- Volume 146, Issue 4, 2000

Staphylococcus aureus is a major cause of bovine mastitis. Since gene expression of many bacteria is known to be regulated by the environment, milk may play an important role in the regulation of the early steps in the pathogenesis of bovine mastitis by S. aureus. To get insight into the response of S. aureus to the milk environment, a Tn917-lacZ mutant library was generated and screened for genes specifically expressed during growth in milk. Twenty-eight mutants were identified and analysed. Four groups of genes were found, involved in cell-wall synthesis, nucleotide synthesis, transcriptional regulation and carbohydrate metabolism. A fifth group contained genes with hypothetical or unknown functions. Many of the genes identified belonged to biosynthetic pathways of S. aureus and other bacterial species which have also been shown to play a role in vivo as determined in murine infection models. Therefore, growth on milk may be an attractive model for the identification of genes preferentially expressed during bovine mastitis.

Vibrio species are Gram-negative bacteria that cause a systemic infection in fish called vibriosis. The authors previously demonstrated that internalization and cytotoxicity are important virulence mechanisms in vibrio–fish epithelial cell interactions. Adherence is a prerequisite for successful internalization. In this study, the adherence capability of two invasive strains [V. anguillarum 811218–5W and G/Virus/5(3)] was compared with that of two non-invasive strains [V. damselae ATCC 33539 and V. anguillarum S2/5/93(2)] using adherence assays in three different types of fish cells (epithelial papillosum of carp, EPC; grunt-fin tissue, GF; and fat-head minnow epithelial cells, FHM). For all four strains there was no significant difference (P>0·05) in the adherence to the different cell lines. V. anguillarum 811218–5W exhibited the highest adherence, followed by G/Virus/5(3) and S2/5/93(2); V. damselae ATCC 33539 showed the lowest adherence. The super-adherence characteristic of V. anguillarum 811218–5W on EPC cells was not affected by inhibitors, sugars, low temperature (4 °C) incubation, or non-biological surfaces such as glass coverslips. The galactose-linked adherence characteristic of V. anguillarum G/Virus/5(3) to the EPC cells was partially inhibited by peptidase treatment of the fish cells, low-temperature incubation, and addition of sugars that contained galactose (such as lactose and N-acetyl-D-galactosamine). De novo synthesis of bacterial protein, viable bacteria and intact carbohydrate structure of vibrios were required for both super-adherence and galactose-linked adherence. These adherence characteristics were also found in ten other invasive vibrios, and galactose-linked adherence was found in nine invasive vibrios.

Aeromonas hydrophila, a normal inhabitant of aquatic environments, is an opportunistic pathogen of a variety of aquatic and terrestrial animals, including humans. A. hydrophila PPD134/91 is defined as virulent whereas PPD35/85 is defined as avirulent on the basis of their different LD50 values in fish. Suppression subtractive hybridization (SSH) was used to identify genetic differences between these two strains. Sixty-nine genomic regions of differences were absent in PPD35/85, and the DNA sequences of these regions were determined. Sixteen ORFs encoded by 23 fragments showed high homology to known proteins of other bacteria. ORFs encoded by the remaining 46 fragments were identified as new proteins of A. hydrophila, showing no significant homology to any known proteins. Among these PPD134/91-specific genes, 22 DNA fragments (21 ORFs) were present in most of the eight virulent strains studied but mostly absent in the seven avirulent strains, suggesting that they are universal virulence genes in A. hydrophila. The PPD134/91-specific genes included five known virulence factors of A. hydrophila: haemolysin (hlyA), protease (oligopeptidase A), outer-membrane protein (Omp), multidrug-resistance protein and histone-like protein (HU-2). Another 47 DNA fragments (44 ORFs) were mainly present in PPD134/91, indicating the heterogeneity among motile aeromonads. Some of these fragments encoded virulence determinants. These included genes for the synthesis of O-antigen and type II restriction/modification system. The results indicated that SSH is successful in identifying genetic differences and virulence genes among different strains of A. hydrophila.