Adapted tolerance to benzalkonium chloride in Escherichia coli K-12 studied by transcriptome and proteome analyses

Abstract

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 (...

Adaptive and Mutational Resistance: Role of Porins and Efflux Pumps in Drug Resistance

“…M35 is also regulated by temperature, being downregulated during growth at 26°C or 42°C compared to growth at 37°C, as well as by osmotic stress and iron limitation. Adaptation to the biocide benzalkonium chloride in E. coli resulted in a reduced presence of several OMPs, such as OmpA, OmpF, and OmpT (23). The authors of that study observed that this conferred increased tolerance to not only this quaternary ammonium compound but also antibiotics such as chloramphenicol, ciprofloxacin, nalidixic acid, ampicillin, and cefotaxime.…”

Nonnative Disulfide Bond Formation Activates the σ 32 -Dependent Heat Shock Response in Escherichia coli

“…Expression of test genes upon stress treatment was normalized against accD expression (first normalization) and expression before stress treatment (second normalization). accD is a gene that has been used as a suitable control in experiments with E. coli exposed to various stress conditions (35). To test the suitability of accD as a gene for normalization, phoP was used as an alternative normalization gene.…”

Small-Molecule Inhibitor of the Shigella flexneri Master Virulence Regulator VirF

“…The gapA and rrsA transcripts were used as internal controls. These are constitutively expressed Shigella genes commonly used to normalize mRNA levels (72)(73)(74). S. flexneri strain SME4331, which carries a null mutation in ipgD, was grown at 30°C overnight in TSB with ampicillin and was then diluted into the same medium to an OD 600 of ϳ0.1, and 1-ml aliquots were further grown at 30°C (with DMSO only) or at 37°C (with SE-1 at 20 or 40 M, or with DMSO only; in all cases, the concentration of DMSO was 0.3%).…”

“…S. flexneri samples grown at 30°C in the absence of SE-1 were included to illustrate the basal expression levels of these genes. The results were normalized to those for the gapA (encoding glyceraldehyde-3-phosphate dehydrogenase) and rrsA (encoding 16S rRNA) genes, two constitutively expressed genes commonly used as qRT-PCR controls in Shigella (72)(73)(74).…”