Comparison of nine extraction methods for bacterial identification using the ONT MinION sequencer.

The Anthrax mailings bioterrorism attack in 2001 revealed the need for universal and rapid microbial forensic analyses on unknown biological evidence. However, the gold standard for bacterial identification includes culturing isolates, which is laborious. Molecular approaches for bacterial identification revolve around 16S ribosomal gene sequencing using Sanger or next generation sequencing (NGS) platforms, but these techniques are laboratory-based and can also be time-consuming. The Oxford Nanopore Technologies (ONT) MinION sequencer can generate long read lengths that span the entire bacterial 16S rRNA gene and accurately identify the species level. This platform can be used in the field, allowing on-site evidence analysis. However, it requires higher quantities of pure DNA compared to other sequencing platforms; thus, the extraction method for bacterial DNA is critical for downstream analysis, which to date are tailored toward a priori knowledge of the species' taxonomic grouping. During an attack, the investigative team may not know what species they are handling; therefore, identifying an extraction method that can handle all bacterial groups and generate clean DNA for the MinION is useful for microbial forensic analysis. The purpose of this study was to identify a "universal" extraction method that can be coupled with ONT MinION sequencing for use in forensic situations for rapid identification. It also evaluated the cloud-based data analysis software provided by ONT, EPI2ME. No "universal" extraction method was identified as optimal for downstream MinION sequencing. However, the DNeasy PowerSoil Kit and Noda et al. Chelex-100 method gave comparable sequencing results and could be used as rapid extraction techniques. This study showed that the ONT 16S Barcoding Kit 1-24 coupled with the 16S FASTQ workflow might not be the best for use in forensic situations where species-level identification needs to be obtained, as most alignments were approximately 89% accurate. In all seven test organisms and nine extraction methods, accurate species identification was only obtained in 63% of the cases.

A Slimy Business: the Future of Fish Skin Microbiome Studies.

Fish skin contains a mucosal microbiome for the largest and oldest group of vertebrates, a location ideal for microbial community ecology and practical applications in agriculture and veterinary medicine. These selective microbiomes are dominated by Proteobacteria, with compositions different from the surrounding water. Core taxa are a small percentage of those present and are currently functionally uncharacterized. Methods for skin sampling, DNA extraction and amplification, and sequence data processing are highly varied across the field, and reanalysis of recent studies using a consistent pipeline revealed that some conclusions did change in statistical significance. Further, the 16S gene sequencing approaches lack quantitation of microbes and copy number adjustment. Thus, consistency in the field is a serious limitation in comparing across studies. The most significant area for future study, requiring metagenomic and metabolomics data, is the biochemical pathways and functions within the microbiome community, the interactions between members, and the resulting effects on fish host health being linked to specific nutrients and microbial species. Genes linked to skin colonization, such as those for attachment or mucin degradation, need to be uncovered and explored. Skin immunity factors need to be directly linked to microbiome composition and individual taxa. The basic foundation has been laid, and many exciting future discoveries remain.

Biochemical but not compositional recovery of skin mucosal microbiome communities after disruption.

BACKGROUND: The microbiomes of animals are complex communities that strongly affect the health of the hosts. Microbiomes on mucosal surfaces have the highest densities and most extensive biochemical exchanges with the hosts. Although antibiotics are potent tools to manage infections, they can disrupt the normal microbiota, causing numerous side effects. MATERIALS AND METHODS: Taking a community ecology approach, mucosal microbiome community responses to five disruptive conditions (two broad-spectrum antibiotics, a biocide, elevated temperature, and rinsing) were analyzed. Skin of the fish Gambusia affinis was the mucosal model. Microbiome recovery was measured by culturable counts, community biochemical profiles, genetic fingerprinting, and community 16S gene sequencing (rinsing condition only). RESULTS: Following all disruptions, the total counts rose and then returned to the pre-treatment (PT) level. This overgrowth was confirmed via direct staining and community metabolic activity measurements. After rinsing, diversity decreased and one taxon dominated (family Aeromonadaceae) temporarily, the findings similar to numerous other studies with antibiotics. While the community did not return to the PT taxonomic composition, the biochemical profile did. CONCLUSION: This suggests that the biochemical pathways in a community are important during recovery, and a return to the original composition is not required to restore original function.

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment.

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host microbiome community diversity and alter the microbial community composition. Many diseases such as antibiotic-associated enterocolitis, inflammatory bowel disease, and metabolic disorders have been linked to a disrupted microbiota. The complex interplay between host, microbiome, and antibiotics needs a tractable model for studying host-microbiome interactions. Our freshwater vertebrate fish serves as a useful model for investigating the universal aspects of mucosal microbiome structure and function as well as analyzing consequential host effects from altering the microbial community. Methods include host challenges such as infection by a known fish pathogen, exposure to fecal or soil microbes, osmotic stress, nitrate toxicity, growth analysis, and measurement of gut motility. These techniques demonstrate a flexible and useful model system for rapid determination of host phenotypes.

Microbiome disruption and recovery in the fish Gambusia affinis following exposure to broad-spectrum antibiotic.

Antibiotics are a relatively common disturbance to the normal microbiota of humans and agricultural animals, sometimes resulting in severe side effects such as antibiotic-associated enterocolitis. Gambusia affinis was used as a vertebrate model for effects of a broad-spectrum antibiotic, rifampicin, on the skin and gut mucosal microbiomes. The fish were exposed to the antibiotic in the water column for 1 week, and then monitored during recovery. As observed via culture, viable counts from the skin microbiome dropped strongly yet returned to pretreatment levels by 1.6 days and became >70% resistant. The gut microbiome counts dropped and took longer to recover (2.6 days), and became >90% drug resistant. The resistance persisted at ~20% of skin counts in the absence of antibiotic selection for 2 weeks. A community biochemical analysis measuring the presence/absence of 31 activities observed a 39% change in results after 3 days of antibiotic treatment. The antibiotic lowered the skin and gut microbiome community diversity and altered taxonomic composition, observed by 16S rRNA profiling. A 1-week recovery period did not return diversity or composition to pretreatment levels. The genus Myroides dominated both the microbiomes during the treatment, but was not stable and declined in abundance over time during recovery. Rifampicin selected for members of the family Comamonadaceae in the skin but not the gut microbiome. Consistent with other studies, this tractable animal model shows lasting effects on mucosal microbiomes following antibiotic exposure, including persistence of drug-resistant organisms in the community.

Concept Inventory Development Reveals Common Student Misconceptions about Microbiology.

Misconceptions, or alternative conceptions, are incorrect understandings that students have incorporated into their prior knowledge. The goal of this study was the identification of misconceptions in microbiology held by undergraduate students upon entry into an introductory, general microbiology course. This work was the first step in developing a microbiology concept inventory based on the American Society for Microbiology's Recommended Curriculum Guidelines for Undergraduate Microbiology. Responses to true/false (T/F) questions accompanied by written explanations by undergraduate students at a diverse set of institutions were used to reveal misconceptions for fundamental microbiology concepts. These data were analyzed to identify the most difficult core concepts, misalignment between explanations and answer choices, and the most common misconceptions for each core concept. From across the core concepts, nineteen misconception themes found in at least 5% of the coded answers for a given question were identified. The top five misconceptions, with coded responses ranging from 19% to 43% of the explanations, are described, along with suggested classroom interventions. Identification of student misconceptions in microbiology provides a foundation upon which to understand students' prior knowledge and to design appropriate tools for improving instruction in microbiology.

Development, Validation, and Application of the Microbiology Concept Inventory.

If we are to teach effectively, tools are needed to measure student learning. A widely used method for quickly measuring student understanding of core concepts in a discipline is the concept inventory (CI). Using the American Society for Microbiology Curriculum Guidelines (ASMCG) for microbiology, faculty from 11 academic institutions created and validated a new microbiology concept inventory (MCI). The MCI was developed in three phases. In phase one, learning outcomes and fundamental statements from the ASMCG were used to create T/F questions coupled with open responses. In phase two, the 743 responses to MCI 1.0 were examined to find the most common misconceptions, which were used to create distractors for multiple-choice questions. MCI 2.0 was then administered to 1,043 students. The responses of these students were used to create MCI 3.0, a 23-question CI that measures students' understanding of all 27 fundamental statements. MCI 3.0 was found to be reliable, with a Cronbach's alpha score of 0.705 and Ferguson's delta of 0.97. Test item analysis demonstrated good validity and discriminatory power as judged by item difficulty, item discrimination, and point-biserial correlation coefficient. Comparison of pre- and posttest scores showed that microbiology students at 10 institutions showed an increase in understanding of concepts after instruction, except for questions probing metabolism (average normalized learning gain was 0.15). The MCI will enable quantitative analysis of student learning gains in understanding microbiology, help to identify misconceptions, and point toward areas where efforts should be made to develop teaching approaches to overcome them.

Mycobacterium avium enters a state of metabolic dormancy in response to starvation.

Members of the Mycobacterium avium complex (MAC) exhibit a highly effective and biphasic response to starvation, losing less than 90% viability after 2 years in deionized water. During the first adaptive phase of 4-7 days, the bacilli exhibit a burst of lipid catabolism, alteration of mycolate modifications, loss of catalase and urease activities, and a decline in sensitivity to antibiotics. There is also a decline in the protein level of alanine tRNA synthetase (AlaS), and an increase in ribonuclease E (Rne) levels. During the following persistence phase, the bacilli become metabolically dormant. However, with return of nutrients, the cells rapidly respond with increased activity, as determined by reduction of a tetrazolium dye. The primary reservoir for MAC is natural and municipal water, and the metabolic dormancy may be analogous to that of other aquatic organisms, such as vibrio. The organized metabolic shutdown that environmental mycobacteria utilize to survive starvation may have evolved into the host-specific dormancy mechanisms of Mycobacterium tuberculosis.

The host effects of Gambusia affinis with an antibiotic-disrupted microbiome.

While serving as critical tools against bacterial infections, antimicrobial therapies can also result in serious side effects, such as antibiotic-associated entercolitis. Recent studies utilizing next generation sequencing to generate community 16S gene profiles have shown that antibiotics can strongly alter community composition and deplete diversity. However, how these community changes in the microbiota are related to the host side effects is still unclear. We have used the freshwater Western mosquitofish (Gambusia affinis) as a tractable vertebrate model system to study host effects following exposure to a broad spectrum antibiotic, rifampicin. After 3days of exposure, the bacterial communities of the mucosal skin and gut microbiomes lost diversity and shifted composition. Compared to unexposed controls, treated fish were more susceptible to a specific pathogen, Edwardsiella ictaluri, yet displayed no survival differences when subjected to a polymicrobial water challenge of soil or feces. Treated fish were more susceptible to osmotic stress from NaCl, but not to the toxin nitrate. Treated fish failed to gain weight as well as controls over one month when fed a matched diet. Because of small sample sizes, pathogen susceptibility and weight gain differences were not statistically significant. This study provides supporting evidence in an experimental laboratory system that an antibiotic can have significant and persistent negative host effects, and provides for future study into the mechanisms of these effects.

Select acetophenones modulate flagellar motility in chlamydomonas.

Acetophenones were screened for activity against positive phototaxis of Chlamydomonas cells, a process that requires co-ordinated flagellar motility. The structure-activity relationships of a series of acetophenones are reported, including acetophenones that affect flagellar motility and cell viability. Notably, 4-methoxyacetophenone, 3,4-dimethoxyacetophenone, and 4-hydroxyacetophenone induced negative phototaxis in Chlamydomonas, suggesting interference with activity of flagellar proteins and control of flagellar dominance.

Mycobacterial ecology of the Rio Grande.

This is the first study to characterize the environmental conditions which contribute to the presence and proliferation of environmental mycobacteria in a major freshwater river. Over 20 different species of environmental mycobacteria were isolated, including the pathogenic M. avium and M. kansasii. Species of the rapidly growing M. fortuitum complex were the most commonly isolated mycobacteria, and one-third of all isolates were not identified at the species level, even by 16S sequencing. PCR restriction analysis of the hsp65 gene was more accurate and rapid than biochemical tests and as accurate as yet less expensive than 16S sequencing, showing great promise as a new tool for species identification of environmentally isolated mycobacteria. Total environmental mycobacteria counts positively correlated with coliform and Escherichia coli counts and negatively correlated with chemical toxicity and water temperature. Environmental mycobacteria can survive in the alkaline conditions of the river despite previous reports that especially acidic conditions favor their presence. A representative river isolate (M. fortuitum) survived better than E. coli O157:H7 at pHs below 7 and above 8 in nutrient broth. The river strain also retained viability at 8 ppm of free chlorine, while E. coli was eliminated at 2 ppm and above. Thus, in vitro studies support environmental observations that a variety of extreme conditions favor the hardy environmental mycobacteria.

Tandem screening of toxic compounds on GFP-labeled bacteria and cancer cells in microtiter plates.

A 96-well fluorescence-based assay has been developed for the rapid screening of potential cytotoxic and bacteriocidal compounds. The assay is based on detection of green fluorescent protein (GFP) in HeLa human carcinoma cells as well as gram negative (Escherichia coli) and gram positive bacteria (Mycobacterium avium). Addition of a toxic compound to the GFP marked cells resulted in the loss of the GFP fluorescence which was readily detected by fluorometry. Thirty-nine distinct naphthoquinone derivatives were screened and several of these compounds were found to be toxic to all cell types. Apart from differences in overall toxicity, two general types of toxic compounds were detected, those that exhibited toxicity to two or all three of the cell types and those that were primarily toxic to the HeLa cells. Our results demonstrate that the parallel screening of both eukaryotic and prokaryotic cells is not only feasible and reproducible but also cost effective.

Health impacts of environmental mycobacteria.

Environmental mycobacteria are emerging pathogens causing opportunistic infections in humans and animals. The health impacts of human-mycobacterial interactions are complex and likely much broader than currently recognized. Environmental mycobacteria preferentially survive chlorination in municipal water, using it as a vector to infect humans. Widespread chlorination of water has likely selected more resistant environmental mycobacteria species and potentially explains the shift from M. scrofulaceum to M. avium as a cause of cervical lymphadenitis in children. Thus, human activities have affected mycobacterial ecology. While the slow growth and hydrophobicity of environmental mycobacteria appear to be disadvantages, the unique cell wall architecture also grants high biocide and antibiotic resistance, while hydrophobicity facilitates nutrient acquisition, biofilm formation, and spread by aerosolization. The remarkable stress tolerance of environmental mycobacteria is the major reason they are human pathogens. Environmental mycobacteria invade protozoans, exhibiting parasitic and symbiotic relationships. The molecular mechanisms of mycobacterial intracellular pathogenesis in animals likely evolved from similar mechanisms facilitating survival in protozoans. In addition to outright infection, environmental mycobacteria may also play a role in chronic bowl diseases, allergies, immunity to other pulmonary infections, and the efficacy of bacillus Calmette-Guerin vaccination.

The relationship of temperature to desiccation and starvation tolerance of the Mycobacterium avium complex.

Mycobacterium avium grew in media at 14-37 degrees C, and persisted at 4 degrees C and 42 degrees C. The bacteria lost approximately 90% viability after 3 months in reverse-osmosis deionized water at 4-37 degrees C. Cooler temperatures lowered the death rate. Death rates also decreased after a 5- to 10-day starvation adaptation period. Alterations of the steady-state levels of different mycolic acid classes, presumably to facilitate thermoadaptation, were found. Following desiccation, M. avium lost viability at a constant rate (half-life of 2.3 days). This implies that bacilli contaminating dry medical surfaces would persist for short periods of time. The remarkable stress survival exhibited by M. avium further suggests persistence in a range of environmental and clinical settings.

Quinones as antimycobacterial agents.

Mycobacterium tuberculosis is a serious worldwide health threat, killing almost 3 million people per year. Other mycobacterial species, especially Mycobacterium avium, are emerging pathogens in the immunocompromised population, most notably AIDS patients. These nontuberculous mycobacteria (NTM) are ubiquitous in the environment, and naturally resistant to many disinfection procedures. Treatment options are limited, and no new antibiotics have been developed against mycobacteria since the 1970s. There is a desperate need for new biocides and antibiotics to prevent and treat mycobacterial infections. A small aromatic compound library has been screened for effectiveness in growth inhibition or killing of mycobacteria. Four species, representing the M. tuberculosis complex, the slow-growing NTM, and the rapid-growing NTM were used. Active compounds had minimal inhibitory concentrations as low as 12.5 microg/mL, with the active component being a quinone. The primarily bactericidal activity observed represents a unique mechanism of action. A fluorescent assay involving M. smegmatis expressing gfp was analyzed as a rapid assay for predicting inhibitory activity, but failed to predict activity well. Our compounds may have significant utility as soluble biocides against mycobacteria and other hardy nosocomial pathogens.