METHODOLOGY: A pair of degenerate primers (Aero F: 5'-YGARATCGAYATCGCCAARCGB-3' and Aero R: 5'-GRCCDATGCTCATRCGRCGGTT-3') was designed that amplified the rpoD gene of 27 Aeromonas species. Subsequently, in silico analysis enabled the differentiation of 25 species using the single restriction endonuclease AluI, while 2 species, A. sanarelli and A. taiwanensis, required an additional restriction endonuclease, HpyCH4IV. Twelve type strains (A. hydrophila ATCC7966T, A. caviae ATCC15468T, A. veronii ATCC9071T, A. media DSM4881T, A. allosaccharophila DSM11576T, A. dhakensis DSM17689T, A. enteropelogens DSM7312T, A. jandaei DSM7311T, A. rivuli DSM22539T, A. salmonicida ATCC33658T, A. taiwanensis DSM24096T and A. sanarelli DSM24094T) were randomly selected from the 27 Aeromonas species for experimental validation.Results/key findings. The twelve type strains demonstrated distinctive RFLP patterns and supported the in silico digestion. Subsequently, 60 clinical and environmental strains from our collection, comprising nine Aeromonas species, were used for screening examinations, and the results were in agreement.
CONCLUSION: This method provides an alternative method for laboratory identification, surveillance and epidemiological investigations of clinical and environmental specimens.
OBJECTIVE: The objectives of this article are to discuss the recent progress in the development of new vaccines against TB; to provide an insight on the mechanism of vaccine-mediated specific immune response stimulation, and to debate on the interaction between vaccines and global interventions to end TB.
METHODS: Effects of APC on expressions of genes encoding catalase (katA), superoxide dismutases (SODs), including sodA and sodM, and alkyl hydroperoxide reductase (ahpC) in S· aureus were quantitated by RT-qPCR in reference to gyrA and 16S rRNA. Corresponding activities of the enzymes were also investigated. The Livak analysis was performed for verification of gene-fold expression data. Effects of APC on intracellular and extracellular reactive oxygen species (ROS) levels were determined using the nitroblue tetrazolium (NBT) reduction assay.
RESULTS: APC-treated S· aureus cells had higher sodA and sodM transcripts at 1.5-fold and 0.7-fold expressions respectively with corresponding increase in total SOD activity of 12.24 U/mL compared to untreated cells, 10.85 U/mL (P<0.05). Expression of ahpC was highest in APC-treated cells with 5.5-fold increased expression compared to untreated cells (P<0.05). Correspondingly, ahpC activity was higher in APC-treated cells at 0.672 (A310nm) compared to untreated cells which was 0.394 (A310nm). In contrast, katA expression was 1.48-fold and 0.33-fold lower respectively relative to gyrA and 16S rRNA. Further, APC-treated cells showed decreased catalase activity of 1.8 ×10-4 (U/L or μmol/(min·L)) compared to untreated cells, which was 4.8 ×10-4 U/L (P<0.05). Absorbance readings (A575nm) for the NBT reduction assay were 0.709 and 0.695 respectively for untreated and treated cells, which indicated the presence of ROS. APC-treated S· aureus cells had lower ROS levels both extracellularly and intracellularly, but larger amounts remained intracellularly compared to extracellular levels with absorbances of 0.457 and 0.137 respectively (P<0.05).
CONCLUSION: APC induced expressions of both sodA and sodM, resulting in increased total SOD activity in S· aureus. Higher sodA expression indicated stress induced intracellularly involving O2- , presumably leading to higher intracellular pools of H2O2. A concommittant decrease in katA expression and catalase activity possibly induced ahpC expression, which was increased the highest in APC-treated cells. Our findings suggest that in the absence of catalase, cells are propelled to seek an alternate pathway involving ahpC to reduce stress invoked by O2- and H2O2. Although APC reduced levels of ROS, significant amounts eluded its antioxidative action and remained intracellularly, which adds to oxidative stress in treated cells.