Bacterial Cytological Profiling as a Tool To Study Mechanisms of Action of Antibiotics That Are Active against Acinetobacter baumannii

Htoo, Htut Htut; Brumage, Lauren; Chaikeeratisak, Vorrapon; Tsunemoto, Hannah; Sugie, Joseph; Tribuddharat, Chanwit; Pogliano, Joe; Nonejuie, Poochit

doi:10.1128/aac.02310-18

Cited by 51 publications

(58 citation statements)

References 76 publications

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“…Ciprofloxacin, rifampicin, and tetracycline were used as inhibitors to replication, transcription, and translation respectively. Fluorescence microscopy results of VP AHPND treated with antibiotics showed that each antibiotic treatment led to a unique morphological change in VP AHPND , similar to what previously observed in gram-negative Escherichia coli and Acinetobactor baumannii treatments 31,42 . Upon ciprofloxacin treatment, inhibition of DNA replication resulted in cell elongation and DNA pooling at the midcell while treatment with rifampicin resulted in DNA decondensation ( Fig.…”

Section: Phage Seahorse Infection Triggers the Condensation Of Host Nsupporting

confidence: 82%

“…3a). The archetypal DNA shape of protein translation inhibition in many studies is a "toroid" 31,42,43 , but DNA shape alteration during very early translation inhibition by an antibiotic (less than one hour) has never been reported. Thus, time-course analysis of toroidal DNA formation in VP AHPND during tetracycline treatment was performed.…”

Section: Phage Seahorse Infection Triggers the Condensation Of Host Nmentioning

confidence: 99%

“…Minimal inhibitory concentrations (MIC) were determined for the following antibiotics: Ciprofloxacin, Rifampicin, and Tetracycline, which were all used in the fluorescence microscopy experiment shown in Table S1. The antibiotics were respectively serially diluted in a 96 well plate using a microdilution method 42 . Overnight cultures of VP AHPND were diluted 100-fold in TSB-1.5% NaCl and allowed to grow on a roller at 30 °C until exponential growth (OD 600 of 0.2) was observed.…”

Section: Minimal Inhibitory Concentrationmentioning

confidence: 99%

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A novel vibriophage exhibits inhibitory activity against host protein synthesis machinery

Thammatinna

Egan

Htoo

et al. 2020

Sci Rep

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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...

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Section: Phage Seahorse Infection Triggers the Condensation Of Host Nsupporting

confidence: 82%

Section: Phage Seahorse Infection Triggers the Condensation Of Host Nmentioning

confidence: 99%

Section: Minimal Inhibitory Concentrationmentioning

confidence: 99%

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A novel vibriophage exhibits inhibitory activity against host protein synthesis machinery

Thammatinna

Egan

Htoo

et al. 2020

Sci Rep

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“…We next asked whether drug treatment of Mtb, like M. smegmatis , elicited well-defined cytological fingerprints. Guided by cytological profiling methods in other bacterial species (7–9), we treated Mtb grown in standard rich growth medium with a high drug dose (3x the 90% inhibitory concentration; IC90) for 17 hours (~1 doubling time). We generated a dataset of morphological features from Mtb treated with 34 antibacterials that encompass a wide range of drug classes and classified the target pathway of each drug according to published findings (Table S1).…”

Section: Resultsmentioning

confidence: 99%

“…In other bacterial systems such as Escherichia coli , Bacillus subtilis , and Acinetobacter baumannii , profiling of cytological changes in response to treatment has yielded a rapid and resource-sparing procedure to determine drug mechanism (7–9). This method, known as bacterial cytological profiling (BCP), groups drugs with similar mechanisms of action by clustering profiles of drug-treated bacteria using multivariate analysis methods such as principal component analysis (PCA) (7–9). BCP is efficient and rapid because these cytological features can be automatically derived from high-throughput images of stained, fixed samples.…”

Section: Introductionmentioning

confidence: 99%

Morphological profiling of tubercule bacilli identifies drug pathways of action

Smith

Pullen

Olson

et al. 2020

Preprint

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34 35 36 Keywords 37 tuberculosis, drug discovery, cell morphology, high throughput 38 39 40 41 42 43 2 Abstract 44Morphological profiling is a method to classify target pathways of antibacterials based on how 45 bacteria respond to treatment through changes to cellular shape and spatial organization. Here, we utilized 46 the cell-to-cell variation in morphological features of Mycobacterium tuberculosis bacilli to develop a rapid 47 profiling platform called Morphological Evaluation and Understanding of Stress (MorphEUS). MorphEUS 48 classified 94% of tested drugs correctly into broad categories according to modes of action previously 49 identified in the literature. In the other 6%, MorphEUS pointed to key off-target or secondary bactericidal 50 activities. We observed cell-wall damaging activity induced by bedaquiline and moxifloxacin through 51 secondary effects downstream from their main target pathways. We implemented MorphEUS to correctly 52 classify three compounds in a blinded study and identified an off-target effect for one compound that was 53 not readily apparent in previous studies. We anticipate that the ability of MorphEUS to rapidly identify 54 pathways of drug action and the proximal cause of bactericidal activity in tubercule bacilli will make it 55 applicable to other pathogens and cell types where morphological responses are subtle and 56 heterogeneous. 58 Significance Statement 59Tuberculosis is a leading cause of death in the world and requires treatment with an arduous 60 multidrug regimen. Many new tuberculosis drugs are in development, and the drug development pipeline 61 would benefit from more rapid methods to learn drug mechanism of action and off-target effects. Here, we 62 describe a high throughput imaging method for rapidly classifying drugs into categories based on the 63 primary and secondary cellular damage called Morphological Evaluation and Understanding of drug-Stress 64 (MorphEUS). We anticipate that MorphEUS will assist in rapidly pinpointing causes of cellular death in 65 response to drug treatment in tuberculosis and other bacterial pathogens. 66 67 68 69 3 Main Text 70 Introduction 71Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), causes more deaths 72 annually than any other infectious agent (1). Tuberculosis treatment is lengthy, lasting between four months 73 to over a year (1). The difficult regimen, rate of relapse, and incidence of drug resistant Mtb has motivated 74 a significant effort to develop new antibacterial compounds that are effective in sterilizing Mtb infection (2). 75Many new drug classes and derivative compounds have been developed (2), but rapidly identifying the 76 primary and secondary pathways of action of each compound is often a protracted process due to the 77 difficulty in generating resistant mutants, and dissecting the broad-reaching metabolic effects of drug 78 treatment leading to death (3). Furthermore, drug action on bacterial cells can elicit dynamic responses in 79 multiple pathways both on-and off-target, s...

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Application and progress of highcontent imaging in molecular biology

Boyang,

Yangyanqiu,

Wenting

et al. 2023

Biotechnology Journal

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Humans have adopted many different methods to explore matter imaging, among which high content imaging (HCI) could conduct automated imaging analysis of cells while maintaining its structural and functional integrity. Meanwhile, as one of the most important research tools for diagnosing human diseases, HCI is widely used in the frontier of medical research, and its future application has attracted researchers’ great interests. Here, the meaning of HCI was briefly explained, the history of optical imaging and the birth of HCI were described, and the experimental methods of HCI were described. Furthermore, the directions of the application of HCI were highlighted in five aspects: protein localization changes, gene identification, chemical and genetic analysis, microbiology, and drug discovery. Most importantly, some challenges and future directions of HCI were discussed, and the application and optimization of HCI were expected to be further explored.

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Bacterial Cytological Profiling as a Tool To Study Mechanisms of Action of Antibiotics That Are Active against Acinetobacter baumannii

Cited by 51 publications

References 76 publications

A novel vibriophage exhibits inhibitory activity against host protein synthesis machinery

A novel vibriophage exhibits inhibitory activity against host protein synthesis machinery

Morphological profiling of tubercule bacilli identifies drug pathways of action

Application and progress of highcontent imaging in molecular biology

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