Liquid-based Assay Research Articles

Frequency of Common and Uncommon BRAF Alterations among Colorectal and Non-Colorectal Gastrointestinal Malignancies.

The predictive and prognostic role of BRAF alterations has been evaluated in colorectal cancer (CRC); however, BRAF alterations have not been fully characterized in non-CRC gastrointestinal (GI) malignancies. In the present study, we report the frequency and spectrum of BRAF alterations among patients with non-CRC GI malignancies. Patients with CRC and non-CRC GI malignancies who underwent somatic tumor profiling via a tissue-based or liquid-based assay were included in this study. Gain-of-function BRAF alterations were defined as pathogenic/likely pathogenic somatic short variants (SVs), copy number amplifications ≥8, or fusions (RNA or DNA). Among 51,560 patients with somatic profiling, 40% had CRC and 60% had non-CRC GI malignancies. BRAF GOF alterations were seen more frequently in CRC (8.9%) compared to non-CRC GI malignancies (2.2%) (p < 0.001). Non-CRC GI malignancies with the highest prevalence of BRAF GOF alterations were bile duct cancers (4.1%) and small intestine cancers (4.0%). Among BRAF GOF alterations, class II (28% vs. 6.8%, p < 0.001) and class III (23% vs. 14%, p < 0.001) were more common in non-CRC GI malignancies. Among class II alterations, rates of BRAF amplifications (3.1% vs. 0.3%, p < 0.001) and BRAF fusions (12% vs. 2.2%, p < 0.001) were higher in non-CRC GI malignancies compared to CRC. Non-CRC GI malignancies demonstrate a distinct BRAF alteration profile compared to CRC, with a higher frequency of class II and III mutations, and more specifically, a higher incidence of BRAF fusions. Future studies should evaluate clinical implications for the management of non-CRC GI patients with BRAF alterations, especially BRAF fusions.

Potential pathogenic germline variant reporting from tumor comprehensive genomic profiling complements classic approaches to germline testing

Existing guidance regarding clinically informed germline testing for patients with cancer is effective for evaluation of classic hereditary cancer syndromes and established gene/cancer type associations. However, current screening methods may miss patients with rare, reduced penetrance, or otherwise occult hereditary risk. Secondary finding of suspected germline variants that may confer inherited cancer risk via tumor comprehensive genomic profiling (CGP) has the potential to help address these limitations. However, reporting practices for secondary finding of germline variants are inconsistent, necessitating solutions for transparent and coherent communication of these potentially important findings. A workflow for improved confidence detection and clear reporting of potential pathogenic germline variants (PPGV) in select cancer susceptibility genes (CSG) was applied to a research dataset from real-world clinical tumor CGP of > 125,000 patients with advanced cancer. The presence and patterns of PPGVs identified across tumor types was assessed with a focus on scenarios in which traditional clinical germline evaluation may have been insufficient to capture genetic risk. PPGVs were identified in 9.7% of tumor CGP cases using tissue- and liquid-based assays across a broad range of cancer types, including in a number of “off-tumor” contexts. Overall, PPGVs were identified in a similar proportion of cancers with National Comprehensive Cancer Network (NCCN) recommendations for germline testing regardless of family history (11%) as in all other cancer types (9%). These findings suggest that tumor CGP can serve as a tool that is complementary to traditional germline genetic evaluation in helping to ascertain inherited susceptibility in patients with advanced cancer.

Pathogen-induced dormancy in liquid limits gastrointestinal colonization of Caenorhabditis elegans

ABSTRACT Colonization is generally considered a prerequisite for infection, but this event is context-dependent, as evidenced by the differing ability of the human pathogen Pseudomonas aeruginosa to efficiently colonize Caenorhabditis elegans on agar but not in liquid . In this study, we examined the impact of the environment, pathogen, host, and their interactions on host colonization. We found that the transition to a liquid environment reduces food uptake by about two-fold. Also expression of specific adhesins was significantly altered in liquid-based assays for P. aeruginosa, suggesting that it may be one factor driving diminished colonization. Unexpectedly, host immune pathways did not appear to play a significant role in decreased colonization in liquid. Although knocking down key immune pathways (e.g. daf-16 or zip-2), either alone or in combination, significantly reduced survival, the changes in colonization were very small. In spite of the limited bacterial accumulation in the liquid setting, pathogenic colonization was still required for the virulence of Enterococcus faecalis. In addition, we found that a pathogen-induced dormancy was displayed by C. elegans in liquid medium after pathogen exposure, resulting in cessation of pharyngeal pumping and a decrease in bacterial intake. We conclude that poor colonization in liquid is likely due to a combination of environmental factors and host-pathogen interactions. These results provide new insights into mechanisms for colonization in different models, enabling pathogenesis models to be fine-tuned to more accurately represent the conditions seen in human infections so that new tools for curbing bacterial and fungal infections can be developed.

Abstract 3351: Identification of novel biomarkers for high grade serous ovarian cancer screening via extracellular vesicle proteomic profiling

Abstract Introduction: Ovarian cancer is a silent killer that rarely shows symptoms and therefore is detected at later stages when treatments are less effective. Developing non-invasive blood tests for pre-symptomatic screening and early detection is crucial. Recent studies support the paradigm that high grade serous carcinomas (HGSOCs), the most common and deadliest form of ovarian cancers, arise from the epithelial cells within the fimbriated end of the fallopian tubes (FT). The goal of this study was to uncover novel protein biomarkers associated with tumor-derived extracellular vesicles (EVs) originating from the FT epithelial cells to advance liquid-based assays for the early detection of HGSOC. We hypothesize that EV-associated proteins (exo-proteins), i.e., lineage biomarkers, shared between the progenitor cells residing in the FT and tumor cells residing in cancerous tissue will allow for detection of the earliest forms of HGSOC. Methods: Differential ultracentrifugation was used to enrich EVs from FT and HGSOC cell lines (n=3 &amp; n=5) and healthy FT and HGSOC tissue explants (n=6 &amp; n=15), respectively. Unbiased proteomic profiling of EVs was performed by LC-MS/MS. Results: We report for the first time 985 proteins that compose the FT/HGSOC EV core proteome. We used bioinformatic pipelines to identify high-quality candidate exo-biomarkers for further validation. We selected exo-proteins that were predicted to have at least one transmembrane domain which could serve as an antigen for immunoaffinity capture. We prioritized proteins that showed a log2 fold-change ≥ -0.58 to identify potential biomarkers present in FT-associated EVs which were elevated as the disease progresses to HGSOC. A ranked list of 45 predicted transmembrane exo-proteins was screened using Simple Western assays for expression in FT and HGSOC cell line-derived EVs. Expression of the top 6 candidate biomarkers (i.e., ACSL4, IGSF8, ITGA2, ITGA5, ITGB3 and MYOF) were confirmed in FT and HGSOC tissue sample by immunohistochemistry using tissue microarray (n=100). Next, we analyzed plasma samples from a small case-control study using our ExoProfile chip (e.g., PMID 31293733) and found these 6 exo-biomarkers, plus folate receptor alpha exhibited higher levels in the HGSOC plasma relative to matched healthy controls. We performed receiver operating characteristic (ROC) analyses and found that all 6 FT/HGSOC shared exo-biomarkers exhibit an overall classification performance ranging from 0.85-0.98 as reflected by the area under the curve (AUC). Furthermore, when we used a linear combination of IGSF8 and ITGA5 based on logistic regression analysis, we achieved a sensitivity of 80% at 99.8% specificity. Conclusion: Validated lineage-associated exo-protein biomarkers can be used in liquid-based assays to detect ovarian cancer while localized to the FT/ovary and when patient outcomes are more favorable. Citation Format: Camille V. Trinidad, Harsh B. Pathak, Shibo Cheng, Rashna Madan, Mihaela E. Sardiu, Leonidas E. Bantis, Yong Zeng, Andrew K. Godwin. Identification of novel biomarkers for high grade serous ovarian cancer screening via extracellular vesicle proteomic profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3351.

Development of miniaturized agar based assays in 96-well microplates applicable to high-throughput screening of industrially valuable microorganisms

High-throughput screening (HTS) is a present-day approach for assaying thousands of cultures in parallel. This miniaturization allows rapid screening of large number of microorganims capable of producing bio-based materials thereby meeting the demands of the ever evolving food, pharmaceutical and cosmetic industry. In this study, agar-based assays for phosphate solubilization, cellulose degradation and lactic acid production were developed in 96-well microplates using Biomek FXP Automated Liquid Handling system. Techno-economic analysis from this study reveals the lower overall cost per assay using HTS as compared to conventional Petri plate assays. Though automated liquid handling workstations have been used to perform liquid-based assays, there are very few studies which report their use for agar-based microplate assays. These findings thus corroborate the establishment of rapid and efficient miniaturized, qualitative agar-based screening methods for identifying microorganisms with potential for commercial application.

A Multiwell-Plate Caenorhabditis elegans Assay for Assessing the Therapeutic Potential of Bacteriophages against Clinical Pathogens.

ABSTRACTIn order to establish phage therapy as a standard clinical treatment for bacterial infections, testing of every phage to ensure the suitability and safety of the biological compound is required. While some issues have been addressed over recent years, standard and easy-to-use animal models to test phages are still rare. Testing of phages in highly suitable mammalian models such as mice is subjected to strict ethical regulations, while insect larvae such as the Galleria mellonella model suffer from batch-to-batch variations and require manual operator skills to inject bacteria, resulting in unreliable experimental outcomes. A much simpler model is the nematode Caenorhabditis elegans, which feeds on bacteria, a fast growing and easy to handle organism that can be used in high-throughput screening. In this study, two clinical bacterial strains of Escherichia coli, one Klebsiella pneumoniae, and one Enterobacter cloacae strain were tested on the model system together with lytic bacteriophages that we isolated previously. We developed a liquid-based assay, in which the efficiency of phage treatment was evaluated using a scoring system based on microscopy and counting of the nematodes, allowing increasing statistical significance compared to other assays such as larvae or mice. Our work demonstrates the potential to use Caenorhabditis elegans to test the virulence of strains of Klebsiella pneumoniae, Enterobacter cloacae, and EHEC/EPEC as well as the efficacy of bacteriophages to treat or prevent infections, allowing a more reliable evaluation for the clinical therapeutic potential of lytic phages.IMPORTANCE Validating the efficacy and safety of phages prior to clinical application is crucial to see phage therapy in practice. Current animal models include mice and insect larvae, which pose ethical or technical challenges. This study examined the use of the nematode model organism C. elegans as a quick, reliable, and simple alternative for testing phages. The data show that all the four tested bacteriophages can eliminate bacterial pathogens and protect the nematode from infections. Survival rates of the nematodes increased from <20% in the infection group to >90% in the phage treatment group. Even the nematodes with poly-microbial infections recovered during phage cocktail treatment. The use of C. elegans as a simple whole-animal infection model is a rapid and robust way to study the efficacy of phages before testing them on more complex model animals such as mice.

Colonization and degradation of polyurethane coatings by Pseudomonas protegens biofilms is promoted by PueA and PueB hydrolases

The ability of bacteria and fungi to degrade polymeric substrates is often assessed in liquid-based assays conducted by exposing polymers to planktonic cells, or cell-free supernatants or enzymes generated from them. These assessments miss the opportunity to specifically examine the role of biofilm formation and physiology in degradative processes. In a previous study, we examined the ability of cell-free supernatants from wildtype and hydrolase-deficient Pseudomonas protegens Pf-5 strains to degrade a commercial polyester polyurethane (PU). In this study, we developed the methodology to conduct spatial-temporal analysis of both biofilm colonization and degradation of polyester PU coatings using the same strains. Wild-type Pf-5 grew as confluent biofilms that produced tendrils containing viable cells; in contrast, Pf-5ΔpueAB, a mutant lacking PueA and PueB hydrolases, colonized the PU only by growing as a confluent biofilm but lacked tendril expansion. Degradation of PU by the wild-type hydrolases may alter the substrata, facilitating colonization by the biofilm. Chemical analysis by in situ Fourier transform infrared (FTIR) and Raman spectroscopies revealed the polyester block of the PU was preferentially degraded to varying degrees by the wild-type Pf-5 and mutant biofilms, and showed degradation due to PueB that was undetectable in liquid-based assays. Raman spectroscopy analysis of biofilms of Pf-5ΔpueAB grown on PU revealed a detectable, but the lowest, level of PU degradation relative to other strains. Degradation of PU by this mutant had previously been undetectable and this study revealed the existence of biofilm-associated hydrolytic mechanisms other than those driven by PueA and PueB. Taken together, our data suggest that both PueA and PueB hydrolases play a role in the colonization and degradation of a model polyester PU by P. protegens biofilms. This study demonstrates the importance of using biofilm-based assays to identify mechanisms of microbiologically-influenced degradation.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans.

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

Feeder-free and serum-free in vitro assay for measuring the effect of drugs on acute and chronic myeloid leukemia stem/progenitor cells

Research on chronic and acute myeloid leukemia (CML/AML) is focused on the development of novel therapeutic strategies to eliminate leukemic stem/progenitor cells that are responsible for drug resistance and disease relapse. Methods to culture hematopoietic stem/progenitor cells (HSPCs) from blood or bone marrow samples are indispensable for investigating disease pathogenesis and delineating drug responses in individual patients. A key challenge in this area is that primary leukemic cells grow poorly in culture or rapidly differentiate and lose their hematopoietic potential. Access to patient samples can also be limiting or cell numbers too low to enable large-scale assays and/or to obtain reproducible quantitative data. Here we describe a feeder cell-free and serum-free liquid culture system for the expansion of CD34+ HSPCs from CML/AML samples and healthy control tissues. Following 7 or 14 days of culture, CD34+ cells are expanded 30- to 65-fold or 400- to 800-fold, yielding a purity of ∼80% and ∼60% CD34+ cells, respectively. This system was adapted to a 96-well format to measure the sensitivity of leukemic and normal HSPCs to cytotoxic drugs after only 7 days. The assay requires only 103 cells per well to determine drug IC50 values and can be performed with uncultured and culture-expanded cells. Importantly, resulting IC50 values strongly correlate with those obtained in the classic colony-forming unit (CFU) assay. Compared with the CFU assay, this novel 96-well liquid-based assay designed specifically for leukemic and normal HSPCs is faster and simpler, with more flexible readout methods for selecting candidates for further drug development.

Ewing sarcoma family of tumors-derived small extracellular vesicle proteomics identify potential clinical biomarkers

Purpose: Ewing Sarcoma Family of Tumors (ESFT), the second most common pediatric osseous malignancy, are characterized by the pathognomonic chromosomal EWS-ETS translocation. Outside of tumor biopsy, no clinically relevant ESFT biomarkers exist. Additionally, tumor burden assessment at diagnosis, monitoring of disease responsiveness to therapy, and detection of disease recurrence are limited to radiographic imaging. To identify new, clinically relevant biomarkers we evaluated the proteome of a subset of ESFT-derived small extracellular vesicles (sEVs).Materials and Methods: We performed the first high quality proteomic study of ESFT-derived sEVs from 5 ESFT cell lines representing the most common EWS-ETS fusion types and identified 619 proteins composing the core ESFT sEV proteome. We compared these core proteins to databases of common plasma-based proteins and sEV-associated proteins found within healthy plasma to identify proteins unique or enriched within ESFT.Results: From these analyses, two membrane bound proteins with biomarker potential were selected, CD99/MIC2 and NGFR, to develop a liquid-based assay enriching of ESFT-associated sEVs and detection of sEV mRNA cargo (i.e., EWS-ETS transcripts). We employed this immuno-enrichment approach to diagnosis of ESFT utilizing plasma (250 μl) from both localized and metastatic ESFT pediatric patients and cancer-free controls, and showed significant diagnostic power [AUC = 0.92, p = 0.001 for sEV numeration, with a PPV = 1.00, 95% CI = (0.63, 1.00) and a NPV = 0.67, 95% CI = (0.30, 0.93)].Conclusions: In this study, we demonstrate utilization of circulating ESFT-associated sEVs in pediatric patients as a source of minimally invasive diagnostic and potentially prognostic biomarkers.

Determination of high-risk HPV status of head and neck squamous cell carcinoma using the Roche cobas HPV test on cytologic specimens and acellular supernatant fluid.

High-risk human papillomavirus-positive (hrHPV+) oropharyngeal squamous cell carcinomas comprise a subset of head and neck squamous cell carcinomas (HNSCCs) with a distinct biology and prognosis. Commonly, the diagnosis of HNSCC is rendered on fine-needle aspiration (FNA). Because cell blocks may be insufficient for determining HPV status using microscopy-based techniques, the ability of liquid-based assays was examined in the current study. The performance of the Roche cobas 4800 platform was evaluated on the FNA material from the cell pellet and corresponding cell-free supernatant fluid specimens of primary and metastatic HNSCCs. These results were compared with the p16 immunostain result from the histologic material obtained from the same patient. Discrepant cases were adjudicated using hrHPV RNA in situ hybridization. A total of 41 samples (23 primary tumors and 18 lymph node metastases) were acquired from 34 patients with HNSCC. Primary tumors included the oropharynx (20 samples), oral cavity (13 samples), larynx (3 samples), and skin (3 samples). In 2 cases, a primary tumor could not be identified. Twenty-three samples (56%) were found to be p16 positive by immunohistochemistry. Twenty-two samples were found to be positive on cobas hrHPV testing from both cell pellet and cell-free supernatant fluid. Two cell-free supernatant fluid specimens yielded indeterminate cobas results. At the time additional hrHPV RNA in situ hybridization analysis was performed, one cobas-positive cell pellet was deemed to be a false-positive result. The sensitivity of the cobas assay was 100% for pellet material and cell-free supernatant fluid, with specificities of 94.7% and 100%, respectively. cobas hrHPV testing of HNSCC specimens demonstrated high concordance with p16 immunohistochemistry on the corresponding cell block and/or tissue specimen. Using the cell-free supernatant fluid in this platform could provide accurate HPV results while conserving material for cytomorphologic analyses.

The Relationship Between Agar Thickness and Antimicrobial Susceptibility Testing.

Antimicrobial susceptibility testing can be done using solid or liquid-based medium. Solid-based assays are easy and inexpensive; they are limited by not being as quantitative as liquid-based assays. Agar depth can influence the accuracy of plate-based assays and it is assumed the basis of this effect is antimicrobial agent diffusion. We tested this assumption by using ETEST® to quantitate the relationship between agar depth and minimum inhibitory concentration and zone of inhibition.

Design and Evaluation of an Aerodynamic Focusing Micro-Well Aerosol Collector.

Aerosol sampling and identification is vital for the assessment and control of particulate matter pollution, airborne pathogens, allergens, and toxins and their effect on air quality, human health, and climate change. In-situ analysis of chemical and biological airborne components of aerosols on a conventional filter is challenging due to dilute samples in a large collection region. We present the design and evaluation of a micro-well (µ-well) aerosol collector for the assessment of airborne particulate matter (PM) in the 0.5-3 micron size range. The design minimizes particle collection areas allowing for in-situ optical analysis and provides an increased limit of detection for liquid-based assays due to the high concentrations of analytes in the elution/analysis volume. The design of the collector is guided by computational fluid dynamics (CFD) modeling; it combines an aerodynamic concentrator inlet that focuses the aspirated aerosol into a narrow beam and a µ-well collector that limits the particle collection area to the µ-well volume. The optimization of the collector geometry and the operational conditions result in high concentrations of collected PM in the submillimeter region inside the µ-well. Collection efficiency experiments are performed in the aerosol chamber using fluorescent polystyrene microspheres to determine the performance of the collector as a function of particle size and sampling flow rate. The collector has the maximum collection efficiency of about 75% for 1 micron particles for the flow rate of 1 slpm. Particles bigger than 1 micron have lower collection efficiencies because of particle bounce and particle loss in the aerodynamic focusing inlet. Collected samples can be eluted from the device using standard pipettes, with an elution volume of 10-20 microliters. The transparent collection substrate and the distinct collection region, independent of particle size, allows for in-situ optical analysis of the collected PM.

Discovery of potential anti-infectives against Staphylococcus aureus using a Caenorhabditis elegans infection model

BackgroundThe limited antibiotic options for effective control of methicillin-resistant Staphylococcus aureus infections has led to calls for new therapeutic approaches to combat this human pathogen. An alternative approach to control MRSA is through the use of anti-infective agents that selectively disrupt virulence-mediated pathways without affecting microbial cell viability or by modulating the host natural immune defenses to combat the pathogen.MethodsWe established a C. elegans – S. aureus liquid-based assay to screen for potential anti-infectives against S. aureus. The assay was utilized to screen 37 natural extracts and 29 synthetic compounds for the ability to extend the lifespan of infected nematodes. Disc diffusion and MIC microdilution tests were used to evaluate the anti-microbial properties of these natural extracts and synthetic compounds whilst in vivo bacterial CFU within the C. elegans gut were also enumerated.ResultsWe screened a total of 37 natural extracts and 29 synthetic compounds for anti-infective properties. The screen successfully revealed 14 natural extracts from six plants (Nypa fruticans, Swietenia macrophylla, Curcuma longa, Eurycoma longifolia, Orthosiphon stamineus and Silybum eburneum) and one marine sample (Faunus ater) that improved the survival of S. aureus-infected worms by at least 2.8-fold as well as 14 synthetic compounds that prolonged the survival of S. aureus-infected nematodes by 4-fold or greater. An anti-microbial screen of all positive hits demonstrated that 8/28 hits had no effect on S. aureus growth. Of these 8 candidates, 5 of them also protected the worms from MRSA infection. We also noted that worms exposed to N. fruticans root and O. stamineus leaf extracts showed reduced intestinal colonization by live S. aureus. This suggests that these extracts could possibly activate host immunity to eliminate the bacteria or interfere with factor/s that prevents pathogen accumulation.ConclusionWe have successfully demonstrated the utility of this liquid-based screen to identify anti-infective substances that prolong S. aureus-infected host survival without affecting bacterial cell viability.

Caenorhabditis elegans Semi-Automated Liquid Screen Reveals a Specialized Role for the Chemotaxis Gene cheB2 in Pseudomonas aeruginosa Virulence

Pseudomonas aeruginosa is an opportunistic human pathogen that causes infections in a variety of animal and plant hosts. Caenorhabditis elegans is a simple model with which one can identify bacterial virulence genes. Previous studies with C. elegans have shown that depending on the growth medium, P. aeruginosa provokes different pathologies: slow or fast killing, lethal paralysis and red death. In this study, we developed a high-throughput semi-automated liquid-based assay such that an entire genome can readily be scanned for virulence genes in a short time period. We screened a 2,200-member STM mutant library generated in a cystic fibrosis airway P. aeruginosa isolate, TBCF10839. Twelve mutants were isolated each showing at least 70% attenuation in C. elegans killing. The selected mutants had insertions in regulatory genes, such as a histidine kinase sensor of two-component systems and a member of the AraC family, or in genes involved in adherence or chemotaxis. One mutant had an insertion in a cheB gene homologue, encoding a methylesterase involved in chemotaxis (CheB2). The cheB2 mutant was tested in a murine lung infection model and found to have a highly attenuated virulence. The cheB2 gene is part of the chemotactic gene cluster II, which was shown to be required for an optimal mobility in vitro. In P. aeruginosa, the main player in chemotaxis and mobility is the chemotactic gene cluster I, including cheB1. We show that, in contrast to the cheB2 mutant, a cheB1 mutant is not attenuated for virulence in C. elegans whereas in vitro motility and chemotaxis are severely impaired. We conclude that the virulence defect of the cheB2 mutant is not linked with a global motility defect but that instead the cheB2 gene is involved in a specific chemotactic response, which takes place during infection and is required for P. aeruginosa pathogenicity.

Formation of Yersinia pseudotuberculosis biofilms on multiple surfaces on Caenorhabditis elegans

A liquid-based assay was used to evaluate the ability of Yersinia pseudotuberculosis to form a bacterial biofilm on the nematode Caenorhabditis elegans. After 3 days of incubation in the liquid assay a biofilm was clearly visible by light microscopy on both the head and vulva region of the worms. At times, the biofilm formation on the vulva appeared to prevent the laying of eggs by the adult hermaphrodite; the eggs would later hatch inside of the worm. One possible explanation for the biofilm formation observed on the vulva may be the increased motion of the cuticle surrounding the vulva when the worm is immersed in a liquid culture. This is the first report of biofilm formation on the vulva of C. elegans.

A liquid-based method for the assessment of bacterial pathogenicity using the nematode Caenorhabditis elegans.

Caenorhabditis elegans has previously been used as an alternative to mammalian models of infection with bacterial pathogens. We have developed a liquid-based assay to measure the effect of bacteria on the feeding ability of C. elegans. Using this assay we have shown that Pseudomonas aeruginosa strain PA14, Burkholderia pseudomallei and Yersinia pestis were able to inhibit feeding of C. elegans strain N2. An increase in sensitivity of the assay was achieved by using C. elegans mutant phm-2, in place of the wild-type strain. Using this assay,P. aeruginosa PA01 inhibited the feeding of C. elegans mutant phm-2. Such liquid-based feeding assays are ideally suited to the high-throughput screening of mutants of bacterial pathogens.

A liquid-based method for the assessment of bacterial pathogenicity using the nematode Caenorhabditis elegans

Caenorhabditis elegans has previously been used as an alternative to mammalian models of infection with bacterial pathogens. We have developed a liquid-based assay to measure the effect of bacteria on the feeding ability of C. elegans. Using this assay we have shown that Pseudomonas aeruginosa strain PA14, Burkholderia pseudomallei and Yersinia pestis were able to inhibit feeding of C. elegans strain N2. An increase in sensitivity of the assay was achieved by using C. elegans mutant phm-2, in place of the wild-type strain. Using this assay, P. aeruginosa PA01 inhibited the feeding of C. elegans mutant phm-2. Such liquid-based feeding assays are ideally suited to the high-throughput screening of mutants of bacterial pathogens.