Fast and reliable screening assay developed to preselect candidate Soft Rot Pectobacteriaceae Tn5 mutants showing resistance to bacteriophage infection

Abstract

We present a simple, fast and inexpensive screening assay to preselect candidate Pectobacterium spp. and Dickeya spp. Tn5 mutants, which carry transposon insertions in genes putatively encoding proteins used by lytic bacteriophages to interact with host cells, for the follow-up studies. The proposed method is fast and costeffective and it does not need any specialized laboratory equipment and/or technical support. The Tn5 mutants are generated using random transposon mutagenesis with the mini-Tn5 transposon. The obtained bacterial mutants are incubated in the presence of viable lytic bacteriophage particles in liquid bacterial growth medium supplemented with resazurin for 12 h at 28°C in a 96-well microtiter plate assay. During the cultivation, the Tn5 mutants that are susceptible to phage infection are lysed. The mutants that are resistant to a viral infection (not lysed after contact with bacteriophages) irreversibly reduce the resazurin violet dye to pink/yellowish-colored resorufin indicating active bacterial metabolism (a positive reaction). The change of the culture color can be observed by eye. The Tn5 mutants that are positive in the screen are selected for sequencing of the Tn5 insertion site directly from bacterial genome. The proposed assay allows generation of a number of immediately-available Tn5 mutants expressing phage-resistant phenotypes that can be later selected for further examinations. As a proof-of-concept, we used this method to evaluate resistance to viral infection of Tn5 mutants of Dickeya solani strain IPO2222 and Pectobacterium parmentieri strain SCC3193 using lytic bacteriophages ɸD5 and ɸA38, respectively.

Application of the Resazurin Cell Viability Assay to Monitor Escherichia coli and Salmonella Typhimurium Inactivation Mediated by Phages

“…Like other studies [ 24 , 49 ], this study demonstrated that the resazurin microplate method is faster and as accurate as the colony-count technique and is more precise and has a lower minimum detection limit than the OD method. As such, this fluorometric method is a good alternative to monitor the concentration of different bacteria during their inactivation by several phages at the same time and, consequently, is a promising technique to screen and preselect new or known phages by their inactivation profile.…”

“…Other colorimetric methods were successfully used in phage screening [ 22 , 49 , 50 , 51 , 52 , 53 ]. Some authors used the tetrazolium-based assay to screen 98 single Acinetobacter baumannii -specific lytic phages to inactivate A. baumannii [ 22 ], others used the resazurin assay to assess the cell activity of Pectobacterium atrosepticum with CRISPR-Cas immunity upon infection with the virulent phages [ 52 ], others used the resazurin assay as a fast screening assay to preselect Dickeya solani and Pectobacterium parmentieri Tn5 mutants in genes coding for proteins used by lytic phages φD5 and φA38 as receptors [ 49 ] and others used the resazurin assay to detect for the presence of phages specific to Streptococcus diacetilactis in cottage cheese samples [ 61 ]. However, these studies only tested the phages’ ability to inactivate or not inactivate the targeted bacterium.…”

“…In addition, it keeps cells intact, which allows for other parallel analyses [ 24 ]. This colorimetric assay has already been used to determine phage host range [ 49 ] and assess bacterial viability after phage treatment [ 22 , 45 , 49 , 50 , 51 , 52 , 53 ]. However, no comparison of the method’s reliability with the most frequently used methods, viable plate count method and optical density measurement, was done to evaluate the effectiveness of bacterial inactivation by phages.…”

Pectobacterium parmentieri SCC 3193 Mutants with Altered Synthesis of Cell Surface Polysaccharides Are Resistant to N4-Like Lytic Bacteriophage ϕA38 (vB_Ppp_A38) but Express Decreased Virulence in Potato (Solanum tuberosum L.) Plants

“…Phage-resistant P. parmentieri Tn5 mutants were preselected as previously described [ 73 ]. To validate their bacteriophage resistance, each Tn5 mutant exhibiting a ϕA38-resistant phenotype in the initial screen was subjected to repeatable phage challenge assays and plaque formation assays as previously described [ 15 ].…”

Fast and reliable procedure developed to generate soft rot Pectobacteriaceae (Pectobacterium spp. and Dickeya spp.) Tn5 mutants resistant to bacteriophage infection

“…2 Host challenge assay of the parental (D. solani IPO2222) strain (marked) and the selected bacteriophage-resistant IPO2222 Tn5 mutants in the absence and presence (+ phiD5) of lytic bacteriophage ϕD5. Per mutant to be analyzed, as described earlier (Czajkowski et al 2019), in duplicates, the wells of the 96-wells plate were inoculated with 50 µl of freshly prepared bacterial cultures (IPO2222 wild type or IPO2222 Tn5 mutants) in LB broth (final cfu ml − 1 of 5 × 10 4 ). Subsequently, 200 µl of bacteriophage ϕD5 suspension in LB broth was added per well (final pfu ml − 1 of 2 × 10 7 ).…”