Detection of antibiotic-resistant bacteria in treated sewage and

their further survival - a case study of the Białka River, Podhale,

southern Poland

Anna Lenart-Boroń

Department of Microbiology

University of Agriculture in Kraków

COST is supported by the

EU Framework Programme

Horizon 2020

ESF provides the COST

Office through an EC

contract

Study site

Białka river

Sewage Treatment Plant – Czarna Góra

Sampling points

Methodology – enumeration and isolation of bacteria

Escherichia coli Enterococcus faecalis Clostridium perfringens

Mesophilic bacteria Psychrophilic bacteria Staphylococcus spp. Fungi

Identification of E. coli

Disc diffusion antibiotic sensitivity test

20 antibiotics tested – basic and extended antibiogram according to the National Reference Centre for Antimicrobial Susceptibility (KORLD)

Antibiotics tested

Basic antibiogram

• AMP – ampicillin 10 µg

• AMC – amoxicillin/clavulanic acid 2:1

• KF – cephalotin 30 µg

• KZ – cephazolin 30 µg

• MA – cefamandole 30 µg

• CN – gentamicin 10 µg

Extended antibiogram

• PRL – piperacillin 100 µg • TIC – ticarcillin 75 µg • TZP – piperacillin/tazobactam 110 µg • FOX – cefoxitin 30 µg • CTX – cefotaxime 30 µg • CAZ – ceftazidime 30 µg • FEP – cefepime 30 µg • ATM – aztreonam 30 µg • CIP – ciprofloxacin 5 µg • AK - amikacin 30 µg • NET – netilmicin 30 µg • TOB – tobramycin 30 µg • TE – tetracycline 30 µg • SXT – trimetoprime/sulfmethaxazole

1.25/23.75 µg

Drug resistance genes Extended Spectrum β-lactamases (ESBLs) – rapidly

evolving group of enzymes, having the ability to hydrolyze third-generation cephalosporins and aztreonam, but are inhibited by clavulanic acid.

Enzyme detected Characteristics

TEM Widespread plasmidic β-lactamase. First reported in E. coli in 1965 and is now the most common β-lactamase in Enterobacteriaceae. Most ESBLs derive from TEM

SHV The second most common β-lactamase, after TEM. Encoded on large plasmids.

CTX-M ESBL enzyme with greater hydrolytic activity against cefotaxime and increased susceptibility to inhibitors (e.g. clavulanate, sulbactam).

Genetic diversity among E. coli strains

PCR amplification of repetitive bacterial DNA elements (rep-PCR) allows for quick, reliable and low cost typing of a wide range of Gram-negative bacteria. This method is characterized by high discriminatory power. Rep-PCR using BOX A1R primer targets naturally occurring multi-copied, conserved, repetitive DNA sequences present in the genomes of most bacteria. BOX A1R primer allows to amplify specific genomic regions between BOX elements (154 bp) and its inverted repeats.

Results – numbers of E. coli

0

1000000

2000000

3000000

4000000

5000000

6000000

Nov.11

2013

Dec.12

2013

Jan.23

2014

Feb.28

2014

Mar.13

2014

Mar.21

2014

Apr.02

2014

May20

2014

Jun.24

2014

Jul.10

2014

Aug.27

2014

Sep.30

2014

Sewage Treatment Plant

0

1000

2000

3000

4000

5000

6000

7000

Nov.11

2013

Dec.12

2013

Jan.23

2014

Feb.28

2014

Mar.13

2014

Mar.21

2014

Apr.02

2014

May20

2014

Jun.24

2014

Jul. 102014

Aug.27

2014

Sep.30

2014

Intake

Results – numbers of E. coli

0

500

1000

1500

2000

2500

3000

Nov.11

2013

Dec.12

2013

Jan.23

2014

Feb.28

2014

Mar.13

2014

Mar.21

2014

Apr.02

2014

May20

2014

Jun.24

2014

Jul.10

2014

Aug.27

2014

Sep.30

2014

Ski resort

0

5000

10000

15000

20000

25000

30000

35000

40000

Nov.11

2013

Dec.12

2013

Jan.23

2014

Feb.28

2014

Mar.13

2014

Mar.21

2014

Apr.02

2014

May20

2014

Jun.24

2014

Jul.10

2014

Aug.27

2014

Sep.30

2014

Trybsz

Results – numbers of E. coli - TNP

Date E. coli fecal E. coli Staphylococcus E. faecalis

Nov. 11 2013 0 0 0 0

Dec. 12 2013 0 0 0 0

Jan. 23 2014 0 0 0 0

Feb. 28 2014 0 0 0 0

Mar. 13 2014 0 0 0 0

Mar. 21 2014 0 0 0 0

Apr. 02 2014 0 0 0 0

May 20 2014 0 0 0 0

Jun. 24 2014 0 0 0 0

Jul. 10 2014 4 5 0 0

Aug. 27 2014 1 0 0 0

Results - number of Enterococcus faecalis - STP

0

200

400

600

800

1000

1200

1400

1600

1800

Nov. 112013

Dec. 122013

Jan. 232014

Feb. 282014

Mar. 132014

Mar. 212014

Apr. 022014

May 202014

Jun. 242014

Jul. 102014

Aug. 272014

Sep. 302014

E. faecalis

Prevalence of bacteria in snow

0

5000

10000

15000

20000

25000

30000

Natural Artificial 1 Artificial 2

E. coli

fecal E. coli

0

5

10

15

20

25

30

Natural Artificial 1 Artificial 2

Enterococcusfaecalis

Staphylococcusspp.

Drug resistance in E. coli

0

10

20

30

40

50

60

70

80

90

AMP AMC KF KZ MA TIC FEP

Per

cen

tage

of

dru

g re

sist

ance

det

ecte

d

Drug resistance in E. coli

25%

40%

10%

25% 3 antibiotics

2 antibiotics

1 antibiotic

sensitive to all

Prevalence of ESBL genes

Genes Prevalence CTX None detected

OXA None detected

SHV None detected

TEM Present in 20% of isolates

Molecular diversity among E. coli

Conclusions • Seasonal changes within the Białka valley

population size due to touristic activity can be tracked by the quality of sewage reaching the river

• STP underestimated – does not include the increase of the population in winter, especially because many stays are illegal/unregistered, therefore difficult to estimate

• Sewage-derived bacteria survive the production of artificial snow and can pose a threat to the health of tourists

• The high antibiotic resistance detected in bacteria from both river water and artificial snow makes the microbial contamination even more dangerous

Thank you for your attention