Optimizing Nanopore Sequencing for Rapid Detection of Microbial Species and Antimicrobial Resistance in Patients at Risk of Surgical Site Infections

Whittle, Emma; Yonkus, Jennifer A.; Jeraldo, Patricio; Alva-Ruiz, Roberto; Kendrick, Michael L.; Grys, Thomas E.; Patel, Robin; Truty, Mark J.; Chia, Nicholas

doi:10.1128/msphere.00964-21

Cited by 19 publications

(11 citation statements)

References 90 publications

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“…This study demonstrated the use of long-read metagenomic sequencing for the detection of ARGs linked directly to bacterial species without the need for genome assembly. The detection of ARGs has been reported from long-read metagenomic sequencing for one study of aspirates from human ventilator-associated pneumonia ( Chen et al, 2023 ) but more typically from samples with substantially less host DNA, such as those recovered from positive blood cultures ( Taxt et al, 2020 ; Liu et al, 2023 ), bile cultures ( Whittle et al, 2022 ), milk cultures ( Ahmadi et al, 2023 ), and urine cultures ( Zhang et al, 2022 ; Ring et al, 2023 ).…”

Section: Discussionmentioning

confidence: 99%

Bacterial enrichment prior to third-generation metagenomic sequencing improves detection of BRD pathogens and genetic determinants of antimicrobial resistance in feedlot cattle

Herman,

Lacoste,

Freeman

et al. 2024

Front. Microbiol.

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IntroductionBovine respiratory disease (BRD) is one of the most important animal health problems in the beef industry. While bacterial culture and antimicrobial susceptibility testing have been used for diagnostic testing, the common practice of examining one isolate per species does not fully reflect the bacterial population in the sample. In contrast, a recent study with metagenomic sequencing of nasal swabs from feedlot cattle is promising in terms of bacterial pathogen identification and detection of antimicrobial resistance genes (ARGs). However, the sensitivity of metagenomic sequencing was impeded by the high proportion of host biomass in the nasal swab samples.MethodsThis pilot study employed a non-selective bacterial enrichment step before nucleic acid extraction to increase the relative proportion of bacterial DNA for sequencing.ResultsNon-selective bacterial enrichment increased the proportion of bacteria relative to host sequence data, allowing increased detection of BRD pathogens compared with unenriched samples. This process also allowed for enhanced detection of ARGs with species-level resolution, including detection of ARGs for bacterial species of interest that were not targeted for culture and susceptibility testing. The long-read sequencing approach enabled ARG detection on individual bacterial reads without the need for assembly. Metagenomics following non-selective bacterial enrichment resulted in substantial agreement for four of six comparisons with culture for respiratory bacteria and substantial or better correlation with qPCR. Comparison between isolate susceptibility results and detection of ARGs was best for macrolide ARGs in Mannheimia haemolytica reads but was also substantial for sulfonamide ARGs within M. haemolytica and Pasteurella multocida reads and tetracycline ARGs in Histophilus somni reads.DiscussionBy increasing the proportion of bacterial DNA relative to host DNA through non-selective enrichment, we demonstrated a corresponding increase in the proportion of sequencing data identifying BRD-associated pathogens and ARGs in deep nasopharyngeal swabs from feedlot cattle using long-read metagenomic sequencing. This method shows promise as a detection strategy for BRD pathogens and ARGs and strikes a balance between processing time, input costs, and generation of on-target data. This approach could serve as a valuable tool to inform antimicrobial management for BRD and support antimicrobial stewardship.

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Section: Discussionmentioning

confidence: 99%

Bacterial enrichment prior to third-generation metagenomic sequencing improves detection of BRD pathogens and genetic determinants of antimicrobial resistance in feedlot cattle

Herman,

Lacoste,

Freeman

et al. 2024

Front. Microbiol.

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“…Murine stool DNA was isolated using the PowerFecal Pro DNA Kit (QiAmp) and sequenced using the 16S Barcoding Kit 1-24 (SQK-16S024) on a GridION (Nanopore). Sequences were aligned using centrifuge ( 46, 47 ). Data was analyzed using RStudio software, and all mouse and human sequence data was removed during analysis.…”

Section: Methodsmentioning

confidence: 99%

Disruption of maternal IgA by prenatal antibiotics precedes intestinalE. colicolonization and late-onset sepsis in neonates

McDonald,

Browning,

Greenfield

et al. 2023

Preprint

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Neonates, most notably prematurely born neonates, are particularly vulnerable to enteric pathogens. It is recommended that all pregnant patients receive antibiotics following Preterm Premature Rupture of Membranes (PPROM) to prolong pregnancy, yet not all antibiotic regimens have been studied for efficacy, especially when considering neonatal outcomes. Here we find, in a clinical cohort of at-risk infants, broad spectrum antibiotic treatment, including cephalosporins, increased the risk of LOS in neonates in comparison to penicillin base treatments. We followed those initial clinical findings with a preclinical model, here showing that prenatal cephalosporin antibiotics, but not penicillins, decreased mammary IgA in dams, and limited the availability of IgA within the lumen of nursing offspring. Decreased IgA correlated with an outgrowth of commensal E.coli and increased colonization and translocation of pathogen E. coli. This increased risk for mortality in a model of LOS that was associated with decreased mammary IgA. We propose maternal administration of select antibiotics could limit maternal IgA availability by disrupting mammary IgA, leading to increased risk of LOS in infants by allowing for pathogen colonization in the neonatal intestine.

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“…Such methods would also use ONT sequencers, due to their rapid turnaround time. The ONT platforms most suited for this kind of rapid diagnosis are the MinION and GridION, which have already been tested with mWGS for human clinical sputum, endotracheal aspirate, blood, urine and other sample types [14][15][16][17][18][19][20][21][22][23]. The MinION's small size (similar to a small smartphone for the Mk1B or a handheld games console for the Mk1C) and relatively low start-up costs could enable its usage in a wide variety of clinical settings.…”

Section: Introductionmentioning

confidence: 99%

Rapid metagenomic sequencing for diagnosis and antimicrobial sensitivity prediction of canine bacterial infections

et al. 2023

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Antimicrobial resistance is a major threat to human and animal health. There is an urgent need to ensure that antimicrobials are used appropriately to limit the emergence and impact of resistance. In the human and veterinary healthcare setting, traditional culture and antimicrobial sensitivity testing typically requires 48–72 h to identify appropriate antibiotics for treatment. In the meantime, broad-spectrum antimicrobials are often used, which may be ineffective or impact non-target commensal bacteria. Here, we present a rapid, culture-free, diagnostics pipeline, involving metagenomic nanopore sequencing directly from clinical urine and skin samples of dogs. We have planned this pipeline to be versatile and easily implementable in a clinical setting, with the potential for future adaptation to different sample types and animals. Using our approach, we can identify the bacterial pathogen present within 5 h, in some cases detecting species which are difficult to culture. For urine samples, we can predict antibiotic sensitivity with up to 95 % accuracy. Skin swabs usually have lower bacterial abundance and higher host DNA, confounding antibiotic sensitivity prediction; an additional host depletion step will likely be required during the processing of these, and other types of samples with high levels of host cell contamination. In summary, our pipeline represents an important step towards the design of individually tailored veterinary treatment plans on the same day as presentation, facilitating the effective use of antibiotics and promoting better antimicrobial stewardship.

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Optimizing Nanopore Sequencing for Rapid Detection of Microbial Species and Antimicrobial Resistance in Patients at Risk of Surgical Site Infections

Abstract: Surgical site infections (SSI) are a significant burden to patients and health care systems. They increase mortality rates, length of hospital stays, and associated health care costs.

Cited by 19 publications

References 90 publications

Bacterial enrichment prior to third-generation metagenomic sequencing improves detection of BRD pathogens and genetic determinants of antimicrobial resistance in feedlot cattle

Bacterial enrichment prior to third-generation metagenomic sequencing improves detection of BRD pathogens and genetic determinants of antimicrobial resistance in feedlot cattle

Disruption of maternal IgA by prenatal antibiotics precedes intestinalE. colicolonization and late-onset sepsis in neonates

Rapid metagenomic sequencing for diagnosis and antimicrobial sensitivity prediction of canine bacterial infections

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