A culture-, amplification-independent, and rapid method for identification of pathogens and antibiotic resistance profile in bovine mastitis milk

Abstract

IntroductionRapid and accurate diagnosis of causative pathogens in mastitis would minimize the imprudent use of antibiotics and, therefore, reduce the spread of antimicrobial resistance. Whole genome sequencing offers a unique opportunity to study the microbial community and antimicrobial resistance (AMR) in mastitis. However, the complexity of milk samples and the presence of a high amount of host DNA in milk from infected udders often make this very challenging.MethodsHere, we tested 24 bovine milk samples (18 mastitis and six non-mastitis) using four different commercial kits (Qiagens’ DNeasy® PowerFood® Microbial, Norgens’ Milk Bacterial DNA Isolation, and Molzyms’ MolYsis™ Plus and Complete5) in combination with filtration, low-speed centrifugation, nuclease, and 10% bile extract of male bovine (Ox bile). Isolated DNA was quantified, checked for the presence/absence of host and pathogen using PCR and sequenced using MinION nanopore sequencing. Bioinformatics analysis was performed for taxonomic classification and antimicrobial resistance gene detection.ResultsThe results showed that kits designed explicitly for bacterial DNA isolation from food and dairy matrices could not deplete/minimize host DNA. Following using MolYsis™ Complete 5 + 10% Ox bile + micrococcal nuclease combination, on average, 17% and 66.5% of reads were classified as bovine and Staphylococcus aureus reads, respectively. This combination also effectively enriched other mastitis pathogens, including Escherichia coli and Streptococcus dysgalactiae. Furthermore, using this approach, we identified important AMR genes such as Tet (A), Tet (38), fosB-Saur, and blaZ. We showed that even 40 min of the MinION run was enough for bacterial identification and detecting the first AMR gene.ConclusionWe implemented an effective method (sensitivity of 100% and specificity of 92.3%) for host DNA removal and bacterial DNA enrichment (both gram-negative and positive) directly from bovine mastitis milk. To the best of our knowledge, this is the first culture- and amplification-independent study using nanopore-based metagenomic sequencing for real-time detection of the pathogen (within 5 hours) and the AMR profile (within 5–9 hours), in mastitis milk samples. These results provide a promising and potential future on-farm adaptable approach for better clinical management of mastitis.

Real-time Taxonomic Characterization of Long-read Mixed-species Sequencing Samples in Sorted Motif Distance Space:Voyager

“…We collected datasets from all available ONT sequencing platforms, flowcells, and relevant sample types to establish expected performance in clinical, animal, and limited environmental community samples, including blood infection data obtained using MinION 14 (ENA PRJEB60525 ) and Flongle 15 (ENA PRJEB49072 ), urine infection data 16 using MinION (ENA PRJEB73819 ) milk mastitis data 2 using MinION (ENA PRJEB53532 ), and environmental mock community data 17 using GridION and PromethION (ENA PRJEB29504). Sample files containing low amounts of data (< 100,000 reads) were excluded.…”

“…The corresponding p-value is computed using the chi 2 cumulative distribution function chisq_cdf, and the likelihood ratio LR as follows: p-value ∼ 1 − chisq_cdf( LR ; DDOF ), where where 20 is an empirical scaling factor, and …”

“…In a clinical context, if patient samples obtained from body fluids, e.g., blood sputum, urine, etc., can be quickly analyzed to characterize pathogens, this will lead to faster and informed treatment, reduced patient time in the hospital, and improved patient outcome 1 . Likewise, fast and user-friendly characterization of infections in the veterinary and livestock sector can relieve the need for preventative antibiotics and prevent the spread of infection to the rest of the herd 2 . Advances in real-time sequencing technologies have the potential to enable such rapid diagnosis in clinical microbiology.…”

Highly sensitive quantitative phase microscopy and deep learning aided with whole genome sequencing for rapid detection of infection and antimicrobial resistance

“…This is due to physicians not being able to diagnose patients accurately in real-time, leading to either prescription of antibiotics for viral infections or a prescription of broad-spectrum drugs that should ideally be kept in reserve. We recently established an effective method for direct sequencing for pathogen identification and antibiotic resistance detection ( Ahmadi et al, 2023 ). The current state-of-the-art diagnostic of infection is mainly based on biochemical analysis of the culture of the clinical material.…”

In Vitro Anti-bacterial Effect of Ox-bile Against Some Important Gram-positive and Gram-negative Bacteria

“…On the other hand, some investigations reported different information and mentioned that E. coli is considered to be bile-resistant or Listeria monocytogenes strains are inherently bile-resistant (2,34). These differences in bile tolerance can be related to the concentration of bile, exposure to various pH and temperatures, or the type and structure of bile (35,36). Other factors include alteration of the cell membrane structure and composition, especially different lipopolysaccharides of the outer membrane, as well as changes in membrane electric charge and hydrophobicity (6).…”