Evolving the EN 1500 test method for alcohol-based hand rub closer to clinical reality by reducing the organic load on hands and enabling product to be applied to dry hands.

BACKGROUND: The methods currently used in Europe and North America to evaluate the bactericidal efficacy of hand hygiene products have some limitations (e.g. selection of test organism, method of contamination), and none of the methods allow prediction of actual clinical efficacy. Therefore, the World Health Organization has proposed the development of methods that better reflect typical clinical reality. METHODS: In Experiment 1, two contamination methods (immersion method according to EN 1500 and low-volume method according to ASTM E2755) were tested with the EN 1500 test organism Escherichia coli using 60% v/v iso-propanol. Experiment 2 compared the two contamination methods with Enterococcus faecalis. Experiment 3 compared the two test organisms using the low-volume contamination method. Data within each experiment were compared using the Wilcoxon test for paired samples, and data from all experiments were combined and fit to linear mixed-effects models. RESULTS: Mixed-effects analysis confirmed that both the test organism and the contamination method impacted the pre-values, and all three factors influenced log10 reductions. Higher pre-values resulted in significantly higher log10 reductions, immersion contributed to significantly higher log10 reductions, and E. coli showed significantly lower log10 reductions. CONCLUSION: An efficacy evaluation against E. faecalis with a low-volume contamination method could be considered as an alternative to the EN 1500 standard. This could help to improve the clinical relevance of the test method by including a Gram-positive organism and reducing the soil load, allowing product application closer to reality.

The chromosomal 2'-N-acetyltransferase of Providencia stuartii: physiological functions and genetic regulation.

Intrinsic chromosomal acetyltransferases involved in aminoglycoside resistance have been identified in a number of bacteria. In Providencia stuartii, a chromosomal acetyltransferase (AAC(2')-Ia) has been characterized in detail. In addition to the ability to acetylate aminoglycosides, the AAC(2')-Ia enzyme has at least one physiological function, which is the acetylation of peptidoglycan. This modification is likely to influence the autolytic system in P. stuartii. The regulation of aac(2')-Ia expression is extremely complex involving at least seven regulatory genes acting in at least two pathways. This complexity in regulation indicates that aac(2')-Ia expression must be tightly controlled in response to different environmental conditions. This presumably reflects the importance of maintaining correct levels of peptidoglycan acetylation. In this review, a summary of data will be presented involving both the physiological and genetic aspects of aac(2')-Ia in P. stuartii.

Measuring residual activity of topical antimicrobials: is the residual activity of chlorhexidine an artefact of laboratory methods?

Residual activity of chlorhexidine gluconate (CHG) was evaluated by pretreating hands with CHG and then touching Staphylococcus aureus dried on to stainless steel discs. By this method, no reduction in bacteria was observed up to 15 min, suggesting that residual CHG does not offer protection against contamination with transient micro-organisms in clinical practice.

Electronic recognition of hand hygiene technique and duration.

We captured 3-dimensional accelerometry data from the wrists of 116 healthcare professionals as they performed hand hygiene (HH). We then used these data to train a k-nearest-neighbors classifier to recognize specific aspects of HH technique (ie, fingertip scrub) and measure the duration of HH events.

aarD, a Providencia stuartii homologue of cydD: role in 2'-N-acetyltransferase expression, cell morphology and growth in the presence of an extracellular factor.

In a search for genes involved in regulation of the 2'-N-acetyltransferase in Providencia stuartii, a mini-Tn5Cm insertion has been isolated in a locus designated aarD. The aarD1::mini-Tn5Cm mutation resulted in a 4.7-fold increase in the levels of beta-galactosidase accumulation from an aac(2')-lacZ transcriptional fusion and a 32-fold increase in the levels of gentamicin resistance in P. stuartii. The wild-type aarD locus was cloned on a 5.0 kb Cla I fragment and complemented the aarD1 mutation. Nucleotide sequence analysis of this fragment identified two large open reading frames whose deduced products displayed significant amino acid identity, 64% and 64%, respectively, to the CydD and CydC proteins of Escherichia coli, which are involved in formation of the cytochrome d oxidase complex. Physical mapping indicated the aarD1::mini-Tn5Cm insertion was within the open reading homologous to CydD. The strain containing the aarD1 mutation was unable to grow in the presence of toluidine blue or on glycerol minimal media in the presence of zinc, suggesting that aarD is functionally equivalent to cydD. Additional phenotypes resulting from the aarD1 mutation included: altered cell morphology, a reduced growth rate and the inability of cells to grow beyond early log phase. Further examination of this phenomenon revealed that the aarD1 mutant was unable to grow in the presence of a self-produced extracellular factor(s). This novel phenotype was limited to P. stuartii as E. coli cydD and delta cydAB::kan mutants were also sensitive to a self-produced extracellular factor.

Identification and analysis of aarP, a transcriptional activator of the 2'-N-acetyltransferase in Providencia stuartii.

The aarP gene has been identified in a search for activators of the 2-N-acetyltransferase [encoded by aac(2')-Ia] in Providencia stuartii. Introduction of aarP into P. stuartii on a multicopy plasmid resulted in a 9.9-fold increase in the accumulation of beta-galactosidase from an aac(2')-lacZ fusion. Northern (RNA) blot analysis demonstrated that this increased aac(2')-Ia expression occurred at the level of mRNA accumulation. The deduced AarP protein was 15,898 Da in size and exhibited significant homology to a number of transcriptional activators in the AraC/XyIS family, including TetD,Rob, MarA, and SoxS. The similarity of AarP to the MarA and SoxS proteins prompted an investigation to determine whether AarP is involved in activation of genes in either the multiple antibiotic resistance (Mar) phenotype or redox stress (SoxRS) system. Introduction of aarP on a multicopy plasmid into either P. stuartii or Escherichia coli conferred a Mar phenotype with higher levels of resistance to tetracycline, chloramphenicol, and ciprofloxacin. Multiple copies of aarP in E. coli also resulted in activation of the endonuclease IV gene (nfo), a gene in the SoxRS regulon of E. coli. The function of aarP in its single-copy state was addressed by using allelic replacement to construct an aarP::Cm disruption, which resulted in a fivefold reduction in the accumulation of aac(2')-Ia mRNA. Analysis of aarP regulation showed that aarP mRNA accumulation was slightly increased by exposure to tetracycline and dramatically increased in cells containing the aarB3 (aar3) mutation, which was previously shown to increase transcription of the aac(2')-Ia gene. (P.N. Rather, E. Oroz, K.J. Shaw, R. Hare, and G. Miller, J. Bacteriol. 175:6492-6498).

Identification and characterization of aarF, a locus required for production of ubiquinone in Providencia stuartii and Escherichia coli and for expression of 2'-N-acetyltransferase in P. stuartii.

Providencia stuartii contains a chromosomal 2'-N-acetyltransferase [AAC(2')-Ia] involved in the O acetylation of peptidoglycan. The AAC(2')-Ia enzyme is also capable of acetylating and inactivating certain aminoglycosides and confers high-level resistance to these antibiotics when overexpressed. We report the identification of a locus in P. stuartii, designated aarF, that is required for the expression of AAC(2')-Ia. Northern (RNA) analysis demonstrated that aac(2')-Ia mRNA levels were dramatically decreased in a P. stuartii strain carrying an aarF::Cm disruption. The aarF::Cm disruption also resulted in a deficiency in the respiratory cofactor ubiquinone. The aarF locus encoded a protein that had a predicted molecular mass of 62,559 Da and that exhibited extensive amino acid similarity to the products of two adjacent open reading frames of unknown function (YigQ and YigR), located at 86 min on the Escherichia coli chromosome. An E. coli yigR::Kan mutant was also deficient in ubiquinone content. Complementation studies demonstrated that the aarF and the E. coli yigQR loci were functionally equivalent. The aarF or yigQR genes were unable to complement ubiD and ubiE mutations that are also present at 86 min on the E. coli chromosome. This result indicates that aarF (yigQR) represents a novel locus for ubiquinone production and reveals a previously unreported connection between ubiquinone biosynthesis and the regulation of gene expression.

Activation of the 2'-N-acetyltransferase gene [aac(2')-Ia] in Providencia stuartii by an interaction of AarP with the promoter region.

The aac(2')-Ia gene in Providencia stuartii encodes a 2'-N-acetyltransferase capable of acetylating both peptidoglycan and certain aminoglycoside antibiotics. Regulation of the aac(2')-Ia gene is influenced in a positive manner by the product of the aarP gene, which encodes a small transcriptional activator of the AraC (XylS) family. In this study, we demonstrate the sequence requirements at the aac(2')-Ia promoter for AarP binding and activation.