Unveiling the efficacy of a bulk Raman spectra-based model in predicting single cell Raman spectra of microorganisms.

In a previous publication, we trained predictive models based on Raman bulk spectra of microorganisms placed on a silicon dioxide protected silver mirror slide to make predictions for new Raman spectra, unknown to the models, of microorganisms placed on a different substrate, namely stainless steel. Now we have combined large sections of this data and trained a convolutional neural network (CNN) to make predictions for single cell Raman spectra. We show that a database based on microbial bulk material is conditionally suited to make predictions for the same species in terms of single cells. Data of 13 different microorganisms (bacteria and yeasts) were used. Two of the 13 species could be identified 90% correctly and five other species 71%-88%. The six remaining species were correctly predicted by only 0%-49%. Especially stronger fluorescence in bulk material compared to single cells but also photodegradation of carotenoids are some effects that can complicate predictions for single cells based on bulk data. The results could be helpful in assessing universal Raman tools or databases.

Genomic variability correlates with biofilm phenotypes in multidrug resistant clinical isolates of Pseudomonas aeruginosa.

The multifactorial nature of Pseudomonas aeruginosa biofilm development and genomic variabilities implicates its resistance to conventional antimicrobials and virulence. Therefore, genetic determinants need to be extensively studied to block the early steps of biofilm or already formed biofilms. In this study, a total of 20 multidrug resistant (MDR) clinical P. aeruginosa isolates were evaluated for their biofilm forming abilities and related genes. Of the isolates tested, all of them showed surface attachment tendencies in nutrient limiting conditions, and classified as strong (SBF = 45%), moderate (MBF = 30%) and weak (WBF = 25%) biofilm formers. Complete genome sequencing of representative strong (DMC-27b), moderate (DMC-20c) and weak biofilm former (DMC-30b) isolates was performed. Analysis of biofilm related genes in the sequenced genomes revealed that, 80 of the 88 biofilm related genes possess 98-100% sequence identity to the reference PAO1 strain. Complete and partial sequence data of LecB proteins from tested isolates indicate that isolates containing PA14-like LecB sequences produced strong biofilms. All of the 7 pel operon protein coding genes in weak biofilm former isolate 30b showed significant nucleotide sequence variation with other tested isolates, and their corresponding proteins are 99% identical with the pel operon proteins of PA7. Bioinformatics analyses identified divergent sequence and structural features that separate PA7 like pel operon proteins from reference PAO1-like pel operon. Congo red and pellicle forming assays revealed that the sequence and structure variations may have interfered with the Pel production pathway and resulted in impaired Pel production in isolate 30b that has a PA7 like pel operon. Expression analysis also showed that both pelB and lecB genes were about 5 to 6 folds upregulated after 24 h in SBF 27b in comparison with WBF 30b. Our findings indicate significant genomic divergence in biofilm related genes of P. aeruginosa strains that affect their biofilm phenotypes.

Virucidal Efficacy of Laundering.

Viruses contribute significantly to the burden of infectious diseases worldwide. Although there are multiple infection routes associated with viruses, it is important to break the chain of infection and thus consider all possible transmission routes. Consequently, laundering can be a means to eliminate viruses from textiles, in clinical settings well as for domestic laundry procedures. Several factors influence the survival and inactivation of microorganisms, including viruses on hard surfaces and textiles. Therefore, textiles should be regarded as potential fomites. While in clinical and industrial settings laundry hygiene is ensured by standardized processes, temperatures of at least 60 °C and the use of oxidizing agents, domestic laundry is not well defined. Thus, the parameters affecting viral mitigation must be understood and prudently applied, especially in domestic laundering. Laundering can serve as a means to break the chain of infection for viral diseases by means of temperature, time, chemistry and mechanical action.

Raman Microscopic Identification of Microorganisms on Metal Surfaces via Support Vector Machines.

An easy, inexpensive, and rapid method to identify microorganisms is in great demand in various areas such as medical diagnostics or in the food industry. In our study, we show the development of several predictive models based on Raman spectroscopy combined with support vector machines (SVM) for 21 species of microorganisms. The microorganisms, grown under standardized conditions, were placed on a silver mirror slide to record the data for model development. Additional data was obtained from microorganisms on a polished stainless-steel slide in order to validate the models in general and to assess possible negative influences of the material change on the predictions. The theoretical prediction accuracies for the most accurate models, based on a five-fold cross-validation, are 98.4%. For practical validation, new spectra (from stainless-steel surfaces) have been used, which were not included in the calibration data set. The overall prediction accuracy in practice was about 80% and the inaccurate predictions were only due to a few species. The development of a database provides the basis for further investigations such as the application and extension to single-cell analytics and for the characterization of biofilms.

Raman Microscopic Analysis of Dry-Surface Biofilms on Clinically Relevant Materials.

Moist/hydrated biofilms have been well-studied in the medical area, and their association with infections is widely recognized. In contrast, dry-surface biofilms (DSBs) on environmental surfaces in healthcare settings have received less attention. DSBs have been shown to be widespread on commonly used items in hospitals and to harbor bacterial pathogens that are known to cause healthcare-acquired infections (HAI). DSBs cannot be detected by routine surface swabbing or contact plates, and studies have shown DSBs to be less susceptible to cleaning/disinfection products. As DSBs are increasingly reported in the medical field, and there is a likelihood they also occur in food production and manufacturing areas, there is a growing demand for the rapid in situ detection of DSBs and the identification of pathogens within DSBs. Raman microspectroscopy allows users to obtain spatially resolved information about the chemical composition of biofilms, and to identify microbial species. In this study, we investigated Staphylococcus aureus mono-species DSB on polyvinylchloride blanks and stainless steel coupons, and dual-species (S. aureus/Bacillus licheniformis) DSB on steel coupons. We demonstrated that Raman microspectroscopy is not only suitable for identifying specific species, but it also enables the differentiation of vegetative cells from their sporulated form. Our findings provide the first step towards the rapid identification and characterization of the distribution and composition of DSBs on different surface areas.

Laundry and textile hygiene in healthcare and beyond.

The elimination of microbial contaminations from textile is an important aspect of laundering apart from the removal of stains and dirt from used and worn textiles. Although the framework for institutional laundering is well regulated to ensure hygienic cleanliness via the use of e.g. high temperatures and bleaching agents, there are several open points, especially in domestic laundering. In both cases, energy efficiency of appliances is a main driver for innovation and has resulted in a general decrease in washing temperatures which in turn can impact the antimicrobial efficacy of laundering. Thus, the different factors influencing the input and removal of microbial cells in the laundering process and possible adverse effects of microbial contaminants in the washing machine and on the textiles as well as suitable counteractions are discussed in this article, focusing on the clinical area but also considering the domestic environment, which will gain importance in the future, e.g. by the increase of elderly and ill persons being cared for at home.

APSES proteins regulate morphogenesis and metabolism in Candida albicans.

Fungal APSES proteins regulate morphogenetic processes, including filamentation and differentiation. The human fungal pathogen Candida albicans contains two APSES proteins: the regulator Efg1p and its homologue Efh1p, described here. Overexpression of EFG1 or EFH1 led to similar phenotypes, including pseudohypha formation and opaque-white switching. An efh1 deletion generated no phenotype under most conditions but caused hyperfilamentation in an efg1 background under embedded or hypoxic conditions. This suggests cooperation of these APSES proteins in the suppression of an alternative morphogenetic signaling pathway. Genome-wide transcriptional profiling revealed that EFG1 and EFH1 regulate partially overlapping sets of genes associated with filament formation. Unexpectedly, Efg1p not only regulates genes involved in morphogenesis but also strongly influences the expression of metabolic genes, inducing glycolytic genes and repressing genes essential for oxidative metabolism. Using one- and two-hybrid assays, we further demonstrate that Efg1p is a repressor, whereas Efh1p is an activator of gene expression. Overall, the results suggest that Efh1p supports the regulatory functions of the primary regulator, Efg1p, and indicate a dual role for these APSES proteins in the regulation of fungal morphogenesis and metabolism.

Evaluation of the virucidal performance of domestic laundry procedures.

Laundering is one of the most important means to ensure a sufficient hygiene standard in the household environment. To evaluate the performance of this process, it is desirable to have methods that mimic the real-life situation as closely as possible. Although methods for the evaluation of the antibacterial and antifungal efficacy of domestic laundry procedures are available, the effect of laundering on viruses is still rather unclear. As the influence of laundry process parameters such as mechanical actions, temperature dynamics or liquor ratio cannot be simulated in vitro by suspension assays, a new in situ test method allowing virus simulation tests in washing machines has been developed. Using this in situ method we could show that conventional household washing detergents have a full virucidal efficiency at 40 degrees C also against non-enveloped surrogate viruses.

Influence of farnesol on the morphogenesis of Aspergillus niger.

Farnesol was the first quorum-sensing regulator to be found in eukaryotic cells. In Candida albicans, a dimorphic fungal human pathogen, farnesol blocks the yeast-to-filamentous growth transition. Here we show that in Aspergillus niger farnesol acts as an inhibitor of conidiation: Colonies grown on media containing farnesol were unable to develop conidia. Although farnesol treated A. niger cultures exhibited a colony morphology resembling the "fluffy" phenotype of A. nidulans, which is caused by a hyperactive G-protein/cAMP pathway, the intracellular level of cAMP in A. niger mycelia grown in presence of farnesol is greatly diminished. Furthermore, whereas inhibiting adenylyl cyclase led to a farnesol-like effect, the addition of external cAMP inhibited conidiation without causing a "fluffy" phenotype. This suggests that the mechanisms regulating conidiation in A. niger and A. nidulans are different.