Application of cryoprotectants for cryopreservation of pathogenic microorganisms (Yersinia pestis, Nocardia farcinia) – #JournalClub no.057

Application of cryoprotectants for cryopreservation of pathogenic microorganisms (Yersinia pestis, Nocardia farcinia)

The importance of pathogenic microorganisms

Pathogenic microorganisms are of great value as biological resources for biosecurity, human health, environmental protection, and renewable energy. Proper preservation of these microbial resources is of paramount importance and requires methods that maintain purity, viability, and integrity while preventing continued degradation.

Investigation of cryopreservation of N. farcinica and Y. pestis

The objective of this study is to investigate cryopreservation technology for two species of bacteria: Nocardia farcinica (Gram-positive) and Yersinia pestis vaccine strains (Gram-negative). This research examines the effects of cryoprotectants (CPAs), freezing temperature, and freeze-thaw cycles on these bacteria during the cryopreservation process.

Effect of cryoprotectancts (CPAs) on bacterial survival

The results show that the addition of CPAs such as glycerol, propylene glycol, sucrose, glucose, L-carnitine, L-proline, and skim milk significantly increased the survival rates of inoculum strains of N. farcinica and Y. pestis. However, it should be noted that high concentrations of CPAs can produce biochemical toxicity in these two pathogens.

Mitigating biochemical toxicity: the role of composite CPAs

The use of composite CPAs not only reduces toxicity but also improves sample survival rates during cryopreservation. The optimal composite CPA for N. farcinica consists of 0.292 M sucrose, 0.62 M L-carnitine, and 2.82 M glycerol, while for Y. pestis it consists of 0.62 M L-carnitine, 8.46 M glycerol, and 0.292 M sucrose.

Optimal conditions: Temperature and freeze-thaw cycles

The results show that the quality of the strains stored at -80°C and -196°C is better. In the freeze-thaw cycles, the two pathogens show a different degree of reduction, with Y. pestis experiencing a greater decrease in survival compared to N. farcinica.

Optimization: uniform distribution of bacteria and the role of nucleation sites

The uniform distribution of bacteria in CPAs facilitates the formation of consistent nucleation sites in the solution system, thus favoring the cryopreservation of strains, as found in experiments using the Differential Scanning Calorimeter.

(Long-term) storage of dried blood spots / DBS for multi-omics analyses -#JournalClub no.055

Multi-omics analysis from archival neonatal dried blood spots: limitations and opportunities

Investigate the feasibility of multi-omics analyses of DBS samples.

Newborn screening programmes (NBS) are implemented in various countries worldwide and involve the analysis of millions of dry blood cards / dried blood spots (DBS) samples per year. The main objective of NBS is to detect adverse health outcomes in newborns at an early stage. In addition to this, DBS samples can also serve as an important resource for population-based research in child and adolescent health. Despite their potential, the feasibility of using archived DBS samples for emerging targeted and untargeted multi-omics analyses has not been explored in the literature. Therefore, this review aims to assess recent developments to identify the opportunities and challenges of applying omics analyses to NBS cards in a research setting.

Search for publications that use DBS for omics analysis

To locate studies using DBS for genomic, proteomic and metabolomic assays, the authors conducted a search of the Medline, Embase and PubMed databases and initially identified 800 records, which were narrowed down to 23 for this review. These studies included one combined genomic/metabolomic, four genomic, three epigenomic, four proteomic and 11 metabolomic studies.

Advantages of using DBS samples for omics analyses

The advantages of using DBS samples include the stability of many biomolecules once plotted on a card, allowing for easy transport and storage. However, for specific measures, factors such as short-term vs. long-term stability, storage temperature and different analyte compositions need to be considered.

Assessment of the stability of biomolecules in DBS samples during sample travel

The sample journey from collection to processing can vary depending on the clinical setting and patient circumstances, but often occurs at room temperature (RT). It is therefore critical to assess the stability of specific biomolecules in DBS samples at room temperature over time. Some studies have shown that certain biomolecules such as carbohydrates, nucleotides and vitamins in DBS cards are sensitive to storage at RT and 37 °C, while others have found no significant differences in metabolite concentration at RT for most clinically relevant compounds.

Recommendations for storage of DBS samples for reliable multi-omics analyses

Current recommendations suggest storing DBS samples at -20 °C or -80 °C for long-term storage of at least two years. However, analyses performed within 28 days of sample collection at room temperature (RT) are also possible, albeit with a gradual decline in metabolite concentration. Different analytes may degrade at different rates at different temperatures, so this should be considered when selecting targets for biobank access.

Challenges in using DBS samples for multi-omics analyses

Despite these challenges, DBS sampling offers several advantages for population-based research, including simplicity, minimal volume requirements and economical transport and storage. The use of omics technology to assess the molecular profiles of neonatal blood samples is very achievable and valuable to the scientific community. Although the technology is likely to evolve, there is already sufficient evidence to validate the current use of high-throughput omics assays, followed by targeted analyses to identify core biomarkers of neonatal disease.

Verwendung von NBS-Proben für große Kohortenstudien mit kontrollierter Lagerung von DBS-Proben.

Use of NBS samples for large cohort studies with controlled storage of DBS samples.

Filter paper / dried blood spots used in the publications

Whatman 903 Protein-Saver-Karten, Dänische DBS-Karten, Guthrie-Karten, Dänische Neugeborenen-DBS-Karten, Whatman FTA-Karte, Ahlstrom Grad 226 Filterpapier, Centocard, HemaSpot-HF Blutentnahmegerät, Karte ImmunoHealth, Glasfaserstreifen ImmunoHealth, schleicher & schuell 2.992, schleicher & schuell 903, Ahlströmer 226

Sample storage and preparation of whole milk samples for determination of microbiota – #JournalClub no.054

Improved assessments of bulk milk micro-biota composition via sample preparation and DNA extraction methods

DNA sequencing is the most common method for studying microbes in humans, animals, food and the environment. The workflow for sample preparation and DNA sequencing consists of numerous steps that are prone to bias and contamination problems. Numerous bacterial species are present in the diverse microbiota of high-quality whole milk. Within the study by Xue et al. presented here, the following bacterial strains were used:

  • Bacillus subtilis
  • Clostridium tyrobutyricum
  • Corynebacterium bovis
  • Enterococcus faecalis
  • Escherichia coli
  • Lactococcus lactis
  • Pseudomonas fluorexcens
  • Staphylococcus aureus
  • Staphylococcus agalactiae

Bakterielle Cell-Mock-Community (BCMC)

Nine strains in BCMC1 were grown to near stationary phase and counted directly under the microscope. The BCMC2 pools just produced were either used immediately for DNA extraction or stored at -80°C. To obtain the bacteria, BCMC1 and BCMC2 were inoculated with ultra-high temperature pasteurised milk and centrifuged at 13,000 for five minutes at 4°C before DNA extraction. They were stored for seven days at -20°C in PBS or PBS containing 25% v/v glycerol.

Methods for cell lysis and DNA purification

It is possible that Escherichia coli and Pseudomonas are more sensitive to DNA shearing and lysis than Gram-positive bacteria. B5 (tapping at 4 m/sec for 10 seconds) and V3 (vortexing at 1800 rpm for 30 seconds) were two of the milder methods tried to break the cells. Three MagMAX DNA purification kits Total, Core and Ultra2 were compared and the Total kit gave the most accurate results.

Impact of the BCMC preparation method on the estimation of community composition

For each BCMC, the 16S rRNA V4 region was sequenced and analysed to determine the proportions that were „observed“. Escherichia and Pseudomonas were significantly lower than expected, and Bacillus and Corynebacterium were significantly higher than expected for BCMC1.

Effects of milk sample volume and cell count on bacterial composition estimates

Larger milk samples resulted in lower alpha diversity and fewer unexpected taxa assignments or bacteria that did not belong to the mock community. Most of these unexpected bacteria were Micrococcus or Tepidimonas.

Effects of the storage method on the detection of sham communities

Regardless of whether the cells were frozen in PBS or in water with 25 % v/v glycerol at -20 °C, the bacterial proportions of BCMC did not change.

Identification and tracking of bacteria

With the help of 16S rRNA gene sequence surveys, it is possible to identify and track bacterial populations. However, bias can occur at almost every stage of the process, from sampling to data analysis and interpretation. The most accurate method for automatic identification of bacteria consists of at least 10 grams of milk, mild cell lysis, proteinase K treatment and magnetic bead-based DNA purification.

Storage conditions of the collected cells and cell lysis method

The mock community remained unchanged despite different freeze-thaw cycles and storage conditions. It can be assumed that other upstream sample preparation steps have a greater influence than this one previously discovered. Although not tested directly, it has been previously demonstrated that liquid milk samples can be frozen directly when rapid cold transfer between collection sites is not possible.

Application of cell lysis and DNA extraction methods to assess the microbiota of raw and pasteurised milk

Although cell lysis techniques can bias the results of 16S rRNA gene amplicon DNA sequencing, they are unlikely to interfere with comparative microbiota studies of different milk samples. The variation within a sample caused by methodological adjustments was significantly less than the variation between samples. Microbial DNA extraction and analysis may require different methodological approaches for different types of cow’s milk samples.

Summary

Examining the microbiota of dairy products requires careful sample preparation and analysis to minimise bias and contamination problems. The use of a bacterial cell mock community (BCMC) can help to verify and improve the accuracy of results. When selecting cell lysis and DNA purification methods, milder techniques should be preferred to avoid bias. Storage conditions of the collected cells should also be carefully controlled to avoid changes in bacterial composition. The use of 16S rRNA gene sequencing can help identify and track bacterial populations, but possible biases must be considered.