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), STP-I (n=74) and STP-O (n=8). Hospital wastewater sampling site
HWW and STP samples Cultivation and identification on selective medium Confirmation using species specific genes uspa Gene E. coli ID gene nuc Gene S. aureus 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 Biochemical Fingerprinting (PhP method) Random Amplified Polymorphic DNA (RAPD) PCR 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 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 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 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 Identical isolates 2, 4 and 9 6 and 7 For each bacterial isolate, it yields a biochemical fingerprint Similarity between the isolates is shown in a dendrogram.
PhP/RAPD C-types E. coli S. aureus Sites Where Bacteria Were Found Weeks where Bacteria were Found W1 W2 W3 W4 W5 W6 W7 W8 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 + - + + + + - + 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 (no. of AB) S. Aureus E.coli 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 (no) 10 8 6 4 2 0 HWW STP Mean resistance to AB (No) 10 8 6 4 2 0 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