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Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year. We report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre-treatment step, the FastDNA® Spin kit, the UltraClean™ and PowerSoil™ soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities. M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25×10(5) cells g(-1) using Griffiths and 4.25×10(6) cells g(-1) using FastDNA® Spin kit.

IncP-1, IncP-7 and IncP-9 plasmids often carry genes encoding enzymes involved in the degradation of man-made and natural contaminants, thus contributing to bacterial survival in polluted environments. However, the lack of suitable molecular tools often limits the detection of these plasmids in the environment. In this study, PCR followed by Southern blot hybridization detected the presence of plasmid-specific sequences in total community (TC-) DNA or fosmid DNA from samples originating from different environments and geographic regions. A novel primer system targeting IncP-9 plasmids was developed and applied along with established primers for IncP-1 and IncP-7. Screening TC-DNA from biopurification systems (BPS) which are used on farms for the purification of pesticide-contaminated water revealed high abundances of IncP-1 plasmids belonging to different subgroups as well as IncP-7 and IncP-9. The novel IncP-9 primer-system targeting the rep gene of nine IncP-9 subgroups allowed the detection of a high diversity of IncP-9 plasmid specific sequences in environments with different sources of pollution. Thus polluted sites are \"hot spots\" of plasmids potentially carrying catabolic genes.

Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year. We report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre-treatment step, the FastDNA® Spin kit, the UltraClean[TM] and PowerSoil[TM] soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities. M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25x10.sup.5 cells g.sup.-1 using Griffiths and 4.25x10.sup.6 cells g.sup.-1 using FastDNA® Spin kit.

Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year. We report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre-treatment step, the FastDNA® Spin kit, the UltraClean[TM] and PowerSoil[TM] soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities. M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25x10.sup.5 cells g.sup.-1 using Griffiths and 4.25x10.sup.6 cells g.sup.-1 using FastDNA® Spin kit.

Infections caused by antimicrobial resistant bacterial pathogens are fast becoming an important global health issue. Strains of Escherichia coli are common causal agents of urinary tract infection and can carry multiple resistance genes. This includes the gene blaCTX-M-15 that encodes for an extended spectrum β-lactamase (ESBL). While studying antimicrobial resistance (AMR) in the environment we isolated several strains of E. coli ST131 downstream of a WWTP in a local river. These isolates were surviving in the river sediment and characterisation proved that a multi-resistant phenotype was evident. Here, we show that E. coli strain 48 (river isolate ST131), provided a protective effect against a third-generation cephalosporin (cefotaxime) for a susceptible E. coli strain 33 (river isolate ST3576) through secretion of a functional ESBL into the growth medium. Furthermore, extracellular ESBL activity was stable for at least 24 h after secretion. Proteomic and molecular genetic analyses identified CTX-M-15 as the major secreted ESBL responsible for the observed protective effect. In contrast to previous studies, OMVs were not the sole route for CTX-M-15 secretion. Indeed, mutation of the Type I secretion system led to a significant reduction in the growth of the ESBL-producing strain as well as a significantly reduced ability to confer protective effect. We speculate that CTX-M-15 secretion, mediated through active secretion using molecular machinery provides a public goods service by facilitating the survival of otherwise susceptible bacteria in the presence of cefotaxime. Competing Interest Statement The authors have declared no competing interest.