Phage cocktails as an alternative to inactivation of Enterobacter cloacae Pereira S.1*, Pereira C.1, Silva Y.J.1, Costa L1 and Almeida A.1 Department of Biology and CESAM, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal *Corresponding author: Email: sarasouto@ua.pt Introduction Enterobacter cloacae is part of the normal flora of the gastrointestinal tract of 40-80% of people. This opportunistic microorganism is capable of causing infection in debilitated and hospitalized patients. E. cloacae is resistant to a broad number of antibiotics therefore infections caused by this bacterium are difficult to control [1]. E. cloacae production of extended spectrum lactamases (ESBL) and metallo-lactamases (MBL) has been reported in different parts of the world [2,3]. The objective of this study is to isolate new bacteriophages to inactivate E. cloacae and evaluate phage survival and the host- phage dynamics in vitro. Material and Methods Bacteria and growth conditions Bacteria grew at 37ºC, pH 7.3 in tryptic soy broth Bacteriophages Phages were isolated from sewage water. Sewage water was filtered through a 0.45 μm pore size polycarbonate membranes. The filtrate was added to double concentrated TSB medium with 1 mL of a fresh culture of E. cloacae. The mixture was incubated at 37ºC for 18 hours and then filtered through a 0.2 μm membrane The titre of the supernatant was determined by the double-layer agar method (Figure 1) using TSA as culture medium. The plates were incubated at 37 °C and examined for the presence of lysis plaques after 12 hours. Three successive single-plaque isolations were performed to obtain a pure phage stock Figure 1 . Soft agar overlay technique Phage survival Bacteriophages were incubated at 25ºC in PBS. Samples were collected at different periods and were quantified by the soft agar overlay technique. The host-phage dynamics Phage therapy assays were performed using the phages separately (E-2, E-3 and E-4) and combined (each phages at the same concentration) using E. cloacae as host at a MOI (multiplicity of infection) of 100. In order to obtain a MOI of 100, 105 CFU mL-1 of the overnight E. cloacae culture and 107 PFU mL-1) of the phage suspension were inoculated in 30 mL of TSB medium and incubated at 25 ºC without agitation for 24 h. The host-phage dynamics was determined in tryptic soy broth, during 24 hours. The experiments were performed at a pH of 7.4 and a temperature of 37°C. For host quantification, aliquots were serially diluted, plated in duplicate and incubated at 37°C for 24 hours. The phage titre was determined by the double-layer method and incubation at 37°C for 18 hours. Results and Discussion Phage survival The concentration of E-2 decreased by two orders of magnitude in the first 105 days. E-3 concentration decreased by one order of magnitude in the first 20 days and reached a plateau until 77 days. Afterwards, the phage titer decreased by three orders of magnitude until 156 days. E-4 concentration only decreased by one order of magnitude after 255 days. Host-phage dynamics Figure 2. Inactivation of E. cloacae by the three phages (E-2, E-3 and E-4) at a MOI of 100 during the 24 h. A. Bacterial concentration: BC – Bacteria control; BP – Bacteria plus phage .B. Phage concentration: PC – phage control; BP – Bacteria plus phage. Values represent the mean of three experiments. Figure 3. Inactivation of E. cloacae by phage cocktail (E-2/E-4) at a MOI of 100 during the 24 h. (A) bacterial concentration: BC – Bacteria control; BP – Bacteria plus phage. (B) Phage concentration: PC – phage control; BP – Bacteria plus phage. Values represent the mean of three experiments. Single phage suspensions reduced the bacteria concentration in ≈ 3 log in 4-6 h (Figure 2). In the presence of phage cocktails there was a reduction of 4.3 log after 4 h of incubation. After 12 h the inactivation was 3.6 log. (Figure 3). Conclusions E-2/E-4 phage cocktail was more effective to inactivate E. cloacae being potential candidates for the control of nosocomial infections. The use of phage cocktails increase the efficacy of phage therapy, one more log of bacteria were inactivated and the inactivation occurred sooner. References 1. Zhou Q, Zhang M, Wang A, Xu J, Yuan Y. Eight-Year Surveillance of Antimicrobial Resistance among Enterobacter Cloacae Isolated in the First Bethune Hospital. Phys Procedia. 2012;33:1194–6. 2 Bell JM, Turnidge JD, Jones RN, Bell JM, Turnidge JD, Jones RN. Prevalence of Extended-Spectrum β -Lactamase-Producing Enterobacter cloacae in the Asia-Pacific Region: Results from the SENTRY Antimicrobial Surveillance Program, 1998 to 2001, 2003. 3. Sianou E, Kristo I, Petridis M, Apostolidis K, Meletis G, Miyakis S. A cautionary case of microbial solidarity: concurrent isolation of VIM-1-producing Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae from an infected wound. Journal of Antimicrobial Chemotherapy. 2012;67(1):244–6. Acknowledgements This work was supported by funding FEDER through COMPETE – Programa Operacional Factores de Competitividade, and by National funding through FCT- Fundação para a Ciência e Tecnologia (Foundation for Science and Technology),within the research project FCOMP-01–0124-FEDER-013934 and project PROMAR 31-03- 05-FEP-0028. The authors also thank the University of Aveiro and the Center for Environmental and Marine Studies (CESAM) for funding (Project Pest-C/MAR/LA0017/2013). Financial support to C. Pereira, and Y. J. Silva was provided by FCT in the form of PhD grants (SFRH/BD/76414/2011 and SFRH/BD/65147/2009) , respectively.