The evaluation of PCR-based techniques for the detection of fecal indicator organisms in environmental samples

Escherichia coli is the indicator organism of choice for the detection of fecal contamination. Standard methods for detecting fecal contamination often rely on detecting fecal coliforms. While this practice if highly efficient in most applications, when used with the sediment-laden waters of Louisiana, free-living soil bacteria, such as Klebsiella pneumoniae were shown to inflate the fecal coliform counts. An accurate assessment of the contamination problem requires methods that detect E. coli. To detect E. coli, several of species-specific genes were tested before the gadAB genes were selected. The polymerase chain reaction (PCR) was chosen to detect these genes using a nested primer array. Using the nested PCR assay, E. coli was routinely detected in laboratory samples down to the single-cell level. This level of detection was deemed essential for a quantitative or semiquantitative assay to determine E. coli numbers. The most beneficial use of PCR-based techniques may be the use of specific fecal indicator organisms to differentiate the sources of fecal contamination. E. coli is the indicator of fecal contamination from essentially all sources posing a risk to human health. The Bacteroides fragilis group (BFG) demonstrates strong potential as a fecal indicator organism of human waste. While the levels of E. coli in humans and cattle are comparable, the levels of BFG are approximately 105 times greater in humans than cattle. By determining the ratio of E. coli to BFG in contaminated water, one should be able to distinguish fecal contamination from humans and common farm animals, such as dairy cattle. This requires the determination of the relative levels of E. coli and BFG and not the absolute levels in the sample. Using the nested PCR method, a region of the 16S rRNA genes specific to members of the BFG was targeted. In the laboratory, B. fragilis cells were detected down to the single-cell level. When environmental water samples were tested, impurities in the template preparations limited the amount of DNA template that could be added to the reactions. Within those limitations the level of detection was comparable to laboratory studies using cultured cell suspensions

Brucella melitensis: the evaluation of a putative hemagglutinin gene\u27s effect on virulence in the caprine model

Brucella melitensis is a facultative intracellular bacterial pathogen that causes abortions in goats and sheep and Malta fever in humans. The zoonotic disease brucellosis causes severe economic losses in the Mediterranean region and parts of Africa, Asia, and Latin America. With the completion of the genomic sequences of B. abortus 2308 and B. melitensis 16M, no classical virulence factors were found; and the chromosomes were virtually identical. However, in B. melitensis, a putative hemagglutinin gene was identified which is absent in B. abortus. The possibility of the hemagglutinin gene being a potential virulence factor was evaluated via gene replacement/deletion in B. melitensis and expression in trans in B. abortus. The hemagglutinin gene was PCR-amplified, cloned into pBBR1MCS-4, and electroporated into B. abortus 2308 yielding B. abortus 2308-QAE. A kanamycin-Region E-kanamycin disrupted gene fragment (KAN-E-KAN) was also generated and electroporated into B. melitensis 16M. The resulting mutants were characterized biochemically to confirm its Brucella origin and screened by antibiotic selective pressure. A colonization study of non-pregnant goats infected with B. abortus 2308, B. melitensis 16M, B. abortus 2308-QAE, or B. melitensis 16MÄE revealed no attenuation of the 16MÄE mutant when compared to 16M at 4, 7, and 21 days post inoculation. The study also showed that both the variant and the mutant were capable of infecting and disseminating throughout the host. All four strains were introduced into the pregnant goat model and evaluated for pathogenicity. Pregnancy/delivery results revealed 27%, 78%, 67%, and 50% abortion rates in goats infected with 2308, 16M, 2308-QAE, and 16MÄE, respectively. Bacterial culture of tissues from 2308, 16M, 2308-QAE, 16MÄE -exposed goats revealed 45 %, 79%, 75%, and 100% colonization of dam/kid pairs, respectively. The expression of the B. melitensis 16M hemagglutinin gene in trans in 2308-QAE revealed a significant (p\u3c0.05) increase in colonization and abortion rates when compared to B. abortus 2308, mimicking the virulence of B. melitensis 16M in pregnant goats. The B. melitensis 16MÄE disruption mutant colonization and abortion rates demonstrated no attenuation in colonization but did show a 28% reduction in abortions when compared to B. melitensis 16M