Ring-fused 2-pyridones effective against multidrug-resistant Gram-positive pathogens and synergistic with standard-of-care antibiotics.

The alarming rise of multidrug-resistant Gram-positive bacteria has precipitated a healthcare crisis, necessitating the development of new antimicrobial therapies. Here we describe a new class of antibiotics based on a ring-fused 2-pyridone backbone, which are active against vancomycin-resistant enterococci (VRE), a serious threat as classified by the Centers for Disease Control and Prevention, and other multidrug-resistant Gram-positive bacteria. Ring-fused 2-pyridone antibiotics have bacteriostatic activity against actively dividing exponential phase enterococcal cells and bactericidal activity against nondividing stationary phase enterococcal cells. The molecular mechanism of drug-induced killing of stationary phase cells mimics aspects of fratricide observed in enterococcal biofilms, where both are mediated by the Atn autolysin and the GelE protease. In addition, combinations of sublethal concentrations of ring-fused 2-pyridones and standard-of-care antibiotics, such as vancomycin, were found to synergize to kill clinical strains of VRE. Furthermore, a broad range of antibiotic resistant Gram-positive pathogens, including those responsible for the increasing incidence of antibiotic resistant healthcare-associated infections, are susceptible to this new class of 2-pyridone antibiotics. Given the broad antibacterial activities of ring-fused 2-pyridone compounds against Gram-positive (GmP) bacteria we term these compounds GmPcides, which hold promise in combating the rising tide of antibiotic resistant Gram-positive pathogens.

Improved antimicrobial spectrum of the N-acetylmuramoyl-L-alanine amidase from Latilactobacillus sakei upon LysM domain deletion.

The gene encoding N-acetylmuramoyl-L-alanine amidase in Latilactobacillus sakei isolated from a fermented meat product was cloned in two forms: its complete sequence (AmiC) and a truncated sequence without one of its anchoring LysM domains (AmiLysM4). The objective of this work was to evaluate the effect of LysM domain deletion on antibacterial activity as well the biochemical characterization of each recombinant protein. AmiC and AmiLysM4 were expressed in Escherichia coli BL21. Using a zymography method, two bands with lytic activity were observed, which were confirmed by LC-MS/MS analysis, with molecular masses of 71 kDa (AmiC) and 66 kDa (AmiLysM4). The recombinant proteins were active against Listeria innocua and Staphylococcus aureus strains. The inhibitory spectrum of AmiLysM4 was broader than AmiC as it showed inhibition of Leuconostoc mesenteroides and Weissella viridescens, both microorganisms associated with food decomposition. Optimal temperature and pH values were determined for both proteins using L-alanine-p-nitroanilide hydrochloride as a substrate for N-acetylmuramoyl-L-alanine amidase activity. Both proteins showed similar maximum activity values for pH (8) and temperature (50 °C). Furthermore, structural predictions did not show differences for the catalytic region, but differences were found for the region called 2dom-AmiLysM4, which includes 4 of the 5 LysM domains. Therefore, modification of the LysM domain offers new tools for the development of novel food biopreservatives.

Novel Lytic Enzyme of Prophage Origin from Clostridium botulinum E3 Strain Alaska E43 with Bactericidal Activity against Clostridial Cells.

Clostridium botulinum is a Gram-positive, anaerobic, spore-forming bacterium capable of producing botulinum toxin and responsible for botulism of humans and animals. Phage-encoded enzymes called endolysins, which can lyse bacteria when exposed externally, have potential as agents to combat bacteria of the genus Clostridium. Bioinformatics analysis revealed in the genomes of several Clostridium species genes encoding putative N-acetylmuramoyl-l-alanine amidases with anti-clostridial potential. One such enzyme, designated as LysB (224-aa), from the prophage of C. botulinum E3 strain Alaska E43 was chosen for further analysis. The recombinant 27,726 Da protein was expressed and purified from E. coli Tuner(DE3) with a yield of 37.5 mg per 1 L of cell culture. Size-exclusion chromatography and analytical ultracentrifugation experiments showed that the protein is dimeric in solution. Bioinformatics analysis and results of site-directed mutagenesis studies imply that five residues, namely H25, Y54, H126, S132, and C134, form the catalytic center of the enzyme. Twelve other residues, namely M13, H43, N47, G48, W49, A50, L73, A75, H76, Q78, N81, and Y182, were predicted to be involved in anchoring the protein to the lipoteichoic acid, a significant component of the Gram-positive bacterial cell wall. The LysB enzyme demonstrated lytic activity against bacteria belonging to the genera Clostridium, Bacillus, Staphylococcus, and Deinococcus, but did not lyse Gram-negative bacteria. Optimal lytic activity of LysB occurred between pH 4.0 and 7.5 in the absence of NaCl. This work presents the first characterization of an endolysin derived from a C. botulinum Group II prophage, which can potentially be used to control this important pathogen.

Building blocks and blueprints for bacterial autolysins.

Bacteria utilize a wide variety of endogenous cell wall hydrolases, or autolysins, to remodel their cell walls during processes including cell division, biofilm formation, and programmed death. We here systematically investigate the composition of these enzymes in order to gain insights into their associated biological processes, potential ways to disrupt them via chemotherapeutics, and strategies by which they might be leveraged as recombinant antibacterial biotherapies. To do so, we developed LEDGOs (lytic enzyme domains grouped by organism), a pipeline to create and analyze databases of autolytic enzyme sequences, constituent domain annotations, and architectural patterns of multi-domain enzymes that integrate peptidoglycan binding and degrading functions. We applied LEDGOs to eight pathogenic bacteria, gram negatives Acinetobacter baumannii, Klebsiella pneumoniae, Neisseria gonorrhoeae, and Pseudomonas aeruginosa; and gram positives Clostridioides difficile, Enterococcus faecium, Staphylococcus aureus, and Streptococcus pneumoniae. Our analysis of the autolytic enzyme repertoires of these pathogens reveals commonalities and differences in their key domain building blocks and architectures, including correlations and preferred orders among domains in multi-domain enzymes, repetitions of homologous binding domains with potentially complementarity recognition modalities, and sequence similarity patterns indicative of potential divergence of functional specificity among related domains. We have further identified a variety of unannotated sequence regions within the lytic enzymes that may themselves contain new domains with important functions.

MMPphg from the thermophilic Meiothermus bacteriophage MMP17 as a potential antimicrobial agent against both Gram-negative and Gram-positive bacteria.

BACKGROUND: New strategies are urgently needed to deal with the growing problem of multidrug-resistant bacterial pathogens. As the natural viruses against bacteria, recently, bacteriophages have received particular attention. Here, we identified and characterized a novel peptidoglycan hydrolase named MMPphg by decoding the complete genome sequence of Meiothermus bacteriophage MMP17, which was isolated in Tengchong hot spring in China and contains a circular genome of 33,172 bp in size and a GC content of 63.4%. FINDINGS: We cloned the MMPphg gene, overproduced and purified the phage lytic protein, which contains a highly conserved M23 metallopeptidase domain and can be activated by Mg2+ and Zn2+. MMPphg is capable of withstanding temperatures up to 70 °C, and preserved more than 80% of its activity after a 30 min treatment between 35 and 65 °C. More interestingly, by disrupting bacterial cells, MMPphg exhibits surprising antimicrobial activity against both Gram-negative and Gram-positive pathogenic bacteria, especially antibiotic-resistant strains such as Escherichia coli O157, Staphylococcus aureus and Klebsiella pneumonia. CONCLUSIONS: In the current age of mounting antibiotic resistance, these results suggest the great potential of MMPphg, the gene product of bacteriophage MMP17, in combating bacterial infections and shed light on bacteriophage-based strategies to develop alternatives to conventional antibiotics for human or veterinary applications.

Characteristics and Lytic Activity of Phage-Derived Peptidoglycan Hydrolase, LysSAP8, as a Potent Alternative Biocontrol Agent for Staphylococcus aureus.

Outbreaks of staphylococcal food poisoning (SFP) causing serious human diseases and economic losses have been reported globally. Furthermore, the spread of Staphylococcus aureus with increased resistance to multiple antimicrobial agents has become a major concern in the food industries and medicine. Here, we isolated an endolysin LysSAP8, as one of the peptidoglycan hydrolases, derived from the bacteriophage SAP8 infecting S. aureus. This endolysin was tagged with a 6×His at the C-terminal of the target protein and purified using affinity chromatography. LysSAP8 demonstrated lytic activity against a broad spectrum of bacteria, which included a majority of the staphylococcal strains tested in this study as well as the methicillin-resistant S. aureus (MRSA); however, no such activity was observed against other gram-positive or gram-negative bacteria. Additionally, LysSAP8 could maintain bactericidal activity until 0.1 nM working concentration and after heat treatment at 37°C for 30 min. The ability of LysSAP8 to lyse cells under varying conditions of temperature (4-43°C), pH (3-9), and NaCl concentrations (0-1,000 mM), and divalent metal ions (Ca2+, Co2+, Cu2+, Mg2+, Mn2+, Hg2+, and Zn2+) was examined. At the optimized condition, LysSAP8 could disrupt approximately 3.46 log CFU/ml of the planktonic cells in their exponential phase of growth within 30 min. In this study, we have suggested that LysSAP8 could be a potent alternative as a biocontrol agent that can be used to combat MRSA.

Modular Assembly of Unique Chimeric Lytic Enzymes on a Protein Scaffold Possessing Anti-Staphylococcal Activity.

Lytic enzymes have been considered as potential alternatives to antibiotics. These enzymes, particularly those that target Gram-positive bacteria, consist of modular cell wall-binding and catalytic domains, which can be shuffled with those of other lytic enzymes to produce unnatural chimeric enzymes. In this work, we report the in vitro shuffling of two different modular domains using a protein self-assembly methodology. Catalytic domains (CD) and cell wall-binding domains (BD) from the bacteriocin lysostaphin (Lst) and a putative autolysin from Staphylococcus aureus (SA1), respectively, were genetically site-specifically biotinylated and assembled with streptavidin to generate 23 permuted chimeras. The specific assembly of a CD (3 equiv) and a BD (1 equiv) from Lst and SA1, respectively [CDL-BDS (3:1)], on a streptavidin scaffold yielded high lytic activity against S. aureus (at least 5.6 log reduction), which was higher than that obtained with either native Lst or SA1 alone. Moreover, at 37 °C, the initial rate of cell lysis was over 3-fold higher than that with free Lst, thereby revealing the unique catalytic properties of the chimeric proteins. In vitro self-assembly of functional domains from modular lytic enzymes on a protein scaffold likely expands the repertoire of bactericidal enzymes with improved activities.

Autolysin mediated adherence of Staphylococcus aureus with Fibronectin, Gelatin and Heparin.

Major autolysin (Atl) of Staphylococcus aureusis a cell surface associated peptidoglycan hydrolase with amidase and glucosaminidase domains. Atl enzymes (amidase and glucosaminidase) are known to participate in biofilm formation and also can bind with host matrices. Earlier studies demonstrated the binding of Atlwithfibronectin, thrombospondin 1, vitronectin and heat shock cognate protein Hsc70. Here, we have shown, Atl mediates attachment of S.aureus to heparin and gelatine as well. The atl mutant strain demonstrated around 2.5 fold decreased adherence with fibronectin, gelatin and heparin coated microtiter plates. The microscopic studies confirmed the reduced binding of atl mutant with them compared to its parental wild type and complemented mutant strains. Amidase and glucosaminidase were expressed as N-terminal histidine tagged proteins from Escherichia coli, purified and refolded. We found refolded amidase bind with fibronectin, gelatin and heparin; whereas refolded glucosaminidase binds with only fibronectin and heparin but not gelatin. These results reemphasize Atl as one of the crucial proteins from Staphylococcus that facilitate their binding with multiple host cellular components during colonization and infection.

Lysibodies are IgG Fc fusions with lysin binding domains targeting Staphylococcus aureus wall carbohydrates for effective phagocytosis.

The cell wall of Gram-positive bacteria contains abundant surface-exposed carbohydrate molecules that are highly conserved within and often across species. The potential therapeutic usefulness of high-affinity antibodies to cell wall carbohydrates is unquestioned, however obtaining such antibodies is challenging due to the poor overall immunogenicity of these bacterial targets. Autolysins and phage lysins are peptidoglycan hydrolases, enzymes that have evolved over a billion years to degrade bacterial cell wall. Such wall hydrolases are modular enzymes, composed of discrete domains for high-affinity binding to cell wall carbohydrates and cleavage activity. In this study, we demonstrate that binding domains from autolysins and lysins can be fused to the Fc region of human IgG, creating a fully functional homodimer (or "lysibody") with high-affinity binding and specificity for carbohydrate determinants on the bacterial surface. Furthermore, we demonstrate that this process is reproducible with three different binding domains specific to methicillin-resistant Staphylococcus aureus (MRSA). Cell-bound lysibodies induced the fixation of complement on the bacterial surface, promoted phagocytosis by macrophages and neutrophils, and protected mice from MRSA infection in two model systems. The lysibody approach could be used to target a range of difficult-to-treat pathogenic bacteria, given that cell wall hydrolases are ubiquitous in nature.

Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk.

Peptidoglycan hydrolases (PGHs) have been suggested as novel therapeutics for the treatment of bovine mastitis. However, activity in the presence of cow's milk is an important requirement for drugs administered into the bovine udder. We have screened a library of >170 recombinant PGHs, including engineered bacteriophage endolysins, for enzymes with activity against Staphylococcus aureus in milk, using a microtiter plate-based protocol. Nine suitable PGH constructs were identified by this approach and further compared in time-kill assays for their efficacy against S. aureus in heat-treated milk. The three most active enzymes (lysostaphin, Ami2638A, and CHAPK_CWT-LST) reduced S. aureus in milk to undetectable numbers within minutes at nanomolar concentrations. Due to their different peptidoglycan cleavage sites, these PGH constructs revealed synergistic activity in most combinations, as demonstrated by checkerboard assays, spot assays, and time-kill experiments. Furthermore, they proved active against a selection of staphylococcal mastitis isolates from different geographical regions when applied individually or in synergistic combination. The most effective PGH combination completely eradicated S. aureus from milk, with no more bacteria being detected within 24 h after addition of the enzymes, corresponding to a reduction of >9 log units compared to the control. Efficacy was also retained at different inoculum levels (3 versus 6 log CFU/ml) and when S. aureus was grown in milk as opposed to broth prior to the experiments. In raw cow's milk, CHAPK_CWT-LST showed reduced efficacy, whereas both Ami2638A and lysostaphin retained their activity, reducing bacterial numbers by >3.5 log units within 3 h.IMPORTANCE Staphylococci and S. aureus in particular are a major cause of bovine mastitis, an inflammation of the mammary gland in cows associated with high costs and risks for consumers of milk products. S. aureus-induced mastitis, commonly treated by intramammary infusion of antibiotics, is characterized by low cure rates and increasing antibiotic resistance in bacteria. Therefore, alternative treatment options are highly desirable. PGHs, including bacteriophage endolysins, rapidly and specifically kill selected pathogens by degrading their cell wall and are refractory to resistance development, therefore holding promise as novel antibacterial agents. This study employed a screening approach to identify PGH constructs with high staphylolytic activity in cow's milk within a large collection of enzymes. Our results suggest that the most promising enzymes identified by this strategy hold potential as novel mastitis therapeutics and support their further characterization in animal models.

Characterization of three autolysins with activity against cereulide-producing Bacillus isolates in food matrices.

Bacillus cereus is a pathogen related with diarrhoeal or emetic food poisoning cases, of which the latter caused by the cereulide-producing isolates are more severe with several reported lethal cases. It is therefore necessary to develop an effective strategy to prevent the propagation of B. cereus in the food supply. In this study, three autolysins from the cereulide-producing B. cereus group isolates, LysIS075, LysF8819.1 and LysCER057, were identified and characterized. The results showed that the three autolysins were highly lytic and bactericidal to the tested cereulide-producing B. cereus group strains and cross-lytic against other tested B. cereus group strains, and they could inhibit the spore germination and propagation of their tested derived emetic strains. Physical and chemical characterization showed that all the three autolysins were alkalophilic with the optimal activity at pH9.0 or 9.5 with one exception of LysF8819.1 also having significant lytic activity at pH5.0, and they all had relative strong lytic activity at 37-50°C during the 30minute assay. However, LysCER057 showed relative susceptibility to thermo-condition. Remarkably, the separate or cock-tail addition of the three autolysins in food matrices (milk and rice porridge) showed effective bactericidal activity within the tested 2h. All the results revealed that the three autolysins might be potential candidates to control emetic B. cereus strains in different applications.

Antibiofilm Activities of a Novel Chimeolysin against Streptococcus mutans under Physiological and Cariogenic Conditions.

Streptococcus mutans often survives as a biofilm on the tooth surface and contributes to the development of dental caries. We investigated the efficacy of ClyR, an engineered chimeolysin, against S. mutans biofilms under physiological and cariogenic conditions. Susceptibility tests showed that ClyR was active against all clinical S. mutans isolates tested as well as S. mutans biofilms that displayed resistance to penicillin. The S. mutans biofilms that formed on hydroxyapatite discs under physiological sugar conditions and cariogenic conditions were reduced ∼2 logs and 3 logs after treatment with 100 µg/ml ClyR, respectively. In comparison, only a 1-log reduction was observed in the chlorhexidine gluconate (ChX)-treated group, and no killing effect was observed in the NaF-treated group. A mouse dental colonization model showed that repeated use of ClyR for 3 weeks (5 µg/day) reduced the number of colonized S. mutans cells in the dental plaques significantly (P < 0.05) and had no harmful effects on the mice. Furthermore, toxicity was not noted at concentrations exceeding those used for the in vitro and in vivo studies, and ClyR-specific antibodies could not be detected in mouse saliva after repeated use of ClyR in the oral cavity. Our data collectively demonstrate that ClyR is active against S. mutans biofilms both in vitro and in vivo, thus representing a preventative or therapeutic agent for use against dental caries.

Characterisation of the antibacterial properties of a bacterial derived peptidoglycan hydrolase (LysCs4), active against C. sakazakii and other Gram-negative food-related pathogens.

Illness caused by the consumption of contaminated food products continues to represent one of the main challenges facing food manufacturers worldwide. Even with current intervention technologies and increased hygiene measures, foodborne illness remains a significant threat to public health. This coupled with the increasing emergence of multidrug resistant pathogens has increased the need for the development of novel technologies for pathogen control. Bacterial derived peptidoglycan hydrolases represent a vast and highly diverse group of enzymes with potential for biocontrol of a range of Gram-positive and Gram-negative foodborne pathogens. In this study, we describe the identification, cloning, expression and purification of a peptidoglycan hydrolase (LysCs4) derived from Cronobacter sakazakii for biocontrol of the aforementioned infant formula pathogen itself. In silico analysis of LysCs4 revealed the gene to display greatest sequence similarity to a putative lysozyme encoded by the lytic Cronobacter phage ES2. Conserved domain analysis of LysCs4 revealed the presence of a single catalytic domain predicted to display O-Glycosyl hydrolase activity and to be a member of the GH24 family. The ability of this enzyme to hydrolyse the peptidoglycan of 25 Gram-negative strains, across 4 different genera, highlights its potential as a novel candidate for biocontrol of C. sakazakii and other Gram-negative food related pathogens.

Murein hydrolase activity of surface layer proteins from Lactobacillus acidophilus against Escherichia coli.

The aim of this study was to investigate the murein hydrolase activities of the surface layer proteins (SLPs) from two strains of Lactobacillus acidophilus using zymography. The influence of these hydrolase activities on Escherichia coli ATCC 43893 was also evaluated by analysing their growth curve, cell morphology and physiological state. After the incubation of E. coli with SLPs, growth was inhibited, the number of viable cells was significantly reduced, examination by transmission electron microscopy showed that the cell wall was damaged and flow cytometry results indicated that the majority of the cells were sublethally injured. All of these results suggested that the SLPs of both L. acidophilus strains possessed murein hydrolase activities that were sublethal to E. coli cells.

Zoocin A and lauricidin in combination reduce Streptococcus mutans growth in a multispecies biofilm.

Dental caries is the most prevalent human infection. It is a multifactorial disease in which the microbial composition of dental plaque plays a major role in the development of clinical symptoms. The bacteria most often implicated in the development of caries are that group of streptococci referred to as the mutans streptococci, in particular Streptococcus mutans and Streptococcus sobrinus. One approach to the prevention of caries is to reduce the numbers of mutans streptococci in plaque to a level insufficient to support demineralization of the tooth. In this study, zoocin A, a peptidoglycan hydrolase, combined with lauricidin, a cell membrane active lipid, was shown over a 72 h period to selectively suppress the growth of S. mutans in a triple species biofilm. Growth of the non-target species Streptococcus oralis and Actinomyces viscosus was not inhibited. In treated systems the amount of extracellular polysaccharide matrix produced was much reduced as determined by use of fluorescein isothiocyanate conjugated wheat germ agglutinin. The pH of treated biofilms remained above neutral as opposed to a value of 4.3 in untreated controls. We conclude that use of antimicrobial compounds that specifically target cariogenic bacteria should be further explored.

LytA, major autolysin of Streptococcus pneumoniae, requires access to nascent peptidoglycan.

The pneumococcal autolysin LytA is a virulence factor involved in autolysis as well as in fratricidal- and penicillin-induced lysis. In this study, we used biochemical and molecular biological approaches to elucidate which factors control the cytoplasmic translocation and lytic activation of LytA. We show that LytA is mainly localized intracellularly, as only a small fraction was found attached to the extracellular cell wall. By manipulating the extracellular concentration of LytA, we found that the cells were protected from lysis during exponential growth, but not in the stationary phase, and that a defined threshold concentration of extracellular LytA dictates the onset of autolysis. Stalling growth through nutrient depletion, or the specific arrest of cell wall synthesis, sensitized cells for LytA-mediated lysis. Inhibition of cell wall association via the choline binding domain of an exogenously added enzymatically inactive form of LytA revealed a potential substrate for the amidase domain within the cell wall where the formation of nascent peptidoglycan occurs.

In vitro destruction of Streptococcus pneumoniae biofilms with bacterial and phage peptidoglycan hydrolases.

Host- and phage-coded cell wall hydrolases have been used to fight Streptococcus pneumoniae growing as planktonic cells in vitro as well as in animal models. Until now, however, the usefulness of these enzymes in biofilm-grown pneumococci has gone untested. The antipneumococcal activity of different cell wall hydrolases produced by S. pneumoniae and a number of its phages was examined in an in vitro biofilm model. The major pneumococcal autolysin LytA, an N-acetylmuramoyl-l-alanine amidase, showed the greatest efficiency in disintegrating S. pneumoniae biofilms. The phage-encoded lysozymes Cpl-1 and Cpl-7 were also very efficient. Biofilms formed by the close pneumococcal relatives Streptococcus pseudopneumoniae and Streptococcus oralis were also destroyed by the phage endolysins but not by the S. pneumoniae autolysin LytA. A cooperative effect of LytA and Cpl-1 in the disintegration of S. pneumoniae biofilms was recorded.

Apoptosis-like death in bacteria induced by HAMLET, a human milk lipid-protein complex.

BACKGROUND: Apoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid. METHODOLOGY/PRINCIPAL FINDINGS: We show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity) to execute cell death. CONCLUSIONS/SIGNIFICANCE: Our results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells.

Recombinant bacteriophage lysins as antibacterials.

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.

Synergistic effects of the Lactobacillus acidophilus surface layer and nisin on bacterial growth.

We have previously described a murein hydrolase activity for the surface layer (S-layer) of Lactobacillus acidophilus ATCC 4356. Here we show that, in combination with nisin, this S-layer acts synergistically to inhibit the growth of pathogenic Gram-negative Salmonella enterica and potential pathogenic Gram-positive bacteria, Staphylococcus aureus and Bacillus cereus. In addition, bacteriolytic effects were observed for the Gram-positive species tested. We postulate that the S-layer enhances the access of nisin into the cell membrane by enabling it to cross the cell wall, while nisin provides the sudden ion-nonspecific dissipation of the proton motive force required to enhance the S-layer murein hydrolase activity.