Chemical sensing of common microorganisms found in biopharmaceutical industries using MIR laser spectroscopy and multivariate analysis.

Mid-infrared laser spectroscopy was used to investigate common bacteria encountered in biopharmaceutical industries. The study involved the detection of bacteria using quantum cascade laser spectroscopy coupled to a grazing angle probe (QCL-GAP). Substrates similar to surfaces commonly used in biopharmaceutical industries were used as support media for the samples. Reflectance measurements were assisted by Multivariate Analysis (MVA) to assemble a powerful spectroscopic technique with classification and identification resources. The species analyzed, Staphylococcus aureus, Staphylococcus epidermidis, and Micrococcus luteus, were used to challenge the technique's capability to discriminate from microorganisms of the same family. Principal Components Analysis and Partial Least Squares-Discriminant Analysis differentiated between the bacterial species, using QCL-GAP-MVA as the reference. Spectral differences in the bacterial membrane were used to determine if these microorganisms were present in the samples analyzed. Results herein provided effective discrimination for the bacteria under study with high sensitivity and specificity.

RING Program: The CFAR Diversity, Equity, and Inclusion Pathway Initiative.

BACKGROUND: Case Western Reserve University (CWRU)/University Hospitals Cleveland Medical Center in Cleveland, OH, and the University of Pittsburgh (Pitt) in Pittsburgh, PA, forged a strategic alliance to form the Rustbelt Center for AIDS Research. The Rustbelt Center for AIDS Research developed a National Institutes of Health-supported diversity, equity, and inclusion pathway initiative termed the "Rustbelt Investigators for the Next Generation (RING) Program" that provides research training experiences for Puerto Rican students that will help them pursue a biomedical research career in HIV. SETTING: The RING Program provides 10-week research training experiences in different disciplines of HIV/AIDS for under-represented minority undergraduate and masters students from 4 campuses (Río Piedras, Mayagüez, Humacao, and Cayey) at the University of Puerto Rico. Mentors are drawn from both CWRU and Pitt. RESULTS: The RING Program recently completed our first wave of recruitment. Recruitment sessions were either virtual or on site at the University of Puerto Rico campuses and included an overview presentation, a Q&A session, and in-person interviews. We interviewed 32 eligible applicants and accepted 10 into the program, of which 9 were female. Five students were matched with faculty at CWRU and 5 with faculty at Pitt. CONCLUSIONS: The RING Program is a comprehensive program in laboratory and implementation science that aims to enhance under-represented Hispanic undergraduate and masters students' passion for pursuing a biomedical research career in HIV.

Microbial Model Construction Activity for Introducing Students to Microbiome and Metagenomic Fields.

Educational activities in biology teaching focused on model construction allow students to make the invisible evident. The development and integration of these pedagogical strategies in emerging sciences is essential and necessary in the classroom of noncollege academia. A two-component pedagogical activity was developed to expose high school students to the emerging disciplines of microbiomes and metagenomics. An introductory talk about microbiomes and host-microbe interactions was designed and presented to high school students at an educational center in the western region of Puerto Rico. After the talk, the students were organized into teams and required to choose one of the microbiome cases discussed to generate an oral presentation and a model describing the microbe's relationship with the host (microbiome interactions). A total of five models were generated by the students, which represented bacterial and yeast interaction with animals and plants. The teaching-learning process was assessed using pre/posttests and model evaluation instruments. The combination of the talk, model construction, and oral presentation increased the general knowledge of the participants by 43% from pre- to posttest. The students' knowledge of the concepts of metagenomics and microbiomes increased 30% and 49%, respectively. The data support that students were able to define and integrate the concepts successfully after the implementation of the educational strategies. This team-based educational model of exposing students to emerging disciplines is necessary to promote the active learning process in current topics in science in a nontraditional way.

Stool microbiome dataset of the critically endangered Puerto Rican parrot (Amazona vittata).

The Puerto Rican parrot (Amazona vittata), endemic to Puerto Rico, is the only native parrot in the United States and is classified as a critically endangered species. There are two captive populations of A. vittata in Puerto Rico located in the Iguaca Aviary in El Yunque National Rainforest and the José L. Vivaldi Aviary in the Río Abajo Forest. To characterize the microbial communities of A. vittata's stool, 21 stool samples from captive birds were collected, DNA extracted and sequenced using Illumina MiSeq. Sequences were processed by removing host sequences (A. vittata genome) and low-quality reads. Taxonomic and functional profiles were generated using MG-RAST. The most abundant domain was Bacteria (96%), followed by Virus (3%), and Eukaryota (0.6%). Among the functions in the microbiome, the most abundant was related to carbohydrates (14%), followed by clustering-based subsystems (12%), protein metabolism (8%), and amino acids and derivatives (7%). This dataset describes the stool microbiome of A. vittata using a metagenomics approach. Data can be used to develop holistic conservation strategies for A. vittata and other endangered birds, as well as to search for bioprospects with potential biomedical and biotechnological applications.

Soil microbiome dataset from Guanica dry forest in Puerto Rico generated by shotgun sequencing.

Guanica dry forest (GDF), located in the southwest area or region of Puerto Rico, is among the most preserved subtropical dry forests in the world [1]. To describe the taxonomic diversity and functional profiles of this environment, metagenomic DNA was extracted from a metagenomic library generated from the GDF. The DNA was shotgun-sequenced using Illumina and analyzed using the MG-RAST server. The diversity profile revealed that the most abundant domain was Bacteria (97.8%) followed by Archaea (1.12%), Eukaryota (1.02%) and Viruses (0.03%). Out of the 50 phyla present, the most abundant was Proteobacteria (41.6%) followed by Actinobacteria (18.7%) and Acidobacteria (7.06%). Moreover, a total of 213 orders, 384 families and 791 genus were identified. The functional profile showed abundance of genes related to Carbohydrates (13.16%), Clustering-based subsystems (13.0%), Amino Acids and Derivatives (9.9%) and Protein Metabolism (8.24%). Furthermore, more specific grouping showed that NULL (21.5%) was the most abundant function group, followed by Plant-Prokaryote DOE project (6.05%), Protein biosynthesis (4.82%), Central carbohydrate metabolism (3.98%), DNA repair (2.72%) and Resistance to antibiotics and toxic compounds (2.66%). This dataset is useful in bioprospecting studies with application in biomedical sciences, biotechnology and microbial, population and applied ecology fields.

Soil metagenome datasets underneath the Arecibo Observatory reflector dish.

The Arecibo Observatory (AO) located in Arecibo, Puerto Rico, is the most sensitive, powerful and active planetary radar system in the world [1]. One of its principal components is the 305 m-diameter spherical reflector dish (AORD), which is exposed to high frequency electromagnetic waves. To unravel the microbial communities that inhabit this environment, soil samples from underneath the AORD were collected, DNA extracted, and sequenced using Illumina MiSeq. Taxonomic and functional profiles were generated using the MG-RAST server. The most abundant domain was Bacteria (91%), followed by Virus (8%), Archaea (0.9%) and Eukaryota (0.9%). The most abundant phylum was Proteobacteria (54%), followed by Actinobacteria (8%), Bacteroidetes (5%) and Firmicutes (4%). In terms of functions, the most abundant among the metagenome corresponded to phages, transposable elements and plasmids (16%), followed by clustering-based subsystems (11%), carbohydrates (10%), and amino acids and derivatives (9%). This is the first soil metagenomic dataset from dish antennas and radar systems, specifically, underneath the AORD. Data can be used to explore the effect of high frequency electromagnetic waves in soil microbial composition, as well as the possibility of finding bioprospects with potential biomedical and biotechnological applications.

Datasets generated by shotgun sequencing of metagenomic libraries of the guajataca water reservoir.

The Guajataca Water Reservoir (GWR) was constructed for irrigation and to bring potable water to the northwestern region of Puerto Rico. The generation of DNA sequencing data from aquatic bodies (AB) using culture-independent approaches allows the investigation of the total microbial diversity as well as the potential anthropogenic impact. Metagenomic libraries were constructed for two GWR sampling sites and genomic information access through shotgun sequencing. After removing the bacterial host cell genome and the library fosmid sequences, the environmental genome was processed through Rapid Annotation using Subsystems Technology for Metagenomes (MG-RAST). The sequences consisted primarily of bacteria (95.70%), followed by viruses (2.94%), other sequences (0.28%) and eukaryote (0.09%). The most abundant species were Enterobacter cloacae (31%), Enterobacter sp. 638 (20%), Enterobacter cancerogenus (10%) and Escherichia coli (11%). Furthermore, the subsystem data showed that 13% of the genes belong to carbohydrates functionality, 12% to clustering-based-subsystems and another 9% related to virulence-disease-and-defense (out of which 8% pertain to genes of antibiotic resistance and toxic compounds). This unique data input will serve as a baseline to a better understanding not only the microbial communities present in the AB, but also the microbial activities with potential application in biotechnological and biomedical fields.

Microbiome dataset from Clara Cave and Empalme Sinkhole waters in Puerto Rico.

Camuy River Cave Park (CRCP) is an underground cave system located at the subtropical karst carved by the Camuy River in the subtropical moist forest of northern Puerto Rico (Nieves-Rivera, 2003) [1]. This article contains a metagenomic dataset from the microbial and functional diversity of Clara Cave and Empalme Sinkhole water samples. The environmental DNA (eDNA) from the samples was extracted following direct Metagenomic DNA Isolation method, followed by Next-Generation-Sequencing technology (Illumina MiSeq). The sequences were submitted to MG-RAST online server for taxonomic profile generation and functional in silico description of the samples. The data consisted of domain Bacteria (96.69%), followed up by Viruses (2.87%), Eukaryotes (0.37%), and Archaea (0.02%). The data distribution by phyla showed Proteobacteria (92.61%), Bacteroidetes (1.66%), Actinobacteria (1.12%), and Firmicutes (0.48%). The subsystem functional data showed that 12.97% of genes were related to clustering-based subsystems, 11.40% to carbohydrates, and 11.0% to amino acids and derivatives. The metagenome dataset generated will provide an understanding and comparison framework of the microbial composition and functional diversity present in caves.

The metagenome of bromeliads phytotelma in Puerto Rico.

Bromeliads tank water or phytotelma is an eutrophic microenvironment where microorganisms have evolved to resist sudden changes in pH and nutritional competition. Metagenomics studies have been poorly studied in bromeliads and environmental DNA (eDNA) characterization for its microenvironment is deficient in Puerto Rico. Therefore, the data represents the microbial communities inhabiting bromeliads phytotelma. eDNA was extracted using Metagenomic DNA Isolation Kit for Water. Next-Generation-Sequencing technology (Illumina MiSeq) was used for sequencing the isolated eDNA. This data provides an insight about diversity and functional depiction of microorganisms inhabiting bromeliads phytotelma. The data of this metagenome is available in the BioSample Submission Portal as Bioproject PRJNA39461 and Sequence Read Archive (SRA) accession number SRP114300. MG-RAST metagenomic analysis server is located under the study ID mgp79812.

The metagenome of Caracolus marginella gut microbiome using culture independent approaches and shotgun sequencing.

Studies underestimate the microbial diversity and genotypic traits in the snails' microbiome. Caracolus marginella, a land snail native to Caribbean islands, can adapt to different environments. Our research focused on the generation of a metagenomic library from C. marginella gut, to further explore the diversity and functional traits. Thirty specimens of C. marginella were collected from the four regions of Puerto Rico. High molecular weight (40 kb) metagenomic libraries were generated using a direct DNA isolation method. DNA was end-repaired and ligated into a pCCFOS1 fosmid vector; then, the cloned DNA was transduced into Escherichia coli EPI300. The master pool library contains approximately 60,200 clones and restriction enzyme digestion showed that 90% of the library contains insert. After removing the fosmid and host genome sequences, 567,015 sequences were analyzed using the MG-RAST online server. The Bacteria domain was the most abundant (82.15%), followed by viruses (16.49%), eukaryotes (0.83%) and archaea (0.31%). The Proteobacteria (51.47%) was predominant in the gut environment, followed by unidentified virus (16.28%), and Actinobacteria (8.52%). Escherichia coli, Streptomyces avermitilis, and Burkholderia sp. were the most abundant species present. Subsystem functional analysis showed that 35.00% of genes belong to transposable elements, 10.00% of genes belong to clustering-based subsystems, 4.00% of genes belong to the production of cofactors and secondary metabolites, and 2.00% resistance to antibiotics and toxic compounds. The data generated in this research is the first metagenomic examination of a snail gut in Puerto Rico, and will serve as a baseline to start understanding of C. marginella gut microbiome.

Unraveling the microbial and functional diversity of Coamo thermal spring in Puerto Rico using metagenomic library generation and shotgun sequencing.

In Puerto Rico, the microbial diversity of the thermal spring (ThS) in Coamo has never been studied using metagenomics. The focus of our research was to generate a metagenomic library from the ThS of Coamo, Puerto Rico and explore the microbial and functional diversity. The metagenomic library from the ThS waters was generated using direct DNA isolation. High molecular weight (40 kbp) DNA was end-repaired, electro eluted and ligated into a fosmid vector (pCCFOS1); then transduced into Escherichia coli EPI300-T1R using T1 bacteriophages. The library consisted of approximately 6000 clones, 90% containing metagenomic DNA. Next-Generation-Sequencing technology (Illumina MiSeq) was used to process the ThS metagenome. After removing the cloning vector, 122,026 sequences with 33.10 Mbps size and 64% of G + C content were annotated and analyzed using the MG-RAST online server. Bacteria showed to be the most abundant domain (95.84%) followed by unidentified sequences (2.28%), viruses (1.67%), eukaryotes (0.15%), and archaea (0.01%). The most abundant phyla were Proteobacteria (95.03%), followed by unidentified (2.28%), unclassified from viruses (1.74%), Firmicutes (0.20%) and Actinobacteria (0.18%). The most abundant species were Escherichia coli, Polaromonas naphthalenivorans, Albidiferax ferrireducens and Acidovorax sp. Subsystem functional analysis showed that 20% of genes belong to transposable elements, 10% to clustering-based subsystems, and 8% to the production of cofactors. Functional analysis using NOG annotation showed that 82.79% of proteins are poorly characterized indicating the possibility of novel microbial functions and with potential biomedical and biotechnological applications. Metagenomic data was deposited into the NCBI database under the accession number SAMN06131862.

Profiling lethal factor interacting proteins from human stomach using T7 phage display screening.

The anthrax lethal factor (LF) is a zinc dependent metalloproteinase that cleaves the majority of mitogen-activated protein kinase kinases and a member of NOD-like receptor proteins, inducing cell apoptosis. Despite efforts to fully understand the Bacillus anthracis toxin components, the gastrointestinal (GI) anthrax mechanisms have not been fully elucidated. Previous studies demonstrated gastric ulceration, and a substantial bacterial growth rate in Peyer's patches. However, the complete molecular pathways of the disease that results in tissue damage by LF proteolytic activity remains unclear. In the present study, to identify the profile of the proteins potentially involved in GI anthrax, protein­protein interactions were investigated using human stomach T7 phage display (T7PD) cDNA libraries. T7PD is a high throughput technique that allows the expression of cloned DNA sequences as peptides on the phage surface, enabling the selection and identification of protein ligands. A wild type and mutant LF (E687A) were used to differentiate interaction sites. A total of 124 clones were identified from 194 interacting­phages, at both the DNA and protein level, by in silico analysis. Databases revealed that the selected candidates were proteins from different families including lipase, peptidase­A1 and cation transport families, among others. Furthermore, individual T7PD candidates were tested against LF in order to detect their specificity to the target molecule, resulting in 10 LF­interacting peptides. With a minimum concentration of LF for interaction at 1 µg/ml, the T7PD isolated pepsin A3 pre­protein (PAP) demonstrated affinity to both types of LF. In addition, PAP was isolated in various lengths for the same protein, exhibiting common regions following PRALINE alignment. These findings will help elucidate and improve the understanding of the molecular pathogenesis of GI anthrax, and aid in the development of potential therapeutic agents.

Microbial community response to addition of polylactate compounds to stimulate hexavalent chromium reduction in groundwater.

To evaluate the efficacy of bioimmobilization of Cr(VI) in groundwater at the Department of Energy Hanford site, we conducted a series of microcosm experiments using a range of commercial electron donors with varying degrees of lactate polymerization (polylactate). These experiments were conducted using Hanford Formation sediments (coarse sand and gravel) immersed in Hanford groundwater, which were amended with Cr(VI) and several types of lactate-based electron donors (Hydrogen Release Compound, HRC; primer-HRC, pHRC; extended release HRC) and the polylactate-cysteine form (Metal Remediation Compound, MRC). The results showed that polylactate compounds stimulated an increase in bacterial biomass and activity to a greater extent than sodium lactate when applied at equivalent carbon concentrations. At the same time, concentrations of headspace hydrogen and methane increased and correlated with changes in the microbial community structure. Enrichment of Pseudomonas spp. occurred with all lactate additions, and enrichment of sulfate-reducing Desulfosporosinus spp. occurred with almost complete sulfate reduction. The results of these experiments demonstrate that amendment with the pHRC and MRC forms result in effective removal of Cr(VI) from solution most likely by both direct (enzymatic) and indirect (microbially generated reductant) mechanisms.

Summer workshop in metagenomics: one week plus eight students equals gigabases of cloned DNA.

We designed a week-long laboratory workshop in metagenomics for a cohort of undergraduate student researchers. During this course, students learned and utilized molecular biology and microbiology techniques to construct a metagenomic library from Puerto Rican soil. Pre-and postworkshop assessments indicated student learning gains in technical knowledge, skills, and confidence in a research environment. Postworkshop construction of additional libraries demonstrated retention of research techniques by the students.

Genotypic characterization of environmental isolates of Cryptococcus gattii from Puerto Rico.

Cryptococcus neoformans and Cryptococcus gattii are found in distinct environments with some overlap around different parts of the world. However, no systematic surveys of these two pathogens have been reported from Puerto Rico, a tropical island uniquely situated between mainland USA and countries in South America. We carried out an exhaustive environmental survey in southwestern Puerto Rico for pathogenic Cryptococcus species. Twenty-two presumptive isolates of C. gattii from cacti and tree detritus were characterized in detail by physiological and molecular methods and seventeen strains were confirmed as C. gattii. Cryptococcus gattii isolates were haploid and majority of them were MATa [corrected] strains. Sixteen out of seventeen C. gattii isolates belonged to VGII/AFLP6 genotype while one isolate was a VGIV/AFLP7 genotype. The results are significant as Puerto Rico strains are distinct from VGIII/AFLP5 strains reported from Southern California, but similar to C. gattii VGII/AFLP6 molecular type implicated in recent outbreaks of cryptococcosis in Pacific Northwest and British Columbia, Canada, but different in its M13 fingerprinting, and a common genotype in South America.

Miniprimer PCR, a new lens for viewing the microbial world.

Molecular methods based on the 16S rRNA gene sequence are used widely in microbial ecology to reveal the diversity of microbial populations in environmental samples. Here we show that a new PCR method using an engineered polymerase and 10-nucleotide "miniprimers" expands the scope of detectable sequences beyond those detected by standard methods using longer primers and Taq polymerase. After testing the method in silico to identify divergent ribosomal genes in previously cloned environmental sequences, we applied the method to soil and microbial mat samples, which revealed novel 16S rRNA gene sequences that would not have been detected with standard primers. Deeply divergent sequences were discovered with high frequency and included representatives that define two new division-level taxa, designated CR1 and CR2, suggesting that miniprimer PCR may reveal new dimensions of microbial diversity.

Learning geomicrobiology as a team using microbial mats, a multidisciplinary approach.

Microbial mats are one of the best suited laminar organo-sedimentary ecosystems for students from different educational backgrounds to visualize the direct relationship between microbes and minerals. We have used tropical hypersaline microbial mats from Puerto Rico as educational tools to promote active learning of geomicrobiology introductory concepts for undergraduate students organized in multidisciplinary teams with biological and geological backgrounds. Besides field trips and independent research projects focused on microbial mats, four intensive workshops and one capstone activity were designed to expose students to the different geomicrobiology subdisciplines (microbiology, molecular biology, geology, and geochemistry). The teaching-learning process was assessed using pre- and posttests, group discussions, activities including Gallery Walks and exquisite cadaver's, case studies, and focal interviews. While the posttest showed a significant difference in conceptual understanding, the Gallery Walk and the capstone activities demonstrated increase in the depth, coherence, and thoughtfulness in answering questions, including a clear integration of the different subdisciplines during their presentations. Finally, the main themes described by the students as important outcomes of their participation in the Research at Undergraduate Institutions: Microbial Observatory (RUI-MO) program were: (i) the opportunity to study and learn new and different science disciplines, (ii) the microbial mats were excellent tools to learn from and integrate different science disciplines, and (iii) working in multidisciplinary teams gave them the opportunity to learn from their peers' discipline backgrounds. To our knowledge this is the first educational initiative that uses tropical hypersaline microbial mats to teach geomicrobiology in a multidisciplinary fashion.

The delta cooperative model: a dynamic and innovative team-work activity to develop research skills in microbiology.

The Delta Cooperative Model (DCM) is a dynamic and innovative teamwork design created to develop fundamentals in research skills. High school students in the DCM belong to the Upward Bound Science and Math (UBSM) program at the Inter American University, Ponce Campus. After workshops on using the scientific method, students were organized into groups of three students with similar research interests. Each student had to take on a role within the group as either a researcher, data analyst, or research editor. Initially, each research team developed hypothesis-driven ideas on their proposed project. In intrateam research meetings, they emphasized team-specific tasks. Next, interteam meetings were held to present ideas and receive critical input. Finally, oral and poster research presentations were conducted at the UBSM science fair. Several team research projects covered topics in medical, environmental, and general microbiology. The three major assessment areas for the workshop and DCM included: (i) student's perception of the workshops' effectiveness in developing skills, content, and values; (ii) research team self- and group participation evaluation, and (iii) oral and poster presentation during the science fair. More than 91% of the students considered the workshops effective in the presentation of scientific method fundamentals. The combination of the workshop and the DCM increased student's knowledge by 55% from pre- to posttests. Two rubrics were designed to assess the oral presentation and poster set-up. The poster and oral presentation scores averaged 83% and 75% respectively. Finally, we present a team assessment instrument that allows the self- and group evaluation of each research team. While the DCM has educational plasticity and versatility, here we document how the this model has been successfully incorporated in training and engaging students in scientific research in microbiology.

Features of Rhodobacter sphaeroides CcmFH.

In this study, the in vivo function and properties of two cytochrome c maturation proteins, CcmF and CcmH from Rhodobacter sphaeroides, were analyzed. Strains lacking CcmH or both CcmF and CcmH are unable to grow under anaerobic conditions where c-type cytochromes are required, demonstrating their critical role in the assembly of these electron carriers. Consistent with this observation, strains lacking both CcmF and CcmH are deficient in c-type cytochromes when assayed under permissive growth conditions. In contrast, under permissive growth conditions, strains lacking only CcmH contain several soluble and membrane-bound c-type cytochromes, albeit at reduced levels, suggesting that this bacterium has a CcmH-independent route for their maturation. In addition, the function of CcmH that is needed to support anaerobic growth can be replaced by adding cysteine or cystine to growth media. The ability of exogenous thiol compounds to replace CcmH provides the first physiological evidence for a role of this protein in thiol chemistry during c-type cytochrome maturation. The properties of R. sphaeroides cells containing translational fusions between CcmF and CcmH and either Escherichia coli alkaline phosphatase or beta-galactosidase suggest that they are each integral cytoplasmic membrane proteins with their presumed catalytic domains facing the periplasm. Analysis of CcmH shows that it is synthesized as a higher-molecular-weight precursor protein with an N-terminal signal sequence.