Bacteriophages can be used as infection control agents: A proof-of concept study involving anti-Acinetobacter baumannii phage

Hospital acquired infections are responsible for morbidity and mortality worldwide. Acinetobacter spp. infections are particularly notorious for complicating patient management in ICU settings. Extremely high mortality rates are associated with Acinetobacter infections because of their resistance to first- and second-line drugs. There is imminent need to develop infections control systems that are specific and environment friendly. Here, we report a proof-of-concept anti-Acinetobacter spp. bacteriophage-based infection control assay that is very target specific as well as innocuous to environment. extensively drug resistant (XDR) Acinetobacter baumannii strain was inoculated at various solid surfaces. A bacteriophage, enriched in the same strain, was applied on the inoculated surfaces. Phenol (carbolic acid) was used as a positive control. We show that bacteriophages can be used as infection control agents. In our assay, they killed XDR Acinetobacter baumannii present on solid surfaces. Our bacteriophage was extremely effective at reducing the CFU of inoculated strain to almost undetectable levels.


Introduction
Different surfaces in healthcare facilities may serve as reserves of pathogens.

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After 24 hours, 12-13 ml suspension from each flask was transferred into pre labelled 15 ml tubes. After 78 centrifugation of 10 minutes at 4200 RPM, the supernatant from each tube was syringe filtered. This 79 filtrate was used for two more rounds of enrichment to increase titers of possible anti-acinetoviruses.

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Plaque assay 81 After three enrichments, suspected bacteriophage containing filtrates were taken and serially diluted ten 82 times (10 -1 through 10 -10 ) in sterile saline solution in ten tubes. An early log phase bacterial inoculum was 83 prepared by adding 300 μl of bacterial suspension in 30 ml LB broth in a flask, followed by the 84 incubating it for 2-4 hours at 37°C. After that, a mixture of bacterial isolate, bacteriophage dilution and 85 soft LB agar (normal solid media contain 1.5% agar) was prepared by adding 0.6% agar in LB broth. For 86 this purpose, 100 μl of early log phase inoculum was added into 20 ml glass tube followed by the addition 87 of 10μl of each of the ten phage dilutions. Then 20 ml of soft LB agar was added. All these tubes were 88 incubated at 37⁰C for 20 minutes for adsorption to take place.

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After that about 8 ml of the mixture from each tube was poured on pre labeled LB agar plates and 90 incubated at 37⁰C for 24 hours or until appearance of plaques on phage positive plates. Plaques were 91 observed and virus titer was calculated by counting plaques on 10 -7 plate (Fig 1).

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Acinetobacter Clearance Assay 93 A 0.5 McFarland inoculum of Acinetobacter baumannii was prepared in 10 ml tube. Three circles of one-94 inch diameter each were drawn on a pre-sterilized lab bench surface (Fig 2) and labeled as "phenol", 95 "phage" and "saline". Then 2ml of bacterial inoculum was poured in each circle. After 20-25 min, 250 μl 96 of 90% phenol, normal saline, and bacteriophage suspension was added on respective inoculated circles.

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Phage was added at 10 MOI (multiplicity of infection: ratio between no. of infectious phage particles and no. of host bacterial cells). Same procedure was adopted for two more surfaces ; top of the incubator and   99  office table. 100 After overnight exposure of inoculated circles with phenol (positive control), saline (negative control) and 101 phage, any remaining bacteria were collected with a moistened sterile swab by rolling it over each circle 102 thoroughly. Then cotton part of swab was aseptically cut into 10ml sterile saline and vortexed to collect 103 bacteria in the saline. Subsequently, 1 µl calibrated loop (SPL Life Sciences, Catalog no. 90001) was 104 dipped into the saline containing inoculum from circles and LB agar plates were semi-quantitatively 105 streaked. Colonies from phenol exposed, saline exposed and phage exposed circles were counted to 106 determine the bactericidal effects of phage (Table 1). gives us infectious titer and hence approximates number of "physically fit" particles. Plates containing 10 -120 7 dilution of phage were used to count plaques as it had isolated and hence countable plaques (Fig 1C).

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All work was done in triplicate and averages were calculated. PFU/ml were extrapolated from the plaque 122 count to be 2x10 12 .

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In this backdrop, we set out to determine potential of our acinetophage isolate in clearing clinically 136 relevant XDR Acinetobacter strain from solid surfaces. For our experiment, we decided to mimic 137 conditions that are common to hospital inhabiting pathogens (Fig 2). We inoculated different solid 138 surfaces with XDR Acinetobacter baumannii strain in known quantities and applied our phage on those 139 surfaces to determine its clearing potential. We infected our bacterial inoculums at 10 MOI because 140 calculations based on Poisson distribution model predict that at 10 MOI almost all bacterial cells receive 141 at least one virus particle. Same volumes of 90% phenol and normal saline were used to serve as positive 142 and negative controls respectively. Purpose was to observe effectiveness of phage as compared to phenol 143 which is an established bactericidal.

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After overnight exposure to phage, phenol and saline, we collected any remaining bacteria from those 150 surfaces and cultured on LB agar plates in quantitative manner using calibrated 1µl loops. Therefore, 151 colony count represented CFU per microliter after the exposure of our bactericidal agents.
We observed that phenol, quite expectedly, was the most efficient antibacterial as its exposure resulted in 153 least number of CFU from all three surfaces; 3, 5 and 2 from lab bench , incubator top and office table   154 respectively (Table 1). Saline exposed surfaces gave much higher colony count (78, 103 and 91 CFU/µl 155 for three surfaces in above mentioned order). Again, it was an expected observation as saline has no 156 antimicrobial potential. Phage exposed surfaces gave 15, 13 and 11 CFU/µl which, although, was higher 157 number when compared to phenol but is significantly smaller than in case of saline treatment (Table 1).
158 Colonies appearing from phenol and phage exposed samples were sporadic (Fig 2: B1