Since the emergence of deadly pathogens and multidrug-resistant bacteria at an alarmingly increased rate, bacteriophages have been developed as a controlling bioagent to prevent the spread of pathogenic bacteria. One of these pathogens, disease-causing Vibrio parahaemolyticus (Vp AHPND ) which induces acute hepatopancreatic necrosis, is considered one of the deadliest shrimp pathogens, and has recently become resistant to various classes of antibiotics. Here, we discovered a novel vibriophage that specifically targets the vibrio host, VP AHPND . The vibriophage, designated Seahorse, was classified in the family Siphoviridae because of its icosahedral capsid surrounded by head fibers and a non-contractile long tail. Phage Seahorse was able to infect the host in a broad range of pH and temperatures, and it had a relatively short latent period (nearly 30 minutes) in which it produced progeny at 72 particles per cell at the end of its lytic cycle. Upon phage infection, the host nucleoid condensed and became toroidal, similar to the bacterial DNA morphology seen during tetracycline treatment, suggesting that phage Seahorse hijacked host biosynthesis pathways through protein translation. As phage Seahorse genome encodes 48 open reading frames with many hypothetical proteins, this genome could be a potential untapped resource for the discovery of phage-derived therapeutic proteins.Vibrio is a genus of motile Gram-negative bacteria that possesses a curved-rod cell shape with a single flagellum. Vibrios are abundant and diverse bacteria that are typically found in marine habitats. The genus Vibrio consists of 14 recognized clades and at least 86 different species 1 . While some of them are not pathogenic, many can cause serious health effects in both human and aquatic life. Due to the continuously rising ocean temperature, the composition of vibrio in the ocean microbiome has been reported to be higher than usual 2-4 . This vibrio-rich environment might increase the incident of a vibrio outbreak in the near future posing risks to global health 2 .Vibrio parahaemolyticus, which is one of the disease-causing Vibrio species, is pathogenic to both humans and marine animals 5 . Consumption of raw seafoods contaminated with the bacteria can cause acute gastroenteritis 5,6 . This opportunistic bacterium is also able to infect through an open wound which can lead to sepsis and, in rare cases, subsequent death in immunocompromised patients 7,8 . Moreover, V. parahaemolyticus that has acquired a plasmid encoding the deadly binary toxins PirA vp /PirB vp is even more virulent 9 . The V. parahaemolyticus strain harboring the plasmid has been found to cause a newly emerging disease in shrimp, known as acute hepatopancreatic necrosis disease (AHPND) 9 . Moreover, the AHPND-causing plasmid is also found to be transferable among other vibrios, increasing the chance of the disease spreading regionally and globally 10 . Unsurprisingly, the spread of AHPND has been reported in many countries, including China, Vietnam, Malaysia, Thailand, Me...

In this study, we sought to determine if an in vivo assay for studying antibiotic mechanisms of action could provide insight into the activity of compounds that may inhibit multiple targets. Thus, we conducted an activity screen of 31 structural analogs of rhodanine-containing pan-assay interference compounds (PAINS). We identified nine active molecules against E. coli and classified them according to their in vivo mechanisms of action. The mechanisms of action of PAINS are generally difficult to identify due to their promiscuity. However, we leveraged bacterial cytological profiling, a fluorescence microscopy technique, to study these complex mechanisms. Ultimately, we found that although some of our molecules promiscuously inhibit multiple cellular pathways, a few molecules specifically inhibit DNA replication despite structural similarity to related PAINS. A genetic analysis of resistant mutants revealed thymidylate kinase (essential for DNA synthesis) as an intracellular target of some of these rhodanine-containing antibiotics. This finding was supported by in vitro activity assays as well as experiments utilizing a thymidylate kinase overexpression system. The analog that demonstrated the lowest IC 50 in vitro and MIC in vivo displayed the greatest specificity for inhibition of the DNA replication pathway, despite containing a rhodamine moiety. While it’s thought that PAINS cannot be developed as antibiotics, this work showcases novel inhibitors of E. coli thymidylate kinase. But perhaps more importantly, this work highlights the utility of bacterial cytological profiling for studying the in vivo specificity of antibiotics and demonstrates that BCP can identify multiple pathways that are inhibited by an individual molecule. Importance: We demonstrate that bacterial cytological profiling is a powerful tool for directing antibiotic discovery efforts because it can be used to determine the specificity of an antibiotic's in vivo mechanism of action. By assaying analogs of PAINS, molecules that are notoriously intractable and non-specific, we (surprisingly) identify molecules with specific activity against E. coli thymidylate kinase. This suggests that structural modifications to PAINS can confer stronger inhibition by targeting a specific cellular pathway. While in vitro inhibition assays are susceptible to false positive results (especially from PAINS), bacterial cytological profiling provides the resolution to identify molecules with specific in vivo activity.

The threat to public health posed by drug-resistant bacteria is rapidly increasing, as some of healthcare’s most potent antibiotics are becoming obsolete. Approximately two-thirds of the world’s antibiotics are derived from natural products produced by Streptomyces encoded biosynthetic gene clusters. Thus, to identify novel gene clusters, we sequenced the genomes of four bioactive Streptomyces strains isolated from the soil in San Diego County and used Bacterial Cytological Profiling adapted for agar plate culturing in order to examine the mechanisms of bacterial inhibition exhibited by these strains. In the four strains, we identified 104 biosynthetic gene clusters. Some of these clusters were predicted to produce previously studied antibiotics; however, the known mechanisms of these molecules could not fully account for the antibacterial activity exhibited by the strains, suggesting that novel clusters might encode antibiotics. When assessed for their ability to inhibit the growth of clinically isolated pathogens, three Streptomyces strains demonstrated activity against methicillin-resistant Staphylococcus aureus. Additionally, due to the utility of bacteriophages for genetically manipulating bacterial strains via transduction, we also isolated four new phages (BartholomewSD, IceWarrior, Shawty, and TrvxScott) against S. platensis. A genomic analysis of our phages revealed nearly 200 uncharacterized proteins, including a new site-specific serine integrase that could prove to be a useful genetic tool. Sequence analysis of the Streptomyces strains identified CRISPR-Cas systems and specific spacer sequences that allowed us to predict phage host ranges. Ultimately, this study identified Streptomyces strains with the potential to produce novel chemical matter as well as integrase-encoding phages that could potentially be used to manipulate these strains.

In this study, we conducted an activity screen of 31 structural analogs of rhodanine-containing pan-assay interference compounds (PAINS). We identified nine active molecules inhibiting the growth of E. coli and classified them according to their in vivo mechanisms of action. The mechanisms of action of PAINS are generally difficult to identify due to their promiscuity. However, we leveraged bacterial cytological profiling, a fluorescence microscopy technique, to study these complex mechanisms. Ultimately, we found that although some of our molecules promiscuously inhibit multiple cellular pathways, a few molecules specifically inhibit DNA replication despite their structural similarity to related PAINS. A genetic analysis of resistant mutants revealed that thymidylate kinase (an enzyme essential for DNA synthesis) is an intracellular target of some of these rhodanine-containing antibiotics. This finding was supported by assays of in vitro activity as well as experiments utilizing a thymidylate kinase overexpression system. The analog that demonstrated the lowest IC50in vitro and MIC in vivo displayed the greatest specificity for the inhibition of DNA replication in E. coli, despite containing a rhodamine moiety. While it's generally thought that PAINS cannot be developed as antibiotics, this work highlights the utility of bacterial cytological profiling for studying the in vivo specificity of antibiotics, and it showcases novel inhibitors of E. coli thymidylate kinase.

17 The threat to public health posed by drug-resistant bacteria is rapidly increasing, as some of 18 healthcare's most potent antibiotics are becoming obsolete. Approximately two-thirds of the 19 world's antibiotics are derived from natural products produced by Streptomyces encoded 20 biosynthetic gene clusters. Thus, in order to identify novel gene clusters, we sequenced the 21 genomes of four bioactive Streptomyces strains isolated from the soil in San Diego County and 22 used Bacterial Cytological Profiling adapted for agar plate culturing in order to examine the 23 mechanisms of bacterial inhibition exhibited by these strains. In the four strains, we identified 24 101 biosynthetic gene clusters. Some of these clusters were predicted to produce previously 25 studied antibiotics; however, the known mechanisms of these molecules could not fully account 26 for the antibacterial activity exhibited by the strains, suggesting that novel clusters might encode 27 antibiotics. When assessed for their ability to inhibit the growth of clinically isolated pathogens, 28 three Streptomyces strains demonstrated activity against methicillin-resistant Staphylococcus 29 aureus. Additionally, due to the utility of bacteriophages for genetically manipulating bacterial 30 strains via transduction, we also isolated four new phages (BartholomewSD, IceWarrior, Shawty, 31 and TrvxScott) against S. platensis. A genomic analysis of our phages revealed nearly 200 32 uncharacterized proteins, including a new site-specific serine integrase that could prove to be a 33 useful genetic tool. Sequence analysis of the Streptomyces strains identified CRISPR-Cas 34 systems and specific spacer sequences that allowed us to predict phage host ranges.35 Ultimately, this study identified Streptomyces strains with the potential to produce novel 36 chemical matter as well as integrase-encoding phages that could potentially be used to 37 manipulate these strains.38 Introduction 39 Antibiotic discovery is an international priority requiring immediate action. 1 The increasing 40 prevalence of multi-drug resistant (MDR) bacterial pathogens has resulted in an increased use 41 of last-resort antibiotics. [1][2][3] Microbes that produce natural products are the most prolific source 42 of clinically approved antibiotics. 4 In particular, soil dwelling Actinobacteria, notably 43 Streptomyces, account for two-thirds of the antibiotics currently on the market. 5-7 Despite 44 intensive studies, however, the full potential of microbes to produce natural products has not 45 been realized. 8 Genome mining studies have shown that microbes encode many biosynthetic 46 gene clusters (BGCs) that have not yet been characterized. 8 It is widely assumed that many of 47 these clusters produce novel natural products and that further characterization of Streptomyces 48 bacteria increases the probability of identifying molecules with unique chemical structures and 49 new mechanisms of action. 9 50 51 In addition to identifying Streptomyces strains containing potentially novel BG...