Heidi Schraft

A comparison of DNA extraction and purification methods to detect Escherichia coli O157:H7 in cattle manure

The extraction of DNA from manure and the subsequent polymerase chain reaction (PCR) amplification of virulence genes to detect pathogens require an effective method of purification. Four different methods were assessed for their effectiveness in extracting and purifying Escherichia coli O157:H7 DNA from cattle manure: phenol/chloroform purification, phenol/chloroform/Sepharose B4 spin columns, phenol/chloroform/polyvinylpolypyrrolidone (PVPP) spun columns, and Mo Bio UltraClean kit. A PCR assay targeting the shiga-like toxin I gene (sltI) was carried out to determine the effectiveness of the four methods in removing PCR inhibitors from the manure samples. All methods were used to extract a manure slurry and the cleanliness of the samples was tested by the PCR with varying concentrations of spiked E. coli O157:H7 target DNA. The PVPP spun columns and the UltraClean kit had the best detection limit, detecting 20 pg of E. coli DNA (about 2x10(3) cells) per 100 mg of manure. The UltraClean kit and the PVPP spun columns also had the best and similar detection limits of 3x10(4) CFU/100 mg manure when E. coli O157:H7 cells were spiked into the manure sample and purified by all four methods. The enrichment of cells after inoculation into manure was performed using tryptic soy broth at 37 degrees C for 5 h. Both the PVPP spun columns and the UltraClean kit methods were used to purify the enriched samples and were able to detect initial inocula of 6 CFU/100 mg manure, indicating that the two methods were highly efficient in purifying DNA from manure samples.

A novel selective growth medium-PCR assay to isolate and detect Sphingomonas in environmental samples

Sphingomonas species can be found ubiquitously in the environment and can be frequently found in surface biofilms. Some Sphingomonas strains are well known for metabolizing complex organic pollutants but some are opportunistic human pathogens. Despite the importance of the Sphingomonas species, a reliable system to isolate this group of bacteria from the environment has not been developed. In this study, a combined streptomycin-piperacillin selective growth medium/polymerase chain reaction (PCR) detection approach is developed to isolate and identify the Sphingomonas bacteria. A total of 72 known Sphingomonas strains (including 21 different Sphingomonas species type strains) and 14 non-Sphingomonas species were tested using a new Sphingomonas-specific growth medium containing 100 and 50 microg/ml streptomycin and piperacillin, respectively. All the Sphingomonas strains showed positive growth on the selective medium and no growth was shown by the non-Sphingomonas species. In addition, two sets of PCR primers targeting the serine palmitoyltransferase gene (spt), a crucial sphingolipid biosynthesis gene, were developed. With the exception of the Sphingomonas subarctica type strain, 71 of the 72 known Sphingomonas samples were amplified positively by either one or both of the spt-specific primers. None of the non-Sphingomonas bacteria were amplified by the spt primers. To verify the effectiveness of this novel approach for use in environmental screening applications the Sphingomonas selective medium was used to isolate 165 potential Sphingomonas isolates, including 101 yellow, 4 orange and 58 unpigmented isolates, from the influent water and biofilm samples of a pulp and paper mill in Northwestern Ontario. Screening of these isolates with the two Sphingomonas spt-PCR primer sets showed that 98% of the yellow isolates and 100% of the orange isolates were positive to the spt-PCR test. None of the unpigmented isolates was positive to the spt-PCR assay. The 16S rDNA of 17% of the spt+ve and -ve isolates were sequenced and analyzed. All of the yellow and orange pigmented isolates were Sphingomonas while none of the unpigmented isolates were Sphingomonas. REP-PCR was performed on 79 Sphingomonas samples randomly selected from the paper mill and hospital isolates and showed that a diverse group of Sphingomonas can be grown or isolated by our Sphingomonas selective growth medium. Therefore, by using the streptomycin-piperacillin selective growth medium in combination with the colour pigmentation and the positive spt-PCR reactions of the isolates, a diverse population of Sphingomonas strains can be isolated and identified from complex microbial communities with high accuracy.

Evaluation of Propidium Monoazide and Quantitative PCR To Quantify Viable Campylobacter jejuni Biofilm and Planktonic Cells in Log Phase and in a Viable but Nonculturable State.

Despite being considered fragile and fastidious, Campylobacter jejuni remains the leading cause of bacterial gastroenteritis in the developed world. C. jejuni survives stresses by forming biofilms or entering a viable but nonculturable (VBNC) state. To investigate the number of viable cells in samples exposed to low nutrient and temperature stress, a novel method, propidium monoazide quantitative PCR (PMAqPCR), was compared with Bac Light biovolume analysis and conventional plate counting for the enumeration of C. jejuni-removed biofilm cells and separately grown planktonic cells in late log phase (24 h). There were no significant differences between viable cell counts obtained using PMAqPCR and those from plate counts or Bac Light biovolume analyses for each sample, confirming that this method provides results consistent with those from accepted enumeration methods (P > 0.05). To induce a VBNC state, C. jejuni planktonic cells and dislodged and washed biofilm cells were separately incubated in phosphate-buffered saline at 4°C for up to 60 days. Even when cells exposed to stress were provided with enrichment in Bolton broth before plating, treated biofilm cells lost culturability by day 10, whereas their planktonic counterparts remained culturable to day 60. The nonculturable biofilm cells remained viable in high numbers to day 60, and viable cell counts from the PMAqPCR (6.15 log cells per ml) were not significantly different from those obtained using the Bac Light assay (6.98 log cells per ml) (P > 0.05), confirming that this novel method is also reliable for cells exposed to stress for extended periods. PMAqPCR shows promise for analysis where C. jejuni exists in biofilms or in the VBNC state. Adopting PMAqPCR in routine monitoring, in conjunction with improved biofilm cell collection methods, will allow for more accurate enumeration of viable and potentially virulent cells, leading to improved sanitation and reduced incidence of infection.

Electrochemical and microbiological characterization of paper mill biofilms

Biofilm samples collected from inside and outside the press and former sections of paper machines in a Northwestern Ontario paper mill for a period of 2 years were characterized microbiologically and electrochemically. Bacterial community profiling was done using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and selected bacterial isolates were identified using 16S rDNA analysis. The bacterial community showed the presence of Proteobacteria, Firmicutes, and Actinobacteria. Sphingomonas sp. was found to be the most common bacterial species, which showed the highest production of extracellular polymeric substances. Bacteria isolated from biofilms showed better adhesion properties than those from water samples. Cyclic voltammetry and electrochemical impedance spectroscopy studies showed that bacteria isolated from biofilms and feed water collected from inside the machine were more easily oxidized than those from outside, suggesting the need for a more rigorous biofilm abatement strategy for inside paper machines.

Efficient bacterial disinfection based on an integrated nanoporous titanium dioxide and ruthenium oxide bifunctional approach

The increasing lack of drinking water around the globe is of great concern. Although UV irradiation, photocatalysis, and electrocatalysis for bacterial disinfection have been widely explored, the synergistic kinetics involved in these strategies have not been reported to date. Herein, we report on an efficient and cost-effective strategy for the remediation of a model bacterium (E. coli), through the integration of photochemistry and electrochemistry based on a bifunctional electrode, which utilizes titanium (Ti) as the substrate, nanoporous titanium dioxide (TiO2) as a photocatalyst, and ruthenium oxide (RuO2) nanoparticles as an electrocatalyst. The nanoporous TiO2 was grown directly onto a Ti substrate via a three-step anodization process, and its photocatalytic activity was significantly enhanced by a facile electrochemical treatment. A high disinfection rate at 0.62 min-1, with >99.999% bacterial removal within 20 min was achieved using the novel TiO2/Ti/RuO2 bifunctional electrode. Complete bacterial disinfection was attained within 30 min as assessed by a spread plate method. Bacterial survival strategies, including a viable but non-culturable state of the bacteria, were also investigated during the bifunctional treatment process. The novel strategy demonstrated in this study has strong potential to be utilized for water purification and wastewater treatment as an advanced environmentally compatible technology.