Curing antibiotic resistance in vivo. Muhammad Kamruzzaman

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1 Curing antibiotic resistance in vivo Muhammad Kamruzzaman

2 Occurrence of resistance -Some bacteria are naturally resistant to certain antibiotics -Gene mutation -Horizontal transfer of antibiotic resistance genes this is the most important mechanism in the Enterobacteriaceae -Over use/misuse of antibiotics can lead to increases resistance

3 Antibiotic resistance problem in Enterobacteriaceae Escherichia coli, Klebsiella pneumoniae dominate the epidemiology of urinary tract infection and lethal sepsis The most important vectors of transmissible AbR are self-transmissible plasmids Resistance genes are often associated with different mobile elements (transposons, integrons etc.) on plasmids Plasmid-borne AbR acquired very quickly and plays major role in dissemination of resistance in and within species AbR resistant Enterobacteriaceae are now termed as Superbugs

4 Plasmid-borne antibiotic resistance Plasmid-borne AbR may be acquired very quickly Day3 swab (Serratia TIM R GEN R ) (bla IMP-4 detected) D30 blood E. coli TIM S GEN S D31 blood E. coli TIM R GEN R (bla IMP-4 plasmid) van Hal et al., J Clin Microbiol. 2009, 47(7):

5 General architecture of conjugative plasmids AbR, virulence genes Partition system, TA system Mobilisation and transfer genes rep, rep control genes

6 Plasmid incompatibility Plasmid replication requires host cell machinery Cross-interference between replication systems of related plasmids make them incompatible This incompatibility ensures that related plasmids do not co-exist in a cell Compatible plasmids Different replicon Cell division Cell division Incompatible plasmids Same replicon

7 Stable maintenance of plasmids Toxin-antitoxin system/ PSK/ addiction system A toxin-antitoxin system is a set of two or more closely linked genes that together encode both a protein 'poison' and a corresponding 'antidote'. Antibiotic resistance, virulence, and other plasmids in bacteria use toxin-antitoxin gene pairs to ensure their persistence during host replication. Hayes F & Van Melderen L (2011), Critical Reviews in Biochemistry and Molecular Biology; 46(5):

8 TAS mechanism in plasmid maintenance Toxin Plasmid is passed on Plasmid is lost Antitoxin Antitoxin degrades Cell death/growth arrest

9 Utilisation of plasmid properties to construct curing plasmid A no antibiotic AT/T B rep Ab R Ab addictive incompatible no Ab AT C Ab non-addictive compatible no Ab

10 Construction of interference plasmid and curing AbR in vitro Reverse engineering AbR plasmid Curing plasmid rep Ab non-addictive incompatible no Ab

11 rep Curing antibiotic resistance in vitro Ab non-addictive incompatible no Ab Target AbR plasmids for curing 1) pel1573 (IncL/M) encodes GEN-R and AbR to almost all available β-lactams (due to the carbapenemase gene bla IMP-4 ) 2) pjie512b (IncI1) resistance to cephalosporin antibiotics and β-lactamase inhibitors (due to AmpC β-lactamase gene bla CMY-2 ) Enterobacteriaceae species tested 1. Escherichia coli 2. Klebsiella pneumoniae 3. Citrobacter freundii 4. Morganella morganii

12 Plasmid curing does not interfere any resident plasmids M1 M kb 48 kb Acquisition and loss of pel1573 from K. pneumoniae with bystander plasmids preserved

13 AbR plasmid curing in mice Special food (D1-3) Transfer into fresh cage CTX D4-6 D7-9 TET Examine target colonization Examine interference plasmid colonization & curing of target plasmid Interference plasmid cleared on day 15 D17-18 Gave CTX to Gr 4 Gr1: Ctl Gr2: Ab Ctl Gr3: Target plasmid only Gr4: Target plasmid and then challenged with interference plasmid Confirmed plasmid curing by PCR and phenotype Examined target bacteria on plate and PCR for resistance genes No target bacteria or resistance genes were detected Bacteria with target plasmid Bacteria with interference plasmid

14 CFU (log 10 value) value) Curing AbR from mice gut A pel1573, CTX pjimk46, TET CTX 8 6 B pjie512b, CTX pjimk56, TET CTX CFU (log C pel1573, CTX D pjie512b, CTX CTX S TET S CTX R (from target plasmid) TET R (from interference plasmid) Days

15 Conclusion and discussions Neutralisation of relevant TA and rep is both necessary and sufficient to design an interference plasmid An interference plasmid can displace a plasmid which is fixed in the accessory genome, and interference plasmid is then itself lost, allows return to the plasmid-free state Plasmid curing by interference plasmid is done without killing bacteria or affecting other plasmid populations and is clearly feasible both in vitro and in mouse gut This approach offers hope of not only rendering resistant pathogen susceptible to therapy but also of a cure for antimicrobial resistance and potentially restoring normal microflora and can also be used in bioremediation of animal and environmental microbiomes.

16 Acknowledgements Prof. Jon Iredell A/Prof. Sally Partridge Dr. Shereen Shoma NHMRC for funding

17 Thank you Questions????

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