal astrovirus infections have revealed both beneficial and detrimental effects for the host. However, further in-depth studies are needed to fully explore the consequences of astrovirus-induced changes in the gut microenvironment as well as the role of the microbiota in astrovirus infection.

Improved yield and accuracy for DNA extraction in microbiome studies with variation in microbial biomass.

A major challenge for microbiome studies is maintaining an even and accurate DNA extraction in the presence of samples with a wide range of bacterial content. Here we compare five DNA extraction methods using replicate stool samples that were diluted to create high and low biomass samples. Our results indicate greater variation in microbiome composition between high and low biomass samples than variation between methods. Many of the extraction methods had reduced yield from low biomass samples; however, our adapted plate column-based extraction method was evenly efficient and captured the largest number of high-quality reads. Based on these results, we have identified a DNA extraction method that ensures adequate yield in metagenomic microbiome studies that have samples with a broad range of bacterial content.

Identification of Leptospira and Bartonella among rodents collected across a habitat disturbance gradient along the Inter-Oceanic Highway in the southern Amazon Basin of Peru.

BACKGROUND: The southern Amazon Basin in the Madre de Dios region of Peru has undergone rapid deforestation and habitat disruption, leading to an unknown zoonotic risk to the growing communities in the area. METHODOLOGY/PRINCIPAL FINDINGS: We surveyed the prevalence of rodent-borne Leptospira and Bartonella, as well as potential environmental sources of human exposure to Leptospira, in 4 communities along the Inter-Oceanic Highway in Madre de Dios. During the rainy and dry seasons of 2014-2015, we captured a total of 97 rodents representing 8 genera in areas that had experienced different degrees of habitat disturbance. Primarily by using 16S metagenomic sequencing, we found that most of the rodents (78%) tested positive for Bartonella, whereas 24% were positive for Leptospira; however, the patterns differed across seasons and the extent of habitat disruption. A high prevalence of Bartonella was identified in animals captured across both trapping seasons (72%-83%) and the relative abundance was correlated with increasing level of land disturbance. Leptospira-positive animals were more than twice as prevalent during the rainy season (37%) as during the dry season (14%). A seasonal fluctuation across the rainy, dry, and mid seasons was also apparent in environmental samples tested for Leptospira (range, 55%-89% of samples testing positive), and there was a high prevalence of this bacteria across all sites that were sampled in the communities. CONCLUSIONS/SIGNIFICANCE: These data indicate the need for increased awareness of rodent-borne disease and the potential for environmental spread along the communities in areas undergoing significant land-use change.

Fitness Landscape of the Immune Compromised Favors the Emergence of Antibiotic Resistance.

Antibiotic resistance can come at a high cost, both in terms of fitness for the pathogen and poorer outcomes for patients. The fitness landscape encountered by bacterial pathogens varies greatly throughout patient populations in terms of host immunity as well as the duration and spectrum of antibiotics encountered. Severely immunocompromised patients present a favorable environment for antibiotic resistance to emerge due to lack of immune-mediated competition and increased opportunities to evolve both on-target and compensatory mutations. Such patients may present unique pathways for antibiotic resistance to emerge.

Evaluating the accuracy of amplicon-based microbiome computational pipelines on simulated human gut microbial communities.

BACKGROUND: Microbiome studies commonly use 16S rRNA gene amplicon sequencing to characterize microbial communities. Errors introduced at multiple steps in this process can affect the interpretation of the data. Here we evaluate the accuracy of operational taxonomic unit (OTU) generation, taxonomic classification, alpha- and beta-diversity measures for different settings in QIIME, MOTHUR and a pplacer-based classification pipeline, using a novel software package: DECARD. RESULTS: In-silico we generated 100 synthetic bacterial communities approximating human stool microbiomes to be used as a gold-standard for evaluating the colligative performance of microbiome analysis software. Our synthetic data closely matched the composition and complexity of actual healthy human stool microbiomes. Genus-level taxonomic classification was correctly done for only 50.4-74.8% of the source organisms. Miscall rates varied from 11.9 to 23.5%. Species-level classification was less successful, (6.9-18.9% correct); miscall rates were comparable to those of genus-level targets (12.5-26.2%). The degree of miscall varied by clade of organism, pipeline and specific settings used. OTU generation accuracy varied by strategy (closed, de novo or subsampling), reference database, algorithm and software implementation. Shannon diversity estimation accuracy correlated generally with OTU-generation accuracy. Beta-diversity estimates with Double Principle Coordinate Analysis (DPCoA) were more robust against errors introduced in processing than Weighted UniFrac. The settings suggested in the tutorials were among the worst performing in all outcomes tested. CONCLUSIONS: Even when using the same classification pipeline, the specific OTU-generation strategy, reference database and downstream analysis methods selection can have a dramatic effect on the accuracy of taxonomic classification, and alpha- and beta-diversity estimation. Even minor changes in settings adversely affected the accuracy of the results, bringing them far from the best-observed result. Thus, specific details of how a pipeline is used (including OTU generation strategy, reference sets, clustering algorithm and specific software implementation) should be specified in the methods section of all microbiome studies. Researchers should evaluate their chosen pipeline and settings to confirm it can adequately answer the research question rather than assuming the tutorial or standard-operating-procedure settings will be adequate or optimal.

The ecology of nasal colonization of Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus: the role of competition and interactions with host's immune response.

BACKGROUND: The first step in invasive disease caused by the normally commensal bacteria Streptococcus pneumoniae, Staphylococcus aureus and Haemophilus influenzae is their colonization of the nasal passages. For any population to colonize a new habitat it is necessary for it to be able to compete with the existing organisms and evade predation. In the case of colonization of these species the competition is between strains of the same and different species of bacteria and the predation is mediated by the host's immune response. Here, we use a neonatal rat model to explore these elements of the ecology of nasal colonization by these occasionally invasive bacteria. RESULTS: When neonatal rats are colonized by any one of these species the density of bacteria in the nasal passage rapidly reaches a steady-state density that is species-specific but independent of inoculum size. When novel populations of H. influenzae and S. pneumoniae are introduced into the nasal passages of neonatal rats with established populations of the same species, residents and invaders coexisted. However, this was not the case for S. aureus - the established population inhibited invasion of new S. aureus populations. In mixed-species introductions, S. aureus or S. pneumoniae facilitated the invasion of another H. influenzae population; for other pairs the interaction was antagonistic and immune-mediated. For example, under some conditions H. influenzae promoted an immune response which limited the invasion of S. pneumoniae. CONCLUSIONS: Nasal colonization is a dynamic process with turnover of new strains and new species. These results suggest that multiple strains of either H. influenzae or S. pneumoniae can coexist; in contrast, S. aureus strains require a host to have no other S. aureus present to colonize. Levels of colonization (and hence the possible risk of invasive disease) by H. influenzae are increased in hosts pre-colonized with either S. aureus or S. pneumoniae.

Hydrogen peroxide-mediated interference competition by Streptococcus pneumoniae has no significant effect on Staphylococcus aureus nasal colonization of neonatal rats.

It has been proposed that the relative scarcity of Staphylococcus aureus and Streptococcus pneumoniae cocolonization in the nasopharynxes of humans can be attributed to hydrogen peroxide-mediated interference competition. Previously it has been shown in vitro that H(2)O(2) produced by S. pneumoniae is bactericidal to S. aureus. To ascertain whether H(2)O(2) has this inhibitory effect in the nasal passages of neonatal rats, colonization experiments were performed with S. aureus and S. pneumoniae. The results of these experiments with neonatal rats are inconsistent with the hypothesis that hydrogen peroxide-mediated killing of S. aureus by S. pneumoniae is responsible for the relative scarcity of cocolonization by these bacteria. In mixed-inoculum colonization experiments and experiments where S. aureus invaded the nasopharynxes of rats with established S. pneumoniae populations, the density of S. aureus did not differ whether the S. pneumoniae strain was H(2)O(2) secreting or non-H(2)O(2) secreting (SpxB). Moreover, the advantage of catalase production by S. aureus in competition with a non-catalase-producing strain (KatA) during nasal colonization was no greater in the presence of H(2)O(2)-producing S. pneumoniae than in the presence of non-H(2)O(2)-producing S. pneumoniae.

Exploring the role of the immune response in preventing antibiotic resistance.

For many bacterial infections, drug resistant mutants are likely present by the time antibiotic treatment starts. Nevertheless, such infections are often successfully cleared. It is commonly assumed that this is due to the combined action of drug and immune response, the latter facilitating clearance of the resistant population. However, most studies of drug resistance emergence during antibiotic treatment focus almost exclusively on the dynamics of bacteria and the drug and neglect the contribution of immune defenses. Here, we develop and analyze several mathematical models that explicitly include an immune response. We consider different types of immune responses and investigate how each impacts the emergence of resistance. We show that an immune response that retains its strength despite a strong drug-induced decline of bacteria numbers considerably reduces the emergence of resistance, narrows the mutant selection window, and mitigates the effects of non-adherence to treatment. Additionally, we show that compared to an immune response that kills bacteria at a constant rate, one that trades reduced killing at high bacterial load for increased killing at low bacterial load is sometimes preferable. We discuss the predictions and hypotheses derived from this study and how they can be tested experimentally.

Within-host evolution for the invasiveness of commensal bacteria: an experimental study of bacteremias resulting from Haemophilus influenzae nasal carriage.

BACKGROUND: Many bacteria responsible for clinically relevant disease reside harmlessly in a large fraction of humans. Three explanations have been proposed to account for why these normally commensal bacteria occasionally cause invasive disease: host susceptibility, stochasticity in the host-bacteria interaction, and the evolution of invasive mutants in colonized hosts. Here we test the third of these hypotheses for the rare invasiveness of commensal bacteria: within-host evolution. METHODS AND RESULTS: Using neonatal rats intranasally colonized with pairs of marked Haemophilus influenzae type b strains, we demonstrate that the resulting bacteremias are derived from single organisms. To test the within-host evolution hypothesis we explored the relative ability of bacteria isolated from the blood and nasal passages of bacteremic rats to colonize the nasopharynx and invade the bloodstream. CONCLUSIONS: Our results provide support for within-host evolution as one but not the sole explanation for the invasiveness of these bacteria. We discuss the implications of these results for both the rare invasiveness of commensal bacteria and the general observation that bacteria isolated from the sites of human invasive disease are almost invariably monoclonal.