Antimicrobial Resistance in the Environment: Towards Elucidating the Roles of Bioaerosols in Transmission and Detection of Antibacterial Resistance Genes.

Antimicrobial resistance (AMR) is continuing to grow across the world. Though often thought of as a mostly public health issue, AMR is also a major agricultural and environmental problem. As such, many researchers refer to it as the preeminent One Health issue. Aerial transport of antimicrobial-resistant bacteria via bioaerosols is still poorly understood. Recent work has highlighted the presence of antibiotic resistance genes in bioaerosols. Emissions of AMR bacteria and genes have been detected from various sources, including wastewater treatment plants, hospitals, and agricultural practices; however, their impacts on the broader environment are poorly understood. Contextualizing the roles of bioaerosols in the dissemination of AMR necessitates a multidisciplinary approach. Environmental factors, industrial and medical practices, as well as ecological principles influence the aerial dissemination of resistant bacteria. This article introduces an ongoing project assessing the presence and fate of AMR in bioaerosols across Canada. Its various sub-studies include the assessment of the emissions of antibiotic resistance genes from many agricultural practices, their long-distance transport, new integrative methods of assessment, and the creation of dissemination models over short and long distances. Results from sub-studies are beginning to be published. Consequently, this paper explains the background behind the development of the various sub-studies and highlight their shared aspects.

Detecting natural adaptation of the Streptococcus thermophilus CRISPR-Cas systems in research and classroom settings.

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems have been adapted into a powerful genome-editing tool. The basis for the flexibility of the tool lies in the adaptive nature of CRISPR-Cas as a bacterial immune system. Here, we describe a protocol to experimentally demonstrate the adaptive nature of this bacterial immune system by challenging the model organism for the study of CRISPR adaptation, Streptococcus thermophilus, with phages in order to detect natural CRISPR immunization. A bacterial culture is challenged with lytic phages, the surviving cells are screened by PCR for expansion of their CRISPR array and the newly acquired specificities are mapped to the genome of the phage. Furthermore, we offer three variants of the assay to (i) promote adaptation by challenging the system using defective viruses, (ii) challenge the system using plasmids to generate plasmid-resistant strains and (iii) bias the system to obtain natural immunity against a specifically targeted DNA sequence. The core protocol and its variants serve as a means to explore CRISPR adaptation, discover new CRISPR-Cas systems and generate bacterial strains that are resistant to phages or refractory to undesired genes or plasmids. In addition, the core protocol has served in teaching laboratories at the undergraduate level, demonstrating both its robust nature and educational value. Carrying out the core protocol takes 4 h of hands-on time over 7 d. Unlike sequence-based methods for detecting natural CRISPR adaptation, this phage-challenge-based approach results in the isolation of CRISPR-immune bacteria for downstream characterization and use.

Evaluation of bacterial contaminants found on unused paper towels and possible postcontamination after handwashing: a pilot study.

BACKGROUND: Bacterial contamination is a concern in the pulp and paper industry. Not only is the machinery contaminated but also can be the end-paper products. Bacterial transmission from unused paper towels to hands and surfaces is not well documented. METHODS: The culturable bacterial community of 6 different unused paper towel brands was determined by culture methods and by sequencing the 16S ribosomal DNA of bacterial contaminants. Next, we investigated the possible airborne and direct contact transmissions of these bacterial contaminants during hand drying after washing. RESULTS: Between 10(2) and 10(5) colony-forming units per gram of unused paper towels were isolated from the different paper towel brands. Bacteria belonging to the Bacillus genus were by far the most abundant microorganisms found (83.0%), followed by Paenibacillus (15.6%), Exiguobacterium (1.6%), and Clostridium (0.01%). Paper towels made from recycled fibers harbored between 100- to 1,000-fold more bacteria than the virgin wood pulp brand. Bacteria were easily transferred to disposable nitrile gloves when drying hands with paper towels. However, no evidence of bacterial airborne transmission was observed during paper towel dispensing. CONCLUSION: This pilot study demonstrated that a large community of culturable bacteria, including toxin producers, can be isolated from unused paper towels and that they may be transferred to individuals after handwashing. This may have implications in some industrial and clinical settings as well as in immunocompromised individuals.

Autofluorescence as a viability marker for detection of bacterial spores.

Recent biological terrorism events have indicated that bacterial spores such as Bacillus anthracis are real threat agents. Real time detection of biological agents is possible with the use of an ultraviolet Fluorescent Aerodynamic Particle Sizer (FLAPS) that measures particles' intrinsic fluorescence. It is important to know whether intrinsic fluorescence could be used to estimate agents' viability. Two categories of Bacillus spore populations can be differentiated by the intensity of intrinsic fluorescence emitted by ultraviolet (UV) stimulation : autofluorescent and non-autofluorescent. This study was performed to determine whether intensity of autofluorescence correlates with spore viability. Spores were analyzed using flow cytometer (equipped with a cell sorter) to mimic optical properties of FLAPS. Autofluorescent and non-autofluorescent spores were sorted according to the intensity of autofluorescence emitted following UV stimulation. Culturability, membrane integrity, membrane potential and dipicolinic acid (DPA) content were assessed. Autofluorescent spores were 1.7 times more culturable than the corresponding non-autofluorescent population. Moreover, a small proportion of autofluorescent spores exhibited extracellular membrane damages. Autofluorescent spores also showed higher membrane potential activity and contained higher levels of DPA. In conclusion, this study documents that the overall viability potential of bacterial spores can be assessed by UV flow cytometry used in the FLAPS technology.