Bacteriological analysis of the water in El Caño Martín Peña

1. Bacteriological analysis of the water in El CañoMartín Peña Detection and quantification of Enteric Pathogenic BacteriaSalmonella and Shigella spp. Mario R. Barrera George M. Rivera Paola Tabaro Mentor: Prof. Mayra Rolón University of the Sacred Heart 1 / march/ 2013

2. Meet the Team George M. Rivera Mario R. Barrera Paola Tabaro Mentor: Prof: Mayra Rolón

3. Meet the Team 2013 2012

4. Caño Martín Peña • Is an invaluable natural habitat for the Metropolitan area of San Juan.

5. Caño Martín Peña + During the recurring years the mangroves and lagoons stretched of the Caño have been contaminated and stretched out into smaller measurements.

6. Caño Martín Peña The deterioration of this area has taken its toll on the residents lifestyles.

7. How to Improve !! • The decontamination of the lagoon that boards the community. • Rehabilitation of the estuary system. • To broaden the community’s knowledge of the microbial presence and the harms that can be encountered.

8. Our objectives • + To detect the presence of pathogenic enteric bacteria such as Salmonella and Shigella SPP. • + To acquire knowledge of the microbial presence found in the Caño Martín Peña.

9. + Refine research skills through different bacteriological techniques. • + Analyze and compare the degree of contamination found with past investigations. • + Raise awareness to the community.

10. Hypothesis • In the past year our fellow classmates were able to detect the presence of Shigella serotypes A,B and C. • The Caño Martín Peña has excessive levels of coliforms, indicators of fecal matter that represent a hazard for the community.

11. Hypothesis + Because of this information we can say there is a high probability of encountering Salmonella and Shigella SPP. Bacteria due to the high coliform levels resulting from water contamination by fecal matter.

12. Coliform bacteria • Rod-shaped Gram negative bacillus. • Lactose fermenters with the production of acid and gas when incubated at 35-37°C. • Easy to culture and their presence is used to indicate fecal contamination by other pathogenic organisms in the water.

13. Enterobacteriaceae • Total coliforms: • Escherichia • Klebsiella • Serratia • Citrobacter • Fecal coliform: • Eschericia coli

14. Escherichia coli

15. Klebsiellapneumoniae

16. Enterobacter

17. Serratiamarcescens

18. Citrobacter

19. Salmonella spp. VsShigella spp. Salmonella spp. Shigella spp. Rod-shaped Gram negative bacteria Non-spore forming and non-motile bacteria • Rod-shaped Gram negative bacillus • Non-spore forming. Motile enterobacteria • Facultative anaerobes • Hydrogen sulfide production which can be detected on a TSI growth media

20. Salmonella Pathogenicity • Most of the infections are due to the ingestion of contaminated food. • Salmonella is responsible for various illnesses such as: • Typhoid fever • Paratyphoid fever • Foodborne illnesses • Enteritis Salmonella also known as food poisoning Salmonella.

21. Shigella Pathogenicity • Most of the infections are caused via ingestion. • The illness is known as Shigellosis. • During infection, Shigella typically causes dysentery . • Invasion of Shigella • Bacterial multiplication • Spreading to epithelial cells • Tissue destruction

22. Investigation Site

23. Station #1 • Width : 8 Ft. • Depth: 2.05 Ft • Observations • Boat nearby • Carton Boxes

24. Station #2 • Width: 3 – 5 Ft • Depth: 4 Ft • Observations • Current of water coming from a pipe. • Fishing traps

25. Station #3 • Width: 8 Ft. • Depth: 2 Ft. • Observations • Samples were taken at the shore, because difficult access. • Garbage bags

26. Methodologies • Dilution of sample • Membrane filtration • Cultivation • SS Agar • MacConkey Agar • EMB • RVS • Identification • Enterotubes

27. Procedure

28. Dilution of sample

29. At the Laboratory

30. Membrane filtration 0.45 μm

31. At the Laboratory

32. Membrane filtration Pure sample Diluted Sample 1:1000

33. Cultivation • At 37º C. • 24 – 48 Hours

34. Selective Mediums Salmonella Shigella Agar MacConkey Agar

35. Selective Mediums RVS EMB

36. Taxonomic Determination:Enterotube System

37. Taxonomic Determination:Enterotube System

38. Enterotube System Citrobacterfreundii Serratiaplymuthica

39. Sampling Dates • M1 – 30/ October/ 2012 • M2 – 28/ November/ 2012 • M3 – 7/ December/ 2012 • M4 – 30/ January/ 2013 • M5 – 11/ February/ 2013

40. Types of Bacteria

41. Types of Bacteria

42. Enterotubes

43. ShigellaEnterotube

44. Findings Number of bacteria/ ml of water

46. Findings on Dilutions • Station #1 • 1 col in 10¯⁷ : 10,000,000 bact/ml • Station #2 • 4 col in 10⁻⁶: 4,000,000 bact/ml • Station #3 • 62 col in 10⁻⁶: 62,000,000 bact/ml

47. Findings on Filtration • Station#1 • Pure > 100 col (TNTC) • Dilute: 5 col * 1000: 5,000 bact/100ml • Station #2 • Pure>100 col (TNTC) • Dilute: 40 col * 1000: 40,000 bact/100ml • Station #3 • Pure>100 col (TNTC) • Dilute: 3 col * 1000: 3,000 bact/100ml Citrobacterfreundi Cedecealapagei Klebsiellapneumoniae Yersinia enterocolitica Citrobacterfreundi Cedecealapagei Enterobacteragglomerans Klebsiellaozaenae Enterobacteragglomerans

48. Conclusions • The high level of contamination is due to: • Stagnant water in some areas. • Sewage and water discharge in to the cano. • Deposition of debris into the community.

49. Conclusions of the Research • From 2004 to 2008 the amount of bacteria were less than a million/ ml of water. • This could be due to the removal of debris • Effort of government and people that lives in the community.

50. + During our Research period we noticed a sudden decrease in the level of bacteria. + Although we were not able to isolate colonies of Salmonella, we did isolate Citrobacterfreundi, Yersinia enterocolitica and Shigella Spp. + Which are indicative of the severity of contamination and a possible source of contamination with pathogens to the residents of nearby areas.