A general overview is presented of the changes in the genetic expression along a time curve through the first 20 min after acidification to pH 4.5 of exponentially growing cultures of the food pathogenic strain Staphylococcus aureus 50583. A newly developed method for statistical significance testing was used to detect significant gene expression responses. Most responses showed an increase or decrease from time zero to 10 min after acidification, and then generally a stabilization in expression level from 10 to 20 min. Increased urease activity appeared to be an important factor in the acid defence, along with proton excretion by NADH dehydrogenase and macromolecule repair mechanisms. Oxidative-stress responses, such as increased expression of thioredoxin genes and upregulation of pentose phosphate pathway genes to generate more reducing power, were also induced. A general reduction in the expression of genes encoding ribosomal proteins and genes involved in nucleotide synthesis, as well as fatty acid and lipoprotein metabolism, reflected the lowered growth rate after acidification. The pH shock did not appear to trigger major virulence responses or biofilm formation. Metal ion regulation and transport were affected by the acid shock, and production of several cofactors such as molybdopterin was increased. Many of the presented observations could be explained, while some represent still-unknown mechanisms. The patterns of regulation were confirmed by quantitive reverse transcriptase PCR (QRT-PCR). Together, these results showed the main responses of S. aureus and will be a good starting point for future, more specific, in-depth studies of specific gene responses that occur in conjunction with the acid-stress defence of S. aureus.
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This paper describes a generalised framework for doing Monte Carlo tests in multivariate linear regression. The rotation methodology assumes multivariate normality and is a true generalisation of the classical multivariate tests-any imaginable test statistic is allowed. The generalised test statistics are dependent on the unknown covariance matrix. Rotation testing handles this problem by conditioning on sufficient statistics.Compared to permutation tests, we replace permutations by proper random rotations. Permutation tests avoid the multinormal assumption, but they are limited to relatively simple models. On the other hand, a rotation test can, in particular, be applied to any multivariate generalisation of the univariate F-test.As an important application, a detailed description of how each single response p-value can be nonconservatively adjusted for multiplicity is given. This method is exact and non-conservative (unlike Bonferroni), and it is a generalisation of the ordinary F-test (except for the computation by simulations). Hence, this paper offers an exact Monte Carlo solution to a classical problem of multiple testing.
Benzalkonium chloride (BC) is a commonly used disinfectant and preservative. This study describes changes in expression level at the transcriptomic and proteomic level for Escherichia coli K-12 gradually adapted to a tolerance level to BC of 7-8 times the initial MIC. Results from DNA arrays and two-dimensional gel electrophoresis for global gene and protein expression studies were confirmed by real-time quantitative PCR. Peptide mass fingerprinting by MALDI-TOF MS was used to identify differentially expressed proteins. Changes in expression level in adapted cells were shown for porins, drug transporters, glycolytic enzymes, ribosomal subunits and several genes and proteins involved in protection against oxidative stress and antibiotics. Adapted strains showed increased tolerance to several antibiotics. In conclusion, E. coli K-12 adapted to higher tolerance to BC acquired several general resistance mechanisms, including responses normally related to the multiple antibiotic resistance (Mar) regulon and protection against oxidative stress. The results revealed that BC treatment might result in superoxide stress in E. coli. INTRODUCTIONQuaternary ammonium compounds (QACs) such as benzalkonium chloride (BC) and cetrimide are frequently used for disinfection and preservation. QACs are relatively stable, non-corrosive compounds with low toxicity and efficacy over a wide pH range. The destructive mechanism of QACs against bacteria is still not known in detail, but is thought to involve a general perturbation of the lipid bilayer in bacterial membranes. This leads to leakage of cytoplasmic material, damaging and ultimately killing the bacterial cell. For a review see Gilbert & Moore (2005). The QACs have been actively deployed since the 1930s and one has not seen a resistance development similar to that seen for antibiotics in the same period. Still, there are numerous reports of QAC resistance (Heir et al., 1999;Langsrud et al., 2003;Wright & Gilbert, 1987), most often associated with acquisition, or hyperexpression, of multi-drug efflux pumps (Li & Nikaido, 2004;Tikhonova & Zgurskaya, 2004), which has also been associated with changes in MIC of therapeutically important antibiotics serving as substrates to such pumps (Langsrud et al., 2004;Poole, 2004Poole, , 2005. Because of their relatively impermeable outer membrane, Gram-negative bacteria are intrinsically more resistant to QACs than Gram-positives (McDonnell & Russell, 1999), but both Gram-negative and Gram-positive BC-resistant bacteria have been isolated from the food industry (Aase et al., 2000;Heir et al., 1995;Langsrud et al., 2003;Soumet et al., 2005).Several studies have linked intrinsic/natural resistance of Gram-negative bacteria to tenside-based disinfectants, such as QACs, to the low permeability of the outer membrane, or broad-spectrum efflux systems (Denyer & Maillard, 2002;Nikaido, 2001). Acquisition of resistance in Escherichia coli has been related mainly to changes in the composition of lipopolysaccharide (LPS) and fatty acids in the membrane (...
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