A revisited two-step microtiter plate assay: Optimization of in vitro multiplicity of infection (MOI) for Coliphage and Vibriophage

Benala, Manikantha; Vaiyapuri, Murugadas; Visnuvinayagam, S.; George, Joshy Chalil; Karthika, R.; George, Iris; Mothadaka, Mukteswar Prasad; Badireddy, Madhusudana Rao

doi:10.1016/j.jviromet.2021.114177

Cited by 12 publications

(6 citation statements)

References 32 publications

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“…In this study, the influence of the factors (phage type, bacterial strain, and MOI) on the observed growth patterns was determined. Previous studies have highlighted the MOI influence on phage therapy, and recently, a fast microtiter plate assay for determination of the optimum MOI for a coliphage was further described [39]. However, in this study, we found the MOI to have a less significant effect on the phage-host growth dynamics outcome compared to the phage species.…”

Section: Discussioncontrasting

confidence: 55%

Classification of In Vitro Phage–Host Population Growth Dynamics

Sørensen

Duchateau

et al. 2021

Microorganisms

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The therapeutic use of bacteriophages (phage therapy) represents a promising alternative to antibiotics to control bacterial pathogens. However, the understanding of the phage–bacterium interactions and population dynamics seems essential for successful phage therapy implementation. Here, we investigated the effect of three factors: phage species (18 lytic E. coli-infecting phages); bacterial strain (10 APEC strains); and multiplicity of infection (MOI) (MOI 10, 1, and 0.1) on the bacterial growth dynamics. All factors had a significant effect, but the phage appeared to be the most important. The results showed seven distinct growth patterns. The first pattern corresponded to the normal bacterial growth pattern in the absence of a phage. The second pattern was complete bacterial killing. The remaining patterns were in-between, characterised by delayed growth and/or variable killing of the bacterial cells. In conclusion, this study demonstrates that the phage–host dynamics is an important factor in the capacity of a phage to eliminate bacteria. The classified patterns show that this is an essential factor to consider when developing a phage therapy. This methodology can be used to rapidly screen for novel phage candidates for phage therapy. Accordingly, the most promising candidates were phages found in Group 2, characterised by growth dynamics with high bacterial killing.

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Section: Discussioncontrasting

confidence: 55%

Classification of In Vitro Phage–Host Population Growth Dynamics

Sørensen

Duchateau

et al. 2021

Microorganisms

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“…The optimum MOI, i.e., the lowest number of phages required to inhibit the growth of the target bacteria, was determined in the second step. The optimum MOI of vibriophage-φLV6 against the luminescent V. harveyi-LV6 host was 79 and was previously determined [56]. On similar lines, the optimum MOIs of vibriophage-φLV6 against five other luminescent vibrio hosts, viz., LV36, LV38, LV40, LV44, and LV45, isolated from shrimp hatcheries, were determined.…”

Section: In Vitro Determination Of Optimum Multiplicity Of Infection ...supporting

confidence: 64%

“…The multiplicity of infection, i.e., the ratio of the number of vibrophage-φLV6 required to lyse luminescent V. harveyi, was determined employing the 2-step microtiter plate assay [56]. Briefly, in the 2-step microtiter assay, a broad range of MOIs (ranging from MOI-0.0001 to MOI-10000) were initially tested for their ability to inhibit the growth of target bacteria.…”

Section: In Vitro Determination Of Optimum Multiplicity Of Infection ...mentioning

confidence: 99%

Genome Characterization and Infectivity Potential of Vibriophage-ϕLV6 with Lytic Activity against Luminescent Vibrios of Penaeus vannamei Shrimp Aquaculture

Benala

Vaiyapuri

Visnuvinayagam

et al. 2023

Viruses

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Shrimp aquaculture, especially during the hatchery phase, is prone to economic losses due to infections caused by luminescent vibrios. In the wake of antimicrobial resistance (AMR) in bacteria and the food safety requirements of farmed shrimp, aqua culturists are seeking alternatives to antibiotics for shrimp health management, and bacteriophages are fast emerging as natural and bacteria-specific antimicrobial agents. This study analyzed the whole genome of vibriophage-ϕLV6 that showed lytic activity against six luminescent vibrios isolated from the larval tanks of P. vannamei shrimp hatcheries. The Vibriophage-ϕLV6 genome was 79,862 bp long with 48% G+C content and 107 ORFs that coded for 31 predicted protein functions, 75 hypothetical proteins, and a tRNA. Pertinently, the vibriophage-ϕLV6 genome harbored neither AMR determinants nor virulence genes, indicating its suitability for phage therapy. There is a paucity of whole genome-based information on vibriophages that lyse luminescent vibrios, and this study adds pertinent data to the database of V. harveyi infecting phage genomes and, to our knowledge, is the first vibriophage genome report from India. Transmission electron microscopy (TEM) of vibriophage-ϕLV6 revealed an icosahedral head (~73 nm) and a long, flexible tail (~191 nm) suggesting siphovirus morphology. The vibriophage-ϕLV6 phage at a multiplicity of infection (MOI) of 80 inhibited the growth of luminescent V. harveyi at 0.25%, 0.5%, 1%, 1.5%, 2%, 2.5%, and 3% salt gradients. In vivo experiments conducted with post-larvae of shrimp showed that vibriophage-ϕLV6 reduced luminescent vibrio counts and post-larval mortalities in the phage-treated tank compared to the bacteria-challenged tank, suggesting the potentiality of vibriophage-ϕLV6 as a promising candidate in treating luminescent vibriosis in shrimp aquaculture. The vibriophage-ϕLV6 survived for 30 days in salt (NaCl) concentrations ranging from 5 ppt to 50 ppt and was stable at 4 °C for 12 months.

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“…The multiplicity of infection (MOI) determination was carried out as the reference research [ 34 – 36 ]. Phage KL01 was incubated with the log-phase culture of K. quasipneumoniae (10 8 CFU/mL) at various proportions (10, 1, 0.1, 0.01, 0.001 and 0.0001) followed by incubation at 37 °C 170 rpm for 5 h. The titers of the phage at different MOI were determined by the double-agar layer plate method.…”

Section: Methodsmentioning

confidence: 99%

“…The one-step growth curve was generated according to a previous report with minor modifications [ 37 ]. According to the optimal MOI 0.1, phage KL01 (~1 × 10 7 PFU/mL)was incubated with exponential phage K. quasipneumoniae (~1 × 10 8 CFU/mL) [ 35 , 36 ]. After incubated at 37 °C for 10 min, the mixture was centrifuged at 12,000 rpm for 5 min at 4 °C to remove the unabsorbed phage in the supernatant by repeated three times.…”

Section: Methodsmentioning

confidence: 99%

Bacterial isolation and genome analysis of a novel Klebsiella quasipneumoniae phage in southwest China’s karst area

Liu,

Wang,

Zhao

et al. 2024

Virol J

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Background Southwest China is one of the largest karst regions in the world. Karst environment is relatively fragile and vulnerable to human activities. Due to the discharge of sewage and domestic garbage, the karst system may be polluted by pathogenic bacteria. The detection of bacterial distribution and identification of phage capable of infecting them is an important approach for environmental assessment and resource acquisition. Methods Bacteria and phages were isolated from karst water in southwest China using the plate scribing and double plate method, respectively. Isolated phage was defined by transmission electron microscopy, one-step growth curve and optimal multiplicity of infection (MOI). Genomic sequencing, phylogenetic analysis, comparative genomic and proteomic analysis were performed. Results A Klebsiella quasipneumoniae phage was isolated from 32 isolates and named KL01. KL01 is morphologically identified as Caudoviricetes with an optimal MOI of 0.1, an incubation period of 10 min, and a lysis period of 60 min. The genome length of KL01 is about 45 kb, the GC content is 42.5%, and it contains 59 open reading frames. The highest average nucleotide similarity between KL01 and a known Klebsiella phage 6939 was 83.04%. Conclusions KL01 is a novel phage, belonging to the Autophagoviridae, which has strong lytic ability. This study indicates that there were not only some potential potentially pathogenic bacteria in the karst environment, but also phage resources for exploration and application.

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A revisited two-step microtiter plate assay: Optimization of in vitro multiplicity of infection (MOI) for Coliphage and Vibriophage

Cited by 12 publications

References 32 publications

Classification of In Vitro Phage–Host Population Growth Dynamics

Classification of In Vitro Phage–Host Population Growth Dynamics

Genome Characterization and Infectivity Potential of Vibriophage-ϕLV6 with Lytic Activity against Luminescent Vibrios of Penaeus vannamei Shrimp Aquaculture

Bacterial isolation and genome analysis of a novel Klebsiella quasipneumoniae phage in southwest China’s karst area

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