Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and lateral flow immunochromatography for rapid identification of ß-lactamase-gene-harboring Enterobacterales in urine specimens: Performance and cost-benefit analyses.

In this single-center prospective study, we evaluated the performance to the MALDI-ToF MS based method in conjunction with lateral flow immunochromatographic (LFIC) in urine specimens for rapid diagnosis of bacterial Urinary Tract Infection (UTI) and detection of carbapenemase and/or extended-spectrum ß- lactamase (ESBL) enzymes produced by the involved bacteria, compared to standard culture, and antimicrobial susceptibility testing/genotypic resistance markers characterization performed on culture-grown colonies. In addition, a cost-benefit analysis comparing this approach against standard procedures was conducted. A total of 324 urines were included in the study, of which 288 (88.9 %) yielded concordant results by the MALDI-ToF MS and conventional culture (Kappa agreement, 0.82; P<0.001). Direct LFIC testing could be carried out in 249/324 urines. Bacterial species carrying ß-lactam genotypic resistance markers were identified in 35 urines (35 CTX-M and 2 OXA-48). Two ESBL-producing Escherichia coli were missed by LFIC (Kappa agreement with standard procedures of 0.96; P<0.001). The cost-benefit analysis indicated that our novel approach resulted in an improvement of clinical outcomes (less need of outpatient care) with a marginal incremental cost (€2.59).

Surface Modification of Bacterial Cellulose for Biomedical Applications.

Bacterial cellulose is a naturally occurring polysaccharide with numerous biomedical applications that range from drug delivery platforms to tissue engineering strategies. BC possesses remarkable biocompatibility, microstructure, and mechanical properties that resemble native human tissues, making it suitable for the replacement of damaged or injured tissues. In this review, we will discuss the structure and mechanical properties of the BC and summarize the techniques used to characterize these properties. We will also discuss the functionalization of BC to yield nanocomposites and the surface modification of BC by plasma and irradiation-based methods to fabricate materials with improved functionalities such as bactericidal capabilities.

The Contemporary Epidemiology, Microbiology and Management of Chronic Osteomyelitis in US Children.

BACKGROUND: While the majority of pediatric osteomyelitis cases are acute in nature, a significant subset present with prolonged symptoms often associated with substantial morbidity. Little data exist to guide clinicians in the management of these infections. We sought to describe the epidemiology, clinical features and management of chronic osteomyelitis (CO) in children. METHODS: We reviewed hospital admissions for CO from 2011 to 2018 at Texas Children's Hospital. Cases were included if symptoms lasted ≥28 days on presentation. Cases were classified as those associated with: (1) a contiguous focus of infection; (2) penetrating trauma; (3) orthopedic hardware; (4) postacute CO (PACO, those occurring after ≥28 days of therapy for acute osteomyelitis); and (5) primary hematogenous CO. RESULTS: One hundred fourteen cases met inclusion criteria. The median patient age was 11.8 years and 35.9% had comorbidities. 70.2% of patients underwent ≥1 surgical procedure. A microbiologic etiology was identified in 72.8% of cases and Staphylococcus aureus was most common (39.4%). Contiguous focus of infection was more often associated with polymicrobial disease with or without Pseudomonas. Postacute CO was caused by S. aureus in 95%. The median duration of total therapy was 210 days. 26.3% of patients experienced treatment failure of which 46% underwent repeat hospital admission/surgery. There was no association between duration of intravenous therapy for CO and treatment failure. CONCLUSIONS: Children with CO represent a diverse group both in terms of pathogenesis and microbiology. Pathogenesis and clinical presentation can provide clues to microbiologic etiology. Prolonged intravenous therapy does not appear to improve outcomes in CO.

Rapid (30-second), equipment-free purification of nucleic acids using easy-to-make dipsticks.

The complexity of current nucleic acid isolation methods limits their use outside of the modern laboratory environment. Here, we describe a fast and affordable method to purify nucleic acids from animal, plant, viral and microbial samples using a cellulose-based dipstick. Nucleic acids can be purified by dipping in-house-made dipsticks into just three solutions: the extract (to bind the nucleic acids), a wash buffer (to remove impurities) and the amplification reaction (to elute the nucleic acids). The speed and simplicity of this method make it ideally suited for molecular applications, both within and outside the laboratory, including limited-resource settings such as remote field sites and teaching institutions. Detailed instructions for how to easily manufacture large numbers of dipsticks in house are provided. Using the instructions, readers can create more than 200 dipsticks in <30 min and perform dipstick-based nucleic acid purifications in 30 s.

MALDI-TOF MS - application in food microbiology.

Microbiological purity control of food products is of great importance in the food industry. Contaminated food is often characterized by a deteriorated taste, smell, and appearance, and when consumed, it can pose a threat to human health and life. Also, contamination incurs huge financial losses to the food industry. Different methods are used for identification of the microorganisms isolated from food, which are based on phenotypic, immunologic, genetic, and spectroscopic techniques. Unfortunately, these methods have the following disadvantages: laborious, time-consuming, requiring a well-trained spectrometer operator with specialist knowledge, or very accurate, but complicated, and extremely expensive. In recent years, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has been gaining increasing importance in the field of food microbiology. Unlike other techniques used for microorganisms identification, MALDI-TOF MS is more rapid, accurate and cost-efficient, and easy to perform. Thus, this method can be applied in the food industry to quickly and accurately identify microorganisms, which is crucial for controlling the quality of food products. The present review aims to discuss the selected applications of MALDI-TOF MS in food microbiology. It mainly focuses on the characteristics of this method and its potential use in the identification and typing of microorganisms including filamentous fungi, yeasts, and bacteria in fermented beverages (beer and wine), honey, dairy products like yogurt and pasteurized milk, pork, and seafood.

Rapid detection of bacterial infection and viability assessment with high specificity and sensitivity using Raman microspectroscopy.

Infectious diseases caused by bacteria still pose major diagnostic challenges in spite of the availability of various molecular approaches. Irrespective of the type of infection, rapid identification of the causative pathogen with a high degree of sensitivity and specificity is essential for initiating appropriate treatment. While existing methods like PCR possess high sensitivity, they are incapable of identifying the viability status of the pathogen and those which can, like culturing, are inherently slow. To overcome these limitations, we developed a diagnostic platform based on Raman microspectroscopy, capable of detecting biochemical signatures from a single bacterium for identification as well as viability assessment. The study also establishes a decontamination protocol for handling live pathogenic bacteria which does not affect identification and viability testing, showing applicability in the analysis of sputum samples containing pathogenic mycobacterial strains. The minimal sample processing along with multivariate analysis of spectroscopic signatures provides an interface for automatic classification, allowing the prediction of unknown samples by mapping signatures onto available datasets. Also, the novelty of the current work is the demonstration of simultaneous identification and viability assessment at a single bacterial level for pathogenic bacteria. Graphical abstract.

The impact of various seed, accessibility and interaction constraints on sRNA target prediction- a systematic assessment.

BACKGROUND: Seed and accessibility constraints are core features to enable highly accurate sRNA target screens based on RNA-RNA interaction prediction. Currently, available tools provide different (sets of) constraints and default parameter sets. Thus, it is hard to impossible for users to estimate the influence of individual restrictions on the prediction results. RESULTS: Here, we present a systematic assessment of the impact of established and new constraints on sRNA target prediction both on a qualitative as well as computational level. This is done exemplarily based on the performance of IntaRNA, one of the most exact sRNA target prediction tools. IntaRNA provides various ways to constrain considered seed interactions, e.g. based on seed length, its accessibility, minimal unpaired probabilities, or energy thresholds, beside analogous constraints for the overall interaction. Thus, our results reveal the impact of individual constraints and their combinations. CONCLUSIONS: This provides both a guide for users what is important and recommendations for existing and upcoming sRNA target prediction approaches.We show on a large sRNA target screen benchmark data set that only by altering the parameter set, IntaRNA recovers 30% more verified interactions while becoming 5-times faster. This exemplifies the potential of seed, accessibility and interaction constraints for sRNA target prediction.

Comparative ecotoxicity assessment of magnetosomes and magnetite nanoparticles.

Magnetite nanoparticles (MNPs) are gaining attention because of their biomedical, environmental and industrial applications. However, they have limited uses because of ecotoxicity. On contrast, bacterially synthesized MNPs such as magnetosomes are found to be biocompatible and less toxic due to the lipid bilayer membrane found around magnetite. In this context, this study compares the physio-chemical properties and toxicology effects of MNPs and magnetosomes in different models such as human red blood cells, macrophage cell lines (RAW 264.7), onion root tips (Allium cepa), Artemia salina (A. salina) and zebrafish embryo (Danio rerio). MNPs showed 38.59% hemolysis whereas the maximum hemolysis induced by magnetosomes was 7.03% for the same concentration (250 µg/ml). The cytotoxicity of MNPs and magnetosomes were 36.01% and 13.4%, respectively, at 250 µg/ml. Onion root tip assay revealed high toxicity when treated with MNPs than magnetosomes. The MNPs were further tested for its toxicity against A. salina and 50% mortality rate was observed. Similarly, notable malformation was seen in zebrafish embryo treated with MNPs. However, magnetosomes did not exhibit any mortality and malformation in A. salina and zebrafish embryo. The study revealed that magnetosomes are safe and do not cause any potential risk to environment compared to synthetic MNPs.Abbreviation: MNPs: Magnetic nanoparticles; ATCC: American Type Culture Collection; MTB: Magnetotactic bacteria; MSR-1: Magnetospirillum gryphiswaldense; DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen; MSGM: Magnetospirillum growth medium; D-PBS: Dulbecco phosphate buffer saline; RBC: Red blood cells; SEM: Scanning electron microscopy; HRTEM: High-resolution transition electron microscope; FTIR: Fourier transform infrared spectroscopy; XRD: X-ray powder diffraction; AFM: Atomic-force microscopy; ZP: Zeta Potential; PSD: Particle Size Distribution; EDX: Energy-dispersive X-ray spectroscopy; PBS: Phosphate buffer saline; DMEM: Dulbecco's modified eagle medium; HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); MTT:3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; DMSO: Dimethyl sulfoxide; ROS: Reactive oxygen species.

Realisation and assessment of a low-cost LED device for contact lens disinfection by visible violet light.

This study presents a device for efficient, low-cost and eye-friendly overnight disinfection of contact lenses by visible violet light as an alternative to disinfection with biocide-containing solutions. Bacterial solutions with one Pseudomonas and one Staphylococcus strain each were irradiated for up to 8 h in commercial transparent contact lens cases by the presented light-emitting diode (LED) device. Samples were taken at different intervals and distributed on agar plates. The surviving bacteria were determined by counting of colony-forming units and compared to the specific requirements of the stand-alone test for contact lens disinfection of the hygiene standard ISO 14729. The concentration of both microorganisms was reduced by three orders of magnitude after less than 4 h of irradiation. The LED current and intensity have not yet been at maximum and could be further increased if necessary for other microorganisms. The presented device fulfils the requirement of the stand-alone test of the contact lens hygienic standard ISO 14729 for the tested Pseudomonas and Staphylococcus strains. According to literature data, the inactivation of Serratia marcescens, Candida albicans and Fusarium solani seems also possible, but may require increased LED current and intensity.

Ecological assessment of groundwater ecosystems disturbed by recharge systems using organic matter quality, biofilm characteristics, and bacterial diversity.

Recharge of aquifers by urban stormwater may trigger significant ecological changes that can be detrimental to the biodiversity and functioning of groundwater ecosystems. Here, the effects of aquifer recharge (AR) on three levels of parameters were investigated: dissolved organic carbon (DOC) quantity and quality, global biofilm characteristics, and diversity changes of bacterial communities. As DOC enrichment by AR can be mitigated by vadose zone (VZ) thickness, three AR sites with thin VZ (< 3 m) and three sites with thick VZ (> 10 m) were selected. For each AR site, clay beads were incubated over a 10-day-long rainy period through wells in recharged and non-recharged groundwaters. Total proteins, dehydrogenase, and hydrolytic activities were monitored from clay beads to assess biofilm development. Bacterial richness on beads was estimated by 16S rRNA-based metabarcoding. AR was found to significantly increase DOC and biodegradable DOC (BDOC) concentrations, biofilm development, and bacterial richness especially in sites with thin VZ. VZ thickness was inversely related to microbial growth indicators and bacterial richness in groundwater, through a control of DOC availability. The proportion of Bacteroidetes 16S rRNA gene reads was higher in recharged groundwater than in non-recharged groundwater, suggesting that this phylum could be used as an indicator of DOC enrichment associated with AR. Quantitative PCR assays for Bacteroides DNA confirmed these trends and showed an enrichment of this bacterial group in DOC-rich aquifer waters. The positive linear relationships between BDOC concentrations and biofilm variables highlighted a strong C-limitation of groundwater impacting bacterial species sorting and activity.

Prospective Study of Microbial Colorants under the Focus of Patent Documents.

BACKGROUND: Colorant-producing microorganisms are quite common in nature. These biomolecules present many biological activities such as antitumoral, antimicrobial and antioxidant, in addition to the various nuances of color, making them of special technological importance to the industrial sectors. OBJECTIVE: This study aims to conduct a technological mapping in the patent, at European patent Office (EPO), in order to evaluate the global panorama of the use of microbial colorants. METHODS: The experimental design was acquired by the keyword-driven approach through the advanced search in the Espacenet database European Patent Office (EPO). The keywords selected were bacteria or fungi* or yeast or algae or microorganism* but not plant* and pigment* or color* or colorant* or dye* and the International Patent Classification code, C09B61, for prospecting of interest. RESULTS: There has been a linear increase in patents developed in the last 20 years, with Japan as the largest depositor in the area. The companies Dainippon Ink and Chemicals and Ajinomoto, both being Japanese, are the largest depositors with 20% of all patents. Among the microorganisms, the filamentous fungi appeared in the first place with 32% of documents and the fungi of the genus Monascus were the most frequently used. CONCLUSION: The use of microbial colorants has been growing among industries, mainly in food sectors, due to the growing demand for products of natural origin. Thus, the increase in research and technological development in the area of microbial colorants can become an economically viable and promising strategy for the various industrial sectors.

The Behavior of Trispyrazolylborato-Metal(II)-Flavonolate Complexes as Functional Models for Bacterial Quercetinase-Assessment of the Metal Impact.

A series of five compounds TpMesMFla (TpMes = hydrotris(3-mesityl)pyrazolylborate; M = Mn, Fe, Co, Ni, Zn; Fla = 3-hydroxyflavonolate) has been synthesized as models for the 2,4-quercetin dioxygenase, QueD. The structures have been determined and the complexes proved to be isomorphous. Considering the structures more closely revealed that they differ in the degree of delocalization in the chelate ring formed through the binding of the two O donors of the flavonolate to the metal center, which is also supported by the results of UV-vis and IR spectroscopic investigations. The resulting trend (Zn/Fe > Co > Mn > Ni) is, however, not in line with the one that was found investigating the redox properties of the complexes by cyclic voltammetry (Zn > Fe > Ni > Co > Mn). Notably, from CV clear-cut information could be derived, as the complexes exhibited exceptionally well-behaved quasi-reversible redox transitions, indicating that the Tp ligand stabilizes the flavonolate radical formed in the oxidation process rather well. The fact that the rates, with which the complexes react with O2 in DMF solution, correlate with the position of the flavonolate redox couples, suggest that these reactions proceed via the initial electron transfer from the flavonolate to O2. After the O2 reaction, salicylic acid was identified as one of the products, the formation of which can be explained by the hydrolysis of the depside that should form upon a dioxygenation similar to the QueD enzyme-catalyzed reaction. 18O labeling experiments confirmed the presence of O2 derived O atoms. Mechanistic inferences based on the above results are discussed.

Assessment of Fuel Quality Parameters and Selection of Bacteria Using PROMETHEE-GAIA Algorithm.

Recently, biodiesel is gaining significant importance due to eco-friendly nature and development of large-scale production methodologies. Biodiesel is a mixture of mono-alkyl esters of fatty acids (FA). During transesterification, the long-chain FAs are combined with methanol to produce fatty acid methyl ester (FAME), the principle component of biodiesel. The biodiesel fuel properties are determined by structural components of FAs such as chain length, degree of unsaturation, and branching of the carbon chain. The fuel quality of biodiesel are evaluated by assessing the properties such as cetane number (CN), iodine value (IV), cold filter plugging point (CFPP), higher heating value (HHV), cloud point (CP), pour point (PP) etc., of FAME. The amount of lipid or fat produced may vary from organism to organism. A particular species may have high biomass with low lipid content and vice versa. So the selection of suitable species/genus by decision analysis software is much needed. Besides various multi-criteria decision analyses, Preference Ranking Organization Method for Enrichment of Evaluation (PROMETHEE) and Graphical Analysis for Interactive Aid (GAIA) analysis is considered as the most promising tool in selecting the prominent biodiesel producing strain. Here we describe the method of evaluating the fuel quality parameters for the produced FAME and selecting the prominent strain through PROMETHEE-GAIA algorithm.

Assessment of leachates from reactive fire-retardant coatings by chemical analysis and ecotoxicity testing.

The environmental compatibility of reactive fire-retardant coatings (intumescent paints) was investigated by a combination of leaching and ecotoxicological tests. Three representative fire-retardant coating systems were tested using two leaching procedures: "Horizontal Dynamic Surface Leaching Test" (DSLT) and the "Intermittent Immersion Test" (IIT). All eluate fractions (8 for DSLT and 9 for IIT) were analyzed for pH, conductivity, concentration of total organic carbon and selected anions und cations. Additionally, a GC-MS screening of selected fractions was conducted for identification of organic compounds. Eluate fractions 1 + 2 and fraction 7 of the DSLT were analyzed in four ecotoxicological tests (algae, daphnia, fish egg, luminescent bacteria) and in one genotoxicity test (umu). Concentration of most analytes was rather low or below limit of detection for many eluates. Analytes detected in eluates of all three products are Zn, Ba, SO42- and PO43-. Release patterns do not indicate a general trend: some compounds show maximum release in the first fractions while for others the maximum was observed in later test stages. Ecotoxic effects in eluates were found, which were higher in the eluate fraction 7 (maximum lowest ineffective dilution for luminescent bacteria (LIDL) 256) than in the eluate fraction 1 + 2 (maximum LIDL = 24). The sensitivity of the test systems was very different with highest effects for luminescent bacteria, followed by algae and daphnia and without effects in the fish egg test and umu test. A biotest battery for the comprehensive assessment is therefore advisable.

Assessment of MC-PDFT Excitation Energies for a Set of QM/MM Models of Rhodopsins.

A methodology for the automatic production of quantum mechanical/molecular mechanical (QM/MM) models of retinal-binding rhodopsin proteins and subsequent prediction of their spectroscopic properties has been proposed recently by some of the authors. The technology employed for the evaluation of the excitation energies is called Automatic Rhodopsin Modeling (ARM), and it involves the use of the complete active space self-consistent field (CASSCF) method followed by a multiconfiguration second-order perturbation theory (in particular, CASPT2) calculation of external correlation energies. Although it was shown that ARM is capable of successfully reproducing and predicting spectroscopic property trends in chromophore-embedding protein sets, practical applications of such technology are limited by the high computational costs of the multiconfiguration perturbation theory calculations. In the present work we benchmark the more affordable multiconfiguration pair-density functional theory (MC-PDFT) method whose accuracy has been recently validated for retinal chromophores in the gas phase, indicating that MC-PDFT could potentially be used to analyze large (e.g., few hundreds) sets of rhodopsin proteins. Here, we test this theory for a set of rhodopsin QM/MM models whose experimental absorption maxima (λ a max) have been measured. The results indicate that MC-PDFT may be employed to calculate λ a max values for this important class of photoresponsive proteins.

Response surface statistical optimization of bacterial nanocellulose fermentation in static culture using a low-cost medium.

This work aimed at the optimization of bacterial nanocellulose (BNC) production by static culture, using Komagataeibacter xylinus BPR 2001 (K. xylinus). Response surface methodology - central composite design was used to evaluate the effect of inexpensive and widely available nutrient sources, namely molasses, ethanol, corn steep liquor (CSL) and ammonium sulphate, on BNC production yield. The optimized parameters for maximum BNC production were % (m/v): molasses 5.38, CSL 1.91, ammonium sulphate 0.63, disodium phosphate 0.270, citric acid 0.115 and ethanol 1.38% (v/v). The experimental and predicted maximum BNC production yields were 7.5 ± 0.54 g/L and 6.64 ± 0.079 g/L, respectively and the experimental and predicted maximum BNC productivity were 0.829 ± 0.046 g/L/day and 0.734 ± 0.079 g/L/day, after 9 days of static culture fermentation, at 30 °C. The effect of surface area and culture medium depth on production yield and productivity were also studied. BNC dry mass production increased linearly with surface area, medium depth and fermentation time. So long as nutrients were still available in the culture media, BNC mass productivity was constant. The results show that a high BNC production yield can be obtained by static culture of K. xylinus BPR 2001 using a low-cost medium. These are promising conditions for the static industrial scale BNC production, since as compared to agitated bioreactors, higher productivities may be reached, while avoiding high capital and operating costs.

Architectural adaptations of microbial fuel cells.

Conventional wastewater treatment consumes a large amount of money worldwide for removal of pollutants prior to its discharge into water body or facilitating reuse. Decreasing energy expenditure during wastewater treatment and rather recovering some value-added products while treating wastewater is an important goal for researchers. Microbial fuel cells (MFCs) are representative bioelectrochemical systems, which offer energy-efficient wastewater treatment. MFCs convert chemical energy of organic matter into electrical energy by using biocatalytic activities. Although MFCs are not truly commercialized, they have potential to make energy-gaining wastewater treatment technologies and represent their capabilities successfully. Over the last decade, MFCs have developed remarkably in almost every dimension including wastewater treatment capabilities, power output, and cost optimization; however, its architectural design is an important consideration for scaling up. Here, we review various architectural advancements and technology up-gradation MFCs have experienced during its journey, to take this technology step forward for commercialization.

A tutorial on uncertainty propagation techniques for predictive microbiology models: A critical analysis of state-of-the-art techniques.

Building mathematical models in predictive microbiology is a data driven science. As such, the experimental data (and its uncertainty) has an influence on the final predictions and even on the calculation of the model prediction uncertainty. Therefore, the current research studies the influence of both the parameter estimation and uncertainty propagation method on the calculation of the model prediction uncertainty. The study is intended as well as a tutorial to uncertainty propagation techniques for researchers in (predictive) microbiology. To this end, an in silico case study was applied in which the effect of temperature on the microbial growth rate was modelled and used to make simulations for a temperature profile that is characterised by variability. The comparison of the parameter estimation methods demonstrated that the one-step method yields more accurate and precise calculations of the model prediction uncertainty than the two-step method. Four uncertainty propagation methods were assessed. The current work assesses the applicability of these techniques by considering the effect of experimental uncertainty and model input uncertainty. The linear approximation was demonstrated not always to provide reliable results. The Monte Carlo method was computationally very intensive, compared to its competitors. Polynomial chaos expansion was computationally efficient and accurate but is relatively complex to implement. Finally, the sigma point method was preferred as it is (i) computationally efficient, (ii) robust with respect to experimental uncertainty and (iii) easily implemented.

A microbial identification framework for risk assessment.

Micro-organisms are increasingly used in a variety of products for commercial uses, including cleaning products. Such microbial-based cleaning products (MBCP) are represented as a more environmentally-friendly alternative to chemically based cleaning products. The identity of the micro-organisms formulated into these products is often considered confidential business information and is not revealed or it is only partly revealed (i.e., identification to the genus, not to the species). That paucity of information complicates the evaluation of the risk associated with their use. The accurate taxonomic identification of those micro-organisms is important so that a suitable risk assessment of the products can be conducted. To alleviate difficulties associated with adequate identification of micro-organisms in MBCP and other products containing micro-organisms, a microbial identification framework for risk assessment (MIFRA) has been elaborated. It serves to provide guidance on a polyphasic tiered approach, combining the data obtained from the use of various methods (i.e., polyphasic approach) combined with the sequential selection of the methods (i.e., tiered) to achieve a satisfactory identity of the micro-organism to an acceptable taxonomic level. The MIFRA is suitable in various risk assessment contexts for micro-organisms used in any commercial product.

Assessment of workers' exposure to microorganisms when using biological degreasing stations.

Biological degreasing stations (BDSs) are used by mechanics. These BDSs use a water-based solution with a microbial degradation process. Occupational exposure during the use of BDSs has not been reported and few studies have identified the bacteria present. The objectives were to measure the concentration of microorganisms during BDSs' use and monitor the bacterial community in the liquid over time. Five mechanical workshops were studied. Six 30-min samples were taken at each workshop over one year. Bioaerosols in the ambient air samples were collected with Andersen impactors near the BDS Bioaerosols in the workers' breathing zone (WBZ) were collected on filters. Fresh bio-degreasing fluids were collected from unopened containers, and used bio-degreasing fluids were collected in the BDS. The results show that the use of BDSs does not seem to increase bioaerosols concentrations in the WBZ (concentrations lower than 480 CFU/m3) and that the bacterial communities (mainly yeasts, Bacillus subtilis and Pseudomonas aeruginosa) in the bio-degreasing fluids change through time and differ from the original community (B. subtilis). This study established that workers using BDSs were exposed to low levels of bioaerosols. No respiratory protection is recommended based on bioaerosols concentrations, but gloves and strict personal hygiene practices are essential.