Developments in Microbial Source Tracking

Transcription

1 "Starcross and the Brunel Pumping Station from the Exe - geograph.org.uk " by Sarah Charlesworth. Licensed under CC BY-SA 2.0 via Commons "Devon UK location map" by Contains Ordnance Survey data Crown copyright and database right. Licensed under CC BY-SA 3.0 via Commons Developments in Microbial Source Tracking Jonathan Porter, Edyta Bartkowska, Rob Bonsor, Andy Gawler, Matilda La Trobe, Rachel Long, Natalie Nicholson, Louise Pearce, Nick Pye, Keith Salter, Steve Wilde National Laboratory Service, Starcross, Devon. Tel jonathan.porter@environment-agency.gov.uk

2 Source Tracking If we know where the bacteria are coming from, we can target our work : humans, ruminants 08-09: dogs, birds 10-12: cows, sheep, pigs

3 Well, that just about wraps things up then. All UK microbiological water quality issues can now be resolved.

4 What does MST data tell you? case study. >40 bathing water samples collected Eight samples had high bacti counts. Agricultural sources dominated seven out of eight. We are NOT able to state ( )/7 = 79% of the faecal bacteria are from ruminant sources. We ARE able to state that faecal bacteria associated with ruminant animals dominated in these samples. Interpret the pattern with local knowledge. This recommendation is still in place. %Hum %Rum

5 MST a work in progress: o bird markers unsatisfactory o bacterial isolates (E. coli) o viruses (and other pathogens) o data analysis o horses

6 MST for sea birds. Catellicoccus marimammalium [Catmar]

7 Example Catmar data, known samples Sample Catmar Water (expected positive) 4.35 Water (expected negative) 1.91 Gull 8.70 Goose 3.35 Swan 3.56 Pheasant 0 Duck 3.84 Pigeon 3.81 Data are log 10 copies /litre or /g wet weight

8 MST a work in progress: o bird markers unsatisfactory o bacterial isolates (E. coli) o viruses (and other pathogens) o data analysis o horses

9 MST using culturable E. coli Simon Davis/DFID

10 MST using culturable E. coli EXPECTED T A G C G G A G C G G A A T C G C C A T C G C C T T

11 BUT SOME ISOLATES G C G G A T A G C G G A T C G C C A T C G C C T T

12 Unclassified isolates 2 main types Animal E. coli 2 mismatches in probe Human E. coli 3 mismatches in forward primer, and final base in forward primer overlaps with probe sequence. One isolate excellent match to E. fergusonii and various Salmonella enterica serovars. Clearly contains the uid gene and phenotypically β-glucuronidase positive.

13 Human + animal E. coli by DNA 5.50 MST using culturable E. coli y = 0.8x R² = Total E. coli by the bathing water directive

14 MST a work in progress: o bird markers unsatisfactory o bacterial isolates (E. coli) o viruses (and other pathogens) o data analysis o horses

15 MST using viruses Adenovirus Norovirus (GI and GII) Enterovirus (coxsackievirus)

16 Observed frequency MST using viruses data Censored data: <X values (also applies to >X, but very few of these) Geometric mean used as general descriptor Cannot substitute zero; have substituted data points one sig. fig. below LOD (e.g. <10 becomes 9; <1 becomes 0.9) Frequency of detection approach used as part of the detailed analysis River water Overall Winter Summer log 10 Virus gene copies per litre

17 Viruses can show exceptional host specificity - huge array of viruses as MST options - lack of basic environmental data on most of them! - not the sole, or ultimate, answer for MST, but useful information? - indicators vs. pathogens Mdk572 licensed under the Creative Commons Attribution-Share Alike 3.0

18 MST a work in progress: o bird markers unsatisfactory o bacterial isolates (E. coli) o viruses (and other pathogens) o data analysis o horses

19 MST data analysis We often get queries asking for guidance on MST data - Partly because it s new - Partly because it is sometimes apparently confused (Rare we get boxes of chocolates...) Data requires more than just general descriptors

20 log 10 MST marker per 100 ml Shellfish waters, south Devon. Data from adjacent catchment consistently suggested ruminant sources were significant. This dataset apparently much more confused HuBac RuBac log 10 Escherichia coli per 100 ml

21 % MST marker 100 Explored 80 the data, looking for patterns %Human %Ruminant 20 0 Early Late

22 48h rainfall a key variable: human when dry, ruminant when wet, but extent of change was sub-catchment specific. Site, rainfall Hum>Rum Mean E. coli (% of samples) /100 ml Site 1, dry Site 2,wet Site 2, dry Site 2, wet Site 3, dry Site 3, wet

23 Exploring MST data: ratios of markers mtbird Catmar Bird/Catmar Duck Gull Pigeon Swan Data are log copies /1000 ml. or /g wet weight

24 Exploratory MST data analysis 2 bathing waters, approx 10 km apart; multiple sample sites for each bathing water. E. coli and IE as standard directive parameters. MST: various data points from: HuBac - human-specific bacteria mthuman - human DNA RuBac ruminant-specific bacteria mtbird - bird DNA Catmar - sea-bird, bacterial Local knowledge birds may be important; wanted to differentiate (ducks and/or pheasants) from (seabirds)

25 Exploratory MST data analysis Basic plots of the data, looking for patterns. Site-specific data has helped the investigation. very strong Catmar signal at one sample point. different sample site with possible pheasant/duck impact: mtbird:catmar anything >1 is non-seabird species; anything < 1 is from seabirds. Local duck and pheasant samples support this claim (mtbird:catmar ,000) Data from water samples all suggest mtbird:catmar <1, therefore bird pollution more likely to be seabirds.

26 Exploratory MST data analysis Can we produce an overall picture? Probably too few data points. All data combined; RuBac decreased over time, HuBac increased. Sorting data according to HuBac: when HuBac is high, HuBac correlates with E. coli. when HuBac is low, Catmar correlates with IE.

27 MST a work in progress: o bird markers unsatisfactory o bacterial isolates (E. coli) o viruses (and other pathogens) o data analysis o horses

28 Linking MST data with the presence of pathogens Bear with me while I ramble...

29 Pathogenic E. coli Diversity of mechanisms causing illness Specific phage carry toxin genes Plasmid DNA carries adherence genes Toxin genes; VT1 and VT2. Adherence gene(s); eae. Typically, E. coli O157:H7 in the UK are VT2 positive; eae positive.

30 CDC: Dr. Patricia Fields, Dr. Collette Fitzgerald. Photo Credit: Janice Carr Pathogenic bacteria Pathogenic bacteria - enrichment cultures (presence/absence) E. coli pathogenicity genes VT1 & VT2 (toxins) eae (attachment) Campylobacter jejuni hipo Campylobacter coli cadf

31 Linking MST data with the presence of pathogens When does bear become bore?

32 Bringing this bit together: Combining MST data and pathogen data MST marker Pathogen a b c d Low Present High Present Low Absent High Absent

33 Associations of pathogens with different sources? HuBac>RuBac eae High HuBac Low HuBac Total Present Absent Total a/b 0.67 c/d 1.47 Odds Ratio 0.45

34 Associations of pathogens with different sources? Shellfish waters, south Devon Pathogenicity marker Odds ratio (High ruminant pollution) VT VT eae 1.56 VT1/VT2/eae 1.63 VT2/eae 2.72

35 Associations of pathogens with different sources? Some jumps in the chain, but an increased likelihood ( 2.7) of finding VT2 and eae when ruminant pollution exceeded human pollution. Many clinical cases of E. coli O157 are VT2/eae positive Not absolute but interesting...

36 Thus emboldened, we marched bravely on... Bacterial pathogen Odds ratio Viral pathogen Odds ratio VT Enterovirus n/a VT2 6 NV GI 0.4 eae 3.8 NV GII 1.0 VT1/VT2/eae 9.2 Adenovirus 0.7 VT2/eae 6 hipo 5.1 cadf 1.5 cadf/hipo 3.9 Odds ratios for the presence of the microbial pathogens when ruminantspecific pollution exceeds human-specific pollution.

37 MST a work in progress: o bird markers unsatisfactory o bacterial isolates (E. coli) o viruses and pathogens o data analysis o horses

38 Image courtesy of Brian Snelson, Hockley, Essex, England, licensed under the Creative Commons Attribution 2.0 Generic license MST for horses. Mitochondrial Bacterial

39 MST a work in progress: o seabird marker Catmar o E. coli isolates a step closer o viruses and pathogens o data analysis slowly gaining confidence o horses mitochondrial and bacterial

40 icrew

41 Thank you!