Bacteriophages and food fermentations

A broad number of food products, commodity chemicals, and biotechnology products are manufactured industrially by large-scale bacterial fermentation of various organic substrates. Because enormous amounts of bacteria are being cultivated each day in large fermentation vats, the risk that bacteriophage contamination rapidly brings fermentations to a halt and cause economical setbacks is a serious threat in these industries. This chapter describes the relationship between bacteriophages and their bacterial hosts in the context of the food fermentation industry. Sources of phage contamination, measures to control their propagation and dissemination, and biotechnological defense strategies developed to restrain phages are discussed. The primary focus will be given to the dairy fermentation industry because it has openly acknowledged the problem of phage and has been working with academia and starter culture companies to develop defense strategies and systems to curtail the propagation and evolution of phages for decades. Other industries will be discussed where appropriate to highlight their similarities and specificities

Microbiological and molecular impacts of AbiK on the lytic cycle of Lactococcus lactis phages of the 936 and P335 species

The lactococcal abortive infection mechanism AbiK was previously shown to be highly effective against the small isometric-headed bacteriophage ul36 of the P335 species, as evidenced by an efficiency of plaquing (e.o.p.) of 10(-6), a 14-fold reduction in the burst size and an efficiency at which centres of infection form (e.c.o.i.) of 0.5%. No phage DNA was detected in the infected AbiK+ cells [Emond, E., Holler, B. J., Boucher, I., Vandenbergh, P. A., Vedamuthu, E. R., Kondo, J. K. & Moineau, S. (1997). Appl Environ Microbiol 63, 1274-1283]. Here, the effects of AbiK are compared on the small isometric-headed phages p2 and P008 (936 species) and on the phage P335 (P335 species). The microbiological impacts of AbiK on p2 were relatively similar to those reported for ul36, with an e.o.p. of 10(6), an 11-fold reduction in the burst size and an e.c.o.i. of 5%. Contrary to phage ul36, replication of phage p2 DNA was observed in the AbiK+ cells. Only immature forms (concatemeric and circular DNA) of phage p2 DNA were found, indicating that the presence of AbiK prevented phage DNA maturation. These distinct molecular consequences of AbiK were also observed for phages P335 and P008, two phages that propagate on the same host. To the knowledge of the authors, this is the first time that different phage responses towards an Abi system have been reported

DNA sequence analysis of three Lactococcus lactis plasmids encoding phage resistance mechanisms

The three Lactococcus lactis plasmids pSRQ700, pSRQ800, and pSRQ900 encode the previously described anti-phage resistance mechanisms LlaDCHI, AbiK, and AbiQ, respectively. Since these plasmids are likely to be introduced into industrial Lactococcus lactis strains used to manufacture commercial fermented dairy products, their complete DNA sequences were determined and analyzed. The plasmids pSRQ700 (7784 bp), pSRQ800 (7858 bp), and pSRQ900 (10,836 bp) showed a similar genetic organization including a common lactococcal theta-type replicon. A second replication module showing features of the pMV158 family of rolling circle replicons was also found on pSRQ700. The theta replication regions of the three plasmids were associated with two additional coding regions, one of which encodes for HsdS, the specificity subunit of the type I restriction/modification system. When introduced into L. lactis IL1403, the HsdS of pSRQ800 and pSRQ900 conferred a weak resistance against phage P008 (936 species). These results indicated that both HsdS subunits can complement the chromosomally encoded type I restriction/modification system in IL1403. The genes involved in the phage resistance systems LlaDCHI, AbiK, and AbiQ were found in close proximity to and downstream of the replication modules. In pSRQ800 and pSRQ900, transfer origins and putative tyrosine recombinases were found upstream of the theta replicons. Genes encoding recombination proteins were also found on pSRQ700. Finally, open reading frames associated with bacteriocin production were found on pSRQ900, but no anti-lactococcal activity was detected. Based on our current knowledge, these three plasmids are safe and suitable for food-grade applications

AbiQ, an abortive infection mechanism from Lactococcus lactis

Lactococcus lactis W-37 is highly resistant to phage infection. The cryptic plasmids from this strain were coelectroporated, along with the shuttle vector pSA3, into the plasmid-free host L. lactis LM0230. In addition to pSA3, erythromycin- and phage-resistant isolates carried pSRQ900, an 11-kb plasmid from L. lactis W-37. This plasmid made the host bacteria highly resistant (efficiency of plaquing <10(-8)) to c2- and 936-like phages. pSRQ900 did not confer any resistance to phages of the P335 species. Adsorption, cell survival, and endonucleolytic activity assays showed that pSRQ900 encodes an abortive infection mechanism. The phage resistance mechanism is limited to a 2.2-kb EcoRV/BclI fragment. Sequence analysis of this fragment revealed a complete open reading frame (abiQ), which encodes a putative protein of 183 amino acids. A frameshift mutation within abiQ completely abolished the resistant phenotype. The predicted peptide has a high content of positively charged residues (pI = 10.5) and is, in all likelihood, a cytosolic protein. AbiQ has no homology to known or deduced proteins in the databases. DNA replication assays showed that phage c21 (c2-like) and phage p2 (936-like) can still replicate in cells harboring AbiQ. However, phage DNA accumulated in its concatenated form in the infected AbiQ+ cells, whereas the AbiQ- cells contained processed (mature) phage DNA in addition to the concatenated form. The production of the major capsid protein of phage c21 was not hindered in the cells harboring AbiQ

Phenotypic and genetic characterization of the bacteriophage abortive infection mechanism abiK from Lactococcus lactis

The natural plasmid pSRQ800 isolated from Lactococcus lactis subsp. lactis W1 conferred strong phage resistance against small isometric phages of the 936 and P335 species when introduced into phage-sensitive L. lactis strains. It had very limited effect on prolate phages of the c2 species. The phage resistance mechanism encoded on pSRQ800 is a temperature-sensitive abortive infection system (Abi). Plasmid pSRQ800 was mapped, and the Abi genetic determinant was localized on a 4.5-kb EcoRI fragment. Cloning and sequencing of the 4.5-kb fragment allowed the identification of two large open reading frames. Deletion mutants showed that only orf1 was needed to produce the Abi phenotype. orf1 (renamed abiK) coded for a predicted protein of 599 amino acids (AbiK) with an estimated molecular size of 71.4 kDa and a pI of 7.98. DNA and protein sequence alignment programs found no significant homology with databases. However, a database query based on amino acid composition suggested that AbiK might be in the same protein family as AbiA. No phage DNA replication nor phage structural protein production was detected in infected AbiK1 L. lactis cells. This system is believed to act at or prior to phage DNA replication. When cloned into a high-copy vector, AbiK efficiency increased 100-fold. AbiK provides another powerful tool that can be useful in controlling phages during lactococcal fermentations

Evaluation of the feasibility and impacts of in situ simulation in emergency medicine—a mixed-method study protocol

Introduction In situ simulation (ISS) consists of performing a simulation in the everyday working environment with the usual team members. The feasibility of ISS in emergency medicine is an important research question, because ISS offers the possibility for repetitive, regular simulation training consistent with specific local needs. However, ISS also raises the issue of safety, since it might negatively impact the care of other patients in the emergency department (ED). Our hypothesis is that ISS in an academic high-volume ED is feasible, safe and associated with benefits for both staff and patients.Methods A mixed-method, including a qualitative method for the assessment of feasibility and acceptability and a quantitative method for the assessment of patients’ safety and participants’ psychosocial risks, will be used in this study.Two distinct phases are planned in the ED of the CHU de Québec-Université Laval (Hôpital de l’Enfant-Jésus) between March 2021 and October 2021. Phase 1: an ISS programme will be implemented with selected ED professionals to assess its acceptability and safety and prove the validity of our educational concept. The number of cancelled sessions and the reasons for cancellation will be collected to establish feasibility criteria. Semistructured interviews will evaluate the acceptability of the intervention. We will compare unannounced and announced ISS. Phase 2: the impact of the ISS programme will be measured with validated questionnaires for the assessment of psychosocial risks, self-confidence and perceived stress among nonselected ED professionals, with comparison between those exposed to ISS and those that were not.Ethics and dissemination The CHU de Québec-Université Laval Research ethics board has approved this protocol (#2020–5000). Results will be presented to key professionals from our institution to improve patient safety. We also aim to publish our results in peer-reviewed journals and will submit abstracts to international conferences to disseminate our findings

Evolution of in-hospital complications.

Evolution of in-hospital complications.</p

Annual trauma admissions over the years.

Annual trauma admissions over the years.</p