The use of PCR-DGGE to determine bacterial fingerprints for poultry and red meat abattoir effluent.

UNLABELLED: Strict legislation and chemical composition monitoring of effluent may be useful, but the data generated do not allow for source tracking, and enforcing legislation remains problematic in the South African setting. These difficulties emphasize the necessity for effluent source traceability. Denaturing gradient gel electrophoresis (DGGE) targeting the V3 region of the 16S rRNA gene was considered as fingerprinting technique for effluent originating from abattoirs slaughtering different animal species. The influence of treatment to remove excess fat from effluent prior to molecular analyses and different PCR approaches on the detection of bacterial diversity were considered. Use of a treatment option to remove fat and a nested PCR approach resulted in up to 51% difference in inter-sample diversity similarity. A robust approach with no pre-treatment to remove PCR inhibitors, such as fat, and direct amplification from genomic DNA yielded optimal/maximal bacterial diversity fingerprints. Repeatable fingerprints were obtained for poultry abattoir effluent over a 4-month period, but profiles for the red meat abattoir varied with maximum similarity detected only 33·2%. Genetic material from faecal indicators Aeromona spp and Clostridium spp were detected. Genera unique to each effluent were present; Anoxybacillus, Patulibacter and Oleispira in poultry abattoir effluent and Porphyromonas and Peptostreptococcus in red meat abattoir effluent. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was the first to demonstrate the application of denaturing gradient gel electrophoresis (DGGE) to construct bacterial diversity fingerprints for high-throughput abattoir effluents. Proved redundancy of fat removal as PCR inhibitor and change in diversity similarity introduced by nested PCR approach. The importance of limiting excessive handling/processing which could lead to misrepresented diversity profiles was emphasized.

Influence of selected antimicrobials on the viability, endotoxicity and lipopolysaccharide composition of Pseudomonas aeruginosa in vitro.

This research focused on the influence of selected antimicrobial agents (AMAs) on the lipopolysaccharide (LPS) composition of Pseudomonas aeruginosa, a common causative agent of nosocomial infections. As LPS has been shown to play a role in attachment and virulence, the research is primarily aimed at shedding light on the response of these organisms to cleaning regimens in healthcare settings using various disinfectants. The endotoxicity and viability of the organisms following disinfection were further investigated via propagation in sublethal concentrations of the selected AMAs. The AMAs included a CIP chlorinated disinfectant, a heavy-duty alkaline detergent and a phenolic handwash solution. The effects of the antimicrobials on LPS both from intact cells and from debris were assessed by gas chromatography-mass spectrometry (GC-MS) analysis and a chromogenic Limulus amoebocyte lysate assay. Results indicated significant changes in the supramolecular structure of the O-polysaccharide when exposed to the AMAs. Adaptations occurred in both the total assessed saccharide and the lipid fractions, especially in the case of the heavy-duty alkaline detergent. Endotoxicity was found to be influenced by changes in the O-chain rather than the lipid fraction. The phenolic handwash and chlorine-based AMA treatments resulted in a slight decrease in the total amount of fatty acids in the LPS compared with saccharides, whereas the heavy-duty alkaline detergent resulted in a notable reduction in total saccharides. Microbial adaptation of the supramolecular structure of LPS may cause a reduction in membrane solubility of these organisms in an aqueous environment, thus affecting the organism's susceptibility to water-soluble AMAs as well as its ability to adhere to charged surfaces.

Genetic organisation of the capsule transport gene region from Haemophilus paragallinarum.

The region involved in export of the capsule polysaccharides to the cell surface of Haemophilus paragallinarum was cloned and the genetic organisation determined. Degenerate primers designed from sequence alignment of the capsule transport genes of Haemophilus influenzae, Pasteurella multocida and Actinobacillus pleuropneumoniae were used to amplify a 2.6 kb fragment containing a segment of the H. paragallinarum capsule transport gene locus. This fragment was used as a digoxigenin labelled probe to isolate the complete H. paragallinarum capsule transport gene locus from genomic DNA. The sequence of the cloned DNA was determined and analysis revealed the presence of four genes, each showing high homology with known capsule transport genes. The four genes were designated hctA, B, C and D (for H. paragallinarum capsule transport genes) and the predicted products of these genes likely encode an ATP-dependent export system responsible for transport of the capsule polysaccharides to the cell surface, possibly a member of a super family designated ABC (ATP-binding cassette) transporters.