Lactococcus Lactis Phage Research Articles

Synergistic antibacterial activity of Lactococcus lactis and Xylella phage MATE 2 for an effective biocontrol strategy against black rot disease in broccoli.

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc), is considered the most destructive disease affecting cruciferous vegetables, resulting in significant losses worldwide. The need for biocontrol agents against Xcc that can reduce reliance on chemical pesticides, enhance sustainability, and ensure crops and environmental health is crucial. Combining phages with other antibacterial agents (i.e., antibiotics and bacteriocins) to treat bacterial infections is gaining increased attention due to the frequently observed synergistic effects. This study introduces for the first time the combination of a lytic phage, i.e., Xylella phage MATE 2 (MATE 2) with nisin-producing Lactococcus lactis subsp. lactis (L. lactis) bacterium as an eco-friendly, cost-effective, and practical strategy for controlling Xcc in cruciferous vegetables. The antibacterial efficacy of MATE 2 and L. lactis, individually and in combination, against Xcc was investigated through a series of in vitro assays and in planta experiments conducted on broccoli plants. The time-killing curves results showed that under conditions of reduced Xcc population concentration (103 CFU/mL), MATE 2 at 108 PFU/mL exerted a persistent inhibitory effect on Xcc growth for 7 days. The Spot assays and v-qPCR analysis showed that both L. lactis and its bacteriocin nisin have significant antibacterial potential to contrast Xcc. Furthermore, combined application of MATE 2 and L. lactis in broccoli plants by foliar spraying generated significant synergistic efficacy in preventing Xcc infections, achieving a 71% reduction in symptoms, compared with 64 and 38% for single applications, respectively. In this study, the positive synergistic effect of the combined application of phage and beneficial bacteria in preventing black rot disease underscores this eco-friendly and cost-effective approach as a promising control measure against plant bacterial diseases.

From lab to technical scale: Ultraviolet C treatment for the reduction of Lactococcus lactis bacteriophage P008 in whey

UV–C irradiation is a powerful non-thermal principle to inactivate lactococcal bacteriophages, which pose a high risk for fermentation failures in dairies. However, milk products limit ray penetration with their turbidity and absorption properties. In reactor design, attention must be paid to uniform and effective dose distribution, which can be achieved by thin fluid layers and/or turbulence. Preliminary tests performed in a lab-scale batch mode irradiation chamber showed filtered whey to be the most suitable application site. In a subsequent larger-scale feasibility study, two continuous flow-through reactors (lab-scale and technical-scale) were investigated. A bacteriophage reduction of 5 log units required a volume-related UV–C dose of 10.6 J mL−1 in the irradiation chamber, but only 1.8 J mL−1 in the continuous lab-scale reactor and 2.4 J mL−1 in the continuous technical-scale reactor. These data show that upscaling is feasible and that an effective bacteriophage inactivation with gentle product treatment is allowed.

An extraordinary dairy phage and its properties: Occurrence, growth, inactivation, survival

Lactococcus lactis phages can be very heat resistant and phage P680 survive pasteurisation. In this work, phage growth, thermal and non-thermal as well as chemical methods were presented together and re-evaluated in the elimination of P680. A fermentation failure within 2 h can occur under the presence of phage P680, which survived heating at 95 °C for 5 min. Up to 4 log units of reduction could be achieved using membrane filtration (cut-off 100 nm). In case of ultraviolet-C treatment, doses up to 0.05 J cm−2 and 4 J cm−2 were required for phage P680 to achieve 6 log units reduction in water and 8 log units reduction in filtered whey. Phage P680 was mostly resistant at pH levels of 3 and 10 (<0.5 log reduction), however, a significant drop of the phage levels was observed at pH values of 2 and 12 (∼6 log units of reduction).

Orthogonal processing strategies to create “phage-free” whey – Membrane filtration followed by thermal or ultraviolet C treatment for the reduction of Lactococcus lactis bacteriophages

Lactococcal bacteriophages can cause fermentation failures in the cheese production (e.g., Cheddar), especially when the by-product whey (≤109 plaque-forming units mL−1 of bacteriophages) is recycled and added to new batches. Using membrane filtration, the bacteriophage titre in whey can be reduced by 4 log units without protein denaturation. To achieve higher bacteriophage reduction (≥9 log units), we attempted an orthogonal process strategy (combination of different technologies supporting each other). Three representative bacteriophages were inactivated in membrane filtered whey by (i) UV-C irradiation and (ii) heat treatment to achieve additional 5 log units reduction. Thermal treatment was only partially successful, as the varying degrees of heat resistance required, in the worst-case, temperature-time combinations above pasteurisation conditions. In contrast, UV-C treatment required doses of maximally 2.25 J cm−2 for successful inactivation, thus representing a promising avenue for the establishment of a novel process for the production of “phage-free” whey.

Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28.

Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here, we describe the biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccφ28. Size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), and negative stain transmission electron microscopy (TEM) indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other double-stranded DNA (dsDNA) phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high-resolution structural studies and rigorous biophysical/biochemical analysis.

Bacteriophages of Lactococcus lactis ssp. and Streptococcus thermоphilus Isolated in Dairy Processing Plants of Ukraine

Bacteriophages of Lactococcus lactis ssp. and Streptococcus thermоphilus Isolated in Dairy Processing Plants of Ukraine

Detection of Lactococcus lactis phage P680, a heat resistant member of the 936 group of phages, by loop-mediated isothermal amplification (LAMP)

The loop-mediated isothermal amplification- (LAMP-) method was adapted for fast and easy detection of phage P680, a heat resistant member of the 936 group of Lactococcus lactis phages. A set of LAMP-primers targeting the major tail protein gene (mtp) were custom designed. The LAMP method for phage P680 detection was tested in water, in native (pH 6.55) and in acid whey (pH 4.60). DNA extraction from whey was optimised by inclusion of Chelex-100™ and a reliable detection of less than 10³ plaque-forming units mL−1 in the complex whey matrix could thus be achieved. By adding SYBR® Safe to the reaction mixture at the end of incubation, a simple visual evaluation of the result as either positive or negative was made possible. Besides the low detection limit, the advantages of LAMP include: (i) an easy to use protocol, (ii) inexpensive equipment, and (iii) a comparably short detection time.

Extreme thermal stability of Lactococcus lactis bacteriophages: Evaluation of phage inactivation in a pilot-plant pasteurizer

Thermal inactivation of highly heat-stable Lactococcus lactis phages P680 and P1532 of the 936 group and P635 of the c2 group was investigated in a pilot-plant pasteurizer after suspension of the phages in raw milk (107 pfu/ml). The heat-sensitive reference phage P008 was tested as a control. The titers of the phages P008 and P635 decreased ≥99.99% after heating for 32 s at 75 °C and 80 °C, respectively. To achieve a similar inactivation of ≥99.99%, phages P680 and P1532 had to be treated for 39 s at temperatures of 95 °C and 97.5 °C, respectively. D- and z-values for thermal inactivation were calculated for each of the phages. When comparing thermal inactivation data of phages in the pilot-plant pasteurizer obtained in this study with literature data on phage heat inactivation in stainless-steel test tubes in a water bath, the pilot-plant pasteurizer was notably more efficient.

Biodiversity of bacteriophages infecting Lactococcus lactis starter cultures

In the current study, we characterized 137 Lactococcus lactis bacteriophages that had been isolated between 1997 and 2012 from whey samples obtained from industrial facilities located in 16 countries. Multiplex PCR grouping of these 137 phage isolates revealed that the majority (61.31%) belonged to the 936 group, with the remainder belonging to the P335 and c2 groups (23.36 and 15.33%, respectively). Restriction profile analysis of phage genomic DNA indicated a high degree of genetic diversity within this phage collection. Furthermore, based on a host-range survey of the phage collection using 113 dairy starter strains, we showed that the c2-group isolates exhibited a broader host range than isolates of the 936 and P335 groups.

Characterization of prophages of Lactococcus garvieae

This report describes the morphological characterization and genome analysis of an induced prophage (PLg-TB25) from a dairy strain of Lactococcus garvieae. The phage belongs to the Siphoviridae family and its morphology is typical of other lactococcal phages. A general analysis of its genome did not reveal similarities with other lactococcal phage genomes, confirming its novelty. However, similarities were found between genes of its morphogenesis cluster and genes of Gram-positive bacteria, suggesting that this phage genome resulted from recombination events that took place in a heterogeneous microbial environment. An in silico search for other prophages in 16 L. garvieae genomes available in public databases, uncovered eight seemingly complete prophages in strains isolated from dairy and fish niches. Genome analyses of these prophages revealed three novel L. garvieae phages. The remaining prophages had homology to phages of Lactococcus lactis (P335 group) suggesting a close relationship between these lactococcal species. The similarity in GC content of L. garvieae prophages to the genomes of L. lactis phages further supports the hypothesis that these phages likely originated from the same ancestor.

Reduction of Lactococcus lactis phage contamination in whey by means of membrane filtration: Impact of phage morphology and of bacterial host cells functioning as “phage fishing tool”

For dairy bacteriophages, the impact of phage morphology and whey components on phage retention efficiencies in filtration processes is not known. Therefore, the influence of (i) five differently shaped Lactococcus lactis phages as well as (ii) differently treated bacterial host cells added to native whey were analysed via cross-flow membrane filtration experiments with polyethersulfone membranes. Filtration efficiency with respect to phage retention and whey protein permeation was investigated. Compared with untreated native whey, phage retention was significantly higher (up to 4.8 log units) in the experiments using whey that was spiked with mechanically disrupted lactococcal host cells. However, the retention of phages commonly found in the dairy environment (i.e., members of the 936, c2, and P335 phage groups) did not correlate with the phage morphology. Results of this model study clearly demonstrate that phage titres can be reduced in whey (containing a sufficient protein concentration) by flat sheet membrane filtration processes.

Identification and Analysis of a Novel Group of Bacteriophages Infecting the Lactic Acid Bacterium Streptococcus thermophilus.

We present the complete genome sequences of four members of a novel group of phages infecting Streptococcus thermophilus, designated here as the 987 group. Members of this phage group appear to have resulted from genetic exchange events, as evidenced by their "hybrid" genomic architecture, exhibiting DNA sequence relatedness to the morphogenesis modules of certain P335 group Lactococcus lactis phages and to the replication modules of S. thermophilus phages. All four identified members of the 987 phage group were shown to elicit adsorption affinity to both their cognate S. thermophilus hosts and a particular L. lactis starter strain. The receptor binding protein of one of these phages (as a representative of this novel group) was defined using an adsorption inhibition assay. The emergence of a novel phage group infecting S. thermophilus highlights the continuous need for phage monitoring and development of new phage control measures. Phage predation of S. thermophilus is an important issue for the dairy industry, where viral contamination can lead to fermentation inefficiency or complete fermentation failure. Genome information and phage-host interaction studies of S. thermophilus phages, particularly those emerging in the marketplace, are an important part of limiting the detrimental impact of these viruses in the dairy environment.

Properties and genomic analysis of Lactococcus garvieae lysogenic bacteriophage PLgT-1, a new member of Siphoviridae, with homology to Lactococcus lactis phages

The lysogenic phage PLgT-1 is highly prevalent in Lactococcus garvieae, which is a serious bacterial pathogen in marine fish. Therefore, information regarding this phage is one of the key factors to predict the evolution of this bacterium. However, many properties of this phage, its complete genome sequence, and its relationship with other viral communities has not been investigated to date. Here, we demonstrated that the phage PLgT-1 was not only induced by an induction agent (Mitomycin C), but could be released frequently during cell division in a nutrient-rich environment or in natural seawater. Integration of PLgT-1 into non-lysogenic bacteria via transduction changed the genotype, resulting in the diversification of L. garvieae. The complete DNA sequence of PLgT-1 was also determined. This phage has a dsDNA genome of 40,273bp with 66 open reading frames (ORFs). Of these, the biological functions of 24 ORFs could be predicted but those of 42 ORFs are unknown. Thus, PLgT-1 is a novel phage with several novel proteins encoded in its genome. The strict MegaBLAST search program for the PLgT-1 genome revealed that this phage had no similarities with other previously investigated phages specific to L. garvieae (WP-2 and GE1). Notably, PLgT-1 was relatively homologous with several phages of Lactococcus lactis and 17 of the 24 predicted proteins encoded in PLgT-1 were homologous with the deduced proteins of various phages from these dairy bacteria. Comparative genome analysis revealed that the L. garvieae phage PLgT-1 was most closely related to the L. lactis phage TP712. However, they differed from each other in genome size and gene arrangement. The results obtained in this study suggest that the lysogenic phage PLgT-1 is a new member of the family Siphoviridae and has been involved in horizontal gene exchange with microbial communities, especially with L. lactis and its phages.

Microbial community dynamics in thermophilic undefined milk starter cultures

Model undefined thermophilic starter cultures were produced from raw milk of nine pasta-filata cheesemaking plants using a selective procedure based on pasteurization and incubation at high temperature with the objective of studying the microbial community dynamics and the variability in performances under repeated (7–13) reproduction cycles with backslopping. The traditional culture-dependent approach, based on random isolation and molecular characterization of isolates was coupled to the determination of pH and the evaluation of the ability to produce acid and fermentation metabolites. Moreover, a culture-independent approach based on amplicon-targeted next-generation sequencing was employed. The microbial diversity was evaluated by 16S rRNA gene sequencing (V1–V3 regions), while the microdiversity of Streptococcus thermophilus populations was explored by using novel approach based on sequencing of partial amplicons of the phosphoserine phosphatase gene (serB). In addition, the occurrence of bacteriophages was evaluated by qPCR and by multiplex PCR. Although it was relatively easy to select for a community dominated by thermophilic lactic acid bacteria (LAB) within a single reproduction cycle, final pH, LAB populations and acid production activity fluctuated over reproduction cycles. Both culture-dependent and -independent methods showed that the cultures were dominated by either S. thermophilus or Lactobacillus delbrueckii subsp. lactis or by both species. Nevertheless, subdominant mesophilic species, including lactococci and spoilage organisms, persisted at low levels. A limited number of serB sequence types (ST) were present in S. thermophilus populations. L. delbrueckii and Lactococcus lactis bacteriophages were below the detection limit of the method used and high titres of cos type S. thermophilus bacteriophages were detected in only two cases. In one case a high titre of bacteriophages was concurrent with a S. thermophilus biotype shift in the culture.This study largely confirms previous data on the composition of undefined thermophilic starters used for the production of traditional cheeses in Italy but it is the first one to systematically address the dynamics of the cultures under a repeated reproduction regime with backslopping.

Lack of the host membrane protease FtsH hinders release of the Lactococcus lactis bacteriophage TP712

The temperate bacteriophage TP712 was unable to plaque on Lactococcus lactis ΔftsH lacking the membrane protease FtsH and complementation in trans restored the WT phenotype. Absence of ftsH did not hinder phage adsorption, phage DNA delivery or activation of the lytic cycle. Thin sections revealed that TP712 virions appeared to be correctly assembled inside the ΔftsH host, but were not released. These virions were infective, demonstrating that a functional host FtsH is required by TP712 to proceed effectively with lysis of the host.

Tale of the unseen phage

Lactococcal phages are among the best characterized phages of Gram-positive bacteria and as such represent excellent models to promote our understanding of phage-host interactions. In recent years, microarray technology has been employed to define the transcriptional profile of phages during infection, while simultaneously uncovering the response of the host to the infection event. The responses to infection by a lytic and a temperate phage of Lactococcus lactis were analyzed, combined with an assessment of the host response to lysogenisation with the temperate phage, Tuc2009. Here, we discuss the lessons learned from such transcriptional profiling studies, focusing on studies relating to phages of Lactococcus lactis, and how this knowledge may be applied to future studies.

Molecular characterization of a new lytic bacteriophage isolated from cheese whey

In this study, we isolated and characterized a lytic Lactococcus lactis bacteriophage from the sera of a failed fermentation. The phage was isolated and cultured in L. lactis subsp. cremoris in M17 medium. The isolated bacteriophage was characterized by multiplex PCR, pulsed-field electrophoresis, DNA restriction digestion, analysis of the N-terminal sequence of the phage major structural protein, transmission electron microscopy and sequencing and analysis of a conserved fragment of its genome. Analysis of the viral genome indicates that its genome is composed of a DNA strand of approximately 48 kb in length, and PCR and microscopy confirmed that IL-P1 belongs to the group of 936-type phages in the family Siphoviridae, which is the most abundant type of lactococcal virus in dairy products worldwide. To our knowledge, this is the first report of a virus within this family that has a presumptive genome larger than 40 kb.

Molecular characterisation and comparison of Lactococcal phages of Pakistan and Germany

Bacteriophages of Lactococcus lactis ssp. diacetylactis isolated in Pakistan and Germany were compared in respect of protein profile and restriction analysis. SDS‐PAGE profile analysis shows similarities amongst the local isolates that have different protein profile when compared with phages from other regions. No marked difference was observed in their DNA homology. The data suggested that the bacteriophages isolated from Pakistan are different from the bacteriophages that have already been characterised from the other parts of the world.

Influence of the suspension media on the thermal treatment of mesophilic lactococcal bacteriophages

The thermal resistances of the Lactococcus lactis phages P008 (a heat-sensitive wide-spread phage) and P680 (a heat-resistant phage) suspended in milk and in solutions supplemented with milk components were studied to elucidate the protective role of milk on phage inactivation. For both phages a decrease of inactivation was observed in casein solution. Furthermore, the inactivation kinetics of the phages in whey, in whey cream (3.5%, 20%, 30% fat) and in whey protein concentrate (0.7%, 5%, 10% protein) were tested. The inactivation experiments in whey cream and in whey protein concentrate having different concentrations of fat and protein revealed that fat had no influence on the inactivation, while, in contrast, the presence of protein had a protective effect.

Thermal inactivation of the heat-resistant Lactococcus lactis bacteriophage P680 in modern cheese processing

The thermal inactivation of the highly thermo-resistant test phage (P680), was investigated in whey, whey protein concentrate (WPC) and whey cream. After a heat treatment at 90 °C for 15 min, only a 6-log reduction was obtained and the phage was still detectable in each medium. Kinetic parameters for the inactivation of the phage were calculated for temperatures ranging from 70 to 90 °C using a non-linear model. With the help of the parameters obtained, the lines of equal effects showing a 9-log inactivation of the phage were calculated. High temperature short time pasteurization was not sufficient for 9-log inactivation of phage P680 in skim milk, whey, WPC or whey cream. Temperature and time combinations ranging from 100 °C for 20 min to 140 °C for 2 s are necessary for a 9-log inactivation of P680.