JM Tyrrell1, M. Woodward2, RA. Howe3, TR. Walsh1 Costs on fitness and pathogenicity of Colisitin & Tigecycline resistance in Multi-drug Resistant (MDR) Klebsiella pneumoniae JM Tyrrell1, M. Woodward2, RA. Howe3, TR. Walsh1  1Department of Medical Microbiology & Infectious Diseases, Cardiff University, Heath Park, Cardiff, Wales, UK. 2Department of Food & Nutritional Studies, University of Reading,UK, RG6 6AP. 3Public Health Wales Microbiology Cardiff , University Hospital of Wales, Heath Park, Cardiff, Wales, UK. Introduction Since its discovery in 2009, the NDM-1 metallo-β-lactamase, carried upon conjugative plasmids by species of Enterobacteriaceae, has disseminated on a global scale. NDM-1 producers are often pan-resistant to front-line antibiotics including carbapenems, aminoglycosides and fluoroquinolones, compromising patient treatment and recovery. Colistin (C) & Tigecycline (T) are frequently the only viable treatment options for NDM-1-positive infections, but resistance to even these is now being reported. Understanding the process and consequences of resistance to C&T on these potential pathogens is thus of great importance. Methods Test Sample: A self optimised passaging method was used to separately increase the C&T MICs of 9 NDM-1-positive Klebsiella pneumoniae (KP). Reversion of C&T Resistance: Once the increased MICs were stabilised, cultures of the C resistant and T resistant ‘mutants’ were re-inoculated into neat broth for 14 days. Every other day cfu counts were carried out with selective pressures at 0, 2, 8, 32 and 128µg/ml with the appropriate microbial (C/T) to deduce the rate at which resistance was being maintained/lost within the bacterial population. Galleria mellonella assay: Galleria mellonella were used as a model of pathogenicity. Larvae were infected with a controlled inocula of each test isolate, administered through the rear-left pro-leg into the larval hemocoel. N=10 larvae were used for each infection challenge. All larvae were incubated at 37ºc. Survival rates scored every 24 hours, death accepted once the larvae no longer responded to touch. Results Figure 1-IR37: (A) % of population growing at 2, 8, 32 & 128µg/ml Colistin over 14 reverse passage events. (B) % population growing at 2 & 8µg/ml Tigecycline over 14 reverse passage events. (C) Survival curves of IR37 WT, CR & TR. A B C Figure 2-IR12: (A) % of population growing at 2, 8, 32 & 128µg/ml Colistin over 14 reverse passage events. (B) % population growing at 2 & 8µg/ml Tigecycline over 14 reverse passage events. (C) Survival curves of IR37 WT, CR & TR. A B C Conclusions The above figures are representative of the results seen for all 9 of our KP test sample. The rates and strategies with which our KP isolates develop resistance to C&T decidedly variable (Table 1). Evolving C&T resistance has a negative impact on the growth rate and virulent capabilities of our KP, as seen in Figures 1c & 2c. There is no correlation between the two factors however. Having reached higher MICs and shown lower rates of population reversion (Figure 1a & b, 2a &b), we may assume that the strategies used in our Colistin ‘mutants’ may be carry less ‘cost’ than the corresponding strategies used by Tigecycline ‘mutants’. K1 HR1 HR2 IR12 IR15 IR17 IR18 IR34 IR37 Colistin WT 0.5 0.25 1 2 CR 128 (13) >128 (21) >128 (16) >128 (24) 128 (4) 128 (11) >128 (10) Tige TR 16 (30) 32 (30) >128 (15) 16 (22) Table 1: Colistin & Tigecycline MICs (µg/ml) of Wild Type (WT) and passaged (CR & TR) KP isolates. Number of passaging events for each ‘mutant’ is shown in brackets Email: tyrrelljm@cardiff.ac.uk