Evaluation of developing maize microbiomes and associations among nitrogen cyclers and key fungal taxa.

More sustainable approaches to agriculture are urgently needed to protect existing resources and optimize crop yields and to provide food for a growing global human population. More sustainable agricultural practices that utilize plant-microbe relationships across cultivation are urgently needed. The main objectives of this study were to track the prokaryotic and fungal microbiomes associated with key growth stages of developing maize to evaluate the relationships among nitrogen cycling bacteria and major fungal genera including those known to contain arbuscular mycorrhizal fungi and other important taxa. Prokaryotic and fungal microbiomes associated with bulk soils, rhizosphere soils and tissues of developing maize were characterized using Illumina MiSeq sequencing. Similarities in microbiome diversity and abundance were compared to sample metadata to explore the influence of external factors on microbiome development. Correlations among target fungal taxa, bulk bacteria and nitrogen cycling bacteria were determined using non-parametric Spearman correlations. Important maize-associated fungal taxa were detected in all samples across growth stages, with Fusarium, Penicillium and Aspergillus fungi comprising up to 4.21, 4.26 and 0.28% of all fungal genera, respectively. Thirteen statistically significant correlations between nitrogen cycling genera and targeted fungal genera were also identified (rS≥0.70 or rS≤-0.70; P<0.05). This study is the first to note a strong positive association among several nitrifying bacteria and Fusarium (R=0.71; P=0.0046), Aspergillus (R=0.71; P=0.0055) and Cladosporium spcies (R=0.74; P=0.0038), suggesting the levels of soil nitrate, nitrite or nitrification intermediates may have large roles in the proliferation of important maize-associated fungi.

A comparative study of gut microbiomes in captive nocturnal strepsirrhines.

Feeding strategy and diet are increasingly recognized for their roles in governing primate gut microbiome (GMB) composition. Whereas feeding strategy reflects evolutionary adaptations to a host's environment, diet is a more proximate measure of food intake. Host phylogeny, which is intertwined with feeding strategy, is an additional, and often confounding factor that shapes GMBs across host lineages. Nocturnal strepsirrhines are an intriguing and underutilized group in which to examine the links between these three factors and GMB composition. Here, we compare GMB composition in four species of captive, nocturnal strepsirrhines with varying feeding strategies and phylogenetic relationships, but nearly identical diets. We use 16S rRNA sequences to determine gut bacterial composition. Despite similar husbandry conditions, including diet, we find that GMB composition varies significantly across host species and is linked to host feeding strategy and phylogeny. The GMBs of the omnivorous and the frugivorous species were significantly more diverse than were those of the insectivorous and exudativorous species. Across all hosts, GMBs were enriched for bacterial taxa associated with the macronutrient resources linked to the host's respective feeding strategy. Ultimately, the reported variation in microbiome composition suggests that the impacts of captivity and concurrent diet do not overshadow patterns of feeding strategy and phylogeny. As our understanding of primate GMBs progresses, populations of captive primates can provide insight into the evolution of host-microbe relationships, as well as inform future captive management protocols that enhance primate health and conservation.

Higher normalized concentrations of tetracycline resistance found in ballast and harbor water compared to ocean water.

Although ballast water is a known vector for the global transport of microorganisms, the Ballast Water Management Convention only sets limits for indicator organisms and does not consider antibiotic resistance genes (ARGs). Herein, we examined the concentration of indicator organisms and prevalence of three ARGs (sul1, tet (M), and vanA) in a total of 53 ballast, 21 harbor, and 8 ocean samples collected in Singapore, China, South Africa, and California. E. coli was found in significantly higher concentrations in ballast samples obtained in Singapore and China compared to South Africa (Singapore, p = 0.040) and California (Singapore, p < 0.001; China, p = 0.038). Harbor samples from China had significantly higher concentrations of E. coli than Singapore (p = 0.049) and California (p = 0.001). When compared to ocean samples, there were significantly higher concentrations of normalized tet(M) in ballast samples from California (p = 0.011) and Singapore (p = 0.019) and in harbor samples from California (p = 0.018), Singapore (p = 0.010), and South Africa (p = 0.008). These findings suggest that microbial loads significantly differ among ports. Furthermore, certain ARGs are enriched in ballast and harbor waters when compared to ocean water, which suggests that ballast waters have the potential to either transport higher concentrations of certain ARGs or that ballast tank conditions may exert selective pressure for some ARGs.

Microbiome composition and implications for ballast water classification using machine learning.

Ballast water is a vector for global translocation of microorganisms, and should be monitored to protect human and environmental health. This study utilizes high throughput sequencing (HTS) and machine learning to examine the bacterial and fungal microbiomes of ballast water to identify associations between 16S and 18S rRNA genes and the fungal ITS region. These sequencing regions were examined using the SILVA v132 and UNITE reference databases. The highest correlation was found between the communities in Silva_16S and UNITE_ITS (0.74). There was a higher proportion of positive inter-kingdom correlations than positive intra-kingdom interactions (p = 0.032). Understanding the reasons for this difference requires additional research under more controlled conditions. Finally, a machine learning model was used to examine the classification accuracy when using each sequencing region and reference database to identify ballast residence time and ballast sample location. There was significantly higher accuracy using SILVA (0.843) compared to UNITE (0.614) (p < 0.001). In the short term, future research with the goal of classifying ballast water samples based on location or ballast water residence time should be performed using the 16S rRNA gene and SILVA reference database. Research to curate other sequencing regions or the UNITE reference database in the aquatic ecosystem may improve the utility of these tools.

Metabarcoding and machine learning analysis of environmental DNA in ballast water arriving to hub ports.

While ballast water has long been linked to the global transport of invasive species, little is known about its microbiome. Herein, we used 16S rRNA gene sequencing and metabarcoding to perform the most comprehensive microbiological survey of ballast water arriving to hub ports to date. In total, we characterized 41 ballast, 20 harbor, and 6 open ocean water samples from four world ports (Shanghai, China; Singapore; Durban, South Africa; Los Angeles, California). In addition, we cultured Enterococcus and E. coli to evaluate adherence to International Maritime Organization standards for ballast discharge. Five of the 41 vessels - all of which were loaded in China - did not comply with standards for at least one indicator organism. Dominant bacterial taxa of ballast water at the class level were Alphaproteobacteria, Gammaproteobacteria, and Bacteroidia. Ballast water samples were composed of significantly lower proportions of Oxyphotobacteria than either ocean or harbor samples. Linear discriminant analysis (LDA) effect size (LEfSe) and machine learning were used to identify and test potential biomarkers for classifying sample types (ocean, harbor, ballast). Eight candidate biomarkers were used to achieve 81% (k nearest neighbors) to 88% (random forest) classification accuracy. Further research of these biomarkers could aid the development of techniques to rapidly assess ballast water origin.

Analyzing trends in ballasting behavior of vessels arriving to the United States from 2004 to 2017.

Maritime shipping transports over 90% of global goods. Ballast water, used to provide vessel stability, has been associated with the introduction of marine invasive species. Thus, understanding ballasting trends is imperative to protecting human and environmental health. This paper examines data from the National Ballast Information Clearinghouse to assess ballasting behavior and shipping trends in the United States. From 2005 to 2017, vessel arrivals have remained relatively constant (annual growth rate of 1.2% per year) while total ballast discharge per vessel has grown at an annual rate of 7.6%. Furthermore, from 2014 to 2016, alternative ballast water management methods have been on the rise, and these treatment options are likely to continue increasing in response to the International Maritime Organization Ballast Water Management Convention that entered into force in September 2017. It is critical that the shipping industry monitors potential cascading impacts on other ballasting behaviors stemming from this shift.

Occurrence of Vibrio species, beta-lactam resistant Vibrio species, and indicator bacteria in ballast and port waters of a tropical harbor.

Ballast water discharges are potential sources for the spread of invasive and pathogenic aquatic organisms. Ballast waters from six ships docked in the Port of Singapore were tested to determine if indictor organisms fell within proposed standards for ballast water discharge according to regulation D-2 of the Ballast Water Management Convention (BWMC) guidelines. Vibrio species were cultured on media supplemented with beta-lactam antibiotics to determine the presence of antibiotic resistant Vibrio species in the ballast waters of these vessels. Indicator organisms were quantified using culture media Colilert-18 and Enterolert in ballast waters of six ships docked in a tropical harbor, with uptake from different geographical locations. Of the six ships, one had ballast water originating from the Persian Gulf, another from the East China Sea, and four from the South China Sea. Two of the six ships which carried ballast waters from the East China Sea and the South China Sea did not meet the D-2 stipulated requirements of the Ballast Water Management Convention for indicator organisms with Enterococci values more than three times higher than the acceptable limit of <100CFU/100mL. Using the most-probable-number-PCR (MPN-PCR) method for Vibrio species detection, non-toxigenic species of V. cholerae (2 MPN/100mL), Vibrio vulnificus (>110 MPN/100mL), and Vibrio parahaemolyticus (2 to >110 MPN/100mL) were detected in at least one of six ballast water samples. Using thiosulfate-citrate-bile salts-sucrose agar (TCBS) supplemented with beta-lactam antibiotics (meropenem, ceftazidime), 11 different Vibrio species, exhibiting resistance to beta-lactam antibiotics were isolated; with Vibrio campbellii (44%) and Vibrio alginolyticus (15%) the most detected antibiotic resistant Vibrio species. A practical approach of prioritized screening of high-risk vessels should be conducted to ensure that the water quality meets D-2 standards prior to discharge.

Water quality at points-of-use in the Galapagos Islands.

Piped drinking water is often considered a gold standard for protecting public health but research is needed to explicitly evaluate the effect of centralized treatment systems on water quality in developing world settings. This study examined the effect of a new drinking water treatment plant (DWTP) on microbial drinking water quality at the point-of-use on San Cristobal Island, Galapagos using fecal indicator bacteria total coliforms and Escherichia coli. Samples were collected during six collection periods before and after operation of the DWTP began from the freshwater sources (n=4), the finished water (n=6), and 50 sites throughout the distribution system (n=287). This study found that there was a significant decrease in contamination by total coliforms (two orders of magnitude) and E. coli (one order of magnitude) after DWTP operation began (p<0.001). However, during at least one post-construction collection cycle, total coliforms and E. coli were still found at 66% and 28% of points-of-use (n=50), respectively. During the final collection period, conventional methods were augmented with human-specific Bacteroides assays - validated herein - with the goal of elucidating possible microbial contamination sources. Results show that E. coli contamination was not predictive of contamination by human wastes and suggests that observed indicator bacteria contamination may have environmental origins. Together these findings highlight the necessity of a holistic approach to drinking water infrastructure improvements in order to deliver high quality water through to the point-of-use.