Biology subjects

Sayeed, M. A.

Publications and source records attributed to Sayeed, M. A..

5 recordsLinked to original sources

bioRxiv2025

A colorimetric method for detecting virulent bacteriophage to Vibrio cholerae in fecal and environmental samples

BackgroundV. cholerae are often infected with vibriophages and these phages may be found associated with V. cholerae bacteria or may be detected independently from the bacteria, especially in environmental water samples. When detected, vibriophages serve as a surrogate for detection of V. cholerae and they also have an important role in the ecology of V. cholerae. Vibriophages can be detected using plaque assays or PCR, but these methods are time-consuming and require specialized laboratory resources. MethodologyTo address limitations of other methods, we developed a simple, rapid, and inexpensive colorimetric assay to detect vibriophage that can be scaled up to quickly and easily to screen a large number of samples. The assay uses resazurin and a bacterium, AC6169 that is susceptible to vibriophages ICP1, 2, and 3. Resazurin is a dye that turns color from blue to pink when added to a culture broth with growing bacteria. When a bacteria-free test sample, such as a Millipore filtered wastewater sample containing vibriophages, is added to a culture broth with AC6169, the bacteria will be lysed by the phages and will not grow, and the color of the broth will remain blue. However, if there are no phages in the sample, the bacteria will grow rapidly, and the culture broth will turn pink. We developed the assay using ICP1 spiked samples of environmental water, stool and frozen bile peptone and found it to be sensitive with a limit of detection of 4 to 40 plaque forming units/ml. ConclusionThis colorimetric assay promises to provide a convenient method to detect vibriophages on a larger scale than was possible earlier to better understand their role as a surrogate for detecting V. cholerae and to better understand their role in the pathogenesis, ecology, and epidemiology of cholera. Author SummaryWe developed a simple and inexpensive colorimetric assay to detect virulent vibriophage in samples of wastewater and stool. The assay uses resazurin, a blue dye and a unique bacterium, AC6169, that is known to be susceptible to vibriophages. Bacteria-free samples (e.g. Millipore filtrates) suspected of having vibriophage are incubated for 2 or 3 hours in a broth with AC6169, following which resazurin is added. After a further 30-minute incubation, the color of the broth is observed. If the sample contains vibriophage, the broth will remain blue, but if there are no vibriophage, the broth will turn pink. The change in color from blue to pink is due to the metabolic reaction from the growing bacteria. When vibriophage are present, they lyse the bacterium AC6169, thus preventing the color change, but if vibriophage are not present, the bacteria multiply rapidly, and the metabolic reaction causes the broth to change color to pink. Because the assay procedure is inexpensive and simple, laboratories in cholera endemic areas should find it convenient for their epidemiologic and clinical surveillance activities.

microbiology↗

bioRxiv2024

Vibrio cholerae lineage and pangenome diversity varies geographically across Bangladesh over one year

Cholera is a diarrhoeal disease caused by Vibrio cholerae. It remains a major public health challenge in the endemic region around the Bay of Bengal. Over decadal time scales, one lineage typically dominates the others and spreads in global pandemic waves. However, it remains unclear to what extent diverse lineages co-circulate during a single outbreak season. Defining the pool of diversity during finer time scales is important because the selective pressures that impact V. cholerae - namely antibiotics and phages - are dynamic on these time scales. To study the nationwide diversity of V. cholerae, we long-read sequenced 273 V. cholerae genomes from seven hospitals over one year (2018) in Bangladesh. Four major V. cholerae lineages were identified: known lineages BD-1, BD-2a, and BD-2b, and a novel lineage that we call BD-3. In 2022, BD-1 caused a large cholera outbreak in Dhaka, apparently outcompeting BD-2 lineages. We show that, in 2018, BD-1 was already dominant in the five northern regions, including Dhaka, consistent with an origin from India in the north. By contrast, we observed a higher diversity of lineages in the two southern regions near the coast. The four lineages differed in pangenome content, including integrative and conjugative elements (ICEs) and genes involved in resistance to bacteriophages and antibiotics. Notably, BD-2a lacked an ICE and is predicted to be more sensitive to phages and antibiotics, but nevertheless persisted throughout the year-long sampling period. Genes associated with antibiotic resistance in V. cholerae from Bangladesh in 2006 were entirely absent from all lineages in 2018-19, suggesting shifting costs and benefits of encoding these genes. Together, our results highlight the dynamic nature of the V. cholerae pangenome and the geographic structure of its lineage diversity.

microbiology↗

bioRxiv2023

Phage predation and antibiotic exposure are inversely associated with disease severity and shape pathogen genetic diversity in cholera patients

Despite an increasingly detailed picture of the molecular mechanisms of phage-bacterial interactions, we lack an understanding of how these interactions evolve and impact disease within patients. Here we report a year-long, nation-wide study of diarrheal disease patients in Bangladesh. Among cholera patients, we quantified Vibrio cholerae (prey) and its virulent phages (predators) using metagenomics and quantitative PCR, while accounting for antibiotic exposure using quantitative mass spectrometry. Virulent phage (ICP1) and antibiotics suppressed V. cholerae to varying degrees and were inversely associated with severe dehydration depending on resistance mechanisms. In the absence of anti-phage defenses, predation was effective, with a high predator:prey ratio that correlated with increased genetic diversity among the prey. In the presence of anti-phage defenses, predation was ineffective, with a lower predator:prey ratio that correlated with increased genetic diversity among the predators. Phage-bacteria coevolution within patients should therefore be considered in the deployment of phage-based therapies and diagnostics. One Sentence SummaryA survey of cholera patients in Bangladesh identifies phage predation as a biomarker of disease severity and driver of coevolution within patients.

microbiology↗

bioRxiv2022

Development of a monoclonal antibody to a vibriophage as a proxy for Vibrio cholerae detection

Cholera is an acute watery diarrheal disease that causes high rates of morbidity and mortality without treatment. Early detection of the etiologic agent of toxigenic Vibrio cholerae is important to mobilize treatment and mitigate outbreaks. Monoclonal antibody (mAb) based rapid diagnostic tests (RDTs) enable early detection in settings without laboratory capacity. However, the odds of an RDT testing positive are reduced by nearly 90% when the common virulent bacteriophage ICP1 is present. We hypothesize that adding a mAb for the common, and specific, virulent bacteriophage ICP1 as a proxy for V. cholerae to an RDT will increase diagnostic sensitivity when virulent ICP1 phage are present. In this study, we used an in-silico approach to identify immunogenic ICP1 protein targets that were conserved across disparate time periods and locations. Specificity of targets to cholera patients with known ICP1 was determined, and specific targets were used to produce mAbs in a murine model. Candidate mAbs to the head protein demonstrated specificity to ICP1 by ELISA and an ICP1 phage neutralization assay. The limit of detection of the final mAb candidate for ICP1 phage particles spiked into cholera stool matrix was 8 x 105 plaque forming units by Western blot analysis. This mAb will be incorporated into a RDT prototype for evaluation in a future diagnostic study to test the guiding hypothesis behind this study.

immunology↗

bioRxiv2020

Gold-standard diagnostics are tarnished by lytic bacteriophage

BackgroundA fundamental clinical and scientific concern is how lytic bacteriophage, as well as antibiotics, impact diagnostic positivity. MethodsCholera was chosen as a model disease to investigate this important question. Patients with diarrheal disease were enrolled at two remote hospitals in Bangladesh. Diagnostic performance was assessed as a function of lytic bacteriophage detection, as well as exposure to the first-line antibiotic azithromycin detected by mass spectrometry. ResultsAmong diarrheal samples positive by nanoliter quantitative PCR for Vibrio cholerae (n=78/849), the odds that a rapid diagnostic test (RDT) or qPCR was positive was reduced by 89% (OR 0.108; 95%CI 0.002-0.872) and 87% (OR 0.130; 95%CI 0.022-0.649) when lytic bacteriophage were detected, respectively. The odds that a rapid diagnostic test (RDT) or qPCR was positive was reduced by more than 99% (OR 0.00; 95% CI: 0.00-0.28) and 89% (OR 0.11; 95% CI: 0.03-0.44) when azithromycin was detected, respectively. ConclusionsEstimations of cholera burden may improve by accommodating for the negative effect of antimicrobial exposure on diagnostic positivity. Furthermore, the findings herein challenge our current approach to interpreting and developing bacterial diagnostics given variable rates of lytic bacteriophage and antibiotic exposure.

microbiology↗