Search

Antibioticresistance and the colonization of bacteria on surfaces,often as biofilms, prolong hospitalization periods, increase mortality,and are thus major concerns for health care providers. There is anurgent need for antimicrobial and antibiofilm surface treatments thatare permanent, can eradicate both biofilms and planktonic pathogensover long periods of time, and do not select for resistant strains.In this study, we have demonstrated a simple, robust, and biocompatiblemethod that utilizes the adhesive property of polydopamine (PDA) tocovalently attach the antimicrobial enzyme lysostaphin (Lst) to avariety of surfaces to generate antibacterial and antibiofilm interfaces.The immobilization of the recombinant Lst onto PDA-coated surfaceswas carried out under physiological conditions, most probably throughthe C-terminal His6-tag fragment of the enzyme, minimizing the lossesof bioagent activity. The modified surfaces were extensively characterizedby X-ray photoelectron spectroscopy and peak force quantitative nanomechanicalmapping (PeakForce QNM) AFM-based method, and the presence of Lston the surfaces was further confirmed immunochemically using anti-Lstantibody. We also found that, in contrast to the physically adsorbedLst, the covalently attached Lst does not leach from the surfacesand maintains its endopeptidase activity to degrade the staphylococcalcell wall, avoiding most intracellular bacterial resistance mechanisms.Moreover, the Lst-coated surfaces kill hospital strains of Staphylococcus aureusin less than 15 min and prevent biofilmformation. This immobilization method should be applicable also toother proteins and enzymes that are recombinantly expressed to includethe His6-tag fragment. [ABSTRACT FROM AUTHOR]

Physiological properties of a selected Staphylococcus epidermidis strain 33 GISK VAN with high resistance to vancomycin and multiple resistance to various antibiotics, as well as decreased sensitivity to lysozyme, lysostaphin, and the low-molecular mass peptide warnerin were studied. The strain was characterized by a thicker cell wall, resulting in considerably decreased rates of vancomycin penetration into the cells and in limited access of the antibiotic to its targets on the bacterial membrane. [ABSTRACT FROM AUTHOR]

The increasing incidence of implant-associated infections induced by Staphylococcus aureus (SA) in combination with growing resistance to conventional antibiotics requires novel therapeutic strategies. In the current study we present the first application of the biofilm-penetrating antimicrobial peptide lysostaphin in the context of bone infections. In a standardized implant-associated bone infection model in mice beta-irradiated lysostaphin-coated titanium plates were compared with uncoated plates. Coating of the implant was established with a poly(D,L)-lactide matrix (PDLLA) comprising lysostaphin formulated in a stabilizing and protecting solution (SPS). All mice were osteotomized and infected with a defined count of SA. Fractures were fixed with lysostaphin-coated locking plates. Plates uncoated or PDLLA-coated served as controls. All mice underwent debridement and lavage on Days 7, 14, 28 to determine the bacterial load and local immune reaction. Fracture healing was quantified by conventional radiography. On Day 7 bacterial growth in the lavages of mice with lysostaphin-coated plates showed a significantly lower count to the control groups. Moreover, in the lysostaphin-coated plate groups complete fracture healing were observed on Day 28. The fracture consolidation was accompanied by a diminished local immune reaction. However, control groups developed an osteitis with lysis or destruction of the bone and an evident local immune response. The presented approach of terminally sterilized lysostaphin-coated implants appears to be a promising therapeutic approach for low grade infection or as prophylactic strategy in high risk fracture care e.g. after severe open fractures. [ABSTRACT FROM AUTHOR]

In this work, a lysostaphin-loaded, control-released, self-setting and injectable porous bone cement with efficient protein delivery was prepared by a novel setting method using hydroxyapatite/chitosan (HA/CS) composite scaffold. The cement samples were made through cementitious reactions by mixing solid powder, a mixture of HA/CS composite particles, lysostaphin, Ca(OH)2, CaCO3 and NaHCO3, with setting liquid containing citric acid, acetic acid, NaH2PO4, CaCl2 and poloxamer. The setting parameters of the cement samples were determined. The results showed that the final setting time was 96.6±5.2 min and the pH value increased from approximately 6.2 to nearly 10 during the setting process and the porosity was 34% at the end. And the microstructure and composition were detected by scanning electron microscopy (SEM), x-ray diffraction and Fourier transform-infrared spectroscopy. For the release behavior of lysostaphin loaded in the cement sample, the in vitro cement extract experiment indicated that about 94.2±10.9% of the loaded protein was released before day 8 and the in vivo Qdot 625 fluorescence tracking experiment showed that the loaded protein released slower than the free one. Then the biocompatibility of the cement samples was evaluated using the methylthiazol tetrazolium assay, SEM and hematoxylin-eosin staining, which suggested good biocompatibility of cement samples with MC 3T3-E1 cells and subcutaneous tissues of mice. Finally the antibacterial activity assay indicated that the loaded lysostaphin had good release ability and strong antibacterial enzymatic activity against methicillin-resistant Staphylococcus aureus. Collectively, all the results suggested that the lysostaphin-loaded self-setting injectable porous bone cement released the protein in a controlled and effective way and the protein activity was well retained during the setting and releasing process. Thus this bone cement can be potentially applied as a combination of artificial bone substitute and controlled-release system for delivery of lysostaphin to treat bone defects and infections. [ABSTRACT FROM AUTHOR]

Staphylococcus simulans biovar staphylolyticus lysostaphin efficiently cleaves Staphylococcus aureus cell walls. The protein is in late clinical trials as a topical anti-staphylococcal agent, and can be used to prevent staphylococcal growth on artificial surfaces. Moreover, the gene has been both stably engineered into and virally delivered to mice or livestock to obtain resistance against staphylococci. Here, we report the first crystal structure of mature lysostaphin and two structures of its isolated catalytic domain at 3.5, 1.78 and 1.26 Å resolution, respectively. The structure of the mature active enzyme confirms its expected organization into catalytic and cell-wall-targeting domains. It also indicates that the domains are mobile with respect to each other because of the presence of a highly flexible peptide linker. The high-resolution structures of the catalytic domain provide details of Zn2+ coordination and may serve as a starting point for the engineering of lysostaphin variants with improved biotechnological characteristics. [ABSTRACT FROM AUTHOR]

Aims: The development of technology for preparing chitosan-protein scaffolds loaded with lysostaphin, which potentially could be used as dressing for wound treatment and soft tissue infections caused by Staphylococcus aureus. Methods and Results: The unique technology of chitosan solubilization using gaseous CO2 instead of organic or inorganic acids was used for the incorporation of lysostaphin, the enzyme that exhibits bactericidal activity against staphylococci, within the structure of chitosan-protein sponges. The developed chitosan-protein scaffolds loaded with lysostaphin revealed high antistaphylococcal activity, which has been confirmed with a large (n = 143) collection of clinical (skin and wound infections) and animal (bovine mastitis) isolates of these bacteria, including MRSA. No change of bactericidal activity of the lyophilized materials has been observed during half-year storage at 4°C. Conclusions: The developed materials are potential candidates for preparing biologically active, antistaphylococcal wound dressing materials. Significance and Impact of the Study: Staphylococci belong to the most popular and most burdensome aetiological factors of wound and soft tissues infections. The developed chitosan-protein scaffolds loaded with lysostaphin could be a possible solution to problems associated with treatment of these infections. [ABSTRACT FROM AUTHOR]

Lysostaphin is an enzyme with bactericidal activity againstStaphylococcus aureusand other staphylococcal species. In spite of many advantages and promising results of preliminary research, the enzyme is still not widely used in medicine, veterinary medicine, or as a food preservative. One of the most important factors limiting application of the enzyme in clinical or technological practice is the high cost of its production. In this study we have determined the optimal conditions for lysostaphin production in a 5-L batch bioreactor. The enzyme production was based on a heterologous,Escherichia coliexpression system designated as pBAD2Lys and constructed earlier in our laboratory. An evident influence of physicochemical conditions of the process (areation, pH and temperature) and composition of the growing media on the amount and activity of produced enzyme was noticed. Efficiency of production of about 13,000 U/L has been achieved in the optimal conditions of the production process: low aeration (400 rpm of mechanical stirrer), pH 6, and temperature 37°C in classical LB medium. Further, about twofold improvement in the production efficiency of the enzyme was achieved as a result of modification of composition of growing media. Finally, more than 80,000 units of lysostaphin were obtained from one (batch) bioreactor with 3 L of culture ofE. coliTOP10F’ transformed with pBAD2Lys plasmid. To the best of our knowledge, this is the most efficient method of production of recombinant lysostaphin inE. coliexpression systems described to date. [ABSTRACT FROM PUBLISHER]

Lysostaphin represents a promising therapeutic agent for the treatment of staphylococcal infections, in particular those of methicillin-resistant Staphylococcus aureus (MRSA). However, conventional expression systems for the enzyme suffer from various limitations, and there remains a need for an efficient and cost-effective production process to facilitate clinical translation and the development of nonmedical applications. While Pichia pastoris is widely used for high-level production of recombinant proteins, there are two major barriers to the production of lysostaphin in this industrially relevant host: lack of expression from the wild-type lysostaphin gene and aberrant glycosylation of the wild-type protein sequence. The first barrier can be overcome with a synthetic gene incorporating improved codon usage and balanced A+T/G+C content, and the second barrier can be overcome by disrupting an N-linked glycosylation sequon using a broadened choice of mutations that yield aglyscosylated and fully active lysostaphin. The optimized lysostaphin variants could be produced at approximately 500 mg/liter in a small-scale bioreactor, and 50% of that material could be recovered at high purity with a simple 2-step purification. It is anticipated that this novel high-level expression system will bring down one of the major barriers to future development of biomedical, veterinary, and research applications of lysostaphin and its engineered variants. [ABSTRACT FROM AUTHOR]

The effect of enzymatic and physical modifications of the surface of two different strains from lactic acid bacteria, Lactobacillus rhamnosus (LGG) and Lactobacillus delbruekii subs. lactis ATCC 4797 (LBD), to stabilize medium-chain triglyceride (MCT) oil based Pickering emulsions were investigated. A section of cell wall degrading enzymes, lysozyme from chicken egg white and human, lysostaphin, mutanolysin from Streptomyces globisporus and proteinase k and the hydrophobic protein zein were used for enzymatic and physical surface modifications. Cell surface modifications were characterized by optical microscopy, scanning electron cryo-microscopy (Cryo-SEM), transmission electron microscopy (TEM), microbial adhesion to hexadecane (MATH) test and zeta potential measurements. The modified cell hydrophobicity in terms of MATH values were increased (around four times) by the enzymatic and physical modifications for LBD and LGG compared to the control. Emulsions stabilized by modified bacterial cells showed higher stability in comparison with unmodified samples, especially for the samples modified with chicken egg lysozyme. Confocal microscopy revealed that the modified bacterial cells were absorbed at the interface between oil and water and preventing the oil particles from coalescence. Thus, modified bacterial cells can be used to formulate food-grade stable Pickering emulsions. Such Pickering emulsions can potentially be clean label alternatives to replace the conventional emulsion preparations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Lysostaphin is a Staphylococcus aureusspecific bacteriolytic enzyme that recognizes and cleaves the pentaglycine linkage of peptidoglycan layers in the cell wall of S. aureus. Its target-specific lytic property to the staphylococcal cell wall has attracted interest for the application of lysostaphin as natural antibiotics against S. aureus. However, studies that consider its applicability in the detection of S. aureus have not been reported yet. Based on this, lysostaphin-mediated peptidoglycan fragmentation principles were implemented in the label-free immunoassay of S. aureus. In this study, peptidoglycan layers of S. aureus were fragmented by lysostaphin and the obtained fragments were quantitatively analyzed by electrochemical impedance spectroscopy (EIS). A linear relationship between the logarithmic value of the S. aureus concentration and the increase in the EIS signal was acquired in a range between 10 and 10 colony-forming units (CFU)/mL. Compared to the whole cell-based analysis, the developed lysostaphin-mediated assay for S. aureus showed a 90% increase in sensitivity because the fragments can interact with the immunoanalytical surface more efficiently than the whole cell. In contrast to previous EIS-based bacterial immunoassays that utilize complex electrodes and/or additional signaling probes such as labeled antibodies for signal amplification, we could detect signals with high sensitivity on conventional electrodes without any signaling probes. In addition, by using the developed method, S. aureus was selectively determined in bacterial samples that also contained 10 CFU/mL of Escherichia coli and Corynebacterium glutamicum. [ABSTRACT FROM AUTHOR]

Objectives To evaluate the in vivo effectiveness of a combination treatment containing ranalexin (a natural antimicrobial peptide) and lysostaphin (an antistaphylococcal endopeptidase) for reducing nasal burden of methicillin-resistant Staphylococcus aureus (MRSA). Methods The community-acquired MRSA strain S. aureus NRS384 (USA300-0114) was used in the present study because it is commonly isolated from human nares and it established consistent and reproducible colonization of cotton rat nares. This model was used to evaluate the efficacy of ranalexin/lysostaphin gels (0.1%–1% w/v; administered intranasally once or once per day for 3 consecutive days) for reducing nasal MRSA burden. Control animals were administered vehicle gel only (0.5% hydroxypropyl methylcellulose) or 2% mupirocin, which is used clinically for nasal decolonization of MRSA. Nasal MRSA burden was assessed at 192 h post-inoculation, which was at least 72 h after the final treatment had been administered. An additional study assessed the efficacy of 0.1% ranalexin/lysostaphin against a mupirocin-resistant MRSA strain (MUP20), which had been selected by serial passage of S. aureus NRS384 through subinhibitory concentrations of mupirocin. Results Gels containing 0.1% ranalexin/lysostaphin consistently reduced median nasal burden of MRSA to an extent similar to or greater than 2% mupirocin. Treatment with 0.1% ranalexin/lysostaphin was also effective against the MUP20 strain. There was evidence for only minimal irritancy in cotton rat nares administered three doses of 0.1% ranalexin/lysostaphin, suggesting that this agent is suitable for short-course therapy such as is employed currently for nasal decolonization with mupirocin. Conclusions Ranalexin/lysostaphin could serve as an alternative to mupirocin for nasal decolonization of MRSA. [ABSTRACT FROM PUBLISHER]

P128 is a chimeric anti-staphylococcal protein having a catalytic domain from a Staphylococcus bacteriophage K tail associated structural protein and a cell wall targeting domain from the Staphylococcus bacteriocin-lysostaphin. In this study, we disclose additional properties of P128 and compared the same with lysostaphin. While lysostaphin was found to get inactivated by heat and was inactive on its parent strain S. simulans biovar staphylolyticus, P128 was thermostable and was lytic towards S. simulans biovar staphylolyticus demonstrating a difference in their mechanism of action. selected mutation studies of the catalytic domain of P128 showed that arginine and cysteine, at 40th and 76th positions respectively, are critical for the staphylolytic activity of P128, although these amino acids are not conserved residues. In comparison to native P128, only the R40s mutant (P301) was catalytically active on zymogram gel and had a similar secondary structure, as assessed by circular dichroism analysis and in silico modeling with similar cell binding properties. Mutation of the arginine residue at 40th position of the P128 molecule caused dramatic reduction in the Vmax (ΔOD600 [mg/min]) value (nearly 270 fold) and the recombinant lysostaphin also showed lesser Vmax value (nearly 1.5 fold) in comparison to the unmodified P128 protein. The kinetic parameters such as apparent Km (Km APP) and apparent Kcat (Kcat APP) of the native P128 protein also showed significant differences in comparison to the values observed for P301 and lysostaphin. [ABSTRACT FROM AUTHOR]

Abstract: Staphylococcus simulans biovar staphylolyticus contains five plasmids, designated pACK1–pACK5. Recently, the nucleotide sequences of three of these plasmids, pACK1, pACK3, and pACK4, were reported. In order to complete the characterization of these five plasmids, the nucleotide sequences of the two remaining plasmids, pACK2 (37683bp) and pACK5 (3191bp), were determined. pACK5 is comprised of two regions, one with 85% identity at the nucleotide level to a region of pWBG1773 and another region with an ORF that shares no significant similarity to sequences previously described in GenBank. pACK2 encodes proteins for cadmium resistance and enhanced biofilm formation. The similarities at the nucleotide level among regions of the plasmids of S. simulans bv. staphylolyticus suggest that these plasmids have undergone multiple intermolecular rearrangements. [Copyright &y& Elsevier]

Lysostaphin (LYS) is an anti-staphylococcal prokaryotic polypeptide that has been used to avoidStaphylococcus aureusmastitis through transgenic or viral vector approaches exogenously expressed in dairy animals. However, glycosylation of lysostaphin expressed in mammalian cells results in a loss of bioactivity. Until now, the mechanism of site-specific glycosylation of lysostaphin causing this loss of bioactivity remains unknown. An immortalized caprine mammary epithelial cell line (CMEC-08-D) was used to study recombinant lysostaphin fused with goatβ-casein, goat lactoferrin (LF) or prokaryotic signal peptides. These constructs were separately ectopically expressed in CMEC-08-D. Results of site-directed mutagenesis show that Asn125but not Asn232is the exact glycosylation site of lysostaphin expressed in CMEC-08-D. In addition, the effect of glycosylation of lysostaphin on its staphylolytic activity was identified through bacterial plate assay. The data indicated that wild type and mutated N232Q-lysostaphin (Asn232to Gln232substitution) lacked staphylolytic activity. In contrast, mutated N125Q (Asn125to Gln125substitution) and N125Q/N232Q-lysostaphin possessed staphylolytic activity. On the other hand, all mutated lysostaphin showed no change in binding ability toS. aureus. This reveals that N-glycosylation at Asn125of lysostaphin expressed in a eukaryotic system greatly decreases lysostaphin bacteriolytic activity but does not affect its binding ability toS. aureus. [ABSTRACT FROM PUBLISHER]

Bacterial cell walls are the guards of cell integrity. They are composed of peptidoglycan that provides rigidity to sustain internal turgor and ensures isolation from the external environment. In addition, they harbor the enzymatic machinery to secure cell wall modulations needed throughout the bacterial lifespan. The main players in this process are peptidoglycan hydrolases, a large group of enzymes with diverse specificities and different mechanisms of action. They are commonly, but not exclusively, found in prokaryotes. Although in most cases, these enzymes share the same molecular function, namely peptidoglycan hydrolysis, they are leveraged to perform a variety of physiological roles. A well-investigated family of peptidoglycan hydrolases is M23 peptidases, which display a very conserved fold, but their spectrum of lytic action is broad and includes both Gram- positive and Gram- negative bacteria. In this review, we summarize the structural, biochemical, and functional studies concerning the M23 family of peptidases based on literature and complement this knowledge by performing large-scale analyses of available protein sequences. This review has led us to gain new insight into the role of surface charge in the activity of this group of enzymes. We present relevant conclusions drawn from the analysis of available structures and indicate the main structural features that play a crucial role in specificity determination and mechanisms of latency. Our work systematizes the knowledge of the M23 family enzymes in the context of their unique antimicrobial potential against drug-resistant pathogens and presents possibilities to modulate and engineer their features to develop perfect antibacterial weapons., Competing Interests: IS holds the patent EP2699254 “The method of proteolysis, peptidase, the composition to be used as a bacteriostatic and bactericidal agent, the mixture and the applications of the active form of LytM from S. aureus or derivatives thereof.” The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Razew, Schwarz, Mitkowski, Sabala and Kaus-Drobek.)

Cutibacterium acnes is a pathogenic bacterium that cause inflammatory diseases of the skin and intervertebral discs. The immune activation induced by C. acnes requires multiple cellular responses in the host. Silkworm, an invertebrate, generates melanin by phenoloxidase upon recognizing bacterial or fungal components. Therefore, the melanization reaction can be used as an indicator of innate immune activation. A silkworm infection model was developed for evaluating the virulence of C. acnes, but a system for evaluating the induction of innate immunity by C. acnes using melanization as an indicator has not yet been established. Here we demonstrated that C. acnes rapidly causes melanization of the silkworm hemolymph. On the other hand, Staphylococcus aureus, a gram-positive bacterium identical to C. acnes, does not cause immediate melanization. Even injection of heat-killed C. acnes cells caused melanization of the silkworm hemolymph. DNase, RNase, and protease treatment of the heat-treated C. acnes cells did not decrease the silkworm hemolymph melanization. Treatment with peptidoglycan-degrading enzymes, such as lysostaphin and lysozyme, however, decreased the induction of melanization by the heat-treated C. acnes cells. These findings suggest that silkworm hemolymph melanization may be a useful indicator to evaluate innate immune activation by C. acnes and that C. acnes peptidoglycans are involved in the induction of innate immunity in silkworms., Competing Interests: The authors have declared that no competing interests exist.

Lysostaphin is a potent bacteriolytic enzyme with endopeptidase activity against the common pathogen Staphylococcus aureus . By digesting the pentaglycine crossbridge in the cell wall peptidoglycan of S. aureus including the methicillin-resistant strains, lysostaphin initiates rapid lysis of planktonic and sessile cells (biofilms) and has great potential for use in agriculture, food industries, and pharmaceutical industries. In the past few decades, there have been tremendous efforts in potentiating lysostaphin for better applications in these fields, including engineering of the enzyme for higher potency and lower immunogenicity with longer-lasting effects, formulation and immobilization of the enzyme for higher stability and better durability, and recombinant expression for low-cost industrial production and in situ biocontrol. These achievements are extensively reviewed in this article focusing on applications in disease control, food preservation, surface decontamination, and pathogen detection. In addition, some basic properties of lysostaphin that have been controversial and only elucidated recently are summarized, including the substrate-binding properties, the number of zinc-binding sites, the substrate range, and the cleavage site in the pentaglycine crossbridge. Resistance to lysostaphin is also highlighted with a focus on various mechanisms. This article is concluded with a discussion on the limitations and future perspectives for the actual applications of lysostaphin.

Abstract: Specific bacteria imaging is highly desirable in clinical diagnostics. Probes enabling rapid and specific diagnostics of bacteria are limited. Current clinical infection diagnostics is time consuming and invasive, relies on microbiological cultures. We investigated the potential of Lysostaphin as a specific probe to label staphylococci in a new labeling protocol. We used azido (N3)-modified Lysostaphin-N3 and DIBO-dye in a two-step bacteria-labeling protocol. N3 and DIBO (di-benzocyclooctyne) are the counterparts of the “click” chemistry. In the first step, Lysostaphin-N3 binds specifically to Staphylococcus aureus. In the second step, N3 clicks to DIBO thus achieving the selective for S. aureus labeling. Such a two-step approach effectively distinguishes S. aureus from Escherichia coli; non-toxic and was proven to work in vivo. The two-step labeling protocol is a promising approach for diagnostic imaging of staphylococcal infections in clinical settings. [Copyright &y& Elsevier]

Staphylococci cause bovine mastitis, with Staphylococcus aureus being responsible for the majority of the mastitis-based losses to the dairy industry (up to $2 billion/annum). Treatment is primarily with antibiotics, which are often ineffective and potentially contribute to resistance development. Bacteriophage endolysins (peptidoglycan hydrolases) present a promising source of alternative antimicrobials. Here we evaluated two fusion proteins consisting of the streptococcal λSA2 endolysin endopeptidase domain fused to staphylococcal cell wall binding domains from either lysostaphin (λSA2-E-Lyso-SH3b) or the staphylococcal phage K endolysin, LysK (λSA2-E-LysK-SH3b). We demonstrate killing of 16 different S. aureus mastitis isolates, including penicillin-resistant strains, by both constructs. At 100 μg/ml in processed cow milk, λSA2-E-Lyso-SH3b and λSA2-E-LysK-SH3b reduced the S. aureus bacterial load by 3 and 1 log units within 3 h, respectively, compared to a buffer control. In contrast to λSA2-E-Lyso-SH3b, however, λSA2-E-LysK-SH3b permitted regrowth of the pathogen after 1 h. In a mouse model of mastitis, infusion of 25 μg of λSA2-E-Lyso-SH3b or λSA2-E-LysK-SH3b into mammary glands reduced S. aureus CFU by 0.63 or 0.81 log units, compared to >2 log for lysostaphin. Both chimeras were synergistic with lysostaphin against S. aureus in plate lysis checkerboard assays. When tested in combination in mice, λSA2-E-LysK-SH3b and lysostaphin (12.5 μg each/gland) caused a 3.36-log decrease in CFU. Furthermore, most protein treatments reduced gland wet weights and intramammary tumor necrosis factor alpha (TNF-α) concentrations, which serve as indicators of inflammation. Overall, our animal model results demonstrate the potential of fusion peptidoglycan hydrolases as antimicrobials for the treatment of S. aureus-induced mastitis. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a common constituent of the bacterial community inhabiting the airways of persons with cystic fibrosis (CF). Culture-independent studies have shown that this species is often present in relatively high abundance and would therefore be expected to exert a pronounced effect on measures of CF airway bacterial community structure. We investigated the impact of DNA extraction method on pyrosequencing-based measures of Staphylococcus abundance and bacterial community structure in 17 sputum samples from five CF patients. Staphylococcus was detected in fewer samples when DNA was extracted using a standard bacterial lysis method compared to when DNA was extracted using a lysis buffer amended with lysostaphin and lysozyme. The standard lysis method resulted in significantly lower measures of Staphylococcus relative abundance and higher levels of community diversity, richness, and evenness compared to the lysostaphin-lysozyme modified method. Measures of community dynamics in serial sputum samples from the same individual were nevertheless highly concordant between the two DNA extraction methods. These results illustrate the impact of DNA preparation method on measures of Staphylococcus abundance and bacterial community structures in studies of the airways microbiota in CF. [ABSTRACT FROM AUTHOR]

Background. Naturally occurring antimicrobial peptides are possibly the “next frontier” in infection prevention. Binding them to mesh could reduce the rate of mesh infections. This study identifies an antimicrobial agent capable of significant antibacterial activity when bound to mesh. Methods. Lysozyme, human beta defensin (HBD-3), human cathelicidin (LL-37), and lysostaphin were adsorbed to polypropylene mesh at various concentrations. Treated meshes were placed in a suspension of 1 × 106Staphylococcus aureus. Antibacterial action was monitored by turbidimetric assay, fluorescent imaging, and a colony counting method. Results. A very high rate of lysis of S aureus cells was observed in the lysostaphin-treated group as measured by optical density; none survived as seen on colony count assays. Optical density for mesh coated with lysozyme, HBD-3, and LL-37 did not differ from untreated controls, with 100% survival rates by colony counts. Conclusion. Lysostaphin had superior antibacterial activity following adsorption to mesh. [ABSTRACT FROM PUBLISHER]

Background: Lysostaphin (LS), a naturally occurring Staphylococcal endopeptidase, has the ability to penetrate biofilm, and has been identified as a potential antimicrobial to prevent mesh infection. The goals of this study were to determine if LS adhered to porcine mesh (PM) can impact host survival, reduce the risk of long-term PM infection, and to analyze lysostaphin bound PM (LS-PM) mesh-fascial interface in an infected field. Methods: Abdominal onlay PMs measuring 3 × 3 cm were implanted in select groups of rats (n = 75). Group assignments were based on bacterial inoculum and presence of LS on mesh. Explantation occurred at 60 d. Bacterial growth and mesh-fascial interface tensile strength were analyzed. Standard statistical analysis was performed. Results: Only one out of 30 rats with bacterial inoculum not treated with LS survived. All 30 LS treated rats survived and had normal appearing mesh, including 20 rats with a bacterial inoculum (106 and 108 CFU). Mean tensile strength for controls and LS and no inoculum samples was 3.47 ± 0.86N versus 5.0 ± 1.0N (P = 0.008). LS groups inoculated with 106 and 108 CFU exhibited mean tensile strengths of 4.9 ± 1.5 N and 6.7 ± 1.6 N, respectively (P = 0.019 and P < 0.001 compared with controls). Conclusion: Rats inoculated with S. aureus and not treated with LS had a mortality of 97%. By comparison, LS treated animals completely cleared S. aureus when challenged with bacterial concentrations of 1 × 106 and 1 × 108 with maintenance of mesh integrity at 60 d. These findings strongly suggest the clinical use of LS-treated porcine mesh in contaminated fields may translate into more durable hernia repair. [ABSTRACT FROM AUTHOR]

Aims: To develop an efficient purification strategy for the bacteriophage lysin CHAPK. To evaluate its antibacterial spectrum, enzymatic properties, optimal reaction conditions and lytic activity against live Staphlyococcus aureus. Methods and Results: Recombinant CHAPK was purified to homogeneity by cation exchange chromatography, with yields of up to 10 mg from 1 l of Escherichia coli culture. The lytic spectrum of CHAPK includes all staphylococcal species and also members of the genera Micrcococcus, Streptococcus, Nesterenkonia, Arthrobacter, Leuconostoc and Carnobacterium. The enzyme was active from pH 6 to 11 with an optimum activity at pH 9, from 5 to 40°C, with an optimum activity at 15°C. When cell lysis by CHAPK and lysostaphin was compared over a concentration range of 2·5-10 μg ml−1 using live Staph. aureus for 5 min at 37°C, CHAPK gave rise to greater turbidity reduction indicating that it works more rapidly than lysostaphin. Conclusions: This study describes in detail the purification and characteristics of the novel phage-derived enzyme CHAPK demonstrating that it has excellent biochemical properties as an anti-staphylococcal agent. Significance and Impact of the Study: Currently, there is a need for new antimicrobial agents due to the increasing worldwide prevalence of antibiotic resistance. Our findings demonstrate the potential for development of CHAPK as an alternative therapeutic against pathogenic staphylococci including MRSA. [ABSTRACT FROM AUTHOR]

Aims: To develop an efficient purification strategy for the bacteriophage lysin CHAPK. To evaluate its antibacterial spectrum, enzymatic properties, optimal reaction conditions and lytic activity against live Staphlyococcus aureus. Methods and Results: Recombinant CHAPK was purified to homogeneity by cation exchange chromatography, with yields of up to 10 mg from 1 l of Escherichia coli culture. The lytic spectrum of CHAPK includes all staphylococcal species and also members of the genera Micrcococcus, Streptococcus, Nesterenkonia, Arthrobacter, Leuconostoc and Carnobacterium. The enzyme was active from pH 6 to 11 with an optimum activity at pH 9, from 5 to 40°C, with an optimum activity at 15°C. When cell lysis by CHAPK and lysostaphin was compared over a concentration range of 2·5-10 μg ml−1 using live Staph. aureus for 5 min at 37°C, CHAPK gave rise to greater turbidity reduction indicating that it works more rapidly than lysostaphin. Conclusions: This study describes in detail the purification and characteristics of the novel phage-derived enzyme CHAPK demonstrating that it has excellent biochemical properties as an anti-staphylococcal agent. Significance and Impact of the Study: Currently, there is a need for new antimicrobial agents due to the increasing worldwide prevalence of antibiotic resistance. Our findings demonstrate the potential for development of CHAPK as an alternative therapeutic against pathogenic staphylococci including MRSA. [ABSTRACT FROM AUTHOR]

Nasal colonization of Staphylococcus aureus ( S.aureus) is known as a significant risk factor for nosocomial infections, and clearance of its nasal colonization greatly reduces the risk. In the present study the preparation and characterizations of the chitosan-o/w cream incorporated with lysostaphin (CCL) were described. It showed that the concentration of incorporated lysostaphin had a direct relationship with its release behavior from the cream. It was rapid at 2 and 3.5 mg lysostaphin/g cream and of a more sustained pattern at 5 mg lysostaphin/g cream. Efficacy of lysostaphin released from the CCL cream to inhibit S.aureus growth was higher than that of lysostaphin delivery routinely treated, as demonstrated by the reduction of the mucociliary transport rate (MTR) in contrast to the control graphite particles ( p < 0.05). Therefore, it is concluded that drug delivery by the CCL holds its potential as a local nasal anti- S.aureus infection. [ABSTRACT FROM AUTHOR]