A quick flow cytometry protocol to assess Helicobacter pylori viability

Alba, Claudio; Marín, Alicia C; McNicholl, Adrián G.; Montalban-Arques, Ana; Mora-Gutiérrez, Irene; Sánchez-Arroyo, Antonio J.; Soler, T; García‐Fresnadillo, David; Alarcón, Teresa; Bernardo, David

doi:10.1016/j.mimet.2020.106043

Cited by 5 publications

(5 citation statements)

References 6 publications

(3 reference statements)

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“…For example, flow cytometry with Live/ Dead bacterial staining was used to measure viability of H. pylori at low density. 59 Moreno-Mesonero et al used a combination of PMA-qPCR and DVC-FISH methods to detect viable H. pylori that internalized in free-living amoebae and lettuce. 60 A new platform was discussed recently for detecting active infection by measuring H. pylori-induced reactive oxygen species.…”

Section: Detection Of Viable Bacteria and Gene Expression Profilesmentioning

confidence: 99%

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Application of molecular techniques inHelicobacter pyloridetection: limitations and improvements

Gong

El‐Omar

2021

Helicobacter

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Application of molecular techniques in Helicobacter pylori detection: limitations and improvements 1 | INTRODUC TI ONHelicobacter pylori (H. pylori), a microaerophilic gram-negative proteobacterium, is the most common human pathogen colonizing the gastric mucosa of over half the world's population. 1,2 Although 70% of infected people are symptomless, H. pylori is recognized as a strong causative factor in many gastrointestinal (GI) diseases such as peptic ulcer, atrophic gastritis, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. 3 The transmission of H. pylori is usually through person-to-person contact or from environmental sources via oral-oral, fecal-oral, and gastro-oral routes. 4 Therefore, its infection rate is linked to familial socioeconomic status, overcrowding, poor hygiene, and environmental contamination in food and water supply. 5 While maintaining a spiral rod-shaped form in a favorable environment, H. pylori may transform into coccoid shape, a viable but non-culturable (VBNC) form with low metabolic activity, to survive under stressful conditions including oxygen deprivation, antibiotic exposure, and epithelial attachment. 6 Treatment of the infection is usually a combination of acid inhibitory agents (eg, Proton Pump Inhibitors PPI) and antibiotics (ie, amoxicillin, clarithromycin, metronidazole, and tetracycline), the choice of which depends on antibiotic sensitivity. 7 Efficient diagnosis is required for successful clinical management to relieve symptoms and to eradicate bacteria. Clinicians have developed numerous H. pylori detection methods, which are divided into two groups based on whether samples are collected by invasive endoscopy. 8 Serology, urea breath test (UBT), and stool antigen test (SAT) are the non-invasive tests carried out on stool, breath, saliva, or serum samples. 9 The invasive tests including endoscopy, histology, culture, and rapid urease test (RUT) are carried out on gastric biopsies. 10 All these conventional tests are based on the presence of different components such as enzymes (UBT, RUT), antigens (SAT), bacteria (culture), and antibodies (serology, histology). 11 Several reviews have discussed their advantages and disadvantages in accuracy, sensitivity, specificity, simplicity, and cost. [12][13][14][15] To improve conventional methods, especially sensitivity, some molecular techniques based on DNA/RNA have been recently used in both invasive and non-invasive H. pylori diagnosis. 16 These new methods include polymerase chain reaction (PCR), 17 real-time PCR, 18 droplet digital PCR (dd-PCR), 19 fluorescent in situ hybridization (FISH), 20 and next-generation sequencing (NGS) including 16S rRNA amplicon sequencing, 16 transcriptomics, 21 and metagenomics. 22 In this issue, Gantuta et al. 23 discussed the advantages of 16S rRNA sequencing in H. pylori diagnosis, which brings to attention the need to review molecular detection tests, especially their shortcomings. This editorial summarizes our current understanding of molecular methods, focusing on ...

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Section: Detection Of Viable Bacteria and Gene Expression Profilesmentioning

confidence: 99%

“…For example, flow cytometry with Live/Dead bacterial staining was used to measure viability of H . pylori at low density 59 …”

Section: Introductionmentioning

confidence: 99%

Application of molecular techniques inHelicobacter pyloridetection: limitations and improvements

Gong

El‐Omar

2021

Helicobacter

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“…The resulting high-resolution microscopic cell images can be further processed using analytical tools like ImageJ, which offers various convenient ready-to-use plugins [6,7]. Additionally, fluorescence-activated cell sorting (FACS) and imaging flow cytometry (IFC) have been developed for applications including cell sorting [8][9][10], quantification [11,12], bacterial viability assessment [13,14], dynamic monitoring of bacterial morphology [15,16] and analysis of the heterogeneity in bacterial communities [16,17]. However, the quality and reproducibility of the results of these tools primarily rely on the employed fluorescent labeling markers for targeted cells [18,19].…”

Section: Introductionmentioning

confidence: 99%

Implementation of Fluorescent-Protein-Based Quantification Analysis in L-Form Bacteria

Tian,

Liu,

Zhang

et al. 2024

Bioengineering

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Cell-wall-less (L-form) bacteria exhibit morphological complexity and heterogeneity, complicating quantitative analysis of them under internal and external stimuli. Stable and efficient labeling is needed for the fluorescence-based quantitative cell analysis of L-forms during growth and proliferation. Here, we evaluated the expression of multiple fluorescent proteins (FPs) under different promoters in the Bacillus subtilis L-form strain LR2 using confocal microscopy and imaging flow cytometry. Among others, Pylb-derived NBP3510 showed a superior performance for inducing several FPs including EGFP and mKO2 in both the wild-type and L-form strains. Moreover, NBP3510 was also active in Escherichia coli and its L-form strain NC-7. Employing these established FP-labeled strains, we demonstrated distinct morphologies in the L-form bacteria in a quantitative manner. Given cell-wall-deficient bacteria are considered protocell and synthetic cell models, the generated cell lines in our work could be valuable for L-form-based research.

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“…Over the last few decades, FCM-based platforms such as the fluorescence-activated cell sorter (FACS) and imaging flow cytometer (IFC) have provided a better solution for cell quantification, biomass estimation, and cell sorting in a more sophisticated way [ 25 , 26 ]. Cell parameters such as light scatter values and specific fluorescent channels are used in FCM to analyze the broad heterogeneity of bacterial communities [ 27 , 28 , 29 ], assess bacterial viability by measuring both live and dead bacteria with fluorescent dyes [ 24 , 30 ], and to monitor bacterial morphological changes in specific conditions [ 29 , 31 ]. Remarkably, a cell shape mutant in Helicobacter pylori was successfully isolated through a single round of FACS enrichment based on low forward scatter (FSC) value, leading to the rapid identification of multiple genes affecting its morphology [ 32 ].…”

Section: Introductionmentioning

confidence: 99%

Cell Sorting-Directed Selection of Bacterial Cells in Bigger Sizes Analyzed by Imaging Flow Cytometry during Experimental Evolution

Tian

Wang

Liu

et al. 2023

IJMS

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Cell morphology is an essential and phenotypic trait that can be easily tracked during adaptation and evolution to environmental changes. Thanks to the rapid development of quantitative analytical techniques for large populations of cells based on their optical properties, morphology can be easily determined and tracked during experimental evolution. Furthermore, the directed evolution of new culturable morphological phenotypes can find use in synthetic biology to refine fermentation processes. It remains unknown whether and how fast we can obtain a stable mutant with distinct morphologies using fluorescence-activated cell sorting (FACS)-directed experimental evolution. Taking advantage of FACS and imaging flow cytometry (IFC), we direct the experimental evolution of the E. coli population undergoing continuous passage of sorted cells with specific optical properties. After ten rounds of sorting and culturing, a lineage with large cells resulting from incomplete closure of the division ring was obtained. Genome sequencing highlighted a stop-gain mutation in amiC, leading to a dysfunctional AmiC division protein. The combination of FACS-based selection with IFC analysis to track the evolution of the bacteria population in real-time holds promise to rapidly select and culture new morphologies and association tendencies with many potential applications.

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A quick flow cytometry protocol to assess Helicobacter pylori viability

Cited by 5 publications

References 6 publications

Application of molecular techniques inHelicobacter pyloridetection: limitations and improvements

Application of molecular techniques inHelicobacter pyloridetection: limitations and improvements

Implementation of Fluorescent-Protein-Based Quantification Analysis in L-Form Bacteria

Cell Sorting-Directed Selection of Bacterial Cells in Bigger Sizes Analyzed by Imaging Flow Cytometry during Experimental Evolution

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