Reversing Bacterial Resistance to Antibiotics by...

of 34 /34
1 Reversing Bacterial Resistance to Antibiotics by Phage- 1 Mediated Delivery of Dominant Sensitive Genes 2 Rotem Edgar, 1,3 Nir Friedman, 2,3 Shahar Molshanski-Mor, and Udi Qimron * 3 Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel 4 Aviv University, Tel Aviv, Israel 5 6 Running title: reversing antibiotic resistance 7 8 Keywords: λ phage, E. coli, tellurite-resistance, rpsL, gyrA 9 10 * Corresponding author. Mailing address: Department of Clinical Microbiology and 11 Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. 12 Phone: 972-3-6405191. Fax: 972-3-6409511. E-mail: [email protected] 13 1 Present address: Epidemiology Division, Sourasky Medical Center, Tel Aviv, Israel 14 2 Present address: Department of Ruminant Sciences, Volcani Center, Bet Dagan, Israel 15 3 These authors contributed equally to this study. 16 Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Appl. Environ. Microbiol. doi:10.1128/AEM.05741-11 AEM Accepts, published online ahead of print on 23 November 2011

Embed Size (px)

Transcript of Reversing Bacterial Resistance to Antibiotics by...

  • 1

    Reversing Bacterial Resistance to Antibiotics by Phage-1

    Mediated Delivery of Dominant Sensitive Genes 2

    Rotem Edgar,1,3 Nir Friedman,2,3 Shahar Molshanski-Mor, and Udi Qimron* 3

    Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel 4

    Aviv University, Tel Aviv, Israel 5


    Running title: reversing antibiotic resistance 7


    Keywords: phage, E. coli, tellurite-resistance, rpsL, gyrA 9


    *Corresponding author. Mailing address: Department of Clinical Microbiology and 11

    Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. 12

    Phone: 972-3-6405191. Fax: 972-3-6409511. E-mail: [email protected] 13

    1 Present address: Epidemiology Division, Sourasky Medical Center, Tel Aviv, Israel 14

    2 Present address: Department of Ruminant Sciences, Volcani Center, Bet Dagan, Israel 15

    3 These authors contributed equally to this study. 16

    Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.05741-11 AEM Accepts, published online ahead of print on 23 November 2011

  • 2


    Pathogen resistance to antibiotics is a rapidly growing problem, leading to an urgent 18

    need for novel antimicrobial agents. Unfortunately, development of new antibiotics 19

    faces numerous obstacles, and a method that will resensitize pathogens to approved 20

    antibiotics therefore holds key advantages. We present a proof-of-principle for a 21

    system that restores antibiotic efficiency by reversing pathogen resistance. This 22

    system uses temperate phages to introduce, by lysogenization, genes rpsL and gyrA 23

    conferring sensitivity in a dominant fashion to two antibiotics, streptomycin and 24

    nalidixic acid, respectively. Unique selective pressure is generated to enrich for 25

    bacteria that harbor the phages encoding the sensitizing constructs. This selection 26

    pressure is based on a toxic compound, tellurite, and therefore does not forfeit any 27

    antibiotic for the sensitization procedure. We further demonstrate a possible way of 28

    reducing undesirable recombination events by synthesizing dominant sensitive genes 29

    with major barriers to homologous recombination. Such synthesis does not 30

    significantly reduce the gene's sensitization ability. Unlike conventional 31

    bacteriophage therapy, the system does not rely on the phage's ability to kill 32

    pathogens in the infected host, but instead, to deliver genetic constructs into the 33

    bacteria, and thus render them sensitive to antibiotics prior to host infection. We 34

    believe that transfer of the sensitizing cassette by the constructed phages will 35

    significantly enrich for antibiotic-treatable pathogens on hospital surfaces. Broad 36

    usage of the proposed system, in contrast to antibiotics and phage therapy, will 37

    potentially change the nature of nosocomial infections toward being more 38

    susceptible to antibiotics rather than more resistant. 39

  • 3



    Bacteria have evolved to overcome a wide range of antibiotics, and resistance 42

    mechanisms against most of the conventional antibiotics have been identified in some 43

    bacteria (7). Accelerated development of newer antibiotics is being overrun by the pace 44

    of bacterial resistance. In the USA, for example, over 70% of hospital-acquired infections 45

    involve bacteria resistant to at least one antibiotic, and in Japan over 50% of the clinical 46

    isolates of Staphylococcus aureus are multidrug-resistant (11). 47

    This increasing threat has revived research into phage therapy. For example, a 48

    clinical phase I and II control trial was recently completed successfully for the treatment 49

    of chronic bacterial ear infections (21). Nevertheless, although phage therapy has been 50

    practiced for several decades in some of the former Soviet Union countries and Poland, 51

    there are still many doubts as to its ability to replace antibiotics. Major concerns over the 52

    use of phage therapy include neutralization of phages by the spleen/liver and by the 53

    immune system, their narrow host range, bacterial resistance to the phage, and lack of 54

    sufficient pharmacokinetic and efficacy studies in humans and animals (1, 11). 55

    A recent study used phages as a genetic tool to increase bacterial susceptibility to 56

    antibiotics. That study used phage M13, of the Gram-negative Escherichia coli, to 57

    genetically target several gene networks, thus rendering the bacteria more sensitive to 58

    antibiotics (10). It demonstrated that disrupting the SOS response by M13-mediated 59

    gene-targeting renders the bacteria several-fold more sensitive to a variety of antibiotics. 60

    It also demonstrated that phage-mediated gene transfer combined with antibiotics 61

    increases the survival of mice infected with pathogenic E. coli. Overall, the study showed 62

  • 4

    that transferring genes by phage M13 weakens the bacteria, and render them more 63

    susceptible to killing by antibiotics. We believed that some aspects of that study required 64

    further modification. First, the transferred genes target a beneficial pathway in bacteria, 65

    and therefore significantly reduce the fitness of the bacteria harboring this phage. 66

    Consequently, negative selection pressure is constantly being applied against transfer of 67

    these genes by the M13 phages. Second, no mechanism to facilitate genetic transfer of the 68

    M13 genes was used: high multiplicity of infection in the experimental settings 69

    compensated for this shortcoming. Nevertheless, such settings cannot be used in field 70

    experiments. Third, the phage was experimentally tested in vivo, in mice, but immune 71

    responses against it were not examined. Therefore, despite the novelty of that study in 72

    terms of unique genetic-targeting by phages, the end result is very similar to conventional 73

    phage-therapy practices, in which phages are used to directly kill the pathogen. 74

    Different approaches make use of phages as "disinfectants" of pathogens present 75

    on edible foods, plants, and farm animals. In addition to increasing the shelf life of these 76

    products, the treatment is intended to prevent occasional outbreaks of disease. The US 77

    Food and Drug Administration recently approved the use of an anti-Listeria phage 78

    cocktail for application on meat and poultry as a preventive measure against Listeria (5). 79

    Other phage cocktails have been approved as food additives in Europe, and many are 80

    currently being developed by phage biotech companies. These applications demonstrate 81

    that phages can be dispersed in the environment and efficiently target pathogens in their 82

    surroundings. 83

    Here we present a proof-of-principle for genetic delivery of constructs using 84

    phages to target pathogens in the environment. In the described system, phages are 85

  • 5

    genetically engineered to reverse the pathogens' drug resistance, thereby restoring their 86

    sensitivity to antibiotics. The phages transfer, by lysogenization into the pathogens, a 87

    drug-sensitizing DNA cassette, which was previously shown to render bacteria sensitive 88

    to agents to which they had acquired resistance (6). Pathogens that are lysogenized by the 89

    designed phages are selected by tellurite because the phage is engineered to contain a 90

    DNA element conferring resistance to this bactericidal agent. Rather than being 91

    administered to patients, the phages are intended for dispersion on hospital surfaces, thus 92

    gradually reversing the occurrence of drug-resistant pathogens and competing with the 93

    resistant pathogens residing in hospitals. This method would thus enable the use of well-94

    established antibiotics against which resistance has been acquired. 95




    Bacterial strains. Bacterial strains used in this study are listed in Table S1 in the 99

    supplemental material, as well as in Tables 1 and 2. 100

    Oligonucleotides. Oligonucleotides used in this study are listed in Table S2 in the 101

    supplemental material. 102

    Isolation of resistant mutants. Over twenty different overnight cultures of a total 103

    of ~1011 E. coli K-12 cells were inoculated on Luria-Bertani (LB)-agar plates containing 104

    50 g/ml streptomycin or 50 g/ml nalidixic acid. Resistant mutants emerged, in both 105

    cases, at a median frequency of ~1 in 109 CFU, and were picked from different cultures, 106

    to reduce the occurrence of sibling mutants. These bacteria were streaked on an agar plate 107

  • 6

    containing the appropriate antibiotic. The rpsL or gyrA genes of resistant mutants 108

    emerging on the plate were PCR-amplified followed by DNA sequencing. 109

    Plasmid construction. Plasmids were constructed using standard molecular 110

    biology techniques. DNA segments were amplified by PCR. Standard digestion of the 111

    PCR products and vector by restriction enzymes was carried out according to the 112

    manufacturer's instructions. Plasmid sequences are provided in the supplemental material. 113

    Phages. Genetic engineering of the different phages was carried out using 114

    gt11/EcoRI/Gigapack III Gold Cloning Kit (Stratagene) according to the 115

    manufacturer's protocols. Briefly, EcoRI-digested arms of phage gt11 were used to 116

    construct the lysogenizing phage carrying the different DNA inserts, encoding a 117

    chloramphenicol-resistance gene. DNA inserts were PCR amplified from plasmids 118

    pRpsL-wt, pRpsL-sil, pRpsL4 (Fig. 1A) using primers 231F/R (Table S2), and digested 119

    with MfeI restriction enzyme, which produces ends that are compatible with EcoRI. 120

    Ligation was carried out using T4 DNA ligase (New England Biolabs). The ligated 121

    products were transformed into E. coli strain Y1088, which supports gt11 growth. 122

    Generated plaques were propagated in E. coli Y1088 or E. coli ymel, which were then 123

    used to lysogenize the hosts. In several cases, phages were further manipulated in a host 124

    which lacks supE, a suppressor gene necessary for phage growth. In such cases, the phage 125

    was transferred by P1-mediated transduction to a permissive host and propagated there. 126

    Phages carrying tellurite resistance were constructed by homologous recombination-127

    based genetic engineering of the tellurite-resistance marker instead of the 128

    chloramphenicol-resistance gene. The tellurite-resistance genes tehAB were amplified 129

    from the E. coli chromosome using primers N1/N2 (Table S2) for -RpsL4-tell, -130

  • 7

    RpsL-wt-tell, -RpsL-sil-tell, -Ctrl-tell, and -GyrA-tell (Fig. 1B). Primers RE22/N2 131

    (Table S2) were used for construction of -2xRpsL-tell. The obtained PCR products were 132

    used for homologous recombination-based genetic engineering as described below. 133

    Sequences of the phage inserts are presented in the supplemental material. 134

    Homologous recombination-based genetic engineering. Homologous 135

    recombination using short-homology flanking ends was performed as described 136

    previously (13). Briefly, an overnight culture of lysogens carrying different DNA inserts 137

    encoding the chloramphenicol-resistance gene was diluted 75-fold in 25 ml LB medium 138

    with appropriate antibiotics and grown at 32C in a shaking water bath to an OD600 of 139

    0.6. Then, half of the culture was heat-induced for recombination function of the 140

    prophage at 42C for exactly 4 min in a shaking water bath. The remaining culture was 141

    left at 32C as the uninduced control. The induced and uninduced samples were 142

    immediately cooled on ice slurry and then pelleted at 3600 g at 0C for 10 min. The pellet 143

    was washed twice in ice-cold ddH2O, then resuspended in 200 l ice-cold ddH2O and 144

    kept on ice until electroporation with ~500 ng of a gel-purified PCR product encoding the 145

    tellurite-resistance genes. A 25-l aliquot of electrocompetent cells was used for each 146

    electroporation in a 0.1-cm cuvette at 25 F, 1.75 kV and 200 . After electroporation, 147

    the bacteria were recovered in 1 ml LB for 1 h in a 32C shaking water bath and 148

    inoculated on selection plates containing 1 to 4 g/ml tellurite. The DNA insertion into 149

    the resulting phages, -RpsL4-tell, -RpsL-wt-tell, -RpsL-sil-tell, -Ctrl-tell, -GyrA-150

    tell, and -2xRpsL-tell, was confirmed by PCR using primer 233F along with 232F or 151

    232R. 152

  • 8

    Lysogenization. Overnight culture of the resistant mutants was diluted 1:100 in 153

    LB with the appropriate antibiotics, 10 mM MgSO4 and 0.2% (W/V) maltose. When the 154

    culture reached an OD600 of 0.60.8, 100 l was mixed with 10 l phage , carrying a 155

    resistance gene, in a 1.5-ml tube and incubated at room temperature for 20 min. Cells 156

    were inoculated on appropriate selection plates and incubated overnight at 32C. 157

    Lysogens emerged on selection plates to which the phage carried a resistance gene. 158

    Lysogenization was validated by plating the lysogens at 42C: lysogens cannot grow at 159

    this temperature because the prophage is induced to its lytic cycle (see Materials and 160

    Methods). 161

    Transductions. Transductions were used to transfer antibiotic-resistance markers 162

    or complete phages between strains (in cases where the strain did not carry suppressor 163

    genes required for growth). P1 lysate was prepared as follows: overnight cultures of 164

    donor strain were diluted 1:100 in 2.5 ml LB + 5 mM CaCl2 + 0.2% (W/V) glucose. After 165

    1 h shaking at 37C (or 32C for lysogens), 107-108 PFU of phage P1 was added. 166

    Cultures were aerated for 1 to 3 h, until lysis occurred. The obtained P1 lysate was used 167

    in transduction where 100 l fresh overnight culture was mixed with 1.25 l of 1 M 168

    CaCl2 and 0 to 100 l P1 phage lysate. After incubation for 30 min at 30C without 169

    shaking, 100 l Na-citrate and 500 l LB were added. Cultures were incubated at 37C or 170

    32C for 45 or 60 min, respectively, then 3 ml of warm LB supplemented with 0.7% agar 171

    was added and the suspension was poured onto a plate containing the appropriate drug. 172

    Transductants obtained on antibiotic plates were streaked several times on selection 173

    plates and verified by PCR for the presence of the transduced DNA fragment. 174

  • 9

    MIC determinations. MIC determination was carried out by following the 175

    procedure described by Wiegand et al. (19). Briefly, bacterial cells were grown overnight 176

    at 32C in LB and diluted to 107109 CFU/ml. The obtained suspension was serially 177

    diluted 10-fold for different spot concentrations, as indicated. Approximately 1 l of 178

    bacterial suspension was then spotted onto selection plates containing different 179

    concentrations of either streptomycin or nalidixic acid along with the appropriate 180

    selection agent (chloramphenicol or tellurite), as indicated, using a 48-pin replicator. 181

    Plates were incubated overnight and photographed using MiniBis Pro (Bio-Imaging 182

    Systems). Photographs were digitally manipulated using GIMP2 software to adjust 183

    contrast. Liquid-based MIC determination assays were carried out by inoculating serial 184

    dilutions of an antibiotic in liquid LB broth with bacterial cultures (OD600 ~0.05) in 96-185

    well microtiter plates. Plates were incubated overnight at 32oC and OD600 was then 186

    measured. The lowest antibiotic concentration at which the relative growth compared to 187

    the "no-drug" control was below 10% was determined as the MIC. 188




    Mutations in the target gene, rpsL, constitute a major resistance mechanism 192

    to streptomycin. Our overall goal in this study is to provide a proof-of-principle for a 193

    genetic system able to restore drug sensitivity to drug-resistant pathogens residing on 194

    hospital surfaces. We chose, as a first step, to use streptomycin as the model drug. 195

    Streptomycin is highly useful as an effective antibiotic against both Gram-negative and 196

    Gram-positive bacteria. For example, streptomycin is a mainstay of tuberculosis therapy. 197

  • 10

    However, streptomycin-resistant Mycobacterium tuberculosis emerge during treatment, 198

    and 24 to 85.2% of them have mutations in either rpsL or rrs (15). The rpsL gene 199

    product, S12, is an essential, highly conserved protein of the 30S small ribosomal 200

    subunit. Most of the acquired resistance to streptomycin is due to specific mutations in 201

    rpsL that prevent the inhibitory binding of streptomycin to the essential rpsL gene 202

    product. We wanted to reproduce these findings in a model bacterium, E. coli, and then to 203

    restore its sensitivity to streptomycin. We therefore inoculated E. coli K-12 on LB-agar 204

    plates containing 50 g/ml streptomycin and selected for resistant mutants. This 205

    procedure fairly simulates the selection of spontaneous drug-resistant-mutant evolution in 206

    hospitals following streptomycin treatment. Resistant colonies emerged with a median 207

    frequency of 1 in 109 CFU. Mutations in rpsL were found in 21 out of 22 resistant 208

    mutants, a frequency that corroborates with that in clinical isolates. As listed in Table 1, 209

    10 mutants harbored a K88R substitution in RpsL, 6 had an R86S substitution, and P42S, 210

    K43L, K43N, R54S, K88E substitutions were each identified once. These mutation types 211

    also corroborate with previous studies, confirming that a major mechanism for 212

    streptomycin resistance relies on mutations in rpsL (e.g. (16, 18)). Therefore, targeting 213

    this resistance mechanism or reversing its effect should prove highly beneficial in 214

    controlling drug-resistant pathogens. 215

    Wild-type (wt) rpsL transformed on a plasmid dominantly confers 216

    streptomycin sensitivity. Lederberg first reported in 1951 that wt rpsL is a dominant 217

    sensitive allele with regard to streptomycin resistance (6). This means that introduction of 218

    a sensitive allele of wt rpsL into a streptomycin-resistant bacterial cell will result in 219

    sensitivity of the bacterium despite the presence of a resistant rpsL allele. These findings 220

  • 11

    were never exploited for clinical practice. To use the dominant sensitivity of rpsL as a 221

    platform for the next sets of experiments which provide proof-of-principle for use of the 222

    constructs in medical settings in the future, we first established the system with our model 223

    strains and plasmids. MICs to streptomycin were determined by agar-plate assay (19). In 224

    this assay, ~104 cells are replica-plated on plates with different drug concentrations. The 225

    lowest concentration at which there is no visible colony-formation is defined as the MIC. 226

    The MICs throughout the study were also measured in a complementary liquid-227

    determination assay, giving a similar readout (not shown). Two representatives of the 228

    most common streptomycin-resistant strains obtained above were taken for further study: 229

    strains Sm6 and Sm13, harboring mutations in rpsL leading to substitutions of R86S and 230

    K88R, respectively. Their MICs to streptomycin were 100 g/ml and 200 g/ml, 231

    respectively, whereas the MIC of the parental strain was 1.56 g/ml. We transformed 232

    these strains with the plasmid pRpsL-wt, encoding the wt rpsL, or a control plasmid, 233

    pRpsL4, encoding a mock gene (a defective rpsL with a 4-bp deletion that disrupts the 234

    reading frame after amino acid 26 of the RpsL protein; see Fig. 1 as well as the 235

    supplemental material for plasmid sequences and maps) under a modified early E. coli 236

    promoter from phage T7. Transformed cells were selected on agar plates supplemented 237

    with 35 g/ml chloramphenicol, as the plasmid encodes chloramphenicol acetyl 238

    transferase, which confers chloramphenicol resistance. The MICs of the transformed 239

    strains to streptomycin were then determined. As shown in Fig. 2A, transformation of the 240

    plasmid encoding wt rpsL, pRpsL-wt, conferred a dominant sensitive phenotype, 241

    restoring the MIC of the resistant mutants Sm6 and Sm13 from 100 g/ml to 12.5 g/ml 242

    and from 200 g/ml to 3.125 g/ml, respectively. A control, streptomycin-sensitive E. 243

  • 12

    coli transformed with these plasmids (pRpsL4 or pRpsL-wt) retained similar MICs to 244

    streptomycin (not shown). These results demonstrate that a wt rpsL allele delivered on a 245

    plasmid into a streptomycin-resistant E. coli renders the cell significantly more sensitive 246

    to streptomycin. 247

    rpsL designed with decreased homology to the wt allele can efficiently restore 248

    streptomycin sensitivity. The system we propose as a proof-of-principle is based on the 249

    rpsL-containing construct being transferred horizontally between strains by 250

    transformation, conjugation or transduction, as described below. Recombination events 251

    between the chromosomal resistant rpsL and the delivered wt rpsL may reduce the 252

    efficiency of the construct because it may eventually recombine with an rpsL copy that 253

    does not confer sensitivity on the transformed strains (nevertheless, there is no danger 254

    that it will confer resistance in sensitive strains as the sensitive allele is dominant). In 255

    order to reduce the undesired recombination events between the incoming allele 256

    conferring sensitivity and the resistant allele in the transformed cell we designed an allele 257

    which cannot undergo homologous recombination with the bacterial copy. Efficient 258

    homologous recombination requires identity between recombining genes. Reduction of 259

    homology from 100% to 90% decreases the frequency of recombination over 40-fold in 260

    E. coli (14). In addition, a minimal efficient processing segment of 23 to 27 bp that is 261

    identical to the invading strand is required for efficient homologous recombination (14). 262

    We synthesized an rpsL gene with silent mutations that maximize the incompatibility of 263

    recombination with the sequence of wt rpsL. Silent substitutions were made in every 264

    possible case, except where codon usage was less than 10% (see the supplemental 265

    material for plasmid sequence). Overall, the genes were identical in only 62% of their 266

  • 13

    sequence, and there was no single minimal efficient process segment between the wt rpsL 267

    and the new rpsL allele, thus providing efficient barriers against homologous 268

    recombination. We designated this allele rpsL-sil, and the plasmid encoding it, pRpsL-sil. 269

    The introduced silent mutations might hamper the folding of the encoded protein or its 270

    expression levels (3). We therefore tested whether this allele, like the wt rpsL, can 271

    dominantly restore sensitivity. As shown in Fig. 2B, dramatic sensitization to 272

    streptomycin was observed, with the MIC values decreasing in Sm6 and Sm13 from 100 273

    g/ml to 25 g/ml and from 200 g/ml to 6.25 g/ml, respectively. The efficiency of 274

    restoration of sensitivity was lower than that observed with the wt rpsL, possibly due to 275

    the product's folding efficiency, as already mentioned. Nevertheless, these results indicate 276

    that both rpsL and rpsL-sil can efficiently restore sensitivity to streptomycin when 277

    expressed from plasmids. 278

    A toxic compound, tellurite, efficiently replaces chloramphenicol as a 279

    selection marker. In the above experiments, chloramphenicol, under the constitutive bla 280

    promoter, was used as a selection and maintenance marker for the rpsL-encoding 281

    plasmids. However, chloramphenicol is not a dispensable antibiotic, and by using it in the 282

    proposed system, sensitivity to streptomycin is restored by forfeiting sensitivity to 283

    chloramphenicol. This outcome is less desirable than one in which drug sensitivity is 284

    restored without forfeiting sensitivities to other drugs. We therefore sought to replace 285

    chloramphenicol with a dispensable, yet efficient, selection substance. A resistance gene 286

    against tellurite (TeO32), a toxic compound, was evaluated. Tellurite is toxic to bacteria 287

    as it forms long-lived sulfur complexes, thus disrupting the thiol balance in the bacterial 288

    cells. Tellurite was once used as a treatment for microbial infections, especially for 289

  • 14

    syphilis, prior to the discovery of antibiotics (22). The tellurite-resistance genes, tehAB, 290

    present naturally in the E. coli chromosome, do not confer resistance to E. coli under their 291

    endogenous promoter due to low transcription (9). Upon expression from an active 292

    promoter (e.g. T7), however, the MIC of tellurite against E. coli increases 50- to 100-293

    fold. The observed MIC of E. coli that either possess or lack the chromosomal tehAB 294

    genes is equal or less than 2 g/ml, compared to a MIC of 128 g/ml in cells harboring 295

    extrachromosomal tehAB with an active promoter (9). Remarkably, all of the known 296

    tellurite-resistance genes, including tehAB, are highly specific to tellurite, showing no 297

    cross-resistance to other compounds (22), and are therefore a relatively safe choice for 298

    our purposes. In addition, the tehAB genes are relatively small (~2.5 kb), making them 299

    easy to clone and genetically transfer. Linking these genes to the antibiotic-sensitizing 300

    DNA cassette would thus constitute an appropriate selectable sensitizing construct. 301

    Plasmids encoding rpsL-sil or the mock gene were constructed, carrying the tellurite-302

    resistance genes, tehAB, instead of the gene encoding chloramphenicol acetyl transferase. 303

    These plasmids were named pRpsL-sil-tell and pRpsL4-tell. The plasmids were 304

    transformed into the streptomycin-resistant strains, Sm6 and Sm13, and the MICs of 305

    these transformed cells to streptomycin were determined. Restoration of sensitivity by 306

    tellurite-based plasmids was comparable to that observed with the chloramphenicol-based 307

    plasmids (Fig. 3). pRpsL-sil-tell sensitized Sm6 from a MIC of 100 g/ml to 1.56 g/ml, 308

    and Sm13 from a MIC of 200 g/ml to 12.5 g/ml. These results indicate that tellurite 309

    can be used instead of the chloramphenicol-resistance marker. They also demonstrate that 310

    tellurite can maintain the plasmids without cross-reactivity with the streptomycin-311

    resistance phenotype. 312

  • 15

    Streptomycin-resistant bacteria lysogenized with phage encoding rpsL 313

    become streptomycin-sensitive. The system is thus shown to restore drug sensitivity 314

    using plasmids as a genetic delivery tool without forfeiting other drugs' efficiencies. The 315

    system is intended for transfer to resistant pathogens residing on hospital surfaces, thus 316

    rendering them treatable by antibiotics. Plasmids are efficiently transferred from host to 317

    host mainly via conjugation. However, establishing a conjugation-based system requires 318

    dissemination of bacteria harboring a conjugative plasmid, which is not desirable from 319

    either regulatory or safety points of view. We therefore evaluated the use of phages as 320

    safer delivery vehicles for the designed constructs. We chose , a model phage which has 321

    been extensively studied, as a gene-delivery tool. This phage can infect its E. coli host 322

    and proceed to the lytic or lysogenic cycle. We used a common phage mutant (gt11, see 323

    Materials and Methods) which is directed to a specific cycle type according to the 324

    ambient temperature, and has a deletion (nin5) designed to allow stable insertion of up to 325

    5 kb of foreign DNA. This phage mutant was engineered to contain wt rpsL, rpsL-sil, or a 326

    mock-rpsL, each linked to the tellurite-resistance genes and designated, respectively, -327

    RpsL-wt-tell, -RpsL-sil-tell, and -RpsL4-tell (see the supplemental material for maps 328

    of the DNA inserts in the phages). One of the streptomycin-resistant strains used above, 329

    Sm13, was lysogenized with the recombinant phages and selected on agar plates 330

    supplemented with 1.5 g/ml tellurite at 32oC, a temperature at which it favors the 331

    lysogenic cycle. The lysogenized bacteria were propagated and their MICs to 332

    streptomycin determined. Lysogenization of Sm13 by the phages resulted in sensitization 333

    of the resistant mutants (Fig. 4A). The MIC value for the -RpsL4-tell lysogen was 200 334

    g/ml, compared to 25 g/ml and 50 g/ml for -RpsL-wt-tell and -RpsL-sil-tell, 335

  • 16

    respectively. Although significant, the sensitization was not as efficient as that observed 336

    using plasmid delivery. 337

    Two copies of the rpsL gene are significantly more efficient than a single copy 338

    in reversing resistance. We suspected that the decreased sensitization observed by 339

    lysogenization relative to plasmid transformation is due to a lower number of rpsL gene 340

    copies introduced by the phage. To test this and improve the sensitization, we cloned 341

    the two different rpsL alleles (wt rpsL and rpsL-sil) into the phage, designated -342

    2xRpsL-tell, and used it to lysogenize resistant strain Sm13 as above. Introduction of two 343

    gene copies dramatically enhanced the sensitization efficiency of the lysogenized strains, 344

    resulting in a significant decrease of the MIC from 200 g/ml to 1.56 g/ml, comparable 345

    to the MIC observed for the sensitive strain (Fig. 4B). As a whole, these results constitute 346

    a proof-of-principle for restoration of sensitivity to streptomycin using a phage that 347

    carries sufficient copies of rpsL, at the "genetic cost" of a resistance marker to a toxic 348

    compound. 349

    Nalidixic acid-resistant bacteria lysogenized with phage encoding gyrA 350

    show restored nalidixic acid sensitivity. The above results demonstrate that 351

    streptomycin resistance can be reversed by the proposed system. We wished to expand 352

    the proof-of-principle to other antibiotics as well, to demonstrate that a "multidrug-353

    sensitivity cassette" can theoretically be used. We therefore chose to target quinolone 354

    resistance, which also manifests dominant sensitivity by the wt allele (12). The quinolone 355

    drug family targets the enzyme gyrase, encoded by gyrA, resulting in DNA-replication 356

    arrest. Mutations in gyrA are observed in a specific region termed "quinolone-resistance-357

    determining region" (QRDR). The wt gyrA allele is dominant sensitive and may therefore 358

  • 17

    reverse resistance (12). Nalidixic acid, the first of the synthetic quinolone family 359

    antibiotics, was used here as a representative of the quinolone family. To test whether the 360

    system can restore sensitivity to quinolone, we isolated spontaneous nalidixic acid-361

    resistant mutants by plating sensitive E. coli on 50 g/ml nalidixic acid. Similar to the 362

    isolation of the streptomycin mutants, we obtained mutants with previously reported 363

    substitutions in the target gene, gyrA (Table 2). We identified five D87G substitutions 364

    and three S83L substitutions in the gyrA gene product. These results corroborate another 365

    study on pathogenic E. coli, which showed that 37 out of 38 isolated quinolone-resistant 366

    gyrA mutants have substitutions at either S83 or D87 or both. Out of 36 pathogenic E. 367

    coli resistant to high levels of nalidixic acid (MIC 256 g/ml), 35 had at least one 368

    mutation in gyrA (4). Here, as with the isolation of streptomycin-resistant mutants, the 369

    fact that most of the spontaneous mutants are located in the target gene highlights the 370

    potential benefit of reversing the effect of these mutations. We next introduced the wt 371

    gyrA expressed from its endogenous promoter, or a control construct, both linked to 372

    tellurite-resistance genes, into phages, designated -GyrA-tell and -Ctrl-tell 373

    respectively. We used these phages to lysogenize a nalidixic acid-resistant strain, Nal2, 374

    harboring a S83L substitution in GyrA. The lysogens were selected on 4 g/ml tellurite 375

    and tested for sensitization by measuring MICs as described above, using nalidixic acid 376

    instead of streptomycin. As shown in Fig. 5, the gyrA construct significantly reversed the 377

    mutant's resistance. The MIC of the resistant mutants decreased twofold when 378

    lysogenized by a gyrA-encoding phage compared to the control phage. The significance 379

    of this sensitization was corroborated by experiments in which we transformed gyrA-380

    encoding plasmids into nalidixic acid-resistant mutants and observed a three orders of 381

  • 18

    magnitude decrease in the number of CFU on 50 g/ml nalidixic acid compared to 382

    resistant cells transformed with a mock plasmid (Fig. S1). Overall, these results indicate 383

    that the proposed system can be used to target nalidixic acid resistance as well as 384

    streptomycin resistance. 385

    Proposed application, safety measures, and advantages of the system. The 386

    proof-of-principle presented here is a step toward solving the major threat of emerging 387

    drug-resistant pathogens, against which we have limited new emerging antibiotic 388

    weapons. It demonstrates that with simple genetic engineering, bacteria can be 389

    resensitized to approved and useful antibiotics. It is suggested that the system be applied 390

    in a simple treatment of hospital surfaces to reverse the resistance of nosocomial 391

    pathogens. Phages against Listeria monocytogenes (ListShield) and E. coli 392

    (EcoShield) are used to spray ready-to-eat food and effectively target the 393

    contaminating pathogen (5). Phages are also used in the USA as effective pesticides on 394

    edible crops, among others (5). Although the above applications used non-genetically-395

    altered phages, they support the safety and efficacy of our proposed spraying of phages 396

    on hospital surfaces. The proposed uses and advantages of the system are presented in 397

    Table 3. Extended transfer of the sensitizing cassette by specifically constructed 398

    lysogenizing phages might enrich for antibiotic-treatable pathogens on hospital surfaces. 399

    This enriched, sensitive population might then interfere with the establishment of newly 400

    introduced resistant pathogens by overtaking their ecological niche. Our approach differs 401

    from conventional phage therapy in the sense that it does not use phages to kill the 402

    pathogens directly. Consequently, there is no selection against the used phage, but rather 403

    selection for pathogens harboring the phage because it contains tellurite resistance. 404

  • 19

    Moreover, the approach avoids the use of phages inside the patient's body, thus 405

    overcoming toxicity issues and other drawbacks of phage therapy, such as phage 406

    neutralization by the spleen and the immune system (11). The presented system makes 407

    use of a temperate phage whose lytic cycle is induced at elevated temperatures (8). This 408

    feature carries added value because in the environment, as long as the temperature is 409

    below 32oC, there is only selective pressure for being lysogenized and taking up the 410

    sensitizing genes linked to the tellurite-resistance marker. However, once the pathogens 411

    are lysogenized, they are less bound to infect humans or other warm-blooded animals, 412

    because such an infection would induce the lytic cycle of the prophage and consequently 413

    kill the bacteria. This added benefit is not essential for a future product, but is an optional 414

    addition. If successful, this approach will render most of the nosocomial infections 415

    treatable by antibiotics, unlike the current situation in which most nosocomial infections 416

    are caused by antibiotic-resistant pathogens. The sensitizing cassette may be expanded to 417

    include other sensitizing genes such as thyA, conferring sensitivity in a dominant way to 418

    trimethoprim (20). Designing several antibiotic-sensitizing genes on a single construct 419

    reduces the probability of spontaneous regaining of resistance against all of these 420

    antibiotics simultaneously. We believe that the provided proof-of-principle can be applied 421

    to different pathogen-phage systems as the extensive co-evolution of phages and bacteria 422

    suggests that it is possible to find lysogenizing phages specific to any bacterium. For 423

    example, the described system could quite simply be modified to target pathogenic E. coli 424

    (e.g. E. coli O104:H4) by carrying out several selection cycles of the described phage 425

    on the desired host, until the phage becomes fully adapted to it. In addition, the dominant 426

    sensitivity of both rpsL and gyrA alleles has been shown to be broad across bacterial 427

  • 20

    species (e.g. (2, 12, 17). Along with the proof-of-principle, we also demonstrate a simple 428

    procedure for creating an efficient barrier against homologous recombination. The 429

    procedure includes replacement of most of the wobble bases, thus reducing the identity 430

    between genes as well as eliminating the minimal efficient processing segment for 431

    homologous recombination, without significantly affecting the translated product. This 432

    procedure can be carried out with currently available technology for basically any desired 433

    gene, as synthetic gene production has become common practice. Further manipulations 434

    to reduce the possibility of tellurite resistance being horizontally transferred without the 435

    sensitization cassette can be designed in a future product. For example, the sensitization 436

    genes can be constructed so that their presence is essential for acquisition of tellurite 437

    resistance: the sensitizing genes would be preferentially positioned before a promoterless 438

    tellurite-resistance gene, making tellurite resistance dependent on expression of the 439

    sensitizing genes. In addition to the system's benefits, which can be improved by 440

    implementing the above safety measures, these phages are simple to prepare and to apply, 441

    constituting a great advantage. Broad usage of the proposed system, in contrast to 442

    antibiotics and phage therapy, will potentially change the nature of nosocomial infections 443

    by making the bacteria more susceptible to antibiotics rather than more resistant. 444





    We thank Nir Osherov for critical reading of the manuscript and Camille 449

    Vainstein for professional language editing. 450

  • 21

    This research was supported by Israel Science Foundation grant 611/10, 451

    Binational Science Foundation grant 2009218, German-Israel Foundation grant 452

    2061/2009, and Marie Curie International Reintegration Grants PIRG-GA-2009-256340 453

    and GA-2010-266717. 454





    1. Brussow, H. 2005. Phage therapy: the Escherichia coli experience. Microbiology 459 151:2133-2140. 460

    2. Drecktrah, D., J. M. Douglas, and D. S. Samuels. 2010. Use of rpsL as a 461 Counterselectable Marker in Borrelia burgdorferi. Appl Environ Microbiol 76:985-987. 462

    3. Kimchi-Sarfaty, C., J. M. Oh, I. W. Kim, Z. E. Sauna, A. M. Calcagno, S. V. Ambudkar, 463 and M. M. Gottesman. 2007. A "silent" polymorphism in the MDR1 gene changes 464 substrate specificity. Science 315:525-528. 465

    4. Komp Lindgren, P., A. Karlsson, and D. Hughes. 2003. Mutation rate and evolution of 466 fluoroquinolone resistance in Escherichia coli isolates from patients with urinary tract 467 infections. Antimicrob Agents Chemother 47:3222-3232. 468

    5. Lang, L. H. 2006. FDA approves use of bacteriophages to be added to meat and poultry 469 products. Gastroenterology 131:1370. 470

    6. Lederberg, J. 1951. Streptomycin resistance; a genetically recessive mutation. J Bacteriol 471 61:549-550. 472

    7. Levy, S. B., and B. Marshall. 2004. Antibacterial resistance worldwide: causes, 473 challenges and responses. Nat Med 10:S122-129. 474

    8. Lieb, M. 1964. Ultraviolet Sensitivity of Escherichia Coli Containing Heat-Inducible 475 Lambda Prophages. Science 145:175-176. 476

    9. Liu, M., and D. E. Taylor. 1999. Characterization of gram-positive tellurite resistance 477 encoded by the Streptococcus pneumoniae tehB gene. FEMS Microbiol Lett 174:385-478 392. 479

    10. Lu, T. K., and J. J. Collins. 2009. Engineered bacteriophage targeting gene networks as 480 adjuvants for antibiotic therapy. Proc Natl Acad Sci U S A 106:4629-4634. 481

    11. Merril, C. R., D. Scholl, and S. L. Adhya. 2003. The prospect for bacteriophage therapy in 482 Western medicine. Nat Rev Drug Discov 2:489-497. 483

    12. Robillard, N. J. 1990. Broad-host-range gyrase A gene probe. Antimicrob Agents 484 Chemother 34:1889-1894. 485

    13. Sharan, S. K., L. C. Thomason, S. G. Kuznetsov, and D. L. Court. 2009. Recombineering: a 486 homologous recombination-based method of genetic engineering. Nat Protoc 4:206-487 223. 488

  • 22

    14. Shen, P., and H. V. Huang. 1986. Homologous recombination in Escherichia coli: 489 dependence on substrate length and homology. Genetics 112:441-457. 490

    15. Shi, R., J. Zhang, C. Li, Y. Kazumi, and I. Sugawara. 2007. Detection of streptomycin 491 resistance in Mycobacterium tuberculosis clinical isolates from China as determined by 492 denaturing HPLC analysis and DNA sequencing. Microbes Infect 9:1538-1544. 493

    16. Sreevatsan, S., X. Pan, K. E. Stockbauer, D. L. Williams, B. N. Kreiswirth, and J. M. 494 Musser. 1996. Characterization of rpsL and rrs mutations in streptomycin-resistant 495 Mycobacterium tuberculosis isolates from diverse geographic localities. Antimicrob 496 Agents Chemother 40:1024-1026. 497

    17. Sung, C. K., H. Li, J. P. Claverys, and D. A. Morrison. 2001. An rpsL cassette, janus, for 498 gene replacement through negative selection in Streptococcus pneumoniae. Appl 499 Environ Microbiol 67:5190-5196. 500

    18. Timms, A. R., H. Steingrimsdottir, A. R. Lehmann, and B. A. Bridges. 1992. Mutant 501 sequences in the rpsL gene of Escherichia coli B/r: mechanistic implications for 502 spontaneous and ultraviolet light mutagenesis. Mol Gen Genet 232:89-96. 503

    19. Wiegand, I., K. Hilpert, and R. E. Hancock. 2008. Agar and broth dilution methods to 504 determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat 505 Protoc 3:163-175. 506

    20. Wong, Q. N., V. C. Ng, M. C. Lin, H. F. Kung, D. Chan, and J. D. Huang. 2005. Efficient 507 and seamless DNA recombineering using a thymidylate synthase A selection system in 508 Escherichia coli. Nucleic Acids Res 33:e59. 509

    21. Wright, A., C. H. Hawkins, E. E. Anggard, and D. R. Harper. 2009. A controlled clinical 510 trial of a therapeutic bacteriophage preparation in chronic otitis due to antibiotic-511 resistant Pseudomonas aeruginosa; a preliminary report of efficacy. Clin Otolaryngol 512 34:349-357. 513

    22. Zannoni, D., F. Borsetti, J. J. Harrison, and R. J. Turner. 2008. The bacterial response to 514 the chalcogen metalloids Se and Te. Adv Microb Physiol 53:1-72. 515

    516 517

  • 23

    TABLES 518


    TABLE 1. E. coli K-12 streptomycin-resistant mutants, Sm1520

    22, isolated on 50 g/ml streptomycin. Substitutions in the 521

    RpsL protein are indicated, as well as the MIC to streptomycin. 522

    523 Streptomycin-resistant


    Substitution in


    MIC (g/ml)

    Sm1 P42S 50

    Sm2 K43L 800

    Sm3 K43N 50

    Sm4 R54S 50

    Sm5 R86S 50

    Sm6 R86S 100

    Sm7 R86S 50

    Sm8 R86S 50

    Sm9 R86S 50

    Sm10 R86S 50

    Sm11 K88E 50

    Sm12 K88R 400

    Sm13 K88R 400

    Sm14 K88R 400

    Sm15 K88R 400

    Sm16 K88R 400

    Sm17 K88R 400

    Sm18 K88R 400

    Sm19 K88R 400

    Sm20 K88R 400

    Sm21 K88R 400

    Sm22 none 50

  • 24

    TABLE 2. E. coli K-12 nalidixic acid-resistant mutants, Nal18, 524

    isolated on 50 g/ml nalidixic acid. Substitutions in the GyrA protein 525

    are indicated, as well as the MIC to nalidixic acid. 526

    527 Nalidixic acid-resistant


    Substitution in


    MIC (g/ml)

    Nal1 S83L 50

    Nal2 S83L 50

    Nal3 S83L 50

    Nal4 D87G 128

    Nal5 D87G 128

    Nal6 D87G 128

    Nal7 D87G 128

    Nal8 D87G 128

  • 25

    TABLE 3. Proposed procedure for using the lysogenizing phages and the selection agent (tellurite) compared to the current 528 cleaning and disinfecting procedures in hospitals. Advantages of this procedure are indicated. 529

    Surface treatment/frequency Current



    procedure Advantage of proposed procedure



    Soap V V

    Renders bacteria drug-sensitive

    Lysogenizing phages X V


    Disinfectant V V

    Selects for drug-sensitive residual bacteria to occupy

    ecological niche

    Tellurite X V

    Frequency Daily V V Repeated usage results in more sensitive clones

  • 26



    FIG. 1. Plasmid (A) and phage (B) maps. The inserts are drawn to scale, with the 532

    relevant genes and genetic elements indicated. CAT - chloramphenicol acetyl transferase, 533

    conferring chloramphenicol resistance; Pamp - bla promoter, P A1* - mutated T7-A1 534

    promoter of T7 phage. The promoter could not be cloned without a mutation, and 535

    therefore we proceeded with the following 1 bp deletion in the promoter sequence 536

    (bolded): 537



    rpsL4 - encodes a truncated RpsL protein, rpsL-wt - encodes the RpsL protein; rpsL-sil 540

    - encodes an RpsL protein, harboring numerous silent mutations, with only 62% identity 541

    to the wt sequence; tehA/tehB - encode proteins which confer tellurite resistance; gyrA-wt 542

    - encodes the Gyrase A protein. 543


    FIG. 2. rpsL encoded by plasmids efficiently sensitizes streptomycin-resistant 545

    mutants. Streptomycin-resistant mutants Sm6 and Sm13, transformed with a plasmid 546

    encoding wt rpsL, pRpsL-wt (A) or rpsL having multiple silent mutations, pRpsL-sil (B), 547

    become more sensitive to streptomycin as compared to mutants transformed with a 548

    plasmid encoding a mock gene, pRpsL4. Serial 10-fold dilutions starting at 105 549

    CFU/spot (from top to bottom) of the different mutants were spotted on plates with the 550

    indicated streptomycin concentrations. Chloramphenicol was supplemented at 35 g/ml 551

    in all plates to maintain the plasmid. Plates were incubated overnight and photographed 552

  • 27

    using MiniBis Pro (Bio-Imaging Systems). A representative experiment out of three is 553

    presented. 554


    FIG. 3. Tellurite-resistance genes efficiently replace chloramphenicol acetyl 556

    transferase as a selection marker. Streptomycin-resistant mutants transformed with a 557

    plasmid encoding wt rpsL as well as a tellurite-resistance gene, pRpsL-sil-tell, become 558

    more sensitive to streptomycin as compared to mutants transformed with a plasmid 559

    encoding a mock gene, pRpsL4-tell. Serial 10-fold dilutions starting at 105 CFU/spot 560

    (from top to bottom) of the different mutants were spotted on plates with the indicated 561

    streptomycin concentrations. Tellurite was supplemented at 1.5 g/ml in all plates to 562

    maintain the plasmid. Plates were incubated overnight and photographed using MiniBis 563

    Pro (Bio-Imaging Systems). A representative experiment out of three is presented. 564


    FIG. 4. rpsL genes introduced by phage sensitize a streptomycin-resistant 566

    mutant. Phage encoding a single copy of either wt rpsL (-RpsL-wt-tell) or rpsL-sil (-567

    RpsL-sil-tell) sensitizes a streptomycin-resistant mutant, Sm13, compared to phage 568

    encoding a mock gene (-RpsL4-tell) (A). Sensitization is significantly enhanced when 569

    the phage carries both copies of rpsL (-2xRpsL-tell) (B). Serial 10-fold dilutions starting 570

    at 105 CFU/spot (from top to bottom) of the different lysogens were spotted on plates 571

    with the indicated streptomycin concentrations. Tellurite was supplemented at 1.5 g/ml 572

    in all plates to maintain the prophage. Plates were incubated overnight and photographed 573

    using MiniBis Pro (Bio-Imaging Systems). A representative experiment out of three is 574

    presented. 575

  • 28


    FIG. 5. gyrA introduced by phage sensitizes a nalidixic acid-resistant mutant. 577

    Phage encoding a single copy of wt gyrA (-GyrA-tell) sensitizes a nalidixic acid-578

    resistant mutant, Nal2, compared to phage encoding a mock gene (-Ctrl-tell). 579

    Triplicates of the different lysogens, at 104 CFU/spot, were spotted on plates with the 580

    indicated streptomycin concentrations. Tellurite was supplemented at 4 g/ml in all 581

    plates. Plates were incubated overnight and photographed using MiniBis Pro (Bio-582

    Imaging Systems). A representative experiment out of three is presented. 583

  • A

    Figure 1


    pRpsL4-tellpRpsL4P amp P amp

    pRpsL-wt-tell pRpsL-wt

    P A1* P A1*


  • A J cI857 (nin5)



    -RpsL4-tell tehA tehBP A1* P amp




    P A1*tehA tehB

    P amp

    -RpsL-wt-tell tehA tehBP A1* P amp



    -RpsL-sil-tell tehA tehBP A1* P amp


    -2xRpsL-tell P amp


    tehA tehBP A1* P amp


    -GyrA-tell tehA tehBP A1* P amp

    rpsL gyrA

    P gyrA

  • 200 streptomycin (g/ml)A 0 1 56 3 125 6 25 12 5 25 50 100

    Figure 2

    2200 streptomycin (g/ml)A


    1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 10 1.56 3.125 6.25 12.5 25 50 100

    B2 - pRpsL-wt


    0 1.56 3.125 6.25 12.5 25 50 200100 streptomycin (g/ml)

    1 pRpsL4Legend:




    1 3 1 3 1 3 1 3 1 3 1 3 1 3 1 3 1 3


    3 - pRpsL-sil1 pRpsL4Legend:

  • Figure 3

    3200 streptomycin (g/ml)


    1 3 1 3 1 3 1 3 1 3 1 3 1 3 1 30 1.56 3.125 6.25 12.5 25 50 100


    3 - pRpsL-sil-tell


    1 pRpsL4-tellLegend:

  • Figure 4


    200 streptomycin (g/ml)A 0 1.56 3.125 6.25 12.5 25 50 10021 321 321 321 321 321 321 321 321 3

    1 - -RpsL4-tell 2 - -RpsL-wt-tell 3 - -RpsL-sil-tell

    B streptomycin (g/ml)1 4 1 4 1 4 1 4 1 4

    0 1.56 3.125 6.25 12.5



    Legend: 1 - -RpsL4-tell 4 - -2XRpsL-tell

  • Figure 5

    21 21 21 21 21 21


    1 - -Ctrl-tell

    Nalidixic acid (g/ml)0 8 16 32 64 128

    2 - -GyrA-tellLegend: