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    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

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    Running title: reversing antibiotic resistance 7

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

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    *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: ehudq@post.tau.ac.il 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

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    ABSTRACT 17

    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

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    INTRODUCTION 40

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    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

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    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

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    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

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    MATERIALS AND METHODS 97

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    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

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    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 C