This document presents a study on antibiotic-resistant bacteria in the Qi-Shan River, assessing bacterial diversity and resistance patterns between upstream and downstream locations. The research involved sampling water and sediment, bacterial isolation, and testing for antibiotic resistance using disk diffusion methods. Key findings indicate the presence of various resistant bacterial species, raising concerns about environmental and public health implications.

1. Antibiotic-resistant
Bacteria along the Qi-Shan river
SPEAKER CHENG-HAN LIU ADVISOR HANS-UWE DAHMS DATE SEP. 25TH, 2019

2. Antibiotics
2
A type of antibacterial medicine
To prevent/ treat bacterial infections
Bactericidal: killing bacteria
Bacteriostatic: inhibiting bacterial growth
Penicillin Tetracycline
Kümmerer 2009 a,b

3. Antibiotic resistance / ABR
3
Antibiotics emerge in environment
Antibiotics kill environmental bacteria
Resistant bacteria survive from antibiotics
Resistant bacteria thrive and spread
Kümmerer 2009 a,b
Ability to resist antibiotics

4. 4
Health issues
River
Domestic sewage
Agricultural runoff
Industrial wastewater
Hospital wastewater
Input
Output
Water consumption
Aquaculture
Irrigation
Infection Uptake
Danner 2019
=
Reservoir

5. 2. Antibiotic resistance
5
Objectives
1. Diversity of bacteria
Antibiotic resistance effect in river water
Moore 2010
Upstream
Downstream
Resistant ?
Susceptible?
River
=
Reservoir

6. Qi-Shan river / 旗山溪
6
Main tributary of the Kao-Ping river
Agriculture irrigation & drainage
楊萬全, 1997
Source of Nan-Hua reservoir
Importance in health issue

7. 1
2
3
4
5
6
7
8
7
22°47'35.5"N 120°27'46.9"E
23°17'17.2"N 120°43'12.0"E
Samples collection
Sampling completed on a single day in September
Sample
container
8 sampling sites (GPS co-ordinates)
Water and sediments
Moore 2010

8. Bacterial identification
8
Flow chart
Bacterial isolation
Antibiotic resistance test

9. Methods & materials
9
Bacterial isolation
Spread plate Quadrant streak
Colony
forming
unit
Agar plate
Spreader
Inoculation loop
Tryptic soy agar
Nutrient agar
LB agar
Samples processed within 48hrs
Plates incubated at 20°C for 48hrs
100μl inoculated; dilution: 100-10-2
Sprute 2019
Agar plate

10. Phylogenetic tree: Evolutionary
relationships among species
Patel 2001 10
Bacterial identification
Methods & materials
Inoculation
Culture
DNA extraction
Isolated DNA
PCR
PCR amplicon
16S rRNA
Universal
Variable
Evolving at constant rate
Multi-copy gene
DNA extraction: Proteinase K
Sanger sequencing

11. (1-2*108 Colony forming units/mL)
Bacteria conc.: OD600 of 0.06
11
Antibiotic resistance test
Disk diffusion
Methods & materials
Inhibition zone
Inoculation
Culture
Plates incubated at 35°C for 24hrs
Swab streak back-and-forth
Minimum inhibitory concentrationAntibiotics
Erythromycin
Gentamicin
Vancomycin
Ciprofloxacin
Amoxicillin
Tetracycline
Trimethoprim
Ceftriaxone
Müller-Hinton agar
Hudzicki 2009

12. 2. Antibiotic resistance
X: Distance from river source
Y: Bacterial resistance
DownstreamUpstream
12
Expected results
1. Diversity of bacteria
• Possible isolated species
• Possibly diverse in downstream
Antibiotic resistance of bacterial populations in the Qi-Shan river
Moore 2010 Ayandiran 2014
Enterobacter spp.
Enterococcus spp.
Staphylococcus aureus
Pseudomonas spp.
Bacillus spp.
Escherichia coli
Upstream
Downstream
1
8

13. 1. Diversity of bacteria
13
Discussion
Naturally intrinsic ABR
Acquired ABR in pathogens
Acquired ABR from exposure
2. Antibiotic resistance
Resistant ?
Susceptible?
Upstream
Downstream
Moore 2010

14. Ubiquitous
Likely transferring resistance gene
Pathogenic
14
Discussion
Diversity of ABR bacteria
Enterobacter spp.
Enterococcus spp.
Staphylococcus aureus
Pseudomonas spp.
Bacillus spp.
Escherichia coli
Which species to test for resistance ?
Common in polluted waters
Considerations:

15. Kümmerer, K., 2009. Antibiotics in the aquatic environment–a review–part I. Chemosphere, 75(4), pp.417-434.
Kümmerer, K., 2009. Antibiotics in the aquatic environment–a review–part II. Chemosphere, 75(4), pp.435-441.
Moore, J.E., Moore, P.J., Millar, B.C., Goldsmith, C.E., Loughrey, A., Rooney, P.J. and Rao, J.R., 2010. The
presence of antibiotic resistant bacteria along the River Lagan. Agricultural Water Management, 98(1),
pp.217-221.
楊萬全, 1997. 高屏溪流域和屏東平原的水資源. 國立臺灣師範大學地理研究報告.
Danner, M.C., Robertson, A., Behrends, V. and Reiss, J., 2019. Antibiotic pollution in surface fresh waters:
occurrence and effects. Science of the Total Environment , 664, pp.793-804.
Sprute, J., 2019. Streaking Agar Plates: 4 Quadrant Streak Method. [online] Microbiology learning: The "why"
ology of microbial testing. Available at: https://microbiologylearning.weebly.com/streaking-agar-plates-4-
quadrant-streak-method.html
Patel, J.B., 2001. 16S rRNA gene sequencing for bacterial pathogen identification in the clinical
laboratory. Molecular Diagnosis, 6(4), pp.313-321.
Hudzicki, J., 2009. Kirby-Bauer disk diffusion susceptibility test protocol [manual].
Ayandiran, T.A., Ayandele, A.A., Dahunsi, S.O. and Ajala, O.O., 2014. Microbial assessment and prevalence of
antibiotic resistance in polluted Oluwa River, Nigeria. The Egyptian Journal of Aquatic Research, 40(3),
pp.291-299.
References

16. Thank you for attention
SPEAKER CHENG-HAN LIU ADVISOR HANS-UWE DAHMS DATE SEP. 25TH, 2019
Q & A