A method to evaluate factors influencing the microbial reduction in domestic dishwashers.

AIMS: To develop a method that is able to determine the microbial reduction in different dishwasher cleaning cycles and differentiate between different program parameters used. METHODS AND RESULTS: Stainless steel biomonitors were contaminated with Micrococcus luteus or Entereococcus faecium and cleaned in a specially programmed household dishwasher with different cleaning temperatures and durations. No detergent, bleach-free detergent or detergent containing activated oxygen bleach was used. The logarithmic reduction (LR) was determined. The microbial reduction depended on the cleaning temperature, the duration of the cleaning cycles and the detergent type used. LR increased with higher temperatures, longer cleaning cycles and use of detergent. CONCLUSIONS: The factors cleaning cycle temperature, cleaning cycle duration, final rinsing temperature and the use of detergent all contributed to the reduction of test-strains in dishwasher cycles. A combination of longer dishwashing cycles and increased temperatures resulted in LRmax of the microbial load. SIGNIFICANCE AND IMPACT OF THE STUDY: Cycles in domestic appliances are very diverse; therefore a standardized method to determine their ability to reduce the microbial load is of great use. The method described here is able to demonstrate the reductions achieved by dishwashing cycles with different parameters and might help to find the necessary balance between energy saving and an acceptable level of hygiene.

Strong correlation of total phenotypic resistance of samples from household environments and the prevalence of class 1 integrons suggests for the use of the relative prevalence of intI1 as a screening tool for multi-resistance.

One of the biggest challenges of health care systems worldwide is the increasing number of pathogenic bacteria resistant to a growing number of antibiotics. In this respect, class 1 integrons which are part of mobile genetic elements can confer several phenotypes including resistance to a broad range of antibiotic classes, heavy metals and biocides. They are linked to common resistance genes and have penetrated pathogenic and commensal bacteria likewise. Therefore its relative prevalence can be a proxy for antimicrobial resistance and anthropogenic pollution. Household environments are areas with a high influx of bacteria from humans, animals and foods, and biocides and detergents are frequently used. In this study we aimed to investigate the relative prevalence of class 1 integrons in household environments, in relation to the number of antibiotic and benzalkonium chloride resistant phenotypes of a sample point, for the validation of the relative prevalence of class 1 integrons as a screening tool for multi-resistance. Kitchen sink and bathroom sink U-bends, dishwasher, washing machines and toothbrushes of 28 households were probed. Copies /mL of class 1 integron integrase gene and 16SrDNA gene were determined by qPCR and bacteria of the respective sample were isolated on ampicillin selective agar plates, followed by the determination of the species and phenotypic resistance profiles. The relative prevalence of class 1 integrons in relation to 16SrDNA was calculated and correlated to phenotypic resistance. Our findings show a high relative prevalence of class 1 integrons in water reticulation systems of household environments and in particular shower U-bends. Furthermore, prevalence of class 1 integrons correlates strongly (rs = 0.95) with total phenotypic resistance at a sample point and suggest that a standardized assay determining the relative prevalence of class 1 integrons could be used as a useful screening tool in the assessment of multi-resistance in environmental samples.

Prevalence of ß-lactamase genes in domestic washing machines and dishwashers and the impact of laundering processes on antibiotic-resistant bacteria.

AIMS: To investigate the prevalence of ß-lactamase genes in domestic washing machines and dishwashers, and the decontamination efficacy of laundering. METHODS AND RESULTS: For the first investigation, swab samples from washing machines (n = 29) and dishwashers (n = 24) were analysed by real-time quantitative PCR to detect genes encoding ß-lactamases. To test the impact of laundering on resistant bacteria, cotton test swatches were artificially contaminated with susceptible and resistant strains of Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus within a second investigation. They were washed in a domestic washing machine with or without activated oxygen bleach (AOB)-containing detergent at 20-50°C. ß-Lactamase genes (most commonly of the AmpC- and OXA-type) were detected in 79% of the washing machines and in 96% of the dishwashers and Pseudomonadaceae dominated the microbiota. The level of bacterial reduction after laundering was ≥80% for all Ps. aeruginosa and Kl. pneumoniae strains, while it was only 37-61% for the methicillin-resistant Staph. aureus outbreak strain. In general, the reduction was tendentially higher for susceptible bacteria than for the resistant outbreak strains, especially for Staph. aureus. CONCLUSIONS: ß-Lactamase genes seem to be frequently present in domestic appliances and may pose a potential risk for cross-contamination and horizontal transfer of genes encoding resistance against clinically important ß-lactams. In general, higher temperatures and the use of AOB can improve the reduction of antibiotic-resistant bacteria, including Staph. aureus which appears to be less susceptible to the decontamination effect of laundering. SIGNIFICANCE AND IMPACT OF THIS STUDY: Data on the presence of antibiotic-resistant bacteria in the domestic environment are limited. This study suggests that ß-lactamase genes in washing machines and dishwashers are frequent, and that antibiotic-resistant strains are generally more resistant to the used washing conditions.

Laundry hygiene-how to get more than clean.

Although laundering should mainly remove stains and dirt from used and worn textiles, the elimination of microbial contamination is an important aim of the laundry process as well. While industrial and institutional laundering employs standardized processes using high temperatures (i.e. 60°C and above) and bleaching agents to ensure a sufficient hygienic reconditioning of textiles, domestic laundering processes are less defined and not always led by purposeful aims. The strive for energy efficiency of household appliances has resulted in a decrease in washing temperatures in Europe during the last decades and convenience aspects led to an increased use of liquid detergents that do not contain bleach which in turn impacts the antimicrobial efficacy of domestic laundering. This review compiles the different factors that influence the input and removal of micro-organisms in the laundering process and discusses the possible adverse effects of microbial contaminants in the washing machine and on the textiles as well as suitable counteractions.

Fabric-skin models to assess infection transfer for impetigo contagiosa in a kindergarten scenario.

Children in community bodies like kindergartens are predisposed to suffer from impetigo. To consider important measures for infection prevention, direct and indirect transmission routes of pathogens must be revealed. Therefore, we studied the role of skin and fabrics in the spread of the impetigo pathogen Staphylococcus aureus and the strain Streptococcus equi (surrogate to Streptococcus pyogenes) in order to assess infection transfer in realistic scenarios. The transmission of test strains was studied with standardized fabric-skin models using a technical artificial skin and fabrics of different fiber types commonly occurring in German kindergartens. In synthetic pus, both test strains persisted on artificial skin and fabrics for at least 4 h. Friction enhanced transfer, depending on the fiber type or fabric construction. In a skin-to-skin setup, the total transfer was higher than via fabrics and no decrease in the transmission rates from donor to recipients could be observed after successive direct skin contacts. Children in kindergartens may be at risk of transmission for impetigo pathogens, especially via direct skin contact, but also by the joint use of fabrics, like towels or handicraft materials. Fabric-skin models used in this study enable further insight into the transmission factors for skin infections on the basis of a practical approach.

Impact of wash cycle time, temperature and detergent formulation on the hygiene effectiveness of domestic laundering.

AIMS: Investigation of the effect of temperature and duration of the laundering process with and without activated oxygen bleach (AOB)-containing detergent on the hygienic effectiveness of laundering. METHODS AND RESULTS: Cotton test swatches were contaminated with Staphylococcus aureus, Enterococcus hirae, Pseudomonas aeruginosa, Candida albicans and Trichophyton mentagrophytes and were washed in a household washing machine using temperatures between 20 and 60°C and different wash cycle times. The logarithmic microbial reduction factor and cross-contamination (i.e. transfer from contaminated to sterile swatches) were used to indicate the hygienic effectiveness of the washing process. For all tested micro-organisms, the temperature needed for decontamination depended on washing time and detergent type. Hygiene effectiveness of laundering was enhanced by inclusion of AOB even at lowest temperatures, except for C. albicans, which was virtually unaffected by AOB. The use of AOB-containing detergents as well as high washing temperatures reduced cross-contamination to sterile swatches included in the load. CONCLUSIONS: Depending on the type of organism, longer wash cycle times or the use of AOB-containing detergents can be used to enhance the hygiene effectiveness of laundering. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates that it is possible to compensate for the loss of hygiene effectiveness of laundering at lower temperatures using detergents with activated oxygen bleach or by extending the wash cycle time.

A potential phosphorylation site for an A-type kinase in the Efg1 regulator protein contributes to hyphal morphogenesis of Candida albicans.

Efg1p in the human fungal pathogen Candida albicans is a member of the conserved APSES class of proteins regulating morphogenetic processes in fungi. We have analyzed the importance for hyphal morphogenesis of a putative phosphorylation site for protein kinase A (PKA), threonine-206, within an Efg1p domain highly conserved among APSES proteins. Alanine substitution of T206, but not of the adjacent T207 and T208 residues, led to a block of hypha formation on solid and in liquid media, while a T206E exchange caused hyperfilamentation. The extent of the morphogenetic defect caused by the T206A mutation depended on hypha-induction conditions. Extragenous suppression of mutations in signaling components, including tpk2 and cek1 mutations, was achieved by wild-type- and T206E-, but not by the T206A-variant-encoding allele of EFG1. All muteins tested were produced at equal levels and at high production levels supported pseudohyphal formation. The results are consistent with a role of Efg1p as a central downstream component of a PKA-signaling pathway including Tpk2p or other PKA isoforms. Threonine-206 of Efg1p is essential as a putative phosphorylation target to promote hyphal induction by a subset of environmental cues.

Distinct and redundant roles of the two protein kinase A isoforms Tpk1p and Tpk2p in morphogenesis and growth of Candida albicans.

TPK1 and TPK2 encode both isoforms of protein kinase A (PKA) catalytic subunits in Candida albicans. Mutants lacking both TPK1 alleles showed defective hyphal morphogenesis on solid inducing media, whereas in liquid hypha, formation was affected slightly. In contrast, tpk2 mutants were only partially morphogenesis defective on solid media, whereas a strong block was observed in liquid. In addition, the yeast forms of tpk2-- but not tpk1-- mutants were completely deficient in invading agar. Because Tpk1p and Tpk2p differ in their N-terminal domains of approximately 80--90 amino acids, while the catalytic portions are highly homologous, the functions of hybrid Tpk proteins with exchanged N-terminal domains were tested. The results demonstrate that the catalytic portions mediate Tpk protein specificities with regard to filamentation, whereas agar invasion is mediated by the N-terminal domain of Tpk2p. Homozygous tpk1 and tpk2 mutants grew normally; however, a tpk2 mutant strain containing a single regulatable TPK1 allele (PCK1p-TPK1) at low expression levels was severely growth defective. It was completely blocked in hyphal morphogenesis and was stress resistant to high osmolarities or temperatures. Thus, both Tpk isoforms in C. albicans share growth functions but, unlike Saccharomyces cerevisiae isoforms, they have positive, specific roles in filament formation in different environments.

Protein kinase A encoded by TPK2 regulates dimorphism of Candida albicans.

External signals induce the switch from a yeast to a hyphal growth form in the fungal pathogen Candida albicans. We demonstrate here that the catalytic subunit of a protein kinase A (PKA) isoform encoded by TPK2 is required for internal signalling leading to hyphal differentiation. TPK2 complements the growth defect of a Saccharomyces cerevisiae tpk1-3 mutant and Tpk2p is able to phosphorylate an established PKA-acceptor peptide (kemptide). Deletion of TPK2 blocks morphogenesis and partially reduces virulence, whereas TPK2 overexpression induces hyphal formation and stimulates agar invasion. The defective tpk2 phenotype is suppressed by overproduction of known signalling components, including Efg1p and Cek1p, whereas TPK2 overexpression reconstitutes the cek1 but not the efg1 phenotype. The results indicate that PKA activity of Tpk2p is an important contributing factor in regulating dimorphism of C. albicans.

Chlamydospore formation in Candida albicans requires the Efg1p morphogenetic regulator.

Chlamydospore formation of the fungal pathogen Candida albicans was found to depend on the Efg1 protein, which regulates the yeast-hyphal transition. Isogenic mutants lacking EFG1 or encoding T206A and T206E variants did not differentiate chlamydospores, while cek1, cph1, or tpk2 mutations had no effect. Furthermore, filamentation of efg1 cph1 double mutants in microaerophilic conditions suggests a novel Efg1p/Cph1p-independent filamentation pathway in C. albicans.