Longitudinal Study of Lactococcus Phages in a Canadian Cheese Factory.

The presence of virulent phages is closely monitored during cheese manufacturing, as these bacterial viruses can significantly slow down the milk fermentation process and lead to low-quality cheeses. From 2001 to 2020, whey samples from cheddar cheese production in a Canadian factory were monitored for the presence of virulent phages capable of infecting proprietary strains of Lactococcus cremoris and Lactococcus lactis used in starter cultures. Phages were successfully isolated from 932 whey samples using standard plaque assays and several industrial Lactococcus strains as hosts. A multiplex PCR assay assigned 97% of these phage isolates to the Skunavirus genus, 2% to the P335 group, and 1% to the Ceduovirus genus. DNA restriction profiles and a multilocus sequence typing (MLST) scheme distinguished at least 241 unique lactococcal phages from these isolates. While most phages were isolated only once, 93 of them (out of 241, 39%) were isolated multiple times. Phage GL7 was isolated 132 times from 2006 to 2020, demonstrating that phages can persist in a cheese factory for long periods of time. Phylogenetic analysis of MLST sequences showed that phages could be clustered based on their bacterial hosts rather than their year of isolation. Host range analysis showed that Skunavirus phages exhibited a very narrow host range, whereas some Ceduovirus and P335 phages had a broader host range. Overall, the host range information was useful in improving the starter culture rotation by identifying phage-unrelated strains and helped mitigating the risk of fermentation failure due to virulent phages. IMPORTANCE Although lactococcal phages have been observed in cheese production settings for almost a century, few longitudinal studies have been performed. This 20-year study describes the close monitoring of dairy lactococcal phages in a cheddar cheese factory. Routine monitoring was conducted by factory staff, and when whey samples were found to inhibit industrial starter cultures under laboratory conditions, they were sent to an academic research laboratory for phage isolation and characterization. This led to a collection of at least 241 unique lactococcal phages, which were characterized through PCR typing and MLST profiling. Phages of the Skunavirus genus were by far the most dominant. Most phages lysed a small subset of the Lactococcus strains. These findings guided the industrial partner in adapting the starter culture schedule by using phage-unrelated strains in starter cultures and removing some strains from the starter rotation. This phage control strategy could be adapted for other large-scale bacterial fermentation processes.

Effect of probiotic bacteria on porcine rotavirus OSU infection of porcine intestinal epithelial IPEC-J2 cells.

Rotavirus infections in nursing or post-weaning piglets are known to cause diarrhea, which can lead to commercial losses. Probiotic supplementation is used as a prophylactic or therapeutic approach to dealing with microbial infections in humans and animals. To evaluate the effect of probiotic bacteria on porcine rotavirus infections, non-transformed porcine intestinal epithelial IPEC-J2 cells were used as an in vitro model, and three different procedures were tested. When cells were exposed to seven probiotics at concentrations of 105, 106, or 107 CFU/mL for 16 h and removed before rotavirus challenge, infection reduction rates determined by flow cytometry were as follows: 15% (106) and 18% (105) for Bifidobacterium longum R0175, 15% (107) and 16% (106) for B. animalis lactis A026, and 15% (105) for Lactobacillus plantarum 299V. When cells were exposed to three selected probiotic strains for 1 h at higher concentrations, that is, 108 and 5 × 108 CFU/mL, before infection with rotavirus, no significant reduction was observed. When the probiotic bacteria were incubated with the virus before cell infection, a significant 14% decrease in the infection rate was observed for B. longum R0175. The results obtained using a cell-probiotics-virus platform combined with flow cytometry analysis suggest that probiotic bacteria can have a protective effect on IPEC-J2 cells before infection and can also prevent rotavirus infection of the cells.

Investigating Lactococcus lactis MG1363 Response to Phage p2 Infection at the Proteome Level.

Phages are viruses that specifically infect and eventually kill their bacterial hosts. Bacterial fermentation and biotechnology industries see them as enemies, however, they are also investigated as antibacterial agents for the treatment or prevention of bacterial infections in various sectors. They also play key ecological roles in all ecosystems. Despite decades of research some aspects of phage biology are still poorly understood. In this study, we used label-free quantitative proteomics to reveal the proteotypes of Lactococcus lactis MG1363 during infection by the virulent phage p2, a model for studying the biology of phages infecting Gram-positive bacteria. Our approach resulted in the high-confidence detection and quantification of 59% of the theoretical bacterial proteome, including 226 bacterial proteins detected only during phage infection and 6 proteins unique to uninfected bacteria. We also identified many bacterial proteins of differing abundance during the infection. Using this high-throughput proteomic datasets, we selected specific bacterial genes for inactivation using CRISPR-Cas9 to investigate their involvement in phage replication. One knockout mutant lacking gene llmg_0219 showed resistance to phage p2 because of a deficiency in phage adsorption. Furthermore, we detected and quantified 78% of the theoretical phage proteome and identified many proteins of phage p2 that had not been previously detected. Among others, we uncovered a conserved small phage protein (pORFN1) coded by an unannotated gene. We also applied a targeted approach to achieve greater sensitivity and identify undetected phage proteins that were expected to be present. This allowed us to follow the fate of pORF46, a small phage protein of low abundance. In summary, this work offers a unique view of the virulent phages' takeover of bacterial cells and provides novel information on phage-host interactions.

Microencapsulation of a Staphylococcus phage for concentration and long-term storage.

In an effort to reduce food safety risks, virulent phages are investigated as antibacterial agents for the control of foodborne pathogens. The aim of this study was to evaluate microencapsulation (ME) as a tool to concentrate and store staphylococcal bacteriophages. As a proof of concept, phage Team1 belonging to the Myoviridae family was microencapsulated in alginate gel particles of 0.5 mm (micro-beads) and 2 mm (macro-beads) of diameter. Gel contraction occurred during the hardening period in the CaCl2 solution, and the diameters of the initial alginate droplets shrunk by 16% (micro-beads) and 44% (micro-beads). As compared to the phage counts in the alginate solution, this contraction resulted in the increase of the phage titers, per g of alginate gel, by factors of 2 (micro-beads) and 6 (micro-beads). The encapsulation yield was highest in the macro-beads. Although phage Team1 was successfully frozen in beads, ME did not improve phage stability to freeze-drying. The addition of glycerol protected the microencapsulated phages during freezing but had no effect on free phage suspensions. Finally, ME improved storage stability at 4 °C but had no impact on freezing or drying over three months of storage.

The impact of Brevibacterium aurantiacum virulent phages on the production of smear surface-ripened cheeses.

Phages are ubiquitous and are particularly abundant in environments where their bacterial hosts thrive, such as those in the cheese industry. Although it is well documented that phages infect lactic acid bacteria, their impact has been notably overlooked on cheese ripening strains, such as Brevibacterium aurantiacum. Here, we aimed to study the impact of B. aurantiacum phages on the production of smear-ripened cheeses. We used model cheeses in industrial settings to monitor the development of the color of the cheese rind as well as of its microbial composition in presence or absence of virulent B. aurantiacum phages. Our results showed that the presence of B. aurantiacum phages significantly slowed down the development of the orange rind color in the model cheeses. In the final days of cheese ripening, phages were also detected in the control curds. By analyzing a hypervariable region of B. aurantiacum phage genomes, we detected phages with tandem repeat patterns that were different from those used in the phage-inoculated cheeses. Our results highlight the risks of using a phage-sensitive strain in smear-ripened cheese production. This is the first study to report on the impact of B. aurantiacum phages on smear-ripened cheeses.

Effect of the homogenization technique on the enumeration of psychrotrophic bacteria in food absorbent pads.

Four methods were tested for enumerating bacteria present in the absorbent pads (AP) used in packaging chicken and other meats. Viable counts were ascertained at day 0 and day 7 (d0 and d7, respectively). Sampling bacterial cells from AP were carried out using a countertop blender, Stomacher, sonication, and blender in combination to sonication. The release of bacterial cells by breaking down the AP with the blender resulted in the highest CFU counts. At d0, a bacterial recovery rate of 94% was obtained with the blender, while the recovery rates using Stomacher or sonication alone were 58% and 73%, respectively. At d7, the Stomacher treatment also gave the lowest colony forming unit (CFU) values in the AP incubated at 7 °C. Sonication of the AP prior to homogenization with the blender did not increase CFU counts. Results suggested that breaking down the AP with a blender gives higher CFU levels than the Stomacher, which is the most commonly used technique for this purpose.

A Lactococcal Phage Protein Promotes Viral Propagation and Alters the Host Proteomic Response During Infection.

The lactococcal virulent phage p2 is a model for studying the Skunavirus genus, the most prevalent group of phages causing milk fermentation failures in cheese factories worldwide. This siphophage infects Lactococcus lactis MG1363, a model strain used to study Gram-positive lactic acid bacteria. The structural proteins of phage p2 have been thoroughly described, while most of its non-structural proteins remain uncharacterized. Here, we developed an integrative approach, making use of structural biology, genomics, physiology, and proteomics to provide insights into the function of ORF47, the most conserved non-structural protein of unknown function among the Skunavirus genus. This small phage protein, which is composed of three α-helices, was found to have a major impact on the bacterial proteome during phage infection and to significantly reduce the emergence of bacteriophage-insensitive mutants.

Targeted Genome Editing of Virulent Phages Using CRISPR-Cas9.

This protocol describes a straightforward method to generate specific mutations in the genome of strictly lytic phages. Briefly, a targeting CRISPR-Cas9 system and a repair template suited for homologous recombination are provided inside a bacterial host, here the Gram-positive model Lactococcus lactis MG1363. The CRISPR-Cas9 system is programmed to cleave a specific region present on the genome of the invading phage, but absent from the recombination template. The system either triggers the recombination event or exerts the selective pressure required to isolate recombinant phages. With this methodology, we generated multiple gene knockouts, a point mutation and an insertion in the genome of the virulent lactococcal phage p2. Considering the broad host range of the plasmids used in this protocol, the latter can be extrapolated to other phage-host pairs.

The CRISPR-Cas app goes viral.

If biology laboratories were smartphones, CRISPR-Cas would be the leading app. Nowadays, technology users rely on apps to communicate, get directions, entertain, and more. Likewise, many life scientists now rely on CRISPR-Cas systems to study the interactions between microbes and their viruses, to track strains as well as to modify and modulate genomes. Considering their high level of polymorphism, CRISPR arrays can increase the resolution of a microbial typing scheme. As dynamic systems, they allow the identification and the tracking of specific sequences, which is highly valuable for epidemiological studies. As a defense mechanism, they offer an opportunity to generate virus-resistant strains or even to construct strains refractory to the acquisition of specific genes. And last but not least, as customizable and transferable tools, CRISPR-Cas systems are particularly promising to fight multi-drug resistant bacteria through the engineering of phages.

Genome Engineering of Virulent Lactococcal Phages Using CRISPR-Cas9.

Phages are biological entities found in every ecosystem. Although much has been learned about them in past decades, significant knowledge gaps remain. Manipulating virulent phage genomes is challenging. To date, no efficient gene-editing tools exist for engineering virulent lactococcal phages. Lactococcus lactis is a bacterium extensively used as a starter culture in various milk fermentation processes, and its phage sensitivity poses a constant risk to the cheese industry. The lactococcal phage p2 is one of the best-studied models for these virulent phages. Despite its importance, almost half of its genes have no functional assignment. CRISPR-Cas9 genome editing technology, which is derived from a natural prokaryotic defense mechanism, offers new strategies for phage research. Here, the well-known Streptococcus pyogenes CRISPR-Cas9 was used in a heterologous host to modify the genome of a strictly lytic phage. Implementation of our adapted CRISPR-Cas9 tool in the prototype phage-sensitive host L. lactis MG1363 allowed us to modify the genome of phage p2. A simple, reproducible technique to generate precise mutations that allow the study of lytic phage genes and their encoded proteins in vivo is described.

An anti-CRISPR from a virulent streptococcal phage inhibits Streptococcus pyogenes Cas9.

The CRISPR-Cas system owes its utility as a genome-editing tool to its origin as a prokaryotic immune system. The first demonstration of its activity against bacterial viruses (phages) is also the first record of phages evading that immunity 1 . This evasion can be due to point mutations 1 , large-scale deletions 2 , DNA modifications 3 , or phage-encoded proteins that interfere with the CRISPR-Cas system, known as anti-CRISPRs (Acrs) 4 . The latter are of biotechnological interest, as Acrs can serve as off switches for CRISPR-based genome editing 5 . Every Acr characterized to date originated from temperate phages, genomic islands, or prophages 4-8 , and shared properties with the first Acr discovered. Here, with a phage-oriented approach, we have identified an unrelated Acr in a virulent phage of Streptococcus thermophilus. In challenging a S. thermophilus strain CRISPR-immunized against a set of virulent phages, we found one that evaded the CRISPR-encoded immunity >40,000× more often than the others. Through systematic cloning of its genes, we identified an Acr solely responsible for the abolished immunity. We extended our findings by demonstrating activity in another S. thermophilus strain, against unrelated phages, and in another bacterial genus immunized using the heterologous SpCas9 system favoured for genome editing. This Acr completely abolishes SpCas9-mediated immunity in our assays.

Detecting natural adaptation of the Streptococcus thermophilus CRISPR-Cas systems in research and classroom settings.

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems have been adapted into a powerful genome-editing tool. The basis for the flexibility of the tool lies in the adaptive nature of CRISPR-Cas as a bacterial immune system. Here, we describe a protocol to experimentally demonstrate the adaptive nature of this bacterial immune system by challenging the model organism for the study of CRISPR adaptation, Streptococcus thermophilus, with phages in order to detect natural CRISPR immunization. A bacterial culture is challenged with lytic phages, the surviving cells are screened by PCR for expansion of their CRISPR array and the newly acquired specificities are mapped to the genome of the phage. Furthermore, we offer three variants of the assay to (i) promote adaptation by challenging the system using defective viruses, (ii) challenge the system using plasmids to generate plasmid-resistant strains and (iii) bias the system to obtain natural immunity against a specifically targeted DNA sequence. The core protocol and its variants serve as a means to explore CRISPR adaptation, discover new CRISPR-Cas systems and generate bacterial strains that are resistant to phages or refractory to undesired genes or plasmids. In addition, the core protocol has served in teaching laboratories at the undergraduate level, demonstrating both its robust nature and educational value. Carrying out the core protocol takes 4 h of hands-on time over 7 d. Unlike sequence-based methods for detecting natural CRISPR adaptation, this phage-challenge-based approach results in the isolation of CRISPR-immune bacteria for downstream characterization and use.

Applications of CRISPR-Cas in its natural habitat.

Key components of CRISPR-Cas systems have been adapted into a powerful genome-editing tool that has caught the headlines and the attention of the public. Canonically, a customized RNA serves to guide an endonuclease (e.g. Cas9) to its DNA target, resulting in precise genomic lesions that can be repaired in a personalized fashion by cellular machinery. Here, we turn to the microbes that are the source of this system to explore many of its other notable applications. These include mining the CRISPR 'memory' arrays for functional genomic data, generation of customized virus-resistant or plasmid-refractory bacterial cells, editing of previously intractable viral genomes, and exploiting the unique properties of a catalytically inactive Cas9, dCas9, to serve as a highly customizable anti-nucleic acid 'antibody'.

Antimicrobial effect of natural preservatives in a cooked and acidified chicken meat model.

The inhibitory effect of Microgard 100, Microgard 300, nisin, Alta 2002, Perlac 1902, sodium lactate and essential oil of mustard on microorganisms experimentally inoculated was screened in an acidified chicken meat model (pH = 5.0) and stored for 2 weeks at a none restrictive growth temperature of 22 degrees C. All antimicrobials tested were used at the highest concentration recommended by their manufacturer. Sausage batter made with mechanically deboned chicken was inoculated with a mixed culture of Escherichia coli ATCC 25922, Brochothrix thermosphacta CRDAV452, and a protective culture Lactobacillus alimentarius BJ33 (FloraCan L-2). A final cell concentration of 3-4 log CFU g (-1) was targeted after cooking at a core temperature of 55 degrees C for each microorganism in order to assess cell count variation effectively. Composition, water activity (a(w)), pH and redox potential of the sausage model was also evaluated. The E. coli population decreased steadily during storage and was close or below detection level (< 1 log CFU g (-1)) for all treatments, including the control, after 14 days. Sodium lactate was most effective against B. thermosphacta; population was 4 log lower than the control after 14 days of storage. When essential oil of mustard was used, aerobic mesophilic bacteria and lactic acid bacteria were significantly lower than the control after 2 days of storage (P < or = 0.05). The other antimicrobial agents tested had no significant effect on the aerobic mesophilic bacteria, E. coli, B. thermosphacta and lactic acid bacteria counts, when compared to the control.

Evidence for Escherichia coli O157:H7 attachment to water distribution pipe materials by scanning electron microscopy.

Scanning electron microscopy observation was used to investigate the adhesion of Escherichia coli O157:H7 on water distribution pipe surfaces such as copper and polyethylene plastic at different contact times and storage temperatures. Our results indicated that E. coli cells could easily attach to both surface types after exposures as short as 1 or 4 h at ambient (20 degrees C) and refrigeration temperatures (4 degrees C). Also, we found that copper surfaces have a higher number of attached E. coli cells than plastic surfaces. The number of cells attached to each type of material depended on the nature of the water distribution pipe surfaces and the length of contact time. In addition, the surface energy value of each surface estimated by contact angle measurements using water, alpha-bromonaphthalene, and dimethyl sulfoxide as wetting agents showed that both copper (41.2 megajoules [MJ] m(-2)) and plastic (45.8 MJ x m(-2)) have a low energy surface. In no cases could evidence of extracellular material be observed on surfaces with either exposure condition.

Attachment of Arcobacter butzleri, a new waterborne pathogen, to water distribution pipe surfaces.

The capability of Arcobacter butzleri to attach to various water distribution pipe surfaces, such as stainless steel, copper, and plastic, was evaluated using scanning electron microscopy. Our results indicated that Arcobacter cells could easily attach to all surface types and the number of attached cells depended on the length of exposure and temperatures (4 and 20 degrees C). Extracellular fibrils were also observed on the stainless steel surface, especially after 72 h of contact times at both refrigeration and ambient temperatures. In addition, the surface energy value of each material was estimated by contact angle measurements using water, alpha-bromonaphthalene, and dimethylsulfoxide. The surface energy of A. butzleri was 58.6 mJ x m(-2) and the surface energy values of the three surfaces studied showed that plastic had a low energy surface (26.1 mJ x m(-2)) as did copper (45.8 mJ x m(-2)) and stainless steel (65.7 mJ x m(-2)).

The PVH as a site of CB1-mediated stimulation of thermogenesis by MC4R agonism in male rats.

The present study was designed to investigate the involvement of the cannabinoid receptor 1 (CB1) in the stimulating effects of the melanocortin-4 receptor (MC4R) agonism on whole-body and brown adipose tissue (BAT) thermogenesis. In a first series of experiments, whole-body and BAT thermogenesis were investigated in rats infused in the third ventricle of the brain with the MC4R agonist melanotan II (MTII) and the CB1 agonist δ9-tetrahydrocannabinol (δ(9)-THC) or the CB1 antagonist AM251. Whole-body thermogenesis was measured by indirect calorimetry and BAT thermogenesis assessed from interscapular BAT (iBAT) temperature. δ(9)-THC blunted the effects of MTII on energy expenditure and iBAT temperature, whereas AM251 tended to potentiate the MTII effects. δ(9)-THC also blocked the stimulating effect of MTII on (14)C-bromopalmitate and (3)H-deoxyglucose uptakes in iBAT. Additionally, δ(9)-THC attenuated the stimulating effect of MTII on the expression of peroxisome proliferator-activated receptor-γ coactivator 1-α (Pgc1α), type II iodothyronine deiodinase (Dio2), carnitine palmitoyltransferase 1B (Cpt1b), and uncoupling protein 1 (Ucp1). In a second series of experiments, we addressed the involvement of the paraventricular hypothalamic nucleus (PVH) in the CB1-mediated effects of MTII on iBAT thermogenesis, which were assessed following the infusion of MTII in the PVH and δ(9)-THC or AM251 in the fourth ventricle of the brain. We demonstrated the ability of δ(9)-THC to blunt MTII-induced iBAT temperature elevation. δ(9)-THC also blocked the PVH effect of MTII on (14)C-bromopalmitate uptake as well as on Pgc1α and Dio2 expression in iBAT. Altogether the results of this study demonstrate the involvement of the PVH in the CB1-mediated stimulating effects of the MC4R agonist MTII on whole-body and BAT thermogenesis.