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SUMMARYIn this study we describe an approach to the characterization of the cultivable heterotrophic bacterial flora of honey bee hives. We assessed the bacterial abundance in brood combs and on hive floors at different times of the year, and identified several isolated bacteria. The number of bacteria recovered from brood combs was notably lower than the number obtained from hive floors. In addition, we observed an increase in bacteria recovered from brood comb samples from winter to summer, probably reflecting the activity of the colony during these seasons. In floor samples bacterial counts were not different among the different sampling times. Most of the identified isolates belonged to the genera Bacillusand Corynebacterium. Spores of Paenibacillus larvaesubspecies larvaewere only found in samples taken during summer of 2002, in accordance with an increase of American foulbrood incidence in Uruguay in the last year. The presence of several micro-organisms such as those identified as belonging to the genus Bacillus, potentially related to those that have biological control capabilities, and the differences between brood combs and floor-recovered bacteria encourage further research on the microbiology of honey bee hives and new strategies of pathogens control.

American foulbrood (AFB), a severe bacterial disease of honeybee brood, has recently been found in Uruguayan apiaries. Detection of the causative agent, Paenibacillus larvaesubspecies larvae, is a very important concern in order to prevent disease dissemination and decrease of honey production. Since spores are the infective forms of this pathogen, in the present work we report the use of polymerase chain reaction (PCR) to detect P. l.subsp. larvaespores from in vitrocultures, larvae with clinical symptoms and experimentally contaminated honey. The set of primers was designed based on the published P. l.subsp. larvae16S rRNA gene. Using this approach we could amplify the pathogen DNA and obtain a great sensitivity and a notable specificity. Detection limit for spore suspension was a 10−2dilution of template DNA obtained from 32 spores, as determined by plate count. For artificially contaminated honey, we could detect the PCR product at a 10−3dilution of template DNA obtained from 170 spores. In addition, when PCR conditions were set to improve specificity, we were able to amplify P. l. subsp. larvaeDNA selectively and no cross-reactions were observed with a variety of related bacterial species, including P. l.subsp. pulvifaciens. Since spore detection is very important to confirm the presence of the disease, this method provides a reliable diagnosis of AFB from infected larvae and contaminated honey in a few hours.

A clinical isolate ofProteus mirabilis(Pr 990) and an isogenic non-flagellate allelic replacement mutant (Pr M9) were tested for their ability to cause infection in the ascending mouse model of urinary tract infection. Wild-type Pr 990 differentiates into swarmer cells in brain-heart infusion broth. Pr M9 neither has flagella nor does it apparently differentiate into swarmer cells after subculturing. The infectivity of both strains from an initial culture and the sixth subculture was assessed in the ascending urinary tract infection mouse model. Infection was ascertained by determining colony forming units from kidney and bladder homogenates from individual mice 7 days after inoculation. In all cases the non-flagellate mutant Pr M9 was at least as infective as Pr 990. Using bacteria from the first culture, Pr M9 infected 61.5% and Pr 990 infected 45.5% of mice tested. The levels of viable counts were similar between the Pr M9 and the Pr 990 infections. Using bacteria from the sixth subculture, Pr M9 infected 75% and Pr 990 infected 76.5% of mice tested. Again viable counts were similar. Pr 990 increased in infectivity from the first to the sixth subculture, whereas Pr M9 did not, but this may be a reflection of the high initial rate of infectivity with first culture Pr M9. These results suggest that neither flagella nor swarmer cells are required forP. mirabilisinfectivity in ascending urinary tract infections in mice.

Our environment is full of potential carcinogens such as UV light, industrial pollutants, pesticides, and food additives, among others. It is estimated that 90% of all carcinogens are also mutagens. The Ames test is one of the most common tests for mutagens. In this problem-based learning activity, undergraduate biology students used the Ames test to screen a substance they provided, to see if it could be considered a mutagen. The idea of surveying substances used in everyday life appealed to our students, and helped engage them in this activity.

We describe a simple and effective method for the extraction of potential mutagens from sediment samples. This method is straightforward and does not require expensive laboratory equipment, thus enabling instructors to analyze the presence of mutagenic substances in a conventional classroom setting. Additionally, we found that students felt encouraged to add the Ames test to the list of analyses they traditionally employ with sediment samples. This link between the environment and the Ames test provided an authentic learning context for students, bridging the gap between the "real-world" and the classroom laboratory, and thus making the educational experience more engaging and meaningful.