Cas3-Derived Target DNA Degradation Fragments Fuel Primed CRISPR Adaptation.

Prokaryotes use a mechanism called priming to update their CRISPR immunological memory to rapidly counter revisiting, mutated viruses, and plasmids. Here we have determined how new spacers are produced and selected for integration into the CRISPR array during priming. We show that Cas3 couples CRISPR interference to adaptation by producing DNA breakdown products that fuel the spacer integration process in a two-step, PAM-associated manner. The helicase-nuclease Cas3 pre-processes target DNA into fragments of about 30-100 nt enriched for thymine-stretches in their 3' ends. The Cas1-2 complex further processes these fragments and integrates them sequence-specifically into CRISPR repeats by coupling of a 3' cytosine of the fragment. Our results highlight that the selection of PAM-compliant spacers during priming is enhanced by the combined sequence specificities of Cas3 and the Cas1-2 complex, leading to an increased propensity of integrating functional CTT-containing spacers.

Insight in lag phase of Listeria monocytogenes during enrichment through proteomic and transcriptomic responses.

The dynamics of the enrichment-based detection procedure of the foodborne pathogen Listeria monocytogenes from food still remains poorly understood. This enrichment is crucial in the reliable detection of this pathogen and more insight into the recovery mechanism during this step is important to advance our understanding of lag phase behaviour during enrichment. In this study we combined transcriptomic and proteomic analyses to better understand the physiological processes within the lag phase of L. monocytogenes during enrichment. Upon transfer of BHI-cultured stationary phase L. monocytogenes cells to half-Fraser enrichment broth (HFB), motility-associated genes and proteins were downregulated, while expression of metal uptake transporters, resuscitation-promoting factors that stimulate growth from dormancy, antibiotic efflux pumps and oxidative stress proteins were upregulated. Next to this, when cells with a heat stress history were cultured in enrichment broth, proteins necessary for recovery were upregulated with functions in DNA-damage repair, protein refolding, cell-wall repair, and zinc transport. Proteomic results pointed to possible factors that support shortening the lag duration, including the addition of 10 µM zinc and the addition of spent HFB containing presumed concentrations of resuscitation-promoting factors. However, these interventions did not lead to biologically relevant reduction of lag phase. Also, when cells were enriched in spent HFB, final cell concentrations were similar to enrichments in fresh HFB, indicating that the enrichment broth seems not to lack critical substrates. Concludingly, this study gives insight into the proteomic changes in the lag phase during enrichment and shows that supplementation of HFB is not the best strategy to optimize the current enrichment method.

Variability in lag duration of Listeria monocytogenes strains in half Fraser enrichment broth after stress affects the detection efficacy using the ISO 11290-1 method.

A collection of 23 Listeria monocytogenes strains of clinical and food origin was tested for their ability to recover and grow out in half Fraser enrichment broth following the ISO 11290-1:2017 protocol. Recovery of sub-lethally heat-injured cells in half Fraser broth was compared to reference cells with no stress pre-treatment. The enrichments were followed over time by plate counts and the growth parameters were estimated with the 3-phase model which described the data best. The reference cells without stress pre-treatment showed a short lag duration, which ranged from 1.4 to 2.7 h. However, significant variation in the ability to recover after 60 °C heat stress was observed among the tested strains and resulted in a lag duration from 4.7 to 15.8 h. A subset of strains was also exposed to low-temperature acid stress, and the lag duration showed to be also stress dependent. Scenario analyses and Monte Carlo simulations were carried out using the growth parameters obtained in the enrichments. This demonstrated that when starting with one cell, the detection threshold for efficient transfer of at least one cell to the secondary enrichment step, i.e. 2 log10 CFU/ml, was not reached by 11 of 23 strains tested (48%) after exposure to 60 °C heat stress. Increasing the incubation time from 24 to 26 h and the transfer volume from 0.1 to 1.0 ml can increase the average probability to transfer at least one cell to the secondary enrichment step from 79.9% to 99.0%. When optimizing enrichment procedures, it is crucial to take strain variability into account as this can have a significant impact on the detection efficacy.

Heterogeneity in single-cell outgrowth of Listeria monocytogenes in half Fraser enrichment broth is affected by strain variability and physiological state.

The behaviour of pathogens at the single-cell level can be highly variable and can thus affect the detection efficacy of enrichment-based detection methods. The outgrowth of single cells of three Listeria monocytogenes strains was monitored after fluorescence-activated single-cell sorting in non-selective brain heart infusion (BHI) broth and selective half Fraser enrichment broth (HFB) to quantify outgrowth heterogeneity and its effect on the detection probability. Single-cell heterogeneity was higher in HFB compared to non-selective BHI and heterogeneity increased further when cells were heat-stressed. The increase in heterogeneity was also strain-dependent because the fast-recovering strain Scott A showed less outgrowth heterogeneity than the slower-recovering strains EGDe and H7962. Modelling of the outgrowth kinetics during the primary enrichment demonstrated that starting at low cell concentrations could fail detection of L. monocytogenes at least partly due to cell heterogeneity. This highlights that it is important to take single-cell heterogeneity into account when optimizing enrichment formulations and procedures when L. monocytogenes contamination levels are low.