Investigating Mechanisms Of Antimicrobial Resistance Using Molecular Techniques Name: Semina Ploumakis; Life Sciences Department. Supervisors: Professor Elizabeth Wellington 1. Introduction Antibiotic-resistance poses an ‘apocalyptic’ threat to human health. Examples include the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals and multidrug-resistant tuberculosis, with its links to poverty, HIV and social exclusion [1]. Little attention has been drawn to the potential role of anthropogenic activity in the evolution of antibiotic resistance in the environment. This is relevant to human health because of the increasing importance of zoonotic diseases as well as the need for predicting emerging resistant pathogens. Bacteria acquire genes from the environment by a process known as horizontal gene transfer. Transposons, integrons or plasmids are capable of transferring antibiotic-resistant genes to other bacteria of the same or different species [2]. Carbapenems and third-generation cephalosporins (3GC’s) are classes of broad-spectrum -lactam antibiotics. Resistance to 3GC’s has cropped up due to the production of extended-spectrum -lactamases (ESBLs) of which the most prevalent are the CTX-M enzymes encoded by bla CTX-M. Carbapenem resistance is relatively new; the predominant gene in the clinic being bla NDM-1. Biocides have the ability to select for genetic elements capable of carrying antibiotic resistance due to co-selection. Quaternary ammonium compounds (QACs) are an example of a widely used biocide. 2. Aim 3. Methods Figure 2: Part 1; Shotgun library construction and analysis. Part 2 – Characterisation of the role of biocide resistance in the selection for 3GC-resistance in the environment.  3GC-resistant isolates found upstream/downstream of a WWTP-impacted river were tested for CTAB (biocide) resistance. These isolates included: Aeromonas media, Citrobacter freundii, Escherichia coli (ST131) and Klebsiella oxytoca.  Rifampicin-resistant and streptomycin-resistant E. coli was used as a recipient strain for solid conjugal mating assays with donor strains initially characterised with biocide + 3GC resistance.  Biocide-resistant transconjugants were subsequently tested for potential antibiotic resistance transferred to them indirectly. This was achieved by streaking them on cefotaxime (3GC) and ciprofloxacin (fluroquinolone) plates followed by overnight incubation at 37°C.  PCR was performed on 6 out of 15 cefotaxime-resistant isolates. 4. Results Part 1: Carbapenem-resistant E.coli plasmid DNA sequences are pending. Figure 3: Finham sewage works on the River Sowe where samples were taken a year apart in late 2009 and early Part 2 : DOWNSTREAM36/40 isolates upstream were resistant to 5 mg ml-1 CTAB. A total of 13/36 were capable of transferring this biocide resistance (including E. coli and R.orthinolytica). UPSTREAM 12/14 isolates upstream were resistant to 5 mg ml-1 CTAB. A total of 7/12 were capable of transferring biocide resistance (including E.coli, K.oxytoca and C. freundii). 15/20 biocide resistant recipients also had associated cefotaxime resistance. Nine of these isolates were also resistant to ciprofloxacin. DNA extraction of resistant colonies The ‘End-It DNA End-Repair Kit’ was used in order to repair genomic DNA fragments containing damaged or incompatible 5’- or 3’-ends. The DNA was then ligated to the E.coli vector pCC1. A technique called ‘drop dialysis’ was used to de-salt the products of library ligation reactions in order to yield more transformants. We then used electro-transformation to modify the genetic properties of the E.coli cells with the use of a strong electric field. Carabapenem resistant E. coli colonies were finally isolated and plasmid DNA was extracted and sent for sequencing (Pending). Gel fractionation DNA electro-elution (5-10 Kb fragments) START END Figure 4 is a PCR image of 6 out of 15 cefotaxime-resistant isolates. Figure 4 Positive controler Negative control Table 1: Results of conjugation assays. IsolatesConjugation DownstreamfrequencyTAXCIPRO Escherichia coli7.00E-07 Escherichia coli2.83E-05 Escherichia coli8.18E-07 Escherichia coli1.07E-06 Escherichia coli4.00E-06  Escherichia coli2.76E-06 Escherichia coli1.14E-05  Escherichia coli1.67E-04  Escherichia coli1.25E-05  Escherichia coli2.00E-07  Escherichia coli3.13E-05  Escherichia coli6.67E-07 Raoultella orthinolytica E-08  Isolates Upstream Escherichia coli6.08E-06  Escherichia coli3.39E-07 Klebsiella oxytoca3.13E-07  Escherichia coli1.71E-08  Citrobacter freundii2.00E-05 Escherichia coli6.67E-07  Escherichia coli2.63E Conclusions Little is known about the environmental antibiotic resistome, however, it is evident that organisms in certain environments harbour antibiotic resistance genes irrespective of the human use of antibiotics. Considering that antibiotic treatment is our foremost method of treating infectious diseases, there are pressing questions that demand solutions if such a precocious medical technology is to remain effective in the years to come [1], [2]. Acknowledgements: University of Warwick, Professor Elizabeth Wellington, Dr. Gregory Amos and everyone in Laboratory C123. Semina Ploumakis contact details: References: [1] - Roger G. Finch (2004). Antibiotic resistance: a view from the prescriber. Nature Reviews Microbiology: 2, [2]- Heather K. Allen, et al (2010). Call of the wild: antibiotic resistance genes in natural environments. Nature Reviews Microbiology: 8, To characterise the genes conferring carbapenem resistance to the following isolates: Aeromonas sp., Escherichia coli, Pseudomonas azotoformans, Pseudomonas gessardii, Pseudomonas marginalis, Pseudomonas putida and Raoultella orthinolytica, isolated from a WWTP-impacted river using shotgun cloning. The next aim was to characterise the role of biocides in the selection and dissemination of 3GC-resistant genes in the environment. Figure 1: Horizontal gene transfer.