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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., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

A total of 177 naturally contaminated water samples were analyzed by membrane filtration according to the Standard Methods for the Examination of Water and Wastewater published by the American Public Health Association. Filters were incubated in parallel on mHPC-agar and 3M Petrifilm Aerobic Count Plates (Petrifilm AC plates) for heterotrophic counts. Fecal coliforms and Escherichia coli were enumerated on mFC-agar and 3M Petrifilm E. coli/Coliform Count Plates (Petrifilm EC plates). Typical colonies on each media type were confirmed following standard procedures. Heterotrophic counts were between 10(3) and 10(4) CFU/mL and the average log10 counts obtained on Petrifilm AC plates were about two-fold lower than on mHPC-agar. Counts for fecal coliforms and E. coli were between 10(2) and 10(3) CFU/mL. Average log10 counts for confirmed fecal coliforms obtained on Petrifilm EC plates were slightly lower than on mFC agar with a correlation coefficient of 0.949. The average log10 counts for confirmed E. coli on Petrifilm EC plates and on mFC agar were statistically not different (P=0.126) with a correlation coefficient of 0.879. Specificity of Petrifilm EC plates and mFC agar was evaluated by comparing typical colony counts with confirmed counts. On mFC agar, counts for typical colonies were by 2 log10 CFU higher than the actual confirmed counts. In contrast, on Petrifilm EC plates typical colony counts were almost identical to confirmed colony counts for both fecal coliforms and E. coli. This comparison illustrates the high specificity of Petrifilm EC plates for enumeration of both fecal coliforms and E. coli in water.

A multiplex PCR-DNA probing assay was developed to detect four major Escherichia coli virotypes. Six highly specific polymerase chain reaction (PCR) primer sets and DIG-labeled chemiluminescent probes were designed to target the Shiga-like toxin I and II genes (stxI and stxII) of verotoxigenic E. coli (VTEC), heat-stable and heat-labile toxin genes of enterotoxigenic E. coli (ETEC), adherence factor (EAF) of enteropathogenic E. coli (EPEC) and a fragment of the invasiveness plasmid (IAL) of enteroinvasive E. coli (EIEC). The primer pairs generate products of 350, 262, 170, 322, 293 and 390 bp in length, respectively. The multiplex primers and probes were tested for specificity against 31 pathogenic E. coli strains, nine nonpathogenic E. coli and non-E.coli enteric and environmental bacterial strains. The results showed a high degree of specificity of the primers and probes for strains from corresponding virotypes and no reaction with the nontarget bacterial strains. The proposed multiplex PCR-DNA probing assay provides rapid and specific detection of four major virotypes of E. coli.

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.