Biological and Synthetic Surfactants Increase Class I Integron Prevalence in Ex Situ Biofilms.

The role of biocides in the spread of antimicrobial resistance (AMR) has been addressed but only a few studies focus on the impact of surfactants on microbial diversity and AMR, although they are common constituents of cleaners, disinfectants, and personal care products and are thus released into the environment in large quantities. In this study, we used a static ex situ biofilm model to examine the development of four biofilms exposed to surfactants and analyzed the biofilms for their prevalence of class I integrons as a proxy for the overall abundance of AMR in a sample. We furthermore determined the shift in bacterial community composition by high-resolution melt analysis and 16S ribosomal RNA (16S rRNA) gene sequencing. Depending on the initial intrinsic prevalence of class I integrons in the respective ex situ biofilm, benzalkonium chloride, alkylbenzene sulfonate, and cocamidopropyl betaine increased its prevalence by up to 6.5× on average. For fatty alcohol ethoxylate and the biosurfactants sophorolipid and rhamnolipid, the mean increase did not exceed 2.5-fold. Across all surfactants, the increase in class I integrons was accompanied by a shift in bacterial community composition. Especially benzalkonium chloride, cocamidopropyl betaine, and alkylbenzene sulfonate changed the communities, while fatty alcohol ethoxylate, sophorolipid, and rhamnolipid had a lower effect on the bacterial biofilm composition.

A loophole in soap dispensers mediates contamination with Gram-negative bacteria.

Liquid soap dispensers are widely used in domestic and clinical settings. In previous studies, the risk of bacterial contamination of refillable systems was pointed out and a bacterial contamination rate of 25%, with values of up to 108 colony-forming units/mL (CFU/mL), was reported. However, the route of contamination remains elusive. To address this point, we determined the microbial contamination of refillable standard pump dispensers and nonrefillable press-dispenser systems. Following the collection of 104 liquid soap dispensers from hotel rooms across Germany, bacterial counts were determined. Isolates of samples containing nonfastidious Gram-negative(lac-) bacteria were further analyzed by the Vitek 2 system for the determination of species. 70.2% of the refillable pump dispensers (mean total bacterial count = 2.2 × 105 CFU/mL) but only 10.6% of the nonrefillable press dispensers, were contaminated (mean total bacterial count = 1.5 × 101 CFU/mL). Of samples containing nonfastidious Gram-negative(lac-) bacteria, Pluralibacter gergoviae was present in 41.7%, Pseudomonads (Pseudomonas aeruginosa and Pseudomonas putida) in 25%, Serratia marcescens in 16.7%, and Klebsiella oxytoca and Pasteurella testudinis in 8.3%. After the initial assessment, we contaminated different dispensing systems with P. aeruginosa/P. gergoviae, to reveal the route of contamination and identied the pressure release of standard pump dispensers as the loophole for microbial contamination.

High resolution ITS amplicon melting analysis as a tool to analyse microbial communities of household biofilms in ex-situ models.

Biofilms are the most common growth types of microorganisms. These complex communities usually consist of different species and are embedded in an extracellular matrix containing polymers, proteins and DNA. This matrix offers protection against different (a)biotic environmental factors and generally increases resistances. Higher resistances against antibiotics are one of the main reasons why biofilms are often associated with healthcare settings. Nevertheless, they are also found in domestic settings, mostly in humid places with abundant nutrients like dishwashers or washing machines. Biofilms in these areas show individual compositions and are influenced for example by temperature, frequency of use or the age of the device. In this study, we introduce a model for the ex-situ cultivation of domestic biofilms from household appliances. Furthermore, we tested the ability of high resolution melting analysis (HRMA) as a tool for analysing these biofilms. Our goal was to maintain a high amount of complexity in the ex-situ biofilms that is characterized by the melting behavior of the contained DNA. Dishwasher and washing machine biofilms were sampled in private households and cultivated for 10 d. After DNA extraction, 16S rDNA was sequenced and melting behavior of the bacterial Internal Transcribed Spacer (ITS) region was analysed. Additionally, testing for independence of continuous new sampling, storage of cultivated biofilms in glycerol stocks and following recultivation of them was done up to three times. Our results show that a high level of complexity could be maintained in the ex-situ biofilms after 10 d of cultivation, although in general the bacterial diversity slightly decreased compared to the original biofilm in most cases. Recultivation of a similar biofilm from glycerol stocks was possible as well with some impact by various factors. Differences in the bacterial composition of biofilms could clearly made visible by HRMA although it was not possible to match peaks to a specific phylogenetic group. Still, HRMA proved to be a less costly and time consuming alternative to sequencing for the characterization of biofilms.

The Household Resistome: Frequency of ß-Lactamases, Class 1 Integrons, and Antibiotic-Resistant Bacteria in the Domestic Environment and Their Reduction during Automated Dishwashing and Laundering.

Households provide a habitat for bacteria originating from humans, animals, foods, contaminated clothes, or other sources. Thus, bacteria carrying antibiotic resistance genes (ARGs) may be introduced via household members, animals, or the water supply from external habitats into private households and vice versa. Since data on antibiotic resistance (ABR) in the domestic environment are limited, this study aimed to determine the abundance of ß-lactamase, mobile colistin resistance, and class 1 integron genes and the correlation of their presence and to characterize phenotypically resistant strains in 54 private households in Germany. Additionally, the persistence of antibiotic-resistant bacteria during automated dishwashing compared to that during laundering was assessed. Shower drains, washing machines, and dishwashers were sampled and analyzed using quantitative real-time PCR. Resistant strains were isolated, followed by identification and antibiotic susceptibility testing using a Vitek 2 system. The results showed a significantly higher relative ARG abundance of 0.2367 ARG copies/16S rRNA gene copies in shower drains than in dishwashers (0.1329 ARG copies/16S rRNA gene copies) and washing machines (0.0006 ARG copies/16S rRNA gene copies). blaCMY-2, blaACT/MIR, and blaOXA-48 were the most prevalent ARG, and intI1 occurred in 96.3% of the households, while no mcr genes were detected. Several ß-lactamase genes co-occurred, and the resistance of bacterial isolates correlated positively with genotypic resistance, with carbapenemase genes dominating across isolates. Antibiotic-resistant bacteria were significantly reduced during automated dishwashing as well as laundering tests and did not differ from susceptible strains. Overall, the domestic environment may represent a potential reservoir of ß-lactamase genes and ß-lactam-resistant bacteria, with shower drains being the dominant source of ABR.IMPORTANCE The abundance of antibiotic-resistant bacteria and ARGs is steadily increasing and has been comprehensively analyzed in natural environments, animals, foods, and wastewater treatment plants. In this respect, ß-lactams and colistin are of particular interest due to the emergence of multidrug-resistant Gram-negative bacteria. Despite the connection of private households to these environments, only a few studies have focused on the domestic environment so far. Therefore, the present study further investigated the occurrence of ARGs and antibiotic-resistant bacteria in shower drains, washing machines, and dishwashers. The analysis of the domestic environment as a potential reservoir of resistant bacteria is crucial to determine whether households contribute to the spread of ABR or may be a habitat where resistant bacteria from the natural environment, humans, food, or water are selected due to the use of detergents, antimicrobial products, and antibiotics. Furthermore, ABR could limit the options for the treatment of infections arising in the domestic environment.

Evaluation of a Novel Rapid Test System for the Detection of Specific IgE to Hymenoptera Venoms.

Background. The Allergy Lateral Flow Assay (ALFA) is a novel rapid assay for the detection of sIgE to allergens. The objective of this study is the evaluation of ALFA for the detection of sIgE to bee venom (BV) and wasp venom (WV) in insect venom allergic patients. Methods. Specific IgE to BV and WV was analyzed by ALFA, ALLERG-O-LIQ, and ImmunoCAP in 80 insect venom allergic patients and 60 control sera. Sensitivity and specificity of ALFA and correlation of ALFA and ImmunoCAP results were calculated. Results. The sensitivity/specificity of ALFA to the diagnosis was 100%/83% for BV and 82%/97% for WV. For insect venom allergic patients, the Spearman correlation coefficient for ALFA versus ImmunoCAP was 0.79 for BV and 0.80 for WV. However, significant differences in the negative control groups were observed. Conclusion. ALFA represents a simple, robust, and reliable tool for the rapid detection of sIgE to insect venoms.

Acid inactivation of prions: efficient at elevated temperature or high acid concentration.

Scrapie prion rods isolated from hamster and non-infectious aggregates of the corresponding recombinant protein rPrP(90-231) were incubated with hydrochloric acid. The amount of PrP and of infectivity that survived incubation in HCl at varying times, acid concentrations and temperatures was quantified by Western blot densitometry and bioassays, respectively. Prion rods and rPrP aggregates showed similar HCl hydrolysis kinetics of PrP, indicating structural homology. For 1 M HCl and 25 degrees C, the rate of PrP hydrolysis follows first-order kinetics at 0.014 h(-1); the rate of infectivity inactivation is 0.54 h(-1). Hydrolysis for 1 h at 25 degrees C was only slightly proportional to HCl concentration up to 5 M, but complete loss of infectivity and PrP reduction to <2 % was observed at 8 M HCl. The temperature dependence of unhydrolysed PrP, as well as infectivity at 1 M HCl for 1 h, showed a slight decrease up to 45 degrees C, but a sigmoidal decrease by several orders of magnitude at higher temperatures. The slow hydrolysis of PrP and inactivation of infectivity by acid treatment at room temperature are attributed to solvent inaccessibility of prion rods and rPrP aggregates, respectively. The more effective hydrolysis and inactivation at temperatures above 45 degrees C are interpreted as thermally induced disaggregation with an activation energy of 50-60 kJ mol(-1). Most importantly, infectivity was always inactivated faster or to a higher extent than PrP was hydrolysed at several incubation times, HCl concentrations and temperatures.

In vitro prion protein conversion in detergent-solubilized membranes.

A fundamental event in the pathogenesis of prion disease is the conversion of PrP(C), a normal glycophosphatidyl-anchored glycoprotein, into an infectious isoform designated PrP(Sc). In a modified version of the protein misfolding cyclic amplification (PMCA) technique [Saborio et al. (2001) Nature 411, 810-813], protease-resistant PrP(Sc)-like molecules (PrPres) can be amplified in vitro in a species- and strain-specific manner from crude brain homogenates, providing a biochemical model of the prion conversion reaction [Lucassen et al. (2003) Biochemistry 42, 4127-4135]. In this study, we investigated the ability of enriched membrane subsets and detergent-solubilized membrane preparations to support PrPres amplification. Membrane fractionation experiments showed that purified synaptic plasma membrane preparations enriched in PrP(C) but largely depleted of late endosomal and lysosomal markers were sufficient to support PrPres amplification. Detergent solubilization experiments showed that a small group of select detergents could be used to produce soluble preparations that contain PrP(C) and fully support PrPres amplification. The stability of PrPres amplification ability in detergent-solubilized supernatants was dependent on detergent concentration. These results lead to the surprising conclusion that membrane attachment is not required for PrP(C) to convert efficiently into PrPres in vitro and also indicate that biochemical purification of PrPres amplification factors from brain homogenates is a feasible approach.

RNA molecules stimulate prion protein conversion.

Much evidence supports the hypothesis that the infectious agents of prion diseases are devoid of nucleic acid, and instead are composed of a specific infectious protein. This protein, PrP(Sc), seems to be generated by template-induced conformational change of a normally expressed glycoprotein, PrP(C) (ref. 2). Although numerous studies have established the conversion of PrP(C) to PrP(Sc) as the central pathogenic event of prion disease, it is unknown whether cellular factors other than PrP(C) might be required to stimulate efficient PrP(Sc) production. We investigated the biochemical amplification of protease-resistant PrP(Sc)-like protein (PrPres) using a modified version of the protein-misfolding cyclic amplification method. Here we report that stoichiometric transformation of PrP(C) to PrPres in vitro requires specific RNA molecules. Notably, whereas mammalian RNA preparations stimulate in vitro amplification of PrPres, RNA preparations from invertebrate species do not. Our findings suggest that host-encoded stimulatory RNA molecules may have a role in the pathogenesis of prion disease. They also provide a practical approach to improve the sensitivity of diagnostic techniques based on PrPres amplification.

In vitro amplification of protease-resistant prion protein requires free sulfhydryl groups.

Prions, the infectious agents of transmissible spongiform encephalopathies, are composed primarily of a misfolded protein designated PrP(Sc). Prion-infected neurons generate PrP(Sc) from a host glycoprotein designated PrP(C) through a process of induced conformational change, but the molecular mechanism by which PrP(C) undergoes conformational change into PrP(Sc) remains unknown. We employed an in vitro PrP(Sc) amplification technique adapted from protein misfolding cyclic amplification (PMCA) to investigate the mechanism of prion-induced protein conformational change. Using this technique, PrP(Sc) from diluted scrapie-infected brain homogenate can be amplified >10-fold without sonication when mixed with normal brain homogenate under nondenaturing conditions. PrP(Sc) amplification in vitro exhibits species and strain specificity, depends on both time and temperature, only requires membrane-bound components, and does not require divalent cations. In vitro amplification of Syrian hamster Sc237 PrP(Sc) displays an optimum pH of approximately 7, whereas amplification of CD-1 mouse RML PrP(Sc) is optimized at pH approximately 6. The thiolate-specific alkylating agent N-ethylmaleimide (NEM) as well as the reversible thiol-specific blockers p-hydroxymercuribenzoic acid (PHMB) and mersalyl acid inhibited PrP(Sc) amplification in vitro, indicating that the conformational change from PrP(C) to PrP(Sc) requires a thiol-containing factor. Our data provide the first evidence that a reactive chemical group plays an essential role in the conformational change from PrP(C) to PrP(Sc).