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.

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Background: The rapid diagnostics of pathogens is essential to prescribe appropriate and early antibiotic therapy. The current methods for pathogen detection require the bacteria to grow in a culture medium, which is time-consuming. This increases the mortality rate and the global burden of antimicrobial resistance. Culture-free detection methods are still under development and are not used in the clinical routine. Therefore decreasing the culture time for accurate detection of infection and resistance is vital for diagnosis. Methods: In this study, we wanted to investigate easy-to-implement factors (in a minimal laboratory set-up), including inoculum size, incubation temperature, and additional supplementation (e.g., vitamin B12 and trace metals), that can significantly reduce the lag time (tlag). These factors were arranged in simple two-level factorial designs using Gram-positive (Escherichia coli and Pseudomonas aeruginosa) and Gram-negative (Staphylococcus aureus and Bacillus subtilis) bacteria, including clinical isolates with known antimicrobial resistance profiles. Blood samples spiked with a clinical isolate of E. coli CCUG17620 were also tested to see the effect of elevated incubation temperature on bacterial growth in blood cultures. Results: We observed that increased incubation temperature (42°C) along with vitamin B12 supplementation significantly reduced the tlag (10 – 115 minutes or 4% - 49%) in pure clinical isolates and blood samples spiked with E. coli CCUG17620. In the case of the blood sample, PCR results also detected bacterial DNA after only 3h of incubation and at three times the CFU/mL. Conclusions: Enrichment of bacterial culture media with growth supplements such as vitamin B12 and increased incubation temperature can be a cheap and rapid method for the early detection of pathogens. This is a proof-of-concept study restricted to a few bacterial strains and growth conditions. In the future, the effect of other growth conditions and difficult-to-culture bacteria should be explored to shorten the lag phase.

Background: Rapid diagnostics of pathogens is essential to prescribe appropriate antibiotic therapy. The current methods for pathogen detection require the bacteria to grow in a culture medium, which is time-consuming. This increases the mortality rate and global burden of antimicrobial resistance. Culture-free detection methods are still under development and are not common in the clinical routine. Therefore, decreasing the culture time for accurately detecting infection and resistance is vital for diagnosis. Methods: This study investigated easy-to-implement factors (in a minimal laboratory set-up), including inoculum size, incubation temperature, and additional supplementation (e.g., vitamin B12 and trace metals), that can significantly reduce the bacterial lag time (tlag). These factors were arranged in simple two-level factorial designs using Gram-positive cocci (Staphylococcus aureus), Gram-positive bacilli (Bacillus subtilis), and Gram-negative bacilli (Escherichia coli and Pseudomonas aeruginosa) bacteria, including clinical isolates with known antimicrobial resistance profiles. Blood samples spiked with a clinical isolate of E. coli CCUG 17620 (Culture Collection University of Gothenburg) were also tested to see the effect of elevated incubation temperature on bacterial growth in blood cultures. Results: We observed that increased incubation temperature (42°C) along with vitamin B12 supplementation significantly reduced the tlag (10 – 115 minutes or 4% - 49%) in pure clinical isolates and blood samples spiked with E. coli CCUG17620. In the case of the blood sample, PCR results also detected bacterial DNA after only 3h of incubation and at three times the CFU/mL. Conclusion: Enrichment of bacterial culture media with growth supplements such as vitamin B12 and increased incubation temperature can be a cheap and rapid method for the early detection of pathogens. This proof-of-concept study is restricted to a few bacterial strains and growth conditions. In the future, the effect of other growth conditions and difficult-to-culture bacteria should be explored to shorten the lag phase.