Alex Grier

The impact of postnatal antibiotics on the preterm intestinal microbiome

Background:Development of the intestinal microbiome in preterm infants has significant impact on infant health. Our objective was to determine if duration of antibiotics within the first 10 and 30 d after birth affects the intestinal microbiome.Methods:Subjects were 24 0/7–31 6/7 wk of gestational age who received ≥50% breast milk and a total of ≥100 ml/kg of feeds by 10 d. Rectal (fecal) swabs were collected at 10 and 30 d and analyzed by 16S rRNA pyrosequencing. At both time points, we examined the rectal microbiome from infants who received only 2 d of antibiotics and those who received at least 7 d of antibiotics.Results:In the 29 infants enrolled in our study, we found a decrease in diversity index from 10 d samples in those who received more antibiotics. Such difference in diversity and richness was not as pronounced in 30 d samples. Firmicutes and Bacteroidetes were most abundant in the 10 d samples. While these two phyla remained dominant in 30 d samples, there was an increase in Proteobacteria and Actinobacteria.Conclusion:Despite antibiotic therapy, neonates continued to acquire bacteria in the gastrointestinal tract. The process of bacterial acquisition is perturbed with the use of antibiotics.

The effect of antibiotics on the microbiome in acute exacerbations of chronic rhinosinusitis

Current treatment of acute exacerbations of chronic rhinosinusitis (CRS) is driven by identification of predominant bacteria using culture‐based methods and determination of antibiotic sensitivities. The objective of this study was to evaluate the response of the sinonasal microbiome to antibiotic therapy in the setting of an acute exacerbation of CRS.

Targeting the gut microbiome to treat the osteoarthritis of obesity

Obesity is a risk factor for osteoarthritis (OA), the greatest cause of disability in the US. The impact of obesity on OA is driven by systemic inflammation, and increased systemic inflammation is now understood to be caused by gut microbiome dysbiosis. Oligofructose, a nondigestible prebiotic fiber, can restore a lean gut microbial community profile in the context of obesity, suggesting a potentially novel approach to treat the OA of obesity. Here, we report that - compared with the lean murine gut - obesity is associated with loss of beneficial Bifidobacteria, while key proinflammatory species gain in abundance. A downstream systemic inflammatory signature culminates with macrophage migration to the synovium and accelerated knee OA. Oligofructose supplementation restores the lean gut microbiome in obese mice, in part, by supporting key commensal microflora, particularly Bifidobacterium pseudolongum. This is associated with reduced inflammation in the colon, circulation, and knee and protection from OA. This observation of a gut microbiome-OA connection sets the stage for discovery of potentially new OA therapeutics involving strategic manipulation of specific microbial species inhabiting the intestinal space.

The Healthy Infant Nasal Transcriptome: A Benchmark Study

Responses by resident cells are likely to play a key role in determining the severity of respiratory disease. However, sampling of the airways poses a significant challenge, particularly in infants and children. Here, we report a reliable method for obtaining nasal epithelial cell RNA from infants for genome-wide transcriptomic analysis, and describe baseline expression characteristics in an asymptomatic cohort. Nasal epithelial cells were collected by brushing of the inferior turbinates, and gene expression was interrogated by RNA-seq analysis. Reliable recovery of RNA occurred in the absence of adverse events. We observed high expression of epithelial cell markers and similarity to the transcriptome for intrapulmonary airway epithelial cells. We identified genes displaying low and high expression variability, both inherently, and in response to environmental exposures. The greatest gene expression differences in this asymptomatic cohort were associated with the presence of known pathogenic viruses and/or bacteria. Robust bacteria-associated gene expression patterns were significantly associated with the presence of Moraxella. In summary, we have developed a reliable method for interrogating the infant airway transcriptome by sampling the nasal epithelium. Our data demonstrates both the fidelity and feasibility of our methodology, and describes normal gene expression and variation within a healthy infant cohort.

Editor’s Highlight: Variation in Methylmercury Metabolism and Elimination Status in Humans Following Fish Consumption

Evaluating the potential for methylmercury (MeHg) toxicity relies on accurately predicting the mercury (Hg) body burden that results from eating fish. Hg body burden is directly determined by the slow elimination kinetics of MeHg in the human body (kel = 0.014 days-1 or t1/2 =50 days). Existing studies on MeHg half-life in humans demonstrate a wide range values (t1/2 = 30 to >150 days) and has lead to uncertainty in the derivation of a regulatory standard for acceptable daily oral intake. The causes of variation in MeHg toxicokinetics in humans remain little explored. Here we characterize variation in human MeHg metabolism and elimination rate (kel) in 37 adult volunteers who consumed 3 fish meals. We determined MeHg elimination rates via longitudinal Hg analysis in single hairs using laser ablation inductively coupled plasma mass spectrometry. We also measured MeHg metabolism (biotransformation) via speciation of fecal Hg. We find an average kel = 0.0157 days-1 (t1/2 = 44 days) amongst a more than 2-fold variation in kel across the cohort (0.0248-0.0112 days-1; t1/2 = 28-62 days). Although MeHg biotransformation varied widely between individuals, it showed a positive association with elimination rates across the cohort. A more than 2-fold change in kel over a period of 2 years was seen in some individuals. In 2 individuals, who received antibiotic for unrelated health issues, elimination rate was seen to slow significantly. Associations of kel with age, body mass index, gender, and fish eating habits were not observed. We establish that a measure of methylmercury metabolism and eliminaiton status (MerMES) can reduce uncertainty in determining an individuals MeHg toxicokinetics subsequent to eating fish.

Neonatal gut and respiratory microbiota: coordinated development through time and space

BackgroundPostnatal development of early life microbiota influences immunity, metabolism, neurodevelopment, and infant health. Microbiome development occurs at multiple body sites, with distinct community compositions and functions. Associations between microbiota at multiple sites represent an unexplored influence on the infant microbiome. Here, we examined co-occurrence patterns of gut and respiratory microbiota in pre- and full-term infants over the first year of life, a period critical to neonatal development.ResultsGut and respiratory microbiota collected as longitudinal rectal, throat, and nasal samples from 38 pre-term and 44 full-term infants were first clustered into community state types (CSTs) on the basis of their compositional profiles. Multiple methods were used to relate the occurrence of CSTs to temporal microbiota development and measures of infant maturity, including gestational age (GA) at birth, week of life (WOL), and post-menstrual age (PMA). Manifestation of CSTs followed one of three patterns with respect to infant maturity: (1) chronological, with CST occurrence frequency solely a function of post-natal age (WOL), (2) idiosyncratic to maturity at birth, with the interval of CST occurrence dependent on infant post-natal age but the frequency of occurrence dependent on GA at birth, and (3) convergent, in which CSTs appear first in infants of greater maturity at birth, with occurrence frequency in pre-terms converging after a post-natal interval proportional to pre-maturity. The composition of CSTs was highly dissimilar between different body sites, but the CST of any one body site was highly predictive of the CSTs at other body sites. There were significant associations between the abundance of individual taxa at each body site and the CSTs of the other body sites, which persisted after stringent control for the non-linear effects of infant maturity. Canonical correlations exist between the microbiota composition at each pair of body sites, with the strongest correlations between proximal locations.ConclusionThese findings suggest that early microbiota is shaped by neonatal innate and adaptive developmental responses. Temporal progression of CST occurrence is influenced by infant maturity at birth and post-natal age. Significant associations of microbiota across body sites reveal distal connections and coordinated development of the infant microbial ecosystem.

Association between Oral Candida and Bacteriome in Children with Severe ECC

Candida albicans is an opportunistic fungal organism frequently detected in the oral cavity of children with severe early childhood caries (S-ECC). Previous studies suggested the cariogenic potential of C. albicans, in vitro and in vivo, and further demonstrated its synergistic interactions with Streptococcus mutans. In combination, the 2 organisms are associated with higher caries severity in a rodent model. However, it remains unknown whether C. albicans influences the composition and diversity of the entire oral bacterial community to promote S-ECC onset. With 16s rRNA amplicon sequencing, this study analyzed the microbiota of saliva and supragingival plaque from 39 children (21 S-ECC and 18 caries-free [CF]) and 33 mothers (17 S-ECC and 16 CF). The results revealed that the presence of oral C. albicans is associated with a highly acidogenic and acid-tolerant bacterial community in S-ECC, with an increased abundance of plaque Streptococcus (particularly S. mutans) and certain Lactobacillus/Scardovia species and salivary/plaque Veillonella and Prevotella, as well as decreased levels of salivary/plaque Actinomyces. Concurrent with this microbial community assembly, the activity of glucosyltransferases (cariogenic virulence factors secreted by S. mutans) in plaque was significantly elevated when C. albicans was present. Moreover, the oral microbial community composition and diversity differed significantly by disease group (CF vs. S-ECC) and sample source (saliva vs. plaque). Children and mothers within the CF and S-ECC groups shared microbiota composition and diversity, suggesting a strong maternal influence on children’s oral microbiota. Altogether, this study underscores the importance of C. albicans in association with the oral bacteriome in the context of S-ECC etiopathogenesis. Further longitudinal studies are warranted to examine how fungal-bacterial interactions modulate the onset and severity of S-ECC, potentially leading to novel anticaries treatments that address fungal contributions.

Microbiome Analysis for Assessments of Treatment Response and Salvage Prognosis in Infected Diabetic Foot Ulcers

Category: Basic Sciences/Biologics, Diabetes Introduction/Purpose: Diabetic foot ulcers (DFUs) contribute to 80% of non-traumatic lower-extremity amputations. Surgeons are often forced to make surgical decision without adequate prognostic information. DFU infections are often polymicrobial, representing complex microbial communities. A microbiota is the ecological community of various microorganisms that share body space. Currently, the methods of detecting an active infection, identifying the pathogenic bacteria within the microbiome, measuring the response to therapy, and assessing prognosis are limited. Using a molecular genomic technique of 16 S rRNA sequencing, our goals are to assess the pathogenic bioburden of DFUs and to monitor the bacterial community changes in response to antibiotic treatment. Our hypothesis is that the microbiome in DFUs responding to debridement and antibiotics treatment is distinct from those that fail to respond. Methods: Patients with type I or II diabetes who presented with an infected DFU were enrolled. Infections were identified using clinical signs. The DFU size was measured and classified using the Wagner classification. Enrolled patients were initially managed with foot salvaging therapy (FST): irrigation and debridement followed by wet-to-dry dressings and 6 weeks of intravenous antibiotic treatment. Superficial and deep DFU samples were obtained and evaluated by 16 S rRNA microbiome analysis and qPCR for bacterial abundance. This was repeated at 4, 8, and 12 weeks following the initiation of FST. At 12 weeks, patients were divided into two groups, healed and non-healed, based on the change in the size of the wound and absence or presence of 12 secondary signs of infection. Alpha- and beta-diversity were measured by the Shannon index and Bray-Curtis dissimilarity index to evaluate changes in the microbiome between the healed and non-healed groups. Results: From July 2015 to August 2016, 21 patients were enrolled and 3 deceased due to medical comorbidities. Of the 18 patients available for follow-up, 10 failed FST and 8 healed. The qPCR and microbiome analysis revealed that the bacterial abundance and diversity of the bacterial community were substantially reduced following debridement and intravenous antibiotic treatment. At the initial enrollment, those group that healed versus non-healed showed significant differences in microbiome composition, with the healed group enriched with Actinomycetales and Staphylococcus, and the non-healed group enriched with Bacteroidales and Streptococcus. At week 4, such differences disappeared and bacterial abundance significantly decreased. New differences were evident at week 8: the healed group was enriched with Actinomycetales and non-healed group with Bacilli. Conclusion: Abundant presence of Bacteroidales and Streptococcus at the initial presentation of infected DFU maybe a poor prognostic sign for healing with FST. Through molecular analysis of the wound microbiome, we can identify pathogens of prognostic value at the initial cultures and assess response to therapy with significant differences at 8 weeks after. Our study provides useful information for counseling patients of treatment prognosis and determining to pursuit further foot salvage versus amputation.

Clinical Application of Immunoassay for Identification of Staphylococcus aureus and Monitoring Treatment Response in Diabetic Foot Infection

Category: Basic Sciences/Biologics, Diabetes Introduction/Purpose: Currently, there is no sensitive and specific diagnostic tool for identifying the active pathogen in a polymicrobial environment such as diabetic foot infection (DFI). In addition, monitoring the success of antibiotic treatment is limited to clinical signs and nonspecific inflammatory markers. Consequently, surgeons are often forced to make interventional decisions without adequate prognostic information. While DFI are generally polymicrobial, about 50% prominently include Staphylococcus aureus (SA). We investigated applicability of utilizing a novel diagnostic immunoassay that measures the patient’s current production of anti-SA antibodies (IgG) to accurately diagnose SA and monitor its pathogenic activity. We hypothesize that 1) compared to standard culture, the immunoassay has a higher sensitivity to detect SA and 2) able monitor changes in pathogenic activity of SA in DFI. Methods: From July 2015 to August 2016, we enrolled 20 diabetic patients with DFU who displayed clinical symptoms and signs of infection which necessitated hospitalization and undertook initial foot salvage therapy (FST): irrigation and debridement followed by wet-to-dry dressings and 6 weeks of intravenous antibiotic treatment. At weeks 0, 4, 8 and 12, the infected DFUs samples were obtained for standard culture and 16 S rRNA microbiome analysis. Whole blood and serum samples were collected to measure the abundance of anti-SA IgG in the serum and in the in vitro secretions of antibody-secreting cells harvested from whole blood in “medium enriched for newly synthesized antibody”. Sensitivity and specificity for detection of SA were compared against the standard culture and microbiome analysis. Preliminary analyses compare the ability of the SA immunoassay to track therapy and its concordance with changes in the microbiome. Results: Of the 20 enrolled patients, 18 were available for at least partial follow-up and only four completed the entire sampling protocol. At the enrollment, 12 patients (60%) were identified positive for SA infection by at least one diagnostic method, while only 8 were diagnosed by standard culture. Six out of 10 SA-positive patients showed polymicrobial growth. The concordance rate for the presence or absence of SA was 85% between the immunoassay and microbiome, 70% between immunoassay and standard culture and 75% between microbiome and standard culture. Comparison of serial samples from the 7 subjects who were SA-positive by both the immunoassay and microbiome analysis demonstrated trends that the two novel assays provide complementary measures of therapeutic success. (Figure 1) Conclusion: Measurement of anti-SA antibodies showed higher sensitivity than standard culture and was able to monitor changes in pathogenic activity of SA in DFI undertaking salvage treatment. This novel immnoassay may serve as an important diagnostic and prognostic tools for monitoring SA infection in polymicrobial DFI. It provides important information for counseling patients of treatment response, prognosis, and determining to pursuit further foot salvage versus amputation. Future study will include expanding immunoassay measure other commonly found organisms in DFI.