Urban Water Security Research Alliance Antibiotic Resistant Bacteria - PowerPoint PPT Presentation

Urban Water Security Research Alliance Antibiotic Resistant Bacteria in Hospital Wastewaters and Sewage Treatment Plants Mohammad Katouli Hospital Wastewater Science Forum, 19-20 June 2012

Antibiotic resistance among bacteria occurs because of: • Increase due to regular use of antimicrobial agents in: – Clinical settings – Farming – Agriculture • Selective pressure

Horizontal gene transfer a common mechanism for developing resistance among bacteria

Antibiotic resistant bacteria (ARB) in hospitals  Reservoirs for large numbers of pathogenic bacteria (resident strains and introduced strains from communities).  High usage of antibiotics to treat infections in patients; selective pressure  Concern with transmission and long term survival in the environment

Dissemination of ARB from hospitals • Routes of dissemination to society: – hospital wastewater, – discharged patients etc. • Antibiotics in wastewater • Excretion in urine and faeces • Direct disposal (Additional selective pressure on bacteria while in wastewater)

Transmission of ABR bacteria into the environment

Sewage treatment plants (STPs) and ABR bacteria Questions: • Are ARB strains found in HWW persistent clones of hospitals? • Can they survive transition to STPs? • Can they survive treatment stages and be released into the environment?

Selected bacteria Staphylococcus aureus (G +ve bacterium) • Causes a wide ranges of diseases in human • e.g. Produces toxin in the blood causing scalded skin syndrome • e.g. Common cause of furuncle (boil), carbuncle etc. Escherichia coli (G -ve bacterium) • Normal inhabitant of the gut • Pathogenic strains cause intestinal (e.g. diarrhoea) or extra intestinal infections (e.g. septicemia, UTI etc.)

Sites and sampling A hospital (H1) with main wastewater outlet pipe and its receiving STP, located in South-east Queensland were sampled for 8 weeks Hospital site: main wastewater outlet pipe STP sites: incoming influent (STP-I) and outlet (STP-O) site E. coli (n=245 isolates UHWW (n= 120), STP-I (n=102) and STP-O (n= 23). S. aureus (n=167) UHWW (n=85), Hospital wastewater STP-I (n=74) and STP-O (n=8). sampling site

HWW and STP samples Cultivation and identification on selective medium Confirmation using species specific genes nuc Gene usp A Gene S. aureus ID gene E. coli ID gene

Disk diffusion method (CLSI) was used to test resistance of bacteria For S. aureus (9 antibiotics) tetracycline (30), amoxycillin-clavulonic acid (AMC), ampicillin (AMP), gentamicin (CN), ciprofloxacin (CIP), chloramphenicol (CHL), amikacin (AK), cefoxitin (FOX) and vancomycin (VAN). For E. coli (16 antibiotics) tazocin (TZP), cefotetan (CTT), cefpodoxime (CPD), cefoxitin (FOX), imipenem (IMI), gentamicin (GEN), nitrofurantoin (NIT), trimethoprim (TMP), sulphafurazole (SF), sulphamethoxazole (RL), tetracycline (TET), ciprofloxacin (CIP), chloramphenicol (C), nalixidic acid (NAL), kanamycin (AK), and norfloxacin (NOR).

Typing Developing a finger print for isolates High-resolution Random Amplified Polymorphic Biochemical Fingerprinting DNA (RAPD) PCR (PhP method) Isolates belonging to the same PhP / RAPD type were considered as members of the same clonal group and classified as common (C) types

Bacterial isolates are inoculated into the microplate seeded with substrates Plates are incubated and read at different time intervals Data are transferred to a computer and processed

Isolate Biochemical fingerprint Identical isolates 1 12 3 21 15 10 24 4 19 10 12 23 2 21 12 9 14 12 21 15 18 12 9 16 3 12 12 15 3 22 3 16 8 23 19 4 2, 4 and 9 4 21 12 9 13 12 21 15 19 12 8 16 5 24 11 7 23 19 12 18 3 7 12 19 6 12 23 14 4 15 5 18 3 17 12 24 6 and 7 7 12 23 14 3 15 5 18 4 17 12 24 8 3 12 23 18 14 11 21 19 3 14 12 9 21 12 9 14 12 21 15 18 11 9 16 10 6 13 23 18 14 12 21 19 3 14 11 For each bacterial isolate, it yields a biochemical fingerprint Similarity between the isolates is shown in a dendrogram .

PhP/RAPD Sites Where Bacteria Were Weeks where Bacteria were Found C-types Found W1 W2 W3 W4 W5 W6 W7 W8 E. coli C1 UHWW and STP-I + + - - - - + + C2 UHWW and STP-I and STP-O + + + + + + + + C3 UHWW and STP-I - - + + - - + + C4 UHWW and STP-I and STP-O - + + + + + + - C5 UHWW and STP-I + - + - + + + - C6 UHWW and STP-I + + - - + + - + C7 UHWW and STP-I and STP-O + - + + + + - + S. aureus C1 UHWW + + + - + - - - C2 UHWW and STP-I + - - - - - - + C3 UHWW and STP-I - + + - + + - - C4 UHWW and STP-I - - - - - + - - C5 UHWW and STP-I and STP-O - - - + + + + - C6 UHWW and STP-I and STP-O - - - - + + + - C7 UHWW and STP-I - - - - - + - -

Prevalence of antibiotic resistant strains in HWW and STP-I and STP-O Resistance S. Aureus E.coli (no. of AB) HWW STP-I STP-O HWW STP-I STP-O None 1 6 0 0 4 0 1 9 13 1 8 14 0 2 7 18 1 8 14 1 3 4 15 0 8 12 2 4 2 8 1 8 16 2 5 27 6 2 19 11 4 6 26 2 2 22 7 4 7 6 4 0 19 8 5 8 2 1 0 15 7 1 9 1 1 1 6 4 3 10 NT NT NT 4 4 0 11 NT NT NT 2 1 1 12 NT NT NT 0 0 0

Prevalence of ABR strains in HWW and STP (STP-I and STP-O) P <0.0001 P <0.0001 Mean antibiotic resistance 10 Mean resistance to AB (No) 10 8 8 6 6 (no) 4 4 2 2 0 0 HWW STP HWW STP site Site S. aureus E. coli

What these data mean?  Persistent hospital strains were found in STP-I (and some in STP-O before released into the receiving water)  Some of these strains were MDR  The number of antibiotics to which strains from HWW were resistant was significantly higher than STP strains  The STP process was effective at removing the sensitive E .coli strains but failed to remove some of the ABR strains  ABR strains belonging to certain types in HWW showed the ability to survive transition to STP and the STP treatment process

Questions to be answered • What proportion of these ARB are sourced from community effluents upstream of the hospital? • Do ABR strains lose their AB resistance genes/survive? • Do HWW always have a higher proportion of ABR strains than the community wastewater? • Do treated hospital wastewater have a less proportion of ABR strains? • If yes, what treatment method is more effective (conventional activated sludge system vs plate membrane bioreactor)

Acknowledgements Collaborators: – Aycan Gundogdu – Jasmin Thompson – Helen Stratton Thanks to: – Project Leader: Kristell Le Corre – Project Champion: Greg Jackson

Urban Water Security Research Alliance THANK YOU www.urbanwateralliance.org.au