E. coli-ΦX174 genotype to phenotype map reveals flexibility and diversity in LPS structures

Extensive efforts have been made to understand the phenotypic diversity of lipopolysaccharide (LPS) structures through deletion and complementation experiments. However, this approach likely underestimates the available phenotypic diversity. To better explore LPS diversity, we generate LPS mutants in Escherichia coli C by selecting for resistance to ΦX174, a bacteriophage that relies solely on binding to core LPS to infect its host. An analysis of 31 E. coli C mutants that are resistant to ΦX174 reveals that each mutant carries at least one mutation in genes linked to core LPS biosynthesis or assembly. Based on which genes are mutated, we predict the core LPS structures of each bacterial mutant, and test our predictions by evolving phages to recognize each evolved LPS structure. We find that phages that evolved to infect the same predicted LPS structure were not always able to cross-infect each other’s host, suggesting that core LPS structure diversity is higher than predicted. Similarly, phage genotype-phenotype maps can be constructed using the bacterial LPS mutant classes. For example, we demonstrate that a combination of two phage mutations leads to loss of the ability to infect wildtype E. coli C. Our results show that phages are a useful tool to study LPS structures, and conversely that the study of LPS structures helps to understand phage evolution and biology.

is only expected to worsen in the coming decades [1][2][3] . An alternative to treating bacterial infections with 36 antibiotics is phage therapy. Major research efforts are being conducted to develop efficient therapies 4-37 6 . An efficient therapy should be able to eliminate a pathogen without selecting for resistance to the 38 phage being used to treat that pathogen. Phage resistance can often evolve by modifying the cell 39 envelop to avoid phage adsorption. The outer membrane of all Gram-negative bacteria is covered in 40 lipopolysaccharides (LPSs), which is consequently the first contact point between a bacterium and its 41 environment 7 . LPS structure determines whether a bacterium will be recognized by the immune 42 system, whether it is susceptible to antibiotics, determines the efficiency of nutrient uptake and, 43 importantly, determines susceptibility to phage infection 8-10 . Hence, LPS structure is an important 44 factor determining the success of phage therapy. 45

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The functions of almost all genes involved in synthesizing and assembling bacterial lipopolysaccharide 47 (LPS) have been eluciated [11][12][13][14][15] . Most Escherichia coli strains produce LPS molecules composed of three 48 parts: the lipid A, the core oligosaccharide (OS; itself consisting of a conserved inner core and an outer 49 core), and the O-antigen chain. Such tripartite LPS molecules are termed "smooth" 12 . Some E. coli 50 strains produce LPS molecules that lack one or more components. LPS molecules lacking the O-51 antigen are classified as "rough", molecules lacking the O-antigen and the OS outer core, as "deep 52 rough" 15,16,11,[17][18][19] . These modifications in LPS structure can lead to a plethora of phenotypic effects, 53 including (i) destabilization of the outer membrane 12 , (ii) changes in the expression of some outer 54 membrane proteins and (iii) modification of intracellular turgor pressure 20 , (iv) increased susceptibility 55 to hydrophobic compounds such as antimicrobial peptides, antibiotics, and bacteriocins 15,16,11,17-19 , (v) 56 altered interactions with the host immune system 21,12 , and (vi) changes in resistance to phages 9,22-24 . 57 58 Phage ΦX174 is a small (~320 Å), tailless coliphage from the Microvirus family. It carries a circular, 59 single-stranded DNA genome of only 5386 bp that encodes 11 genes 25,26 . To enter a host cell, ΦX174 60 relies solely on attaching to the core OS by one of its spike proteins before migrating and irreversibly 61 adsorbing to the host's cell surface via its capsid [27][28][29] . ΦX174 does it with a high degree of specificity: 62 in one study, among 783 different E. coli isolates, only six (0.8 %) could be infected by wildtype 63 ΦX174 30 . In the laboratory ΦX174 infects -and hence is usually grown on -E. coli C, which produces 64 rough type (i.e., lacking in O-antigen) LPS molecules [31][32][33][34] (Figs. 1 and S1).

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Here, we explore the evolutionary potential of E. coli C to become resistant to infection by ΦX174 74 and, conversely, the potential of ΦX174 mutants to infect phage-resistant E. coli C hosts with modified 75 LPS structures. We first generate a set of 31 E. coli C mutants that are resistant to infection by ΦX174 76 wildtype and determine that each mutant carries at least one mutation affecting LPS biosynthesis. 77 78 The currently accepted model for predicting LPS structure posits that the presence or absence of each 79 LPS gene leads to a single LPS phenotype. Based on the position of each mutation, the one-gene-one-80 phenotype model allows us to predict the LPS structure of each of our LPS mutants. Our 31 E. coli C 81 mutants collectively produce LPS molecules with seven distinct structures. To test the accuracy of 82 these predictions, we evolved and genotypically characterized a set of sixteen ΦX174 genotypes that 83 can, between them, infect all initially resistant 31 E. coli C mutants. If the LPS structure predictions 84 are correct then all bacteria predicted to carry the same LPS structure should be susceptible to the 85 assembled downstream of the missing component are also absent from the final LPS structure. Under 118 the assumption that each mutation in an LPS gene leads to a complete lack of function, this LEGO ® 119 block principle was used to predict the LPS structure and type of each of the 31 E. coli C phage-120 resistant mutants (Figs. 2, S1, S2 and Table S1). A total of seven emergent LPS structures were 121 forecast from our set of mutants (Fig. 2). Compared with the E. coli C wildtype LPS, three of these 122 seven LPS structures carry alterations in the outer-core OS, and hence are predicted to be rough type 123 (as is wildtype E. coli C; ten mutants). The remaining four LPS structures differ from wildtype LPS in 124 both the inner-and outer-core OS, and are presumed to be of the deep rough type (twenty mutants) 125 (see Table S1). The LPS structure and type of the remaining mutant (E. coli C R25) was not predicted, 126 because the effect of the phage-resistance mutation on LPS biosynthesis remains unclear; specifically, 127 R25 carries a nonsense mutation in rfaH. The rfaH gene encodes a transcriptional antiterminator that 128 regulates the waa operon 14,40 , meaning that any number of LPS biosynthetic genes could potentially be 129 affected by this mutation. two groups depending on their degree of truncation and/or loss of phosphate groups: the "deep rough" 134 phenotype with a completely truncated outer core or a loss of phosphate groups (twenty E. coli C mutants), and 135 the "rough" phenotype with smaller LPS truncations located in the outer core (ten E. coli C mutants). The LPS 136 phenotype of the final E. coli C mutant (R25) could not be predicted (see text). The products of genes in red 137 are also involved in LPS biosynthesis and, although no mutations were identified in these genes during this 138 study, are also potential targets for phage-resistance. Details on the functions of gmhA, gmhB, gmhD, hldE, galU, 139 and galE are provided in Fig. S2. "?P": No information was found on the phosphorylation of Hep(I) in the 140 absence of waaF; (P): only 40% of the hexose phosphorylation was observed 39 ; R#: the number gave to each 141 E. coli C resistant strain; "*, **, ***, °, °°": identical symbols indicate that different genes are inactivated but are 142 expected to lead to the same LPS core structure.

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The LPS structure predictions above can be tested by evolving wildtype ΦX174 to specifically infect 145 E. coli C cells with modified LPS structures. That is, a phage that can infect a given modified LPS 146 structure is expected to be able to infect all bacterial strains displaying this LPS type, regardless of the 147 underlying mutations. Alternatively, the inability of mutant phages to cross-infect bacterial strains of 148 the same predicted LPS structural class (but via different mutations) would indicate that these 149 mutations actually lead to distinct LPS structures. 150 151 ΦX174 evolves to overcome host resistance, a process that can be accelerated by host diversity 152 Phages that can infect resistant E. coli C mutants were evolved by serially transferring wildtype ΦX174 153 on each of the 31 resistant E. coli C strains ( Fig. 3A and Methods). To reduce the complexity of the 154 experiment, bacterial cultures were not allowed to co-evolve. Instead, phages were inoculated into 155 fresh, exponentially growing host cultures at each transfer. The experiment gave rise to twelve evolved 156 phages that were collectively able to infect 21 (of the 31) initially resistant E. coli C strains (these display 157 the "easy" resistant phenotype; see Table S2). However, even after 21 transfers, ten (of 31) resistant 158 E. coli C strains remained uninfected (the "hard" resistant phenotype; see Table S2). To increase the 159 chances of evolving phages capable of infecting these strains, two further evolution experiments were 160 designed (Figs. 3B and 3C). In both of these experiments, phage diversity was increased by 161 combining all evolved phage strains into a phage cocktail 41,42 . Phage diversity was maintained from 162 one transfer to another by adding all corresponding resistant strains to the host culture (see Methods). 163 The phage cocktail was serially transferred once per day in the second evolution experiment, while 164 four transfers per day were performed in the third evolution experiment. At the end of the third 165 evolution experiment, while six E. coli C resistant strains remained uninfected by their own phage, all 166 resistant strains could be cross-infected by at least one evolved of the phage mutant. Our method of 167 isolating re-infecting phage is simple and effective, and allowed the successful retrieval of phages that 168 can target any of the 31 resistant phenotypes. 169 170 Increasing both phage and host diversities in the second and third evolution experiments accelerated 171 the adaptation of ΦX174. This result seemingly contradicts recent research by Sant et al.,which 172 elegantly shows that higher host diversity decreases the rate at which phages evolve 43 . By adapting 173 phage ΦJB01 (a phage that likely uses the LPS to infect its E. coli host 43 ) to a set of different E. coli 174 strains, the authors demonstrate that adaptation to resistant (non-permissive) strains is faster in 175 evolution experiments where ΦJB01 is exposed to only one permissive and one non-permissive host, 176 than when two non-permissive and one permissive strain are used. Similar to the two-strains 177 experiment of Sant et al. 43 , in our first evolution experiment, we evolved phage ΦX174 in the presence 178 of one permissive and one non-permissive E. coli C strain. While the experiment quickly gave rise to 179 phages able to infect most of the non-permissive strains (Fig. 4A), some phage-resistant hosts could 180 only be infected when both host and phage diversities were increased ( Fig. 4B-C To identify the mutation(s) responsible for the re-infection of the resistance mutants, we performed 217 whole genome re-sequencing on the 16 evolved phages (isogenic isolates) that successfully overcame 218 the corresponding resistance in one of the three evolution experiments (see Methods). We found 219 mutations in F and/or H genes in all 16 (Table S2) and discuss their molecular details in depth in the 220 subsequent section. Notably, all hard resistant E. coli C strains -those whose resistance was overcome 221 in the second and/or third evolution experiments -are only infected by phage carrying four mutations 222 (see Tables S1 & S2). The evolutionary emergence of a multi-mutation phage is expected to require 223 a large number of transfers in a two-strain evolution experiment (Fig. 4B). However, this process is 224 significantly accelerated by propagating a diverse mixture of phages that can overcome easy resistance, 225 some of which may act as evolutionary intermediates (i.e., carry a subset of mutations required to 226 overcome hard resistance). The mutational sets required to overcome hard resistance can subsequently 227 emerge in a single mutational step, either by recombination 44 or by de novo mutation (Fig. 4C). For 228 example, hard resistant strain E. coli C R22 (a galU mutant; see Table S1) is overcome in the second 229 evolution experiment by ΦX174 R22 T1, a phage that carries four mutations (three in F, one in H; 230 see Table S2). Each of these mutations was present in the phage cocktail used to initiate the 231 evolutionary lineage: ΦX174 R18 T4 (which overcomes the easy resistance of E. coli C R18) contains 232 three of the four mutations, and the final mutation is found in ΦX174 R10 T6 (which overcomes the 233 easy resistance in E. coli C R10). Thus, adaptation to an easy resistant LPS structure (e.g., that of E. (populations) that failed to infect their corresponding resistant E. coli C strains (see Table S3). Of the 255 three controls and 15 resistant lines (the phage infecting R5 was removed from the mutational 256 analyses, see Text S1) that remained uninfected by their own phage, a total of 33 mutations were 257 detected, but only ten were unique. All of these mutations are located in the F gene, except one in the 258    Table S2). Parallel evolution in phages that evolved to infect E. coli C strains with the 300 same predicted LPS structure generally support the predicted classes of LPS structure. However, 301 analyses in the following section demonstrate that LPS structure predictions generally underestimate 302 LPS diversity. 303 304

Evolved phages can be used to discriminate between LPS phenotypes 305
To determine precisely which of the 31 initially resistant E. coli C strains each of the 16 evolved phages 306 can infect (i.e., the infection pattern of each phage), a cross-infection assay was performed (Fig. S3). 307 This involved measuring the infectivity of all possible host-phage combinations by two distinct 308 methods: (i) phage spotting on host lawns, and (ii) host spotting on phage lawns. In cases where a 309 mismatch between the two results was observed, standard plaque assays were used as a tie breaker 310 (see Methods). If our predictions -which are based on current literature in the field -of seven distinct 311 LPS structures among the 31 initially resistant E. coli C strains are accurate (see Fig. 2 and Table S1), 312 then all phages that evolved to infect a mutant with a given predicted LPS structure are expected to 313 be capable of cross-infecting all other host strains with the same predicted LPS structure. That is, the 314 infection patterns in Fig. S3 would be identical for each class (colour) of E. coli C mutant. To visually 315 determine whether infection patterns at least cluster according to predicted LPS structure, a 316 hierarchical agglomerative clustering analysis was performed on the infection matrix in Fig. 6. Of the 317 six predicted LPS structures that occur in at least two E. coli C mutants, three form unbroken clusters 318 with respect to infectivity pattern (cyan, light purple, and dark purple highlighting in Fig. 6) and three 319 do not (magenta, yellow, green highlighting). Each group is discussed in more detail below. 320 321 LPS predictions that cluster by infection pattern. Firstly, ΦX174 mutants R22 T1 and R28 T1 -isogenic 322 phages that evolved to infect resistant strains with mutations in galU (E. coli C R22) and waaG (E. coli 323 C R28) -can cross-infect all eight E. coli C mutants predicted to exhibit the same LPS structure (E. coli 324 C R9, R16, R17, R22, R23, R28, R30, R35; highlighted in cyan in Figs. 6 and S4). Secondly, both E. 325 coli C waaW mutants (R2 and R34; light purple) are cross-infected by the same three evolved phage 326 genotypes (R6 T12, R13 T6, R25 T4). Thirdly, both E. coli C waaF mutants (R12 and R14; dark purple) 327 are infected by a near-identical set of seven (R12) or six (R14) phages. In each of these cases, phage 328 infectivity patterns appear to be a good indicator of host LPS phenotype (and, to some extent, 329 underlying host genotype).  (Fig. 6). Hence, it is likely that the LPS structure of R7 differs from that of the other 352 waaO mutants. The difference could conceivably result from residual (or altered) WaaO function; E. 353 coli C R7 carries an in-frame deletion of a 6-bp repeat in waaO, which alters two WaaO residues, but 354 leaves the remainder of the protein intact (see Table S1). While it may be possible that the LPS 355 structure of R7 resembles that of galU/waaG mutants (Figs. 2 and S4 in Figs. 6 and S4) also do not fit with the LPS structure prediction; R6 is infected by a single phage 360 that evolved directly on host R6 (phage R6 T12 from the third evolution experiment), while R11 is 361 cross-infected by two different phages (phage R13 T6 from the first evolution experiment, and R25 362 T4 from the third). The cluster map analysis indicates that R11 displays an LPS phenotype comparable 363 to the rough LPS phenotype of R33, a waaT mutant (Fig. 6). E. coli C R6 was the last resistant strain 364 to be infected by an evolved phage, and the evolved phage infecting R6 cannot cross-infect R11. 365 Therefore, the location and nature of the mutation in waaP strongly impact the LPS structure, possibly 366 by affecting the number of phosphorylated hexoses in the inner core LPS (see Fig. S1). 367 368 Thirdly, eight deep rough E. coli C mutants are predicted to have the shortest possible LPS structure 369 ( Fig. 2; green highlighting in Figs. 6 and S4). Yet the combination of phages that can infect each of 370 them is remarkably different, with these E. coli C strains clustering into four different phenotypic sub-371 groups (Fig. 6). The first sub-group consists of E. coli C R8, R10, R18, and R26. Each of these strains 372 can be cross-infected by their corresponding evolved phages regardless of their genotypes (a mixture 373 of waaC or hldE mutants). However, none of the four evolved phages can cross-infect E. coli C R27 374 or R31 (hldE mutants). E. coli C R27 and R31 constitute the second sub-group, in which both bacteria 375 and their corresponding evolved phages acquired identical genotypes and phenotypes. E. coli C R5 376 (gmhB mutant) and E. coli C R32 (hldE mutant) fall into a third and fourth sub-groups. They are the 377 only deep-rough mutants for which the corresponding phage lineages failed to adapt during the first 378 evolution experiment. Only a very narrow but different set of evolved phages can infect them 379 (especially for R32), and none of them belongs to the first or second deep rough LPS phenotypic 380 group. 381 382 The phenotypic differences observed in the four groups are not due to the involvement of hldE, waaC, 383 and gmhB, since a total loss of function in any of these genes always leads to a complete truncation of 384 the inner core LPS (Fig. S2B). E. coli C R8, R10, R18, and R26 belong to the same phenotypic group 385 despite having either hldE or waaC mutated, while neither R27, R31, or R32 (hldE mutants) belong to 386 this group (Table S1, Fig. 6). The involvement of the gmhB gene in E. coli C R5 might be an exception. 387 Previous research demonstrated that the deletion of gmhB only caused a partial defect in the synthesis 388 of the LPS core, resulting in the formation of heptoseless and heptose-rich forms 55 . However, the lack 389 of other resistant strains carrying mutations in gmhB in our dataset makes it unclear whether any 390 mutation in gmhB leads to the same mix of a unique deep rough and the WT phenotypes or if multiple 391 deep rough phenotypes can appear and persist. Nonetheless, the phenotype exhibited by R5 is 392 strikingly close to the one of E. coli C WT (Fig. 6) Another major finding of our study, namely that increasing phage and host diversity allows the 435 infection of hard resistant phenotypes (see The main safety advantage of ΦX174 has also been one of the greatest issues for the potential of a 456 therapeutic agent: its high host specificity 30 . Here we show that evolution experiments can easily 457 extend the host range of ΦX174 to formerly resistant strains, unlocking its potential as a powerful 458 therapeutic. Hence, due to therapeutic potential and potential for knowledge transfer, we believe that 459  : ΦX174 R4 T7, ΦX174 R5 T2, ΦX174 R8 T6, ΦX174 R10 T6, ΦX174 R13 T6, ΦX174  525   R18 T4, ΦX174 R19 T1, ΦX174 R20 T5, ΦX174 R21 T1, ΦX174 R24 T1, ΦX174 R26 T1, ΦX174  526  R27 T1, ΦX174 R29 T1, and ΦX174 R31 T1. Phages infecting R5 and R19 were used for the cocktail 527 but removed from the mutational analysis (see Text S1 and Table S4). Each lineage was transferred 528 daily for up to four days, on non-evolving host cultures containing (i) E. coli C wildtype, (ii) the 14 529 host strains (see Text S1 and Table S4) for which resistance had been overcome (permissive hosts), 530 and (iii) an excess of the still-resistant strain of interest (non-permissive host). For the non-permissive 531 host, we used R6 (waaP/pssA mutant), R22 (galU mutant), R25 (rfaH mutant), or R28 (waaG mutant; 532 see Table S1). E. coli C R22 and R28 were chosen as representatives of the galU and waaG mutants, 533 respectively. Each bacterial strain (permissive and non-permissive) was grown separately in LB liquid 534 culture for one hour (37˚C, 250 rpm), then pooled at roughly equal number of colony forming units 535 The resistant strain of interest was added in excess to give a final volume of 4 ml. Infected cultures 536 were initiated by adding the phage cocktail (MOIinput ~ 0.1), shaken at 250 rpm, 37˚C, for 5 hours (see 537 Protocol from the second evolution experiment was adjusted to reduce the amount of time necessary 545 to retrieved the desired phages. We increased the number of lineages' transfers per day from one to 546 four, for a total of four days (16 transfers). E. coli C R6 (waaP/pssA mutant) and R25 (rfaH mutant, 547 see Table S1) were used as non-permissive hosts. After reaching their exponential log phase, bacterial 548 strains (except the non-permissive hosts) were mixed depending on their growth ( phase culture in 4 ml SSA. Then, 3 µl of each undiluted evolved phage lysate (between 10 7 and 10 9 573 pfu ml -1 ) was dropped at the surface. Method 2. Each top agar overlay was prepared by mixing a volume 574 of each phage lysate in 4 ml SSA at a final concentration of ~10 7 pf ml -1 . Then, 3 µl of both undiluted 575 and ten-fold diluted of each E. coli C strain (from overnight culture) were dropped onto the surface 576 with a pipette. Plates were incubated inverted at 37˚C for ~17 hours. E. coli C wildtype was used as a 577 positive control (permissive strain), and E. coli K-12 MG1655 as a negative control (non-permissive 578 strain). A bacterial host strain was classified as sensitive only when signs of lysis were detected using 579 both methods, in at least two (of three) replicates per method. A phage-bacterium combination that 580 yielded different results between the two methods was tested in standard plaque assays (see 581 Supplementary Methods). Finally, the host strain was classified as sensitive if plaques was observed 582 at both dilutions. 583 584 Data visualisation. The hierarchical agglomerative clustering analysis (Clustermap Fig. 6), the 585 heatmap (Fig. S3), and growth curves (Fig. S4) were generated using the default settings of the 586 Seaborn library (version 0.11.2) for Python (version 3.7.4). All schematic drawings (Figs. 1-4, S1 and 587 S2) were created with BioRender.com. The Lollipop plot (Fig. 5A)  Bacteria and phage isolates that were excluded from downstream analyses. We removed four bacterial strains (E. 629 coli C R1, R3, R15, and R19) and two evolved phage strains (ΦX174 R5 T2 and ΦX174 R19 T1) from 630 both mutational and phenotypical analyses. The reasons behind these exclusions are outlined below. 631 632 Bacteria. We found that both E. coli C R3 and R15 were not isogenic. While whole genome re-633 sequencing from their respective glycerol stocks showed only a single mutation in galE, whole genome 634 re-sequencing performed on colony re-streaks showed that additional mutations were systematically 635 associated with the single mutation in galE (Tables S4 and S5). E. coli C R1 displayed an unstable 636 resistant phenotype using the spotting assay method 1 (see section Determination of the evolved 637 phages' host range by spotting assays). We performed phenotypic assays by plating top agar 638 overlays from overnight cultures of E. coli C R1 without phage. All overnight cultures were started 639 with a randomly picked single colony obtained from the glycerol stock of R1. We observed that 640 colonies of R1 made different lawn types: either "smooth" (no bacterial aggregate) or "granulous" 641 (presence of numerous bacterial aggregates), which could potentially impact phage infectivity. Finally, 642 E. coli C R19 was only partially resistant to ΦX174 wildtype. ΦX174 wildtype was still able to infect 643 and produced a few small clear plaques on a lawn of R19. It carries a single mutation yajC (locus tag 644 B6N50_17610), which encodes a periplasmic protein 75 with a putative preprotein translocase subunit 645 (Pfam e-value 2.3e-26) (see Table S4). While no link to the LPS biosynthesis or assembly has yet been 646 defined, yajC could conceivably play a role in the injection of phage DNA into the bacterium's 647 cytoplasmic membrane 76,77 . 648 649 In our matrices (Figs. 6 & S3), E. coli C R32 cannot be infected by any evolved phages from the first 650 evolution experiment. It is because the phage infecting R5 was the only phage that could infect R32, 651 but was removed from the subsequent analysis (see below). 652 653 Phage. Since we removed R19 from the final analysis, we also removed its corresponding evolved phage 654 obtained during the first evolution experiment (ΦX174 R19 T1). We also removed the phage infecting 655 R5 (ΦX174 R5 T2) because it was not isogenic (confirmed by Sanger Sequencing). Details on their 656 respective mutations (in the H gene) can be found in Table S4.     . 2). "Easy" or "Hard" LPS 738 structure phenotypes?: easy LPS phenotypes were overcome or could be cross-infected after the first phage evolution experiment, while hard 739 LPS phenotypes required the second or third phage evolution experiment to be infected. "→": indicates 5'à3' direction. "INS": insertion.          For both methods, phage lysate spots were dried for at least 30 minutes under sterile laminar flow, 885 and the plates subsequently incubated inverted at 37˚C for ~17 hours. E. coli C wildtype was used as 886 a positive control (permissive strain), and E. coli K-12 MG1655 as a negative control (non-permissive 887 strain). A bacterial host strain was classified as sensitive only when signs of lysis were detected using 888 both methods, in at least two (of three) replicates per method. A phage-bacterium combination that 889 yielded different results between the two methods was tested in standard plaque assays as follows: a 890 top agar overlay was prepared by mixing 100 µl of the phage lysate diluted to 10 -1 and 10 -6 with 200 891 µl of the resistant E. coli C strain from stationary phase culture in 4 ml SSA, then poured on LB plates 892 and dried for at least 15 minutes. The presence or absence of plaques was assessed the next day. 893 Finally, the host strain was classified as sensitive if plaques was observed at both dilutions. 894 895 896