Todd P. Primm

Health Impacts of Environmental Mycobacteria

SUMMARY 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.

Acetophenones with selective antimycobacterial activity

Aims:  Mycobacteria are a serious cause of infections in humans, with limited treatment options, as no new antibiotics have been developed against mycobacteria since the 1960s. In this study, the antimycobacterial activity of a small library of acetophenone (AP) compounds was analysed.

Recent Advances in Methodologies for the Discovery of Antimycobacterial Drugs

Mycobacteria, especially M. tuberculosis, have remained a worldwide dominant cause for human morbidity (~10 million annual cases) and mortality (3 million deaths annually) since ancient times. An estimated one-third of living humans are latently infected with M. tuberculosis. Despite this, there have been no new drugs specifically developed against mycobacteria since the 1960s. Because of a slow growth rate, biosafety concerns, and other issues, drug discovery by in vitro screening of natural and synthetic compounds has been limited in the past. However, a number of new techniques and technologies for whole cell antimycobacterial screening have been developed recently. This review examines and compares these methods, discusses common issues in screening with mycobacteria, and considers potential future developments in the field.

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.

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.

Green Fluorescent Protein as a Biosensor for Toxic Compounds

In this brief review, we present recent results in the development of fluorescence-based assays for the detection of compounds with cytotoxic, anticancer and anti-microbial properties. As other reviews have explored various aspects related to these topics, this review will focus on the use of the Green Fluorescent Protein (GFP) for the detection of potentially toxic and/or therapeutic compounds. Since high-throughput screening of chemical compounds can be both expensive and laborious, development of low cost and efficient cell-based assays to determine biological activity should greatly enhance the early screening process. In our recent studies, we have developed a couple of GFP-based assays for the rapid screening of compounds with cytotoxic and bacteriocidal properties. As will be described in more detail in subsequent sections, a new 96-well assay has recently been developed that allows for the simultaneous screening of test compounds on gram positive and negative bacteria as well as mammalian (human cancer) cells. Our results demonstrate that both mammalian cells and bacteria can be analyzed in tandem to rapidly determine which compounds are specifically toxic to one of these cell types. The parallel screening of both eukaryotic and prokaryotic cells was found to be feasible, reproducible, and cost effective.

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.

Nucleotide Manipulatives to Illustrate the Central Dogma.

The central dogma is a core concept that is critical for introductory biology and microbiology students to master. However, students often struggle to conceptualize the processes involved, and fail to move beyond simply memorizing the basic facts. To encourage critical thinking, we have designed a set of magnetic nucleotide manipulatives that allow students to model DNA structure, along with the processes of replication, transcription, and translation.

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.