Proof of Concept for Recombinant Cellular Controls in Quantitative Molecular Pathogen Detection

ABSTRACTIn this study, we present the concept of internal sample process controls (ISPCs) to monitor the efficiency of an analytical chain using sample preparation and quantitative PCR (qPCR). A recombinantListeria monocytogenesΔprfA(targeted deletion) strain containing a competitive artificial single-copy genomic target was applied to naturally contaminated samples to demonstrate its analytical suitability as an ISPC.

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Abstract 11119: Real-Time Nanopore Sequencing for Bacterial Pneumonia After Out-of-Hospital Cardiac Arrest, a Pilot Proof-of-Concept Study

Circulation ◽

10.1161/circ.144.suppl_2.11119 ◽

2021 ◽

Vol 144 (Suppl_2) ◽

Author(s):

Alexandra Weissman ◽

Mariam Bramah Lawani ◽

Thomas Rohan ◽

Clifton W CALLAWAY

Keyword(s):

Patient Care ◽

Pathogen Detection ◽

Bacterial Pathogen ◽

Improve Patient Care ◽

Antibiotic Administration ◽

Nanopore Sequencing ◽

Proof Of Concept ◽

Chi Square ◽

Significant Difference ◽

Improve Patient

Introduction: Pneumonia is common after OHCA but is difficult to diagnose in the first 72 hours following ROSC, this results in early untargeted antibiotic administration based on non-specific imaging and laboratory findings. Antibiotic resistance is rising, is influenced by untargeted antibiotic administration, and can increase patient morbidity and mortality as well as healthcare costs. Precision methods of bacterial pathogen detection in OHCA patients are needed to improve patient care. This proof-of-concept pilot study aimed to assess feasibility of bacterial pathogen sequencing and comparability of sequencing results to clinical culture after OHCA. Methods: Blood and bronchoalveolar lavage (BAL) were obtained from residual clinical specimens collected within 12 hours of ROSC. Bacterial DNA was extracted using the Qiagen PowerLyzer PowerSoil DNA kit, sequenced using the MinION nanopore sequencer, and analyzed with Oxford Nanopore Technologies’ EPI2ME bioinformatics software. Sequencing results were compared to culture results using McNemar’s chi-square statistic. Study-defined pneumonia was based on presence of at least two characteristics within 72 hours of ROSC: fever (temperature ≥38°C); persistent leukocytosis >15,000 or leukopenia <3,500 for 48 hours; persistent chest radiography infiltrates for 48 hours per clinical radiology read; bacterial pathogen cultured. Results: We enrolled 38 consecutive OHCA subjects: mean age 61.8 years (18.0); 16 (42%) female; 25 (66%) White, 7 (18%) Black, 6 (16%) “Other” race; 7 subjects (18%) survived and 31 (82%) died; 16 (42%) subjects had pneumonia. Sequencing results were available in 12 hours while culture results were available in 48-72 hours after collection. There was a non-significant difference in the proportion of the same pathogens identified for each method per McNemar’s chi-square: p = 0.38, difference of 0.095 (-0.095, 0.286). Conclusions: Nanopore sequencing detects pathogenic bacteria comparable to clinical microbiologic culture and in less time. This technology can produce a paradigm shift in early bacterial pathogen detection in OHCA survivors, which can improve patient care. The technology is applicable to other patient populations and for viral and fungal pathogens.

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Construction, Characterization, and Use of Two Listeria monocytogenes Site-Specific Phage Integration Vectors

Journal of Bacteriology ◽

10.1128/jb.184.15.4177-4186.2002 ◽

2002 ◽

Vol 184 (15) ◽

pp. 4177-4186 ◽

Cited By ~ 320

Author(s):

Peter Lauer ◽

Man Yin Nora Chow ◽

Martin J. Loessner ◽

Daniel A. Portnoy ◽

Richard Calendar

Keyword(s):

Listeria Monocytogenes ◽

Lethal Dose ◽

Attachment Site ◽

Donor Cell ◽

Single Copy ◽

Cell Extract ◽

Site Specific ◽

Specific Phage ◽

Serotype 1 ◽

Serotype 4B

ABSTRACT Two site-specific shuttle integration vectors were developed with two different chromosomal bacteriophage integration sites to facilitate strain construction in Listeria monocytogenes. The first vector, pPL1, utilizes the listeriophage U153 integrase and attachment site within the comK gene for chromosomal insertion. pPL1 contains a useful polylinker, can be directly conjugated from Escherichia coli into L. monocytogenes, forms stable, single-copy integrants at a frequency of ∼10−4 per donor cell, and can be used in the L. monocytogenes 1/2 and 4b serogroups. Methods for curing endogenous prophages from the comK attachment site in 10403S-derived strains were developed. pPL1 was used to introduce the hly and actA genes at comK-attBB′ in deletion strains derived from 10403S and SLCC-5764. These strains were tested for second-site complementation in hemolysin assays, plaquing assays, and cell extract motility assays. Unlike plasmid-complemented strains, integrated pPL1-complemented strains were fully virulent in the mouse 50% lethal dose assay. Additionally, the PSA phage attachment site on the L. monocytogenes chromosome was characterized, and pPL1 was modified to integrate at this site. The listeriophage PSA integrates in the 3′ end of an arginine tRNA gene. There are 17 bp of DNA identity between the bacterial and phage attachment sites. The PSA prophage DNA sequence reconstitutes a complete tRNAArg gene. The modified vector, pPL2, was integration proficient at the same frequency as pPL1 in common laboratory serotype 1/2 strains as well as serotype 4b strains.

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EIS-Based Biosensors in Foodborne Pathogen Detection with a Special Focus on Listeria monocytogenes

Methods in Molecular Biology - Foodborne Bacterial Pathogens ◽

10.1007/978-1-4939-9000-9_7 ◽

2018 ◽

pp. 87-101 ◽

Cited By ~ 1

Author(s):

Palmiro Poltronieri ◽

Elisabetta Primiceri ◽

Rajeswaran Radhakrishnan

Keyword(s):

Listeria Monocytogenes ◽

Pathogen Detection ◽

Foodborne Pathogen ◽

Special Focus ◽

Foodborne Pathogen Detection

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Rapid sample preparation with Lyse-It® for Listeria monocytogenes and Vibrio cholerae

PLoS ONE ◽

10.1371/journal.pone.0201070 ◽

2018 ◽

Vol 13 (7) ◽

pp. e0201070 ◽

Cited By ~ 3

Author(s):

Tonya M. Santaus ◽

Shan Li ◽

Paula Ladd ◽

Amanda Harvey ◽

Shannon Cole ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Sample Preparation ◽

Vibrio Cholerae

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The Platelet Fraction Is a Novel Reservoir to Detect Lyme Borrelia in Blood

Biology ◽

10.3390/biology9110366 ◽

2020 ◽

Vol 9 (11) ◽

pp. 366

Author(s):

Victoria P. Sanderson ◽

Iain L. Mainprize ◽

Lisette Verzijlenberg ◽

Cezar M. Khursigara ◽

Melanie K. B. Wills

Keyword(s):

Pathogen Detection ◽

Bacterial Culture ◽

Direct Detection ◽

Laboratory Culture ◽

Detection Methods ◽

Clinical Samples ◽

Blood Collection ◽

Processing Conditions ◽

Proof Of Concept ◽

Clinical Detection

Serological diagnosis of Lyme disease suffers from considerable limitations. Yet, the technique cannot currently be replaced by direct detection methods, such as bacterial culture or molecular analysis, due to their inadequate sensitivity. The low bacterial burden in vasculature and lack of consensus around blood-based isolation of the causative pathogen, Borrelia burgdorferi, are central to this challenge. We therefore addressed methodological optimization of Borrelia recovery from blood, first by analyzing existing protocols, and then by using experimentally infected human blood to identify the processing conditions and fractions that increase Borrelia yield. In this proof-of-concept study, we now report two opportunities to improve recovery and detection of Borrelia from clinical samples. To enhance pathogen viability and cultivability during whole blood collection, citrate anticoagulant is superior to more commonly used EDTA. Despite the widespread reliance on serum and plasma as analytes, we found that the platelet fraction of blood concentrates Borrelia, providing an enriched resource for direct pathogen detection by microscopy, laboratory culture, Western blot, and PCR. The potential for platelets to serve as a reservoir for Borrelia and its diagnostic targets may transform direct clinical detection of this pathogen.

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A long-amplicon quantitative PCR assay with propidium monoazide to enumerate viable Listeria monocytogenes after heat and desiccation treatments

Food Microbiology ◽

10.1016/j.fm.2019.103310 ◽

2020 ◽

Vol 86 ◽

pp. 103310 ◽

Cited By ~ 2

Author(s):

Martin Laage Kragh ◽

Mikala Thykier ◽

Lisbeth Truelstrup Hansen

Keyword(s):

Listeria Monocytogenes ◽

Quantitative Pcr ◽

Propidium Monoazide ◽

Pcr Assay ◽

Quantitative Pcr Assay

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Physical and antibiotic stresses require activation of the RsbU phosphatase to induce the general stress response in Listeria monocytogenes

Microbiology ◽

10.1099/mic.0.041202-0 ◽

2010 ◽

Vol 156 (9) ◽

pp. 2660-2669 ◽

Cited By ~ 23

Author(s):

Ji-Hyun Shin ◽

Margaret S. Brody ◽

Chester W. Price

Keyword(s):

Listeria Monocytogenes ◽

Protein Interactions ◽

Single Copy ◽

Nutritional Stress ◽

Serine Phosphorylation ◽

Intestinal Lumen ◽

Food Borne ◽

Multiple Challenges ◽

Stress Signals ◽

Food Borne Infections

Among pathogenic strains of Listeria monocytogenes, the σ B transcription factor has a pivotal role in the outcome of food-borne infections. This factor is activated by diverse stresses to provide general protection against multiple challenges, including those encountered during gastrointestinal passage. It also acts with the PrfA regulator to control virulence genes needed for entry into intestinal lumen cells. Environmental and nutritional signals modulate σ B activity via a network that operates by the partner switching mechanism, in which protein interactions are controlled by serine phosphorylation. This network is well characterized in the related bacterium Bacillus subtilis. A key difference in Listeria is the presence of only one input phosphatase, RsbU, instead of the two found in B. subtilis. Here, we aim to determine whether this sole phosphatase is required to convey physical, antibiotic and nutritional stress signals, or if additional pathways might exist. To that end, we constructed L. monocytogenes 10403S strains bearing single-copy, σ B-dependent opuCA–lacZ reporter fusions to determine the effects of an rsbU deletion under physiological conditions. All stresses tested, including acid, antibiotic, cold, ethanol, heat, osmotic and nutritional challenge, required RsbU to activate σ B. This was of particular significance for cold stress activation, which occurs via a phosphatase-independent mechanism in B. subtilis. We also assayed the effects of the D80N substitution in the upstream RsbT regulator that activates RsbU. The mutant had a phenotype consistent with low and uninducible phosphatase activity, but nonetheless responded to nutritional stress. We infer that RsbU activity but not its induction is required for nutritional signalling, which would enter the network downstream from RsbU.

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