Valéria M. GodinhoFernanda M. S. NascimentoSilvana Q. SilvaMarcos von Sperling

Characterization of

pathogenic bacteria in a

UASB-polishing pond system

using molecular techniques Federal University of Minas Gerais – UFMGBrazil

8th IWA Specialist Group Conference on Waste StabilizationPondsDATE: April 26 to 30, 2009 - VENUE: Belo Horizonte/MG

INTRODUCTION

Pathogenic bacteria in wastewater systems

• UASB-Polishing ponds

• Organic and inorganic matter removal

• Pathogenic bacteria decay

• pH, UV, T, DO ...

• Evaluation of pathogenic bacterial decay

• Indirectly by faecal (thermotolerant) coliforms and E. coli

INTRODUCTION

Pathogenic bacteria in sewage

INTRODUCTION

Pathogenic bacteria analysis

INTRODUCTION

Pathogenic bacteria analysis

• Traditional techniques

• Cultivation and Isolation

• Limitations:

• Specific culture media and growth conditions

• Similar physiology

• E. coli and Shigella

• Fastidious organisms

• Campylobacter and Helicobacter

INTRODUCTION

Pathogenic bacteria analysis

• Molecular techniques

• Polymerase Chain Reaction method (PCR)

INTRODUCTION

Pathogenic bacteria analysis

• Molecular techniques

• Fluorescent In situ hybridization (FISH)

INTRODUCTION

Pathogenic bacteria analysis

• Literature

• PCR and FISH have been applied to investigatepathogenic microorganisms in different types ofsamples, including water and wastewater samples

(Lee et al., 2006; Peng et al., 2002, Alonso et al., 2006).

AIM

To investigate the occurrence of potential pathogenic bacteria

(Escherichia coli, Salmonella enterica subsp. enterica-ser-

Typhimurium, Shigella spp., Enterococcus spp., Helicobacter

pylori, Staphylococcus aureus) in a wastewater plant

composed by a UASB reactor followed by polishing ponds

using molecular techniques.

METHODS

Description of the wastewater treatment plant

Center for Research and Trainning in Sanitation (CePTS)

UFMG - COPASA

Figure 1 – Flowsheet of the treatment system

WWTP – composed by UASB followed by serial ponds

METHODS

Pathogenic bacteria detection

Raw sewage UASB Pond 1

Pathogenic bacteriacharacterization DNA extraction (Egli et al. 2003)

PCR amplification (specific primers)

Samplesconcentration 4,000 rpm for 20 min filtered at 8 µm and 1.2 µm

2L centrifuged at 4,000 rpm for 20 min

Resuspended in PBS

Oligonucleotides (primers) used for pathogenic bacteria investigation

Target bacteria Name Sequence 5’ 3’

Amplicon size(bp)

Reference

Primers for PCR

E. coliL-uid739 tggtaattaccgacgaaaacggc

840 Bej (2004)Bej et al. (1991)R-uid578 gtggcgaaatattcccgtgcact

S. enterica subsp. enterica

L-himA cgtgctctggaaaacggtgag123 Bej et al. (1994)

R-himA cgtgctgtaataggaatatcttca

Enterococcus spp.Ent1 tactgacaaaccattcatgatg

112 Ke et al. (1999)Ent2 aacttcgtcaccaacgcgaac

Shigella dysenteriaeL-phoBR attgaagccgcgccgacgcaa

152 Bej (2004)R-phoBR cgttgcctgacaccttgaggg

Helicobacter pylori16SHP1 gcaatcagcgtcagtaatgttc

521 Lu et al. (2002)16SHP2 gctaagagatcagcctatgtcc

Staphylococcus aureus

nuc1 gcgattgatggtgatacggtt270 Burtscher and

Wuertz (2003)nuc2 agccaagccttgacgaactaaagc

METHODS

Pathogenic bacteria detection

PCR Strategy

PositiveOK

Negative• ↑ [sample• ↓ [sample] dilution••

“Nested PCR”,

Positive, butinespecific

• ↓ [sample] dilution

METHODS

Escherichia coli quantification

Raw sewage Pond 1

FISH 200 mL 4,000 rpm for 20 min filtered at 8 µm andcentrifuged at 4,000 rpm for 20 min

Resuspended in PBS

Fixation in 4% paraformaldehyde (Amann et al.1990)

Hybridization conditions with probe ECO 1167: 30% formamide, 46oC 2h (Hann et al. 2001)

Epifluorescence visualization of 15 microscopic fields

Colilert 100 mL IDEXX Quanti-Tray®/2000 - Colilert® system

RESULTS

Detection of pathogenic bacteria

DNA extraction

DNA extractions from samples and pure culture of a) Escherichia coli, b) Salmonella spp. c) Enterococcus spp., d) Shigella dysenteriae, e) Staphylococcus aureus

DNA from Helicobacter pylori was obtained from Hospital das Clínicas - UFMG

RS UASB P1

a b C d e

RESULTS

Detection of pathogenic bacteria by PCR

Optimization of PCR conditions for each pairof primers with positive and negative controls

a) Escherichia coli, b) Enterococcus faecallis,

c) Helicobacter pylori, d) Salmonella sppe) Shigella sonnei

f) Staphylococcus aureus,

Negative controls:Salmonella (a, b),

E. coli (c, d, f), Yersiniaenterocolítica (e).

RESULTS

Detection of pathogenic bacteria by PCR

d

RSP1RS

d

RS

RS

P1RS

P1RS

d

ca db

RS P1 RS P1 P1P1

RSRS

PCR products from amplification of DNA from: a) Escherichia coli, b) Salmonella entericasubsp. enterica, c) Enterococcus spp., d) Shigella dysenteriae.

Bacteria RS UASB P1Escherichia coli + + +S. enterica subsp. enterica + + +Enterococcus spp. + + -Shigella dysenteriae + + -Helicobacter pylori - - -Staphylococcus aureus - - -

Detection of pathogenic bacteria

DISCUSSION

Detection of pathogenic bacteria by PCR

Limit of detection for PCR: 102 or 103 cells/mL (Sharma and Carlson, 2000).

RESULTS

E. coli quantification by FISH

Control experimentPure culture of Escherichia coli visualized under a) phase contrast microscopy

b) Epifluorescence with DAPI, c) Epifluorescence with probe Eco1167.

a b c

RESULTS

E. coli quantification by FISH

RS – E. coli + probe ECO 1167

P1 – E. coli + probe ECO 1167

Statistics Sampling Raw sewage Pond 1

Mean(cells/100mL)

1 3.92x107 3.82x106

2 9.78x107 4.17x106

3 2.46x107 3.56x106

Standard deviation (cells/100mL)

1 9.50x106 3.52x105

2 1.91x107 6.73x105

3 7.07x106 1.05x106

Coefficient of variation

1 0.24 0.09

2 0.20 0.16

3 0.29 0.29

Quantification of E. coli by FISH technique

RESULTS

E. coli quantification by FISH

Sampling Raw sewage Pond 1

Mean(MPN/100mL)

1 3.1 x107 4.6 x105

2 3.2 x108 1.8 x106

3 2.1 x107 9.8 x104

Quantification of E. coli by Colilert

DISCUSSION

E. Coli quantification

• Estimated removal efficiency of E. coli – 85 to 95% for FISH – 98 to 99% for Colilert®– Ponds in series are able to reduce considerably the number of E. coli cells during the

treatment. • Previous data based on Colilert® showed that the complete WWT plant was able

to reduce 99.99% of E. coli cells (103 to 104 MPN/100mL in the final effluent (von Sperling and Mascarenhas, 2005; von Sperling, 2008).

• Difference in the efficiency of E. coli removal methodological– FISH - quantification is based on counting individual cells – Colilert® - based in MPN table.

• It is believed that counting individual cells is more precise for estimating microorganisms than cultivation techniques

– however ….

DISCUSSION

E. Coli quantification

• FISH bias (regarding to polishing ponds effluent samples)– Presence of autofluorescent algae overestimation of cell

number.• Exclusion by cell size

– Morphology of bacterial cells may change in response of low levels of available substrate present in polishing ponds.

• In this study the cells of E. coli in the P1 effluent were smaller than E. coli cells from the RS

– Algae removal and sample concentration• Need to satisfy the detection limit of the technique (103 to 104

cells/mL, Amann et al., 1995). – Probes based on ribosomal RNA

• Fluorescent signal is proportional on ribosome content

CONCLUSIONS

• E. coli and Salmonella enterica subsp. enterica, present in the RS were not completely removed in the investigated system

• Enterococcus spp. and Shigella dysenteriae were not detected in the P1 effluent, suggesting that the number of cells were considerably reduced along the treatment.

• Helicobacter pylori and Staphylococcus aureus were not detected in the RS neither in UASB and P1 effluents

– the number of cells from these species is probably not high enough to be detected by PCR (102 or 103 cells/mL, Sharma and Carlson, 2000).

• E. coli measurements by FISH (RS:107 and P1:106 ) were up to 2 log units higher than the Colilert technique

CONCLUSIONS

• PCR is a good method for pathogenic bacteria detection but gives only qualitative results

• FISH technique for evaluating E. coli decay …– low levels of pathogenic bacteria in polishing pond effluents

(usually is in the range of the detection limit of the technique)– Probe signal depends on the amount of ribosomal RNA inside the

cells, which is proportional to the cell activity• Are they active enough to be detectable?

• Future studies: Real Time PCR (quantitative PCR)

ACKNOWLEDGEMENTS

• UFMG

• PROSAB

• FINEP

• CNPq

• FAPEMIG