Candidate biomarkers of antibiotic resistance for the monitoring of wastewater and the downstream environment.

Urban wastewater treatment plants (UWTPs) are essential for reducing the pollutants load and protecting water bodies. However, wastewater catchment areas and UWTPs emit continuously antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), with recognized impacts on the downstream environments. Recently, the European Commission recommended to monitor antibiotic resistance in UWTPs serving more than 100 000 population equivalents. Antibiotic resistance monitoring in environmental samples can be challenging. The expected complexity of these systems can jeopardize the interpretation capacity regarding, for instance, wastewater treatment efficiency, impacts of environmental contamination, or risks due to human exposure. Simplified monitoring frameworks will be essential for the successful implementation of analytical procedures, data analysis, and data sharing. This study aimed to test a set of biomarkers representative of ARG contamination, selected based on their frequent human association and, simultaneously, rare presence in pristine environments. In addition to the 16S rRNA gene, ten potential biomarkers (intI1, sul1, ermB, ermF, aph(3'')-Ib, qacEΔ1, uidA, mefC, tetX, and crAssphage) were monitored in DNA extracts (n = 116) from raw wastewater, activated sludge, treated wastewater, and surface water (upstream and downstream of UWTPs) samples collected in the Czech Republic, Denmark, Israel, the Netherlands, and Portugal. Each biomarker was sensitive enough to measure decreases (on average by up to 2.5 log-units gene copy/mL) from raw wastewater to surface water, with variations in the same order of magnitude as for the 16S rRNA gene. The use of the 10 biomarkers allowed the typing of water samples whose origin or quality could be predicted in a blind test. The results show that, based on appropriate biomarkers, qPCR can be used for a cost-effective and technically accessible approach to monitoring wastewater and the downstream environment.

Microbial Contamination of Photographic and Cinematographic Materials in Archival Funds in the Czech Republic.

In this study we investigated the microbial contamination of 126 samples of photographic and cinematographic materials from 10 archival funds in the Czech Republic. Microorganisms were isolated from the light-sensitive layer by swabbing it with a polyurethane sponge. Microbial isolates were identified by MALDI-TOF MS (bacteria) or by phenotype testing and microscopy (fungi). Bacterial contamination was more abundant and more diverse than fungal contamination, and both were significantly associated with archives. The most frequently isolated fungal genera were Cladosporium, Eurotium, Penicillium, Aspergillus and Alternaria. The most frequently isolated bacteria were Gram-positive genera such as Staphylococcus, Micrococcus, Kocuria, Streptococcus and Bacillus. This bacterial and fungal diversity suggests that air is the main vehicle of contamination. We also analysed the impact of the type of material used for the carrier (paper, baryta paper, cellulose acetate and nitrate or glass) or the light-sensitive layer (albumen, gelatine, collodion and other) on the level and diversity of microbial contamination. Carriers such as polyester and cellulose nitrate may have a negative impact on bacterial contamination, while paper and baryta paper may have a partially positive impact on both fungal and bacterial contamination.

Monitoring antibiotic resistance genes in wastewater environments: The challenges of filling a gap in the One-Health cycle.

Antibiotic resistance (AR) is a global problem requiring international cooperation and coordinated action. Global monitoring must rely on methods available and comparable across nations to quantify AR occurrence and identify sources and reservoirs, as well as paths of AR dissemination. Numerous analytical tools that are gaining relevance in microbiology, have the potential to be applied to AR research. This review summarizes the state of the art of AR monitoring methods, considering distinct needs, objectives and available resources. Based on the overview of distinct approaches that are used or can be adapted to monitor AR, it is discussed the potential to establish reliable and useful monitoring schemes that can be implemented in distinct contexts. This discussion places the environmental monitoring within the One-Health approach, where two types of risk, dissemination across distinct environmental compartments, and transmission to humans, must be considered. The plethora of methodological approaches to monitor AR and the variable features of the monitored sites challenge the capacity of the scientific community and policy makers to reach a common understanding. However, the dialogue between different methods and the production of action-oriented data is a priority. The review aims to warm up this discussion.

Evaluation of the Antimicrobial Efficacy of N-Acetyl-l-Cysteine, Rhamnolipids, and Usnic Acid-Novel Approaches to Fight Food-Borne Pathogens.

In the food industry, the increasing antimicrobial resistance of food-borne pathogens to conventional sanitizers poses the risk of food contamination and a decrease in product quality and safety. Therefore, we explored alternative antimicrobials N-Acetyl-l-cysteine (NAC), rhamnolipids (RLs), and usnic acid (UA) as a novel approach to prevent biofilm formation and reduce existing biofilms formed by important food-borne pathogens (three strains of Salmonella enterica and two strains of Escherichia coli, Listeria monocytogenes, Staphylococcus aureus). Their effectiveness was evaluated by determining minimum inhibitory concentrations needed for inhibition of bacterial growth, biofilm formation, metabolic activity, and biofilm reduction. Transmission electron microscopy and confocal scanning laser microscopy followed by image analysis were used to visualize and quantify the impact of tested substances on both planktonic and biofilm-associated cells. The in vitro cytotoxicity of the substances was determined as a half-maximal inhibitory concentration in five different cell lines. The results indicate relatively low cytotoxic effects of NAC in comparison to RLs and UA. In addition, NAC inhibited bacterial growth for all strains, while RLs showed overall lower inhibition and UA inhibited only the growth of Gram-positive bacteria. Even though tested substances did not remove the biofilms, NAC represents a promising tool in biofilm prevention.

Antimicrobial Properties of Palladium and Platinum Nanoparticles: A New Tool for Combating Food-Borne Pathogens.

Although some metallic nanoparticles (NPs) are commonly used in the food processing plants as nanomaterials for food packaging, or as coatings on the food handling equipment, little is known about antimicrobial properties of palladium (PdNPs) and platinum (PtNPs) nanoparticles and their potential use in the food industry. In this study, common food-borne pathogens Salmonella enterica Infantis, Escherichia coli, Listeria monocytogenes and Staphylococcus aureus were tested. Both NPs reduced viable cells with the log10 CFU reduction of 0.3-2.4 (PdNPs) and 0.8-2.0 (PtNPs), average inhibitory rates of 55.2-99% for PdNPs and of 83.8-99% for PtNPs. However, both NPs seemed to be less effective for biofilm formation and its reduction. The most effective concentrations were evaluated to be 22.25-44.5 mg/L for PdNPs and 50.5-101 mg/L for PtNPs. Furthermore, the interactions of tested NPs with bacterial cell were visualized by transmission electron microscopy (TEM). TEM visualization confirmed that NPs entered bacteria and caused direct damage of the cell walls, which resulted in bacterial disruption. The in vitro cytotoxicity of individual NPs was determined in primary human renal tubular epithelial cells (HRTECs), human keratinocytes (HaCat), human dermal fibroblasts (HDFs), human epithelial kidney cells (HEK 293), and primary human coronary artery endothelial cells (HCAECs). Due to their antimicrobial properties on bacterial cells and no acute cytotoxicity, both types of NPs could potentially fight food-borne pathogens.

Application of Nanopore Sequencing (MinION) for the Analysis of Bacteriome and Resistome of Bean Sprouts.

The aspiration these days is to apply rapid methods for parallel analysis of bacteriome and resistome of food samples to increase food safety and prevent antibiotic resistance genes (ARGs) spreading. In this work, we used nanopore sequencing (NS) to determine the diversity and dynamics of the microbiome and resistome in two types of bean sprouts. We proved that NS provided an easy, quick, and reliable way to identify the microbiome and resistome of a food sample also. The species diversity obtained by NS and by cultivation methods with MALDI-TOF MS identification was comparable. In both samples, before and after cultivation (30 °C, 48 h), the dominant part of bacteriome formed Gammaproteobacteria (Enterobacteriaceae, Erwiniaceae, Pseudomonadaceae, Moraxellaceae) and then Firmicutes (Streptococcaceae). The diversity and abundance of single ARGs groups were comparable for both samples despite bacteriome differences. More than 50% of the detected ARGs alignments were mutations conferring resistance to aminoglycosides (16S rRNA), resistance to fluoroquinolones (gyrA, gyrB, parC, parD) and elfamycin (EF-Tu). ARGs encoding efflux pumps formed more than 30% of the detected alignments. Beta-lactamases were represented by many variants, but were less abundant.

The effect of gold and silver nanoparticles, chitosan and their combinations on bacterial biofilms of food-borne pathogens.

The antimicrobial activity of gold and silver nanoparticles (AuNPs, AgNPs), chitosan (CS) and their combinations was established by determining the minimum inhibitory concentration for planktonic (MICPC80) and biofilm growth (MICBC80), for biofilm formation (MICBF80), metabolic activity (MICBM80) and reduction (MICBR80), and for the metabolic activity of preformed biofilm (MICMPB80). Biofilms were quantified in microtitre plates by crystal violet staining and metabolic activity was evaluated by the MTT assay. Chitosan effectively suppressed biofilm formation (0.31-5 mg ml-1) in all the tested strains, except Salmonella enterica Infantis (0.16-2.5 mg ml-1) where CS and its combination with AgNPs induced biofilm formation. Nanoparticles inhibited biofilm growth only when the highest concentrations were used. Even though AuNPs, AgNPs and CS were not able to remove biofilm mass, they reduced its metabolic activity by at least 80%. The combinations of nanoparticles with CS did not show any significant positive synergistic effect on the tested target properties.

Transcriptomic and metabolic responses of Staphylococcus aureus in mixed culture with Lactobacillus plantarum, Streptococcus thermophilus and Enterococcus durans in milk.

Staphylococcus aureus is a major food-borne pathogen due to the production of enterotoxin and is particularly prevalent in contaminated milk and dairy products. The lactic acid bacteria (LAB) are widely used as biocontrol agents in fermented foods which can inhibit pathogenic flora. In our work, we investigated the influence of three strains of LAB (Lactobacillus plantarum, Streptococcus thermophilus and Enterococcus durans) on the relative expression of three enterotoxin genes (sea, sec, sell) and eight virulence and/or regulatory genes (sarA, saeS, codY, srrA, rot, hld/RNAIII, agrA/RNAII, sigB) in two S. aureus strains (MW2 and Sa1612) in TSB and reduced-fat milk (1.5 %) at 30 °C over a 24-h period. The tested LAB and S. aureus strains proved to be mutually non-competitive or only slightly competitive during co-cultivation. In addition, under the above-mentioned conditions, differential gene expression between the S. aureus MW2 and Sa1612 strains was well documented. S. aureus growth was changed in mixed culture with LAB; however, its effect on the repression of sea and sec expression correlated with production of these virulence factors. In comparison, the presence of LAB strains generally inhibited the expression of sec, sell, sarA, seaS, agrA/RNAII and hld/RNAIII genes. The effect of LAB strains presence on the expression of sea, codY, srrA, rot and sigB genes was medium, time, LAB and S. aureus strain specific. SEA and SEC production was significantly reduced in milk compared to TSB in pure culture. After the 24-h cultivation, S. aureus MW2 and Sa1612 SEC production was 187 and 331 times lower in milk compared to TSB, respectively (0.07 and 0.39 ng/mL in milk, versus 13.1 and 129.2 ng/mL in TSB, respectively). At the same time S. aureus MW2 and Sa1612 SEA production was 77 and 68 times lower in milk compared to TSB, respectively (0.99 and 0.17 ng/mL in milk, versus 76.4 and 11.5 ng/mL in TSB, respectively). This study has revealed new insights into the interaction between S. aureus and LAB (L. plantarum, S. thermophilus, E. durans) on the level of the expression and/or production of S. aureus enterotoxins, regulatory and virulence genes in different media, including milk. This study provides data which may improve the quality of food production.