Laboratory activity to teach about the proliferation of salmonella in vegetables

We designed a three-week laboratory experience that can complement any Microbiology teaching laboratory to expand students’ knowledge of the ecology of human enteric pathogens outside of their animal hosts. Through their participation in this laboratory activity, students learned that vegetative and reproductive plant parts could be a natural habitat for enteric bacteria such as non-typhoidal strains of Salmonella enterica. This field was recently brought to the forefront of the scientific and public interest by outbreaks of human illness linked to the consumption of fresh fruits and vegetables. Students were encouraged to develop their own testable hypotheses to compare proliferation of Salmonella enterica sv Typhimurium LT2 in different vegetables: cherry and regular size tomatoes, onions, lettuce, yellow and red bell peppers (Escherichia coli can be substituted for BSL1 laboratories). Upon completion of the laboratory experience, students were able to: 1) Develop testable hypotheses addressing the ability of a human pathogen Salmonella enterica to colonize and proliferate in vegetables; 2) Determine that different vegetables support the growth of Salmonella to a different extent; 3) Conduct statistical analysis and identify any significant differences. The teaching-learning process was assessed with a pre/post test, with an average increase in content understanding from ~15% to 85%. We also measured students’ proficiency while conducting specific technical tasks, revealing no major difficulties while conducting the experiments. Students indicated satisfaction with organization and content of the practices. 100% of the students agreed that the exercises improved their knowledge of this subject

Salmonella and tomatoes

Outbreak information linking fresh tomato fruit to illnesses is reviewed in this chapter. While tomato fruit appear to support substantial proliferation of certain serovars of Salmonella enterica, detection of this pathogen in tomato plants prior to harvest is rare, and reports of Salmonella existence in tomato fruit still attached to field-grown plants are virtually non-existent. The bacterium is sensitive to UV and can be outcompeted by the native phytomicrobiota, which may explain its absence in field-grown crops. However, the persistence of certain serovars in fields and ponds of certain production areas is noted. Together with evidence of bacteria becoming internalized in tomato fruit during crop development likely through natural apertures, the presence of S. enterica in and around production fields suggests that an unusual weather event could lead to Salmonella contamination of fruit prior to harvest. The bacterium appears physiologically adaptive toward proliferation in tomato fruit. Once inside tomatoes, Salmonella is capable of sensing the availability of nutrients and physiological state of the fruit and differentially regulates specific genes. However, because Salmonella is an efficient nutrient scavenger, removal of multiple metabolic and regulatory genes was required to reduce its fitness within the fruit. Plants do not appear to recognize human enterics as pathogens, and their defenses treat them as endophytes

Factors affecting formation of large calcite crystals (≥1mm) in Bacillus subtilis 168 biofilm

B4 is the most common medium used in general organomineralization studies and has been used to assay or to characterize mineral precipitation potential. In an exercise for the optimization of the laboratory conditions of crystal precipitation in vitro, we used Bacillus subtilis 168 as a type strain and its isogenic mutants. While literature is mainly focused on observing generic precipitation, we investigated the requirement to obtain large crystals (≥1mm), which could be advantageous in wide-ranging implications for bioconsolidation of soil, sand, stone, and cementitious materials. Calcite crystals are visible on B4 agar plates within 7 days at 37˚C after inoculum of B. subtilis 168 strain. In this study we show that to form large crystals with a diameter ≥1mm several conditions must be met: i) Reduced amount of B4 medium into the Petri plate improve crystal formation. 55mm Petri plates contained only 4mL of B4 agar medium reached a plateau in 6 days at 37ºC. High moisture and presence of water condense would decrease crystal formation. ii) Inoculation of cells using a rod instead of a circular shaped spot. When the same number of B. subtilis cells was streaked, rod-shape biofilm significantly fostered crystal precipitation, while spot-shape prevented precipitation. iii) When more than one biofilm is present within the same plate, mutual interactions can affect precipitation in each biofilm. iv) Spherical nucleation sites are identified as initial step during the formation of large calcite crystal

Resisting antimicrobial resistance: lessons from fungus farming ants

Attine ants use antimicrobials produced by commensal bacteria to inhibit parasites on their fungal gardens. However, in this agricultural system, antimicrobial use does not lead to overwhelming resistance, as is typical in clinical settings. Mixtures of continually evolving antimicrobial variants could support these dynamics. [Abstract copyright: Copyright © 2019 Elsevier Ltd. All rights reserved.

Exopolymeric substances (EPS) from Bacillus subtilis: polymers and genes encoding their synthesis

Bacterial exopolymeric substances (EPS) are molecules released in response to the physiological stress encountered in the natural environment. EPS are structural components of the extracellular matrix in which cells are embedded during biofilm development. The chemical nature and functions of these EPS are dependent on the genetic expression of the cells within each biofilm. Although some bacterial matrices have been characterized, understanding of the function of the EPS is relatively limited, particularly within the Bacillus genus. Similar gaps of knowledge exist with respect to the chemical composition and specific roles of the macromolecules secreted by Bacillus subtilis in its natural environment. In this review, the different EPS from B. subtilis were classified into four main functional categories: structural (neutral polymers), sorptive (charged polymers), surface-active and active polymers. In addition, current information regarding the genetic expression, production and function of the main polymers secreted by B. subtilis strains, particularly those related to biofilm formation and its architecture, has been compiled. Further characterization of these EPS from B. subtilis remains a challenge

Fitness of antibiotic-resistant bacteria in the environment: a laboratory activity

In this laboratory experiment, we propose an opportunity for students to broaden their understanding of the ecology of antibiotic-resistant and sensitive waterborne bacteria. Antibiotics can be found in rivers or soil as a consequence of agricultural practices or as a result of human use. Concentrations of antibiotics in the environment may range from a few ng to μg L-1. Such concentrations can affect the selection and fitness of resistant bacteria. In this laboratory activity, students learn how to set up a fitness experiment by using an isogenic pair of antibiotic-resistant and sensitive bacteria in the presence or absence of selective pressure. Microcosms were generated by using filtered river water containing populations of resistant and sensitive bacteria. Competition of both populations was measured in the presence or absence of antibiotics. Students appreciated the use of microcosms for in vitro experiments and the extent to which the fitness of resistant and sensitive bacteria changed in the presence and/or absence of a selective pressure in river water. Student learning was measured by using different types of assessments: multiple-choice, true/false, fill in the blanks, laboratory skills observations, and laboratory reports. After the laboratory activity, the percentage of correct answers significantly rose from ~20% to ~85%. Laboratory skills were also evaluated during the exercises, showing no major issues during the experiment. Students showed proficiency in analyzing the complexity of fitness data by reaching a mean of 5.57 (standard error 0.57) over a maximum score of 7 points

Laboratory activity to effectively teach introductory geomicrobiology concepts to non-geology majors

We have designed a three-week experiment that can complement any microbiology course, to teach main geomicrobiology concepts for non-geology majors. One of the most difficult concepts for non-geology majors to comprehend is how bacteria serve as a platform for different mineralization reactions. In our three-week laboratory practice, students learn the main principles and conditions required for an induced bacterial mineralization. Upon completion of the laboratory experience, students will: 1) learn how microbial-induced mineralization (such as calcium carbonate formation) is affected by differential media and growth conditions; 2) understand how bacterial physiology affects any induced in situ or in vitro mineralization; 3) comprehend how growing conditions and bacterial physiologies interrelate, resulting in differential crystal formation. The teaching-learning process was assessed using a pre-/posttest with an increase from 26% to 76% in the number of positive answers from the students. We also measured the students' proficiency while conducting specific technical tasks, revealing no major difficulties while conducting the experiments. A final questionnaire was provided with satisfactory evaluations from the students regarding the organization and content of the practices. 84-86% of the students agreed that the exercises improved their knowledge in geomicrobiology and would like to attend similar laboratories in the future. Such response is the best indicator that the laboratory practice can be implemented in any undergraduate/graduate microbiology course to effectively teach basic geomicrobiology concepts to non-geology majors