Donor-derived cell-free DNA predicts allograft failure and mortality after lung transplantation.

BACKGROUND: Allograft failure is common in lung-transplant recipients and leads to poor outcomes including early death. No reliable clinical tools exist to identify patients at high risk for allograft failure. This study tested the use of donor-derived cell-free DNA (%ddcfDNA) as a sensitive marker of early graft injury to predict impending allograft failure. METHODS: This multicenter, prospective cohort study enrolled 106 subjects who underwent lung transplantation and monitored them after transplantation for the development of allograft failure (defined as severe chronic lung allograft dysfunction [CLAD], retransplantation, and/or death from respiratory failure). Plasma samples were collected serially in the first three months following transplantation and assayed for %ddcfDNA by shotgun sequencing. We computed the average levels of ddcfDNA over three months for each patient (avddDNA) and determined its relationship to allograft failure using Cox-regression analysis. FINDINGS: avddDNA was highly variable among subjects: median values were 3·6%, 1·6% and 0·7% for the upper, middle, and low tertiles, respectively (range 0·1%-9·9%). Compared to subjects in the low and middle tertiles, those with avddDNA in the upper tertile had a 6·6-fold higher risk of developing allograft failure (95% confidence interval 1·6-19·9, p = 0·007), lower peak FEV1 values, and more frequent %ddcfDNA elevations that were not clinically detectable. INTERPRETATION: Lung transplant patients with early unresolving allograft injury measured via %ddcfDNA are at risk of subsequent allograft injury, which is often clinically silent, and progresses to allograft failure. FUND: National Institutes of Health.

Metagenome tracking biogeographic agroecology: Phytobiota of tomatoes from Virginia, Maryland, North Carolina and California.

Describing baseline microbiota associated with agricultural commodities in the field is an important step towards improving our understanding of a wide range of important objectives from plant pathology and horticultural sustainability, to food safety. Environmental pressures on plants (wind, dust, drought, water, temperature) vary by geography and characterizing the impact of these variable pressures on phyllosphere microbiota will contribute to improved stewardship of fresh produce for both plant and human health. A higher resolution understanding of the incidence of human pathogens on food plants and co-occurring phytobiota using metagenomic approaches (metagenome tracking) may contribute to improved source attribution and risk assessment in cases where human pathogens become introduced to agro-ecologies. Between 1990 and 2007, as many as 1990 culture-confirmed Salmonella illnesses were linked to tomatoes from as many as 12 multistate outbreaks (Bell et al., 2012; Bell et al., 2015; Bennett et al., 2014; CDC, 2004; CDC, 2007; Greene et al., 2005a; Gruszynski et al., 2014). When possible, source attribution for these incidents revealed a biogeographic trend, most events were associated with eastern growing regions. To improve our understanding of potential biogeographically linked trends in contamination of tomatoes by Salmonella, we profiled microbiota from the surfaces of tomatoes from Virginia, Maryland, North Carolina and California. Bacterial profiles from California tomatoes were completely different than those of Maryland, Virginia and North Carolina (which were highly similar to each other). A statistically significant enrichment of Firmicutes taxa was observed in California phytobiota compared to the three eastern states. Rhizobiaceae, Sphingobacteriaceae and Xanthobacteraceae were the most abundant bacterial families associated with tomatoes grown in eastern states. These baseline metagenomic profiles of phyllosphere microbiota may contribute to improved understanding of how certain ecologies provide supportive resources for human pathogens on plants and how components of certain agro-ecologies may play a role in the introduction of human pathogens to plants.

HISTONE DEACETYLASE 19 and the flowering time gene FD maintain reproductive meristem identity in an age-dependent manner.

The shoot apical meristem (SAM) undergoes developmental transitions that include a shift from vegetative to reproductive growth. This transition is triggered by flowering time genes, which up-regulate floral meristem (FM) identity genes that, in turn, control flower development by activating floral organ identity genes. This cascade of transcriptional activation is refined by repression mechanisms that temporally and spatially restrict gene expression to ensure proper development. Here, we demonstrate that HISTONE DEACETYLASE 19 (HDA19) maintains the identity of the reproductive SAM, or inflorescence meristem (IM), late in Arabidopsis thaliana development. At late stages of growth, hda19 IMs display a striking patterning defect characterized by ectopic expression of floral organ identity genes and the replacement of flowers with individual stamenoid organs. We further show that the flowering time gene FD has a specific function in this regulatory process, as fd hastens the emergence of these patterning defects in hda19 growth. Our work therefore identifies a new role for FD in reproductive patterning, as FD regulates IM function together with HDA19 in an age-dependent fashion. To effect these abnormalities, hda19 and fd may accentuate the weakening of transcriptional repression that occurs naturally with reproductive meristem proliferation.

Circulating cell-free DNA as a biomarker of tissue injury: Assessment in a cardiac xenotransplantation model.

BACKGROUND: Observational studies suggest that cell-free DNA (cfDNA) is a biomarker of tissue injury in a range of conditions including organ transplantation. However, the lack of model systems to study cfDNA and its relevance to tissue injury has limited the advancements in this field. We hypothesized that the predictable course of acute humoral xenograft rejection (AHXR) in organ transplants from genetically engineered donors provides an ideal system for assessing circulating cfDNA as a marker of tissue injury. METHODS: Genetically modified pig donor hearts were heterotopically transplanted into baboons (n = 7). Cell-free DNA was extracted from pre-transplant and post-transplant baboon plasma samples for shotgun sequencing. After alignment of sequence reads to pig and baboon reference sequences, we computed the percentage of xenograft-derived cfDNA (xdcfDNA) relative to recipient by counting uniquely aligned pig and baboon sequence reads. RESULTS: The xdcfDNA percentage was high early post-transplantation and decayed exponentially to low stable levels (baseline); the decay half-life was 3.0 days. Post-transplantation baseline xdcfDNA levels were higher for transplant recipients that subsequently developed graft loss than in the 1 animal that did not reject the graft (3.2% vs 0.5%). Elevations in xdcfDNA percentage coincided with increased troponin and clinical evidence of rejection. Importantly, elevations in xdcfDNA percentage preceded clinical signs of rejection or increases in troponin levels. CONCLUSION: Cross-species xdcfDNA kinetics in relation to acute rejection are similar to the patterns in human allografts. These observations in a xenotransplantation model support the body of evidence suggesting that circulating cfDNA is a marker of tissue injury.

Late manifestation of alloantibody-associated injury and clinical pulmonary antibody-mediated rejection: Evidence from cell-free DNA analysis.

BACKGROUND: Antibody-mediated rejection (AMR) often progresses to poor health outcomes in lung transplant recipients (LTRs). This, combined with the relatively insensitive clinical tools used for its diagnosis (spirometry, histopathology) led us to determine whether clinical AMR is diagnosed significantly later than its pathologic onset. In this study, we leveraged the high sensitivity of donor-derived cell-free DNA (ddcfDNA), a novel genomic tool, to detect early graft injury after lung transplantation. METHODS: We adjudicated AMR and acute cellular rejection (ACR) in 157 LTRs using the consensus criteria of the International Society for Heart and Lung Transplantation (ISHLT). We assessed the kinetics of allograft injury in relation to ACR or AMR using both clinical criteria (decline in spirometry from baseline) and molecular criteria (ddcfDNA); percent ddcfDNA was quantitated via shotgun sequencing. We used a mixed-linear model to assess the relationship between and ddcfDNA levels and donor-specific antibodies (DSA) in AMR+ LTRs. RESULTS: Compared with ACR, AMR episodes (n = 42) were associated with significantly greater allograft injury when assessed by both spirometric (0.1 liter vs -0.6 liter, p < 0.01) and molecular (ddcfDNA) analysis (1.1% vs 5.4%, p < 0.001). Allograft injury detected by ddcfDNA preceded clinical AMR diagnosis by a median of 2.8 months. Within the same interval, spirometry or histopathology did not reveal findings of allograft injury or dysfunction. Elevated levels of ddcfDNA before clinical diagnosis of AMR were associated with a concurrent rise in DSA levels. CONCLUSION: Diagnosis of clinical AMR in LTRs lags behind DSA-associated molecular allograft injury as assessed by ddcfDNA.

Using a Control to Better Understand Phyllosphere Microbiota.

An important data gap in our understanding of the phyllosphere surrounds the origin of the many microbes described as phyllosphere communities. Most sampling in phyllosphere research has focused on the collection of microbiota without the use of a control, so the opportunity to determine which taxa are actually driven by the biology and physiology of plants as opposed to introduced by environmental forces has yet to be fully realized. To address this data gap, we used plastic plants as inanimate controls adjacent to live tomato plants (phyllosphere) in the field with the hope of distinguishing between bacterial microbiota that may be endemic to plants as opposed to introduced by environmental forces. Using 16S rRNA gene amplicons to study bacterial membership at four time points, we found that the vast majority of all species-level operational taxonomic units were shared at all time-points. Very few taxa were unique to phyllosphere samples. A higher taxonomic diversity was consistently observed in the control samples. The high level of shared taxonomy suggests that environmental forces likely play a very important role in the introduction of microbes to plant surfaces. The observation that very few taxa were unique to the plants compared to the number that were unique to controls was surprising and further suggests that a subset of environmentally introduced taxa thrive on plants. This finding has important implications for improving our approach to the description of core phytobiomes as well as potentially helping us better understand how foodborne pathogens may become associated with plant surfaces.

Comparison of Microbial Communities Isolated from Feces of Asymptomatic Salmonella-Shedding and Non-Salmonella Shedding Dairy Cows.

In the United States Salmonella enterica subsp. enterica serotypes Kentucky and Cerro are frequently isolated from asymptomatic dairy cows. However, factors that contribute to colonization of the bovine gut by these two serotypes have not been identified. To investigate associations between Salmonella status and bacterial diversity, as well as the diversity of the microbial community in the dairy cow hindgut, the bacterial and archaeal communities of fecal samples from cows on a single dairy farm were determined by high-throughput sequencing of 16S rRNA gene amplicons. Fecal grab samples were collected from two Salmonella-positive cows and two Salmonella-negative cows on five sampling dates (n = 20 cows), and 16S rRNA gene amplicons from these samples were sequenced on the Illumina MiSeq platform. A high level of alpha (within) and beta diversity (between) samples demonstrated that microbial profiles of dairy cow hindguts are quite diverse. To determine whether Salmonella presence, sampling year, or sampling date explained a significant amount of the variation in microbial diversity, we performed constrained ordination analyses (distance based RDA) on the unifrac distance matrix produced with QIIME. Results indicated that there was not a significant difference in the microbial diversity associated with Salmonella presence (P > 0.05), but there were significant differences between sampling dates and years (Pseudo-F = 2.157 to 4.385, P < 0.05). Based on these data, it appears that commensal Salmonella infections with serotypes Cerro and Kentucky in dairy cows have little or no association with changes in the abundance of major bacterial groups in the hindgut. Rather, our results indicated that temporal dynamics and other undescribed parameters associated with them were the most influential drivers of the differences in microbial diversity and community structure in the dairy cow hindgut.

The impact of systemic and copper pesticide applications on the phyllosphere microflora of tomatoes.

BACKGROUND: Contamination of tomatoes by Salmonella can occur in agricultural settings. Little is currently understood about how agricultural inputs such as pesticide applications may impact epiphytic crop microflora and potentially play a role in contamination events. We examined the impact of two materials commonly used in Virginia tomato agriculture: acibenzolar-S-methyl (crop protectant) and copper oxychloride (pesticide) to identify the effects these materials may exert on baseline tomato microflora and on the incidence of three specific genera; Salmonella, Xanthomonas and Paenibacillus. RESULTS: Approximately 186 441 16S rRNA gene and 39 381 18S rRNA gene sequences per independent replicate were used to analyze the impact of the pesticide applications on tomato microflora. An average of 3 346 677 (634 892 974 bases) shotgun sequences per replicate were used for metagenomic analyses. CONCLUSION: A significant decrease in the presence of Gammaproteobacteria was observed between controls and copper-treated plants, suggesting that copper is effective at suppressing growth of certain taxa in this class. A higher mean abundance of Salmonella and Paenibacillus in control samples compared to treatments may suggest that both systemic and copper applications diminish the presence of these genera in the phyllosphere; however, owing to the lack of statistical significance, this could also be due to other factors. The most distinctive separation of shared membership was observed in shotgun data between the two different sampling time-points (not between treatments), potentially supporting the hypothesis that environmental pressures may exert more selective pressures on epiphytic microflora than do certain agricultural management practices.