Development of a sensitive and false-positive free PMA-qPCR viability assay to quantify VBNC Escherichia coli and evaluate disinfection performance in wastewater effluent.

The detection and quantification of viable Escherichia coli cells in wastewater treatment plant effluent is very important as it is the main disinfection efficacy parameter for assessing its public health risk and environmental impact. The aim of this study was to develop a sensitive and false-positive free propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) assay to quantify the viable but non-culturable (VBNC) E. coli present in secondary wastewater effluent after chlorine disinfection. The qPCR target was the E. coli uidA gene, and native Taq was used to eliminate false positives caused by the presence of contaminant E. coli DNA in recombinant Taq polymerase reagents. Due to issues with qPCR inhibitors in wastewater, this study explored several pre-DNA extraction treatment methods for qPCR inhibitor removal. PMA-qPCR validation was done using salmon testes DNA (Sketa DNA) as an exogenous control added directly to the wastewater samples and amplified using a separate qPCR assay. After disinfection of secondary effluent with 2ppm chlorine at the plant, the mean Log10 CFU reduction in E. coli was 2.85 from a mean CFU of 3.48/10mL compared to 0.21 Log10 CCE mean reduction of the uidA gene from a mean CCE of 3.16/10mL. The VBNC cell concentrations were calculated as 2.32 Log10/10mL by subtracting the colony forming units (CFU) obtained from membrane filtration from the calculated CFU equivalent (CCE) values obtained from PMA-qPCR. These results demonstrate the effective use of a PMA-qPCR method for the quantification of the E. coli uidA gene and indicate there are high numbers (2.01×103CCE/100mL) of VBNC E. coli cells leaving the wastewater treatment plant in the final effluent after chlorine treatment. VBNC bacterial cells are of concern as they have the potential to resuscitate and grow, regain virulence, affect natural microbiome in the discharge sites, and pass on antimicrobial resistant genes to other microorganisms.

Decontamination of indoor air to reduce the risk of airborne infections: Studies on survival and inactivation of airborne pathogens using an aerobiology chamber.

BACKGROUND: Although indoor air can spread many pathogens, information on the airborne survival and inactivation of such pathogens remains sparse. METHODS: Staphylococcus aureus and Klebsiella pneumoniae were nebulized separately into an aerobiology chamber (24.0 m3). The chamber's relative humidity and air temperature were at 50% ± 5% and 20°C ± 2°C, respectively. The air was sampled with a slit-to-agar sampler. Between tests, filtered air purged the chamber of any residual airborne microbes. RESULTS: The challenge in the air varied between 4.2 log10 colony forming units (CFU)/m3 and 5.0 log10 CFU/m3, sufficient to show a ≥3 log10 (≥99.9%) reduction in microbial viability in air over a given contact time by the technologies tested. The rates of biologic decay of S aureus and K pneumoniae were 0.0064 ± 0.00015 and 0.0244 ± 0.009 log10 CFU/m3/min, respectively. Three commercial devices, with ultraviolet light and HEPA (high-efficiency particulate air) filtration, met the product efficacy criterion in 45-210 minutes; these rates were statistically significant compared with the corresponding rates of biologic decay of the bacteria. One device was also tested with repeated challenges with aerosolized S aureus to simulate ongoing fluctuations in indoor air quality; it could reduce each such recontamination to an undetectable level in approximately 40 minutes. CONCLUSIONS: The setup described is suitable for work with all major classes of pathogens and also complies with the U.S. Environmental Protection Agency's guidelines (2012) for testing air decontamination technologies.

Aptamer-based viability impedimetric sensor for bacteria.

The development of an aptamer-based viability impedimetric sensor for bacteria (AptaVISens-B) is presented. Highly specific DNA aptamers to live Salmonella typhimurium were selected via the cell-systematic evolution of ligands by exponential enrichment (SELEX) technique. Twelve rounds of selection were performed; each comprises a positive selection step against viable S. typhimurium and a negative selection step against heat killed S. typhimurium and a mixture of related pathogens, including Salmonella enteritidis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Citrobacter freundii to ensure the species specificity of the selected aptamers. The DNA sequence showing the highest binding affinity to the bacteria was further integrated into an impedimetric sensor via self-assembly onto a gold nanoparticle-modified screen-printed carbon electrode (GNP-SPCE). Remarkably, this aptasensor is highly selective and can successfully detect S. typhimurium down to 600 CFU mL(-1) (equivalent to 18 live cells in 30 µL of assay volume) and distinguish it from other Salmonella species, including S. enteritidis and S. choleraesuis. This report is envisaged to open a new venue for the aptamer-based viability sensing of a variety of microorganisms, particularly viable but nonculturable (VBNC) bacteria, using a rapid, economic, and label-free electrochemical platform.

Aptamer-based impedimetric sensor for bacterial typing.

The development of an aptamer-based impedimetric sensor for typing of bacteria (AIST-B) is presented. Highly specific DNA aptamers to Salmonella enteritidis were selected via Cell-SELEX technique. Twelve rounds of selection were performed; each comprises a positive selection step against S. enteritidis and a negative selection step against a mixture of related pathogens, including Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Citrobacter freundii, to ensure the species-specificity of the selected aptamers. After sequencing of the pool showing the highest binding affinity to S. enteritidis, a DNA sequence of high affinity to the bacteria was integrated into an impedimetric sensor via self-assembly onto a gold nanoparticles-modified screen-printed carbon electrode (GNPs-SPCE). Remarkably, this aptasensor is highly selective and can successfully detect S. enteritidis down to 600 CFU mL(-1) (equivalent to 18 CFU in 30 µL assay volume) in 10 min and distinguish it from other Salmonella species, including S. typhimurium and S. choleraesuis. This report is envisaged to open a new venue for the aptamer-based typing of a variety of microorganisms using a rapid, economic, and label-free electrochemical platform.

Experimental evaluation of an automated endoscope reprocessor with in situ generation of peracetic acid for disinfection of semicritical devices.

OBJECTIVE: To evaluate the effectiveness of a high-level disinfection solution generated inside an endoscope processing system for decontaminating external and internal surfaces of experimentally contaminated heat-sensitive medical devices. METHODS: The American Society for Testing and Materials Simulated-Use Test protocol (E1837-02), which incorporates a soil load in each inoculum, was used to evaluate the efficacy of the system when processing 4 common types of endoscopes contaminated separately with 5 types of nosocomial pathogens: Pseudomonas aeruginosa (ATCC 15442), spores of Clostridium difficile (ATCC 9689), a glutaraldehyde-resistant strain of Mycobacterium chelonae, a vancomycin-resistant strain of Enterococcus faecalis, and a methicillin-resistant strain of Staphylococcus aureus. Rinse solution samples from channels and from surfaces of the processed endoscopes were tested for any microbicidal residues. RESULTS: For all organisms tested, the baseline level of contamination of the endoscopes ranged from 5 log(10) to greater than 7 log(10) at each external surface site and internal channel. All tests showed reductions in viability of the test organisms to undetectable levels. All rinse solution samples from external and internal sites of the endoscopes proved to be free of any residual microbicidal activity. CONCLUSIONS: The endoscope reprocessor, with its processor-generated high-level disinfection solution, successfully reduced the numbers of selected, clinically relevant pathogens to undetectable levels both in the channels and on the outside surfaces of the 4 representative endoscopes tested in this study.