Bacteriophage vB_EcoM_112 (formerly e11/2) is an Escherichia coli phage with specificity for the O157:H7 serotype. The vB_EcoM_112 genome sequence shares high degrees of similarity with the phage T4 genome sequence.
Listeria monocytogenes is a virulent food-borne pathogen most often associated with the consumption of “ready-to-eat” foods. The organism is a common contaminant of food processing plants where it may persist for extended periods of time. A commonly used approach for the control of Listeria monocytogenes in the processing environment is the application of biocides such as quaternary ammonium compounds. In this study, the transcriptomic response of a persistent strain of L. monocytogenes (strain 6179) on exposure to a sub-lethal concentration of the quaternary ammonium compound benzethonium chloride (BZT) was assessed. Using RNA-Seq, gene expression levels were quantified by sequencing the transcriptome of L. monocytogenes 6179 in the presence (4 ppm) and absence of BZT, and mapping each data set to the sequenced genome of strain 6179. Hundreds of differentially expressed genes were identified, and subsequent analysis suggested that many biological processes such as peptidoglycan biosynthesis, bacterial chemotaxis and motility, and carbohydrate uptake, were involved in the response of L. monocyotogenes to the presence of BZT. The information generated in this study further contributes to our understanding of the response of bacteria to environmental stress. In addition, this study demonstrates the importance of using the bacterium's own genome as a reference when analysing RNA-Seq data.
Listeria monocytogenes; RNA-Seq; benzethonium chloride; transcriptome; gene expression; biocide stress
Lactococci isolated from non-dairy sources have been found to possess enhanced metabolic activity when compared to dairy strains. These capabilities may be harnessed through the use of these strains as starter or adjunct cultures to produce more diverse flavor profiles in cheese and other dairy products. To understand the interactions between these organisms and the phages that infect them, a number of phages were isolated against lactococcal strains of non-dairy origin. One such phage, ΦL47, was isolated from a sewage sample using the grass isolate L. lactis ssp. cremoris DPC6860 as a host. Visualization of phage virions by transmission electron microscopy established that this phage belongs to the family Siphoviridae and possesses a long tail fiber, previously unseen in dairy lactococcal phages. Determination of the lytic spectrum revealed a broader than expected host range, with ΦL47 capable of infecting 4 industrial dairy strains, including ML8, HP and 310, and 3 additional non-dairy isolates. Whole genome sequencing of ΦL47 revealed a dsDNA genome of 128, 546 bp, making it the largest sequenced lactococcal phage to date. In total, 190 open reading frames (ORFs) were identified, and comparative analysis revealed that the predicted products of 117 of these ORFs shared greater than 50% amino acid identity with those of L. lactis phage Φ949, a phage isolated from cheese whey. Despite their different ecological niches, the genomic content and organization of ΦL47 and Φ949 are quite similar, with both containing 4 gene clusters oriented in different transcriptional directions. Other features that distinguish ΦL47 from Φ949 and other lactococcal phages, in addition to the presence of the tail fiber and the genome length, include a low GC content (32.5%) and a high number of predicted tRNA genes (8). Comparative genome analysis supports the conclusion that ΦL47 is a new member of the 949 lactococcal phage group which currently includes the dairy Φ949.
Lactococcus lactis; non-dairy; phage; tail fiber; genome
New antibacterial agents are urgently needed for the elimination of biofilm-forming bacteria that are highly resistant to traditional antimicrobial agents. Proliferation of such bacteria can lead to significant economic losses in the agri-food sector. This study demonstrates the potential of the bacteriophage-derived peptidase, CHAPK, as a biocidal agent for the rapid disruption of biofilm-forming staphylococci, commonly associated with bovine mastitis. Purified CHAPK applied to biofilms of Staphylococcus aureus DPC5246 completely eliminated the staphylococcal biofilms within 4 h. In addition, CHAPK was able to prevent biofilm formation by this strain. The CHAPK lysin also reduced S. aureus in a skin decolonization model. Our data demonstrates the potential of CHAPK as a biocidal agent for prevention and treatment of biofilm-associated staphylococcal infections or as a decontaminating agent in the food and healthcare sectors.
In this study, whole-genome microarrays were used to gain insights into the global molecular response of Lactococcus lactis subsp. lactis IL1403 at an early stage of infection with the lytic phage c2. The bacterium differentially regulated the expression of 61 genes belonging to 14 functional categories, including cell envelope processes (12 genes), regulatory functions (11 genes), and carbohydrate metabolism (7 genes). The nature of these genes suggests a complex response involving four main mechanisms: (i) induction of membrane stress proteins, (ii) d-alanylation of cell wall lipoteichoic acids (LTAs), (iii) maintenance of the proton motive force (PMF), and (iv) energy conservation. The phage presence is sensed as a membrane stress in L. lactis subsp. lactis IL1403, which activated a cell wall-targeted response probably orchestrated by the concerted action of membrane phage shock protein C-like homologues, the global regulator SpxB, and the two-component system CesSR. The bacterium upregulated genes (ddl and dltABCD) responsible for incorporation of d-alanine esters into LTAs, an event associated with increased resistance to phage attack in Gram-positive bacteria. The expression of genes (yshC, citE, citF) affecting both PMF components was also regulated to restore the physiological PMF, which was disrupted following phage infection. While mobilizing the response to the phage-mediated stress, the bacterium activated an energy-saving program by repressing growth-related functions and switching to anaerobic respiration, probably to sustain the PMF and the overall cell response to phage. To our knowledge, this represents the first detailed description in L. lactis of the molecular mechanisms involved in the host response to the membrane perturbations mediated by phage infection.
Pseudomonas aeruginosa is a common cause of infection in the lungs of patients with cystic fibrosis (CF). In addition, biofilm formation and antibiotic resistance of Pseudomonas are major problems that can complicate antibiotic therapy. We evaluated the efficacy of using bacteriophages to kill the pathogen in both biofilms and in the murine lung. We isolated and characterized two phages from a local wastewater treatment plant, a myovirus (ϕNH-4) and a podovirus (ϕMR299-2). Both phages were active against clinical isolates of P. aeruginosa. Together, the two phages killed all 9 clinical isolate strains tested, including both mucoid and nonmucoid strains. An equal mixture of the two phages was effective in killing P. aeruginosa NH57388A (mucoid) and P. aeruginosa MR299 (nonmucoid) strains when growing as a biofilm on a cystic fibrosis bronchial epithelial CFBE41o- cell line. Phage titers increased almost 100-fold over a 24-h period, confirming replication of the phage. Furthermore, the phage mix was also effective in killing the pathogen in murine lungs containing 1 × 107 to 2 × 107
P. aeruginosa. Pseudomonas was effectively cleared (reduced by a magnitude of at least 3 to 4 log units) from murine lungs in 6 h. Our study demonstrates the efficacy of these two phages in killing clinical Pseudomonas isolates in the murine lung or as a biofilm on a pulmonary cell line and supports the growing interest in using phage therapy for the control and treatment of multidrug-resistant Pseudomonas lung infections in CF patients.
Given the rise in antibiotic resistance, nonantibiotic therapies are required for the treatment of infection. This is particularly true for the treatment of Pseudomonas infection in patients with cystic fibrosis. We have identified two bacterial viruses (bacteriophages) that can kill Pseudomonas growing on human lung cells and in an animal model of lung infection. The use of bacteriophages is particularly appropriate because the killing agent can replicate on the target cell, generating fresh copies of the bacteriophage. Thus, in the presence of a target, the killing agent multiplies. By using two bacteriophages we can reduce the risk of resistant colonies developing at the site of infection. Bacteriophage therapy is an exciting field, and this study represents an important demonstration of efficacy in validated infection models.
The four-plasmid complement of the raw milk cheese isolate Lactococcus lactis subsp. lactis biovar diacetylactis DPC3901 was sequenced, and some genetic features were functionally analyzed. The complete sequences of pVF18 (18,977 bp), pVF21 (21,739 bp), pVF22 (22,166 bp), and pVF50 (53,876 bp) were obtained. Each plasmid contained genes not previously described for Lactococcus, in addition to genes associated with plant-derived lactococcal strains. Most of the novel genes were found on pVF18 and encoded functions typical of bacteria associated with plants, such as activities of plant cell wall modification (orf11 and orf25). In addition, a predicted high-affinity regulated system for the uptake of cobalt was identified (orf19 to orf21 [orf19-21]), which has a single database homolog on a plant-derived Leuconostoc plasmid and whose functionality was demonstrated following curing of pVF18. pVF21 and pVF22 encode additional metal transporters, which, along with orf19-21 of pVF18, could enhance host ability to uptake growth-limiting amounts of biologically essential ions within the soil. In addition, vast regions from pVF50 and pVF21 share significant homology with the plant-derived lactococcal plasmid pGdh442, which is indicative of extensive horizontal gene transfer and recombination between these plasmids and suggests a common plant niche for their hosts. Phenotypes associated with these regions include glutamate dehydrogenase activity and Na+ and K+ transport. The presence of numerous plant-associated markers in L. lactis DPC3901 suggests a plant origin for the raw milk cheese isolate and provides for the first time the genetic basis to support the concept of the plant-milk transition for Lactococcus strains.
The aim of this study was to use comparative modeling to predict the three-dimensional structure of the CHAPK protein (cysteine, histidine-dependent amidohydrolase/peptidase domain of the LysK endolysin, derived from bacteriophage K). Iterative PSI-BLAST searches against the Protein Data Bank (PDB) and nonredundant (nr) databases were used to populate a multiple alignment for analysis using the T-Coffee Expresso server. A consensus Maximum Parsimony phylogenetic tree with a bootstrap analysis setting of 1,000 replicates was constructed using MEGA4. Structural templates relevant to our target (CHAPK) were identified, processed in Expresso and used to generate a 3D model in the alignment mode of SWISS-MODEL. These templates were also processed in the I-TASSER web server. A Staphylococcus saprophyticus CHAP domain protein, 2K3A, was identified as the structural template in both servers. The I-TASSER server generated the CHAPK model with the best bond geometries when analyzed using PROCHECK and the most logical organization of the structure. The predicted 3D model indicates that CHAPK has a papain-like fold. Circular dichroism spectropolarimetry also indicated that CHAPK has an αβ fold, which is consistent with the model presented. The putative active site maintained a highly conserved Cys54-His117-Glu134 charge relay and an oxyanion hole residue Asn136. The residue triplet, Cys-His-Glu, is known to be a viable proteolytic triad in which we predict the Cys residue is used in a nucleophilic attack on peptide bonds at a specific site in the pentaglycine cross bridge of staphylococcal cell wall peptidoglycan. Use of comparative modeling has allowed approximation of the 3D structure of CHAPK giving information on the structure and an insight into the binding and active site of the catalytic domain. This may facilitate its development as an alternative antibacterial agent.
bacteriophage; CHAP; endolysin; in silico; peptidase; staphylococcus
This study investigated the effect of bacteriophages (phages) e11/2 and e4/1c against Escherichia coli O157:H7 in an ex vivo rumen model and in cattle in vivo. In the ex vivo rumen model, samples were inoculated with either 103 or 106 CFU/ml inoculum of E. coli O157:H7 and challenged separately with each bacteriophage. In the presence of phage e11/2, the numbers of E. coli O157:H7 bacteria were significantly (P < 0.05) reduced to below the limit of detection within 1 h. Phage e4/1c significantly (P < 0.05) reduced E. coli O157:H7 numbers within 2 h of incubation, but the number of surviving E. coli O157:H7 bacteria then remained unchanged over a further 22-h incubation period. The ability of a phage cocktail of e11/2 and e4/1c to reduce the fecal shedding of E. coli O157:H7 in experimentally inoculated cattle was then investigated in two cattle trials. Cattle (yearlings, n = 20 for trial one; adult fistulated cattle, n = 2 for trial two) were orally inoculated with 1010 CFU of E. coli O157:H7. Animals (n = 10 for trial one; n = 1 for trial two) were dosed daily with a bacteriophage cocktail of 1011 PFU for 3 days postinoculation. E. coli O157:H7 and phage numbers in fecal and/or rumen samples were determined over 7 days postinoculation. E. coli O157:H7 numbers rapidly declined in all animals within 24 to 48 h; however, there was no significant difference (P > 0.05) between the numbers of E. coli O157:H7 bacteria shed by the phage-treated or control animals. Phages were recovered from the rumen but not from the feces of the adult fistulated animal in trial two but were recovered from the feces of the yearling animals in trial one. While the results from the rumen model suggest that phages are effective in the rumen, further research is required to improve the antimicrobial effectiveness of phages for the elimination of E. coli O157:H7 in vivo.
With the increasing worldwide prevalence of antibiotic resistant bacteria, bacteriophage endolysins (lysins) represent a very promising novel alternative class of antibacterial in the fight against infectious disease. Lysins are phage-encoded peptidoglycan hydrolases which, when applied exogenously (as purified recombinant proteins) to Gram-positive bacteria, bring about rapid lysis and death of the bacterial cell. A number of studies have recently demonstrated the strong potential of these enzymes in human and veterinary medicine to control and treat pathogens on mucosal surfaces and in systemic infections. They also have potential in diagnostics and detection, bio-defence, elimination of food pathogens and control of phytopathogens. This review discusses the extensive research on recombinant bacteriophage lysins in the context of antibacterials, and looks forward to future development and potential.
lysin; endolysin; bacteriophage; pathogen; antibacterial; infection; lytic; enzyme
The endolysin LysK derived from staphylococcal phage K has previously been shown to have two enzymatic domains, one of which is an N-acetylmuramoyl-L-alanine amidase and the other a cysteine/histidine-dependant amidohydrolase/peptidase designated CHAPk. The latter, when cloned as a single-domain truncated enzyme, is conveniently overexpressed in a highly-soluble form. This enzyme was shown to be highly active in vitro against live cell suspensions of S. aureus. In the current study, the IVIS imaging system was used to demonstrate the effective elimination of a lux labeled S. aureus from the nares of BALB/c mice.
Staphylococcus; decolonization; lysin; bacteriophage; nasal
A truncated derivative of the phage endolysin LysK containing only the CHAP (cysteine- and histidine-dependent amidohydrolase/peptidase) domain exhibited lytic activity against live clinical staphylococcal isolates, including methicillin-resistant Staphylococcus aureus. This is the first known report of a truncated phage lysin which retains high lytic activity against live staphylococcal cells.
The recently sequenced genome of Lactobacillus helveticus DPC4571  revealed a dairy organism with significant homology (75% of genes are homologous) to a probiotic bacteria Lb. acidophilus NCFM . This led us to hypothesise that a group of genes could be determined which could define an organism's niche.
Taking 11 fully sequenced lactic acid bacteria (LAB) as our target, (3 dairy LAB, 5 gut LAB and 3 multi-niche LAB), we demonstrated that the presence or absence of certain genes involved in sugar metabolism, the proteolytic system, and restriction modification enzymes were pivotal in suggesting the niche of a strain. We identified 9 niche specific genes, of which 6 are dairy specific and 3 are gut specific. The dairy specific genes identified in Lactobacillus helveticus DPC4571 were lhv_1161 and lhv_1171, encoding components of the proteolytic system, lhv_1031 lhv_1152, lhv_1978 and lhv_0028 encoding restriction endonuclease genes, while bile salt hydrolase genes lba_0892 and lba_1078, and the sugar metabolism gene lba_1689 from Lb. acidophilus NCFM were identified as gut specific genes.
Comparative analysis revealed that if an organism had homologs to the dairy specific geneset, it probably came from a dairy environment, whilst if it had homologs to gut specific genes, it was highly likely to be of intestinal origin.
We propose that this "barcode" of 9 genes will be a useful initial guide to researchers in the LAB field to indicate an organism's ability to occupy a specific niche.
Mobile genetic elements are major contributing factors to the generation of genetic diversity in prokaryotic organisms. For example, insertion sequence (IS) elements have been shown to specifically contribute to niche adaptation by promoting a variety of genetic rearrangements. The complete genome sequence of the cheese culture Lactobacillus helveticus DPC 4571 was determined and revealed significant conservation compared to three nondairy gut lactobacilli. Despite originating from significantly different environments, 65 to 75% of the genes were conserved between the commensal and dairy lactobacilli, which allowed key niche-specific gene sets to be described. However, the primary distinguishing feature was 213 IS elements in the DPC 4571 genome, 10 times more than for the other lactobacilli. Moreover, genome alignments revealed an unprecedented level of genome stability between these four Lactobacillus species, considering the number of IS elements in the L. helveticus genome. Comparative analysis also indicated that the IS elements were not the primary agents of niche adaptation for the L. helveticus genome. A clear bias toward the loss of genes reported to be important for gut colonization was observed for the cheese culture, but there was no clear evidence of IS-associated gene deletion and decay for the majority of genes lost. Furthermore, an extraordinary level of sequence diversity exists between copies of certain IS elements in the DPC 4571 genome, indicating they may represent an ancient component of the L. helveticus genome. These data suggest a special unobtrusive relationship between the DPC 4571 genome and its mobile DNA complement.
Lacticin 3147 is a broad-spectrum two-peptide lantibiotic whose genetic determinants are located on two divergent operons on the lactococcal plasmid pMRC01. Here we introduce each of 14 subclones, containing different combinations of lacticin 3147 genes, into MG1363 (pMRC01) and determine that a number of them can facilitate overproduction of the lantibiotic. Based on these studies it is apparent that while the provision of additional copies of genes encoding the biosynthetic/production machinery and the regulator LtnR is a requirement for high-level overproduction, the presence of additional copies of the structural genes (i.e., ltnA1A2) is not.
Two-component regulatory systems are one primary mechanism for environmental sensing and signal transduction. Annotation of the complete genome sequence of the probiotic bacterium Lactobacillus acidophilus NCFM revealed nine two-component regulatory systems. In this study, the histidine protein kinase of a two-component regulatory system (LBA1524HPK-LBA1525RR), similar to the acid-related system lisRK from Listeria monocytogenes (P. D. Cotter et al., J. Bacteriol. 181:6840-6843, 1999), was insertionally inactivated. A whole-genome microarray containing 97.4% of the annotated genes of L. acidophilus was used to compare genome-wide patterns of transcription at various pHs between the control and the histidine protein kinase mutant. The expression pattern of approximately 80 genes was affected by the LBA1524HPK mutation. Putative LBA1525RR target loci included two oligopeptide-transport systems present in the L. acidophilus genome, other components of the proteolytic system, and a LuxS homolog, suspected of participating in synthesis of the AI-2 signaling compound. The mutant exhibited lower tolerance to acid and ethanol in logarithmic-phase cells and poor acidification rates in milk. Supplementation of milk with Casamino Acids essentially restored the acid-producing ability of the mutant, providing additional evidence for a role of this two component system in regulating proteolytic activity in L. acidophilus.
Two genes, bshA and bshB, encoding bile salt hydrolase enzymes (EC 126.96.36.199) were identified in the genome sequence of Lactobacillus acidophilus NCFM. Targeted inactivation of these genes via chromosomal insertion of an integration vector demonstrated different substrate specificities for these two enzymes.
Insertional inactivation of the plasmid-encoded determinants for Mg2+ and Co2+ transport, orf18/corA, provides a tool for screening recombinant clones in Lactococcus, based on the observation that overexpression of orf18/corA results in cell growth inhibition on certain concentrations of CoCl2. The lacticin 3147 immunity gene, ltnI, was used to insertionally inactivate orf18/corA. The resulting clones were capable of growth on concentrations of CoCl2 that were inhibitory to the parent strain. Since only 3 of 17 lactococcal starters naturally harbor corA, the system has potential as a screen for selecting recombinant lactococcal clones.
Lactococcus lactis DPC4275 is a bacteriocin-producing transconjugant of the industrial starter strain DPC4268. Strain DPC4275 was generated through conjugal transfer by mating DPC4268 with L. lactis MG1363 containing the 60-kb plasmid pMRC01, which encodes the genetic determinants for the lantibiotic lacticin 3147 and for a phage resistance mechanism of the abortive infection type. The many significant applications of this strain prompted a genetic analysis of its apparently unstable bacteriocin-producing phenotype. Increased levels of lacticin 3147 produced by DPC4275 were associated with the appearance of an 80-kb plasmid, designated pMRC02, which was derived from DNA originating from pMRC01 (60 kb) and a resident DPC4268 proteinase plasmid, pMT60 (60 kb). Indeed, pMRC02 was shown to be derived from the insertion of a 17-kb fragment of pMRC01, encompassing the lacticin 3147 operon, into pMT60. The presence of pMRC02 at a high copy number was found to correlate with increased levels of lacticin 3147 in DPC4275 compared to the wild-type containing pMRC01. Subsequent transfer of pMRC02 into the plasmid-free strain MG1363 by electroporation allowed a direct phenotypic comparison with pMRC01, also studied in the MG1363 background. Plasmid pMRC02 displayed phage resistance similar to that by pMRC01, although it was less potent, as demonstrated by a larger plaque size for phage c2 infection of MG1363(pMRC02). While this locus is flanked by IS946 elements, the sequencing of pMT60-pMRC01 junction sites established that this event was unlikely to be insertion sequence mediated and most probably occurred by homologous recombination followed by deletion of most of pMRC01. This was not a random occurrence, as nine other transconjugants investigated were found to have the same junction sites. Such derivatives of commercial strains producing increased levels of bacteriocin could be exploited as protection cultures for food applications.
Lacticin 3147 is a broad-spectrum bacteriocin produced by Lactococcus lactis subsp. lactis DPC3147 (M. P. Ryan, M. C. Rea, C. Hill, and R. P. Ross, Appl. Environ. Microbiol. 62:612–619, 1996). Partial purification of the bacteriocin by hydrophobic interaction chromatography and reverse-phase fast protein liquid chromatography revealed that two components are required for full activity. Lacticin 3147 is bactericidal against L. lactis, Listeria monocytogenes, and Bacillus subtilis; at low concentrations of the bacteriocin, bactericidal activity is enhanced when target cells are energized. This finding suggests that the presence of a proton motive force promotes the interaction of the bacteriocin with the cytoplasmic membrane, leading to the formation of pores at these low lacticin 3147 concentrations. These pores were shown to be selective for K+ ions and inorganic phosphate. The loss of these ions resulted in immediate dissipation of the membrane potential and hydrolysis of internal ATP, leading to an eventual collapse of the pH gradient at the membrane and ultimately to cell death. Our results suggest that lacticin 3147 is a pore-forming bacteriocin which acts on a broad range of gram-positive bacteria.