Characterization of Yersinia pestis Phage Lytic Activity in Human Whole Blood for the Selection of Efficient Therapeutic Phages

Abstract

The global increase in multidrug-resistant (MDR) pathogenic bacteria has led to growing interest in bacteriophage (“phage”) therapy. Therapeutic phages are usually selected based on their ability to infect and lyse target bacteria, using in vitro assays. In these assays, phage infection is determined using target bacteria grown in standard commercial rich media, while evaluation of the actual therapeutic activity requires the presence of human blood. In the present work, we characterized the ability of two different Yersinia pestis lytic phages (ϕA1122 and PST) to infect and kill a luminescent Y. pestis EV76 strain suspended in Brain Heart Infusion (BHI)-rich medium or in human whole blood, simulating the host environment. We found that the ability of the phages to infect and lyse blood-suspended Y. pestis was not correlated with their ability to infect and lyse BHI-suspended bacteria. While the two different phages exhibited efficient infective capacity in a BHI-suspended culture, only the PST phage showed efficient lysis ability against blood-suspended bacteria. Therefore, we recommend that for personalized phage therapy, selection of phage(s) for efficient treatment of patients suffering from MDR bacterial infections should include prior testing of the candidate phage(s) for their lysis ability in the presence of human blood.

Phage resistance accompanies reduced fitness of uropathogenicE. coliin the urinary environment

“…Conventionally, bacteria-phage interactions are studied in bacteriologic medium, however, more recent work has begun to assess these interactions in the context of the host environment including ex vivo blood or in vivo tissues(43, 59, 60). To assess how phage activity could be influenced by the urinary environment, we compared 18h bacterial growth curves in LB medium and human urine.…”

Reporter-Phage-Based Detection and Antibiotic Susceptibility Testing of Yersinia pestis for a Rapid Plague Outbreak Response

“…We conducted the diagnostic assay using different bacterial origins, including Y. pestis colonies suspended in rich growth media, spiked into and grown in human whole-blood culture (in Bactec aerobic + bottles) or spiked into PBS-suspended environmental (asphalt) samples. It should be mentioned that to identify Y. pestis spiked into and grown in human blood culture, the removal of blood components prior to phage administration was needed as these components inhibit φA1122 infection [29,31]. Thus, we used the serum separation tubes (SSTs) for separating the cellular and plasma fractions from the bacteria.…”

“…Experiments in this work were conducted using the nonvirulent Y. pestis strains Kimberley53∆70∆10 and EV76 [31]. Kimberley53∆70∆10 is resistant to streptomycin.…”

“…Bacteriophage stock was prepared by the agar overlay method [34]. Phage titration was performed by serial 10-fold dilutions in SM buffer (0.1 M NaCl, 8 mM MgSO 4 , 50 mM Tris-HCl pH 7.5 and 0.01% gelatin solution), followed by spot assay or plaque assay [31]. The phage stock was stored at 4 • C in the dark until use.…”

“…We found that in the presence of blood, the detection of the luminescent signal was delayed, and a longer incubation time of 100 min was required for MIC determination (Figure S2A). We assumed that blood components block phage infection, as we recently published for the parent phage, φA1122 [31]. Therefore, we tested whether elimination of blood components would increase the phage-derived luminescent signal.…”