Effects of recombinant lysostaphin on cytotoxicity and interleukin-8 level in normal human epidermal keratinocytes cell line

The normal human epidermal keratinocyte (NHEK) was used to evaluate the cytotoxicity of recombinant lysostaphin. As determined with the Neutral Red (NR) cytotoxicity assay, the midpoint toxicity value (NR50) after 48 h exposure was 16 ± 0.4 g lysostaphin/l. Lysostaphin cytotoxicity effect is much less than the surface active agent, sodium laurate. However, the NR50 value after 48-h exposure was 1.9 ± 0.02 g/l for S. aureus lysate derived from the bacterial lytic action of lysostaphin. A linear increase in interleukin-8 (IL-8) level in NHEK cells from resting levels of 65 ± 3 pg/ml to peak of 760 ± 15 pg/ml during the first 9 hours was noted for the cells treated with 800 mg lysostaphin/l. S. aureus lysate has the same effect on the induction of IL-8 levels. The induced rises in IL-8 were lysostaphin and S. aureus lysate concentration dependence. © Rapid Science Ltd. 1998     

Lysostaphin lysis procedure for detection of Staphylococcus aureus by the firefly bioluminescent ATP method

The objective of this study was to examine the use of lysostaphin as an ATP-extracting agent for the estimation of Staphylococcus aureus cell number by a rapid bioluminescent ATP method. The results of the study showed that lysostaphin (22 U/ml) was able to lyse most of the S. aureus cells (greater than 99.9%) at room temperature in 1 min; ATP of S. aureus cells extracted by the lysostaphin lysis procedure was stable for 24 h in the presence of EDTA; there was a linear relationship between the ATP content and the number of S. aureus cells (ranging from 10(4) to 10(6) CFU/ml); and the lysis of S. aureus cells by lysostaphin allowed estimation of the number of S. aureus cells in mixed cultures and in meat samples.     

Lysostaphin lysis procedure for detection of Staphylococcus aureus by the firefly bioluminescent ATP method.

The objective of this study was to examine the use of lysostaphin as an ATP-extracting agent for the estimation of Staphylococcus aureus cell number by a rapid bioluminescent ATP method. The results of the study showed that lysostaphin (22 U/ml) was able to lyse most of the S. aureus cells (greater than 99.9%) at room temperature in 1 min; ATP of S. aureus cells extracted by the lysostaphin lysis procedure was stable for 24 h in the presence of EDTA; there was a linear relationship between the ATP content and the number of S. aureus cells (ranging from 10(4) to 10(6) CFU/ml); and the lysis of S. aureus cells by lysostaphin allowed estimation of the number of S. aureus cells in mixed cultures and in meat samples.     

Direct observation of Staphylococcus aureus cell wall digestion by lysostaphin

The advent of Staphylococcus aureus strains that are resistant to virtually all antibiotics has increased the need for new antistaphylococcal agents. An example of such a potential therapeutic is lysostaphin, an enzyme that specifically cleaves the S. aureus peptidoglycan, thereby lysing the bacteria. Here we tracked over time the structural and physical dynamics of single S. aureus cells exposed to lysostaphin, using atomic force microscopy. Topographic images of native cells revealed a smooth surface morphology decorated with concentric rings attributed to newly formed peptidoglycan. Time-lapse images collected following addition of lysostaphin revealed major structural changes in the form of cell swelling, splitting of the septum, and creation of nanoscale perforations. Notably, treatment of the cells with lysostaphin was also found to decrease the bacterial spring constant and the cell wall stiffness, demonstrating that structural changes were correlated with major differences in cell wall nanomechanical properties. We interpret these modifications as resulting from the digestion of peptidoglycan by lysostaphin, eventually leading to the formation of osmotically fragile cells. This study provides new insight into the lytic activity of lysostaphin and offers promising prospects for the study of new antistaphylococcal agents.     

Effect of D-alanylation of (lipo)teichoic acids of Staphylococcus aureus on host secretory phospholipase A2 action before and after phagocytosis by human neutrophils

Invading bacteria such as Staphylococcus aureus induce mobilization of professional phagocytes (e.g., neutrophils) and extracellular antibacterial proteins (e.g., group IIA phospholipase A2 (gIIA PLA2)). Accumulation of gIIA PLA2 in inflammatory fluids confers potent extracellular antistaphylococcal activity and at lower concentrations promotes bacterial phospholipid degradation during phagocytosis of S. aureus by human neutrophils. D-alanylation of (lipo) teichoic acids of S. aureus increases bacterial resistance to gIIA PLA2 approximately 100-fold, raising the possibility that the resistance of ingested S. aureus to related gV and gX secretory PLA2 present in human neutrophil granules depends on D-alanylation mediated by the dlt operon. However, we show that isogenic wild-type and dltA S. aureus are equally resistant to gV/X PLA2 during phagocytosis and when exposed to the purified enzymes. The fates of wild-type and dltA S. aureus exposed to serum and human neutrophils differed significantly only when extracellular gIIA PLA2 was also present before phagocytosis. The extreme potency of the gIIA PLA2 toward dltA S. aureus suggests that even small amounts of this extracellular enzyme mobilized early in inflammation could contribute substantially to the overall cytotoxicity of acute inflammatory exudates toward S. aureus when D-alanylation of (lipo)teichoic acids is limiting.     

<Emphasis Type="Italic">Staphylococcus simulans</Emphasis> recombinant lysostaphin: Production,purification, and determination of antistaphylococcal activity

Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by SigmaAldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.     

Photodynamic effect of protoporphyrin diarginate (PPArg2) on methicillin-resistant Staphylococcus aureus and human dermal fibroblasts

The worldwide rise in the antibiotic resistance of bacteria forces the development of alternative antimicrobial treatments. A potential approach is photodynamic inactivation (PDI). The aim of the present study was to determine the phototoxicity of protoporphyrin diarginate (PPArg(2)) against methicillin-resistant Staphylococcus aureus and human dermal fibroblasts. Different concentrations (0 to 20 microM) of PPArg(2) and light dose of 6 J cm(-2) were tested. Cell viability was evaluated using the methylthiazoletetrazolium (MTT) assay. Incubation with 10 microM followed by illumination yielded a 3.6 log(10)-unit reduction in the viable count for Staphylococcus aureus. At the same experimental conditions, only 22.5% of the fibroblasts were photoinactivated. Protoporphyrin diarginate at concentrations up to 20 microM demonstrated no toxicity towards S. aureus or fibroblasts when not irradiated. These results suggest that the protoporphyrin diarginate exerts a high bactericidal effect against methicillin-resistant S. aureus strain without harming eukaryotic cells.     

Bacteriostatic activity of human lactoferrin against Staphylococcus aureus is a function of its iron-binding properties and is not influenced by antibiotic resistance

The in vitro antistaphylococcal activity of lactoferrin and the antibiotic resistance of clinical Staphylococcus aureus isolates obtained from three different sites of infection were examined. Antibiotic, but not lactoferrin resistance correlated with selective antibiotic pressure, and nosocomial and most community isolates were antibiotic resistant, whereas only a third of each group was resistant to lactoferrin. The antimicrobial activity of lactoferrin, both in defined medium and in normal human plasma serum, was dependent upon its ferrochelating properties. Therapeutic approaches based on the use of ferrochelating agents such as lactoferrin combined with antimicrobial drugs may help to counteract the reduced efficacy of current antibiotics.     

Effect of tumor necrosis factor-alpha on intracellular Staphylococcus aureus in vascular endothelial cells.

Although tumor necrosis factor-alpha (TNF-alpha) is an important host factor against intracellular bacteria, little is known about the effect of TNF-alpha on the persistence of intracellular Staphylococcus aureus in vascular endothelial cells. It was investigated whether recombinant human TNF-alpha influences the survival of intracellular S. aureus (ATCC 29213) in human umbilical vein endothelial cells (HUVEC) under a condition with an antistaphylococcal agent, and its mechanism. The HUVECs were incubated with TNF-alpha, oxacillin, or both in 24-well plates for up to 48 h following internalization of S. aureus (10(6) CFU well(-1)) into HUVECs for 1 h. TNF-alpha (1 ng mL(-1)) significantly reduced the number of intracellular S. aureus in HUVECs, and TNF-alpha plus oxacillin eliminated more intracellular S. aureus in HUVEC than oxacillin alone. The LDH viability assay and quantification of apoptosis using photometric enzyme-immunoassay showed that TNF-alpha preferentially induced cell death and apoptosis of HUVECs infected with S. aureus compared with noninfected HUVECs. These results indicate that TNF-alpha helps antistaphylococcal antibiotics to eliminate intracellular S. aureus in vascular endothelial cells, partly because TNF-alpha preferentially induces apoptosis of endothelial cells infected by S. aureus.     

Lysostaphin in Experimental Renal Infections

By use of a renal staphylococcal infection model in mice, single intravenous doses of lysostaphin ranging from 1.56 to 50 mg/kg were effective in: (i) controlling the staphylococcal population of kidneys, (ii) reducing the mortality rate, and (iii) clearing high numbers of kidneys of infection. Semisynthetic penicillins and other antistaphylococcal antibiotics given in the same manner did not have significant activity. Only by the administration of a long-acting, depot form of penicillin (Bicillin) could results comparable to those seen with lysostaphin be obtained. The results of this study suggest that lysostaphin may be useful in staphylococcal septicemias in preventing the establishment of new foci of infection.     

Bacteriostatic activity of serum against staphylococci

Cybulska, Janina (State Institute of Hygiene, Warsaw, Poland), and J. Jeljaszewicz. Bacteriostatic activity of serum against staphylococci. J. Bacteriol. 91:953-962. 1966.-Antistaphylococcal activity of normal serum against strains exhibiting various patterns of coagulase, clumping-factor, and staphylokinase production is not connected with the presence of these factors. Purified coagulase does not influence this property of serum. Coagulase-negative strains with clumping-factor activity grow in normal serum as typical pathogenic staphylococci. Serum bacteriostatic activity against staphylococci may be reversed by several nonspecific factors, such as sterile broth, supernatant fluids of coagulase-negative strains, and ammonium sulfate precipitates of culture supernatant fluids of various staphylococci. Immune sera with a high agglutinating titer for staphylococcal cells do not prevent growth of serum-resistant strains; serum-susceptible strains are inhibited as in normal serum control. Activation or blocking of the serum fibrinolytic system does not influence serum bacteriostatic activity. The growth rate of serum-resistant strains is identical in serum and in Todd-Hewitt broth; serum-susceptible strains are inhibited to the inoculum level, but decreases and increases in viable count are noted during a 24-hr observation period. Observations made with sera of 10 animal species clearly demonstrated differences in serum bacteriostatic activity, mouse serum being completely noninhibitory and cat serum only weakly inhibitory. The technique of quantitative determination of serum susceptibility of staphylococci is described, and the importance of serum antistaphylococcal activity in vitro is discussed. Experimental staphylococcal infection produced in rabbits by intravenous injection of different Staphylococcus aureus strains did not result in significant changes in serum antistaphylococcal activity. The technique of experimental infection used caused chronic infection, with a peak on the 14th day; this was proved by means of a newly developed 5'-nucleotidase test. At the same time, sera of infected animals exhibited slight inhibitory properties, which returned to initial values 1 week later. Infection was produced by strains recognized as nonpathogenic and was inhibited in vitro by sera from both normal and infected rabbits. It is concluded that antistaphylococcal activity of serum should be considered as an "in vitro" phenomenon, which seems to have no importance in defense mechanisms of rabbits infected intravenously with staphylococci.     

Bactericidal activity of the serum against Staphylococcus aureus grown in the presence of subinhibitory doses of cefamandole and gentamicin

Growth of S. aureus in the presence of subinhibitory concentrations of cefamandole alone (at 1/5 the MIC) or with gentamicin (both at 1/5 the MIC) produced large, globular cells with reduced viability (50%) as compared to untreated bacteria. Gentamicin (1/5 the MIC) did not modify bacteria and also reduced viability of inoculum (57% vs. controls). Cefamandole treated bacteria were more susceptible to the rat serum bactericidal activity then control bacteria. The growth of S. aureus in the presence of cefamandole plus gentamicin did not modify further bacterial susceptibility to serum opsonins. The data suggest that even at subinhibitory concentrations cefamandole may exert an antibacterial effect by acting in cooperation with serum factors.     

Target cell specificity of a bacteriocin molecule: a C-terminal signal directs lysostaphin to the cell wall of Staphylococcus aureus.

Microbial organisms secrete antibiotics that cause the selective destruction of specific target cells. Although the mode of action is known for many antibiotics, the mechanisms by which these molecules are directed specifically to their target cells hitherto have not been described. Staphylococcus simulans secretes lysostaphin, a bacteriolytic enzyme that cleaves staphylococcal peptidoglycans in general but that is directed specifically to Staphylococcus aureus target cells. The sequence element sufficient for the binding of the bacteriocin as well as of hybrid indicator proteins to the cell wall of S.aureus consisted of 92 C-terminal lysostaphin residues. Targeting to the cell wall of S.aureus occurred either when the hybrid indicator molecules were added externally to the bacteria or when they were synthesized and exported from their cytoplasm by an N-terminal leader peptide. A lysostaphin molecule lacking the C-terminal targeting signal was enzymatically active but had lost its ability to distinguish between S.aureus and S.simulans cells, indicating that this domain functions to confer target cell specificity to the bacteriolytic molecule.     

Neomycin and paromomycin inhibit 30S ribosomal subunit assembly in Staphylococcus aureus

A number of different antibiotics that prevent translation by binding to the 50S ribosomal subunit of bacterial cells have recently been shown to also prevent assembly of this subunit. Antibacterial agents affecting 30S particle activities have not been examined extensively for effects on small subunit formation. The aminoglycoside antibiotics paromomycin and neomycin bind specifically to the 30S ribosomal subunit and inhibit translation. These drugs were examined in Staphylococcus aureus cells to see whether they had a second inhibitory effect on 30S particle assembly. A 3H-uridine pulse and chase assay was used to examine the kinetics of subunit synthesis in the presence and absence of each antibiotic. 30S subunit formation was inhibited by both compounds. At 3 microg/mL each antibiotic reduced the rate of 30S formation by 80% compared with control cells. Both antibiotics showed a concentration-dependent inhibition of particle formation, with a lesser effect on 50S particle formation. For neomycin, the IC50 for 30S particle formation was equal to the IC50 for inhibition of translation. Both antibiotics reduced the viable cell number with an IC50 of 2 microg/mL. They also inhibited protein synthesis in the cells with different IC50 values (2.5 and 1.25 microg/mL). This is the second demonstration of 30S ribosomal subunit-specific antibiotics that prevent assembly of the small subunit.     

Cloning of lysozyme and lysostaphin genes of Staphylococcus aureus and their expression in Bacillus subtilis cells

The gene of microbial lysozyme (lyz) of S. aureus 118 and the gene of lysostaphin (lzf) of S. aureus RN 3239 were cloned and their expression in B. subtilis cells was shown. Lysozyme production in B. subtilis recombinant clone pLF14-Lyz, obtained as the result of cloning, was 2.5-fold greater than lysozyme production in S. aureus wild strain 118. Lysostaphin production in B. subtilis recombinant strain pLF14-Lzf which had inherited the cloned genes was approximately equal to lysostaphin production observed in S. aureus initial strain RN 3239. The production of lysozyme and lysostaphin in the cells of B. subtilis recombinant strains was observed at 30 degrees C and pH 5.5, while in S. aureus initial strains 118 and RN 3239 bacteria produced lysozyme and lysostaphin at 37 degrees C and pH 7.5 respectively.     

Antibacterial activity of hinokitiol against both antibiotic‐resistant and ‐susceptible pathogenic bacteria that predominate in the oral cavity and upper airways

Hinokitiol, a component of the essential oil isolated from Cupressaceae, possesses antibacterial and antifungal activities and has been used in oral care products. In this study, the antibacterial activities of hinokitiol toward various oral, nasal and nasopharyngeal pathogenic bacteria, including Streptococcus mutans, Streptococcus sobrinus, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Fusobacterium nucleatum, methicillin‐resistant and ‐susceptible Staphylococcus aureus, antibiotic‐resistant and ‐susceptible Streptococcus pneumoniae, and Streptococcus pyogenes were examined. Growth of all these bacterial strains was significantly inhibited by hinokitiol, minimal inhibitory concentrations of hinokitiol against S. mutans, S. sobrinus, P. gingivalis, P. intermedia, A. actinomycetemcomitans, F. nucleatum, methicillin‐resistant S. aureus, methicillin‐susceptible S. aureus, antibiotic‐resistant S. pneumoniae isolates, antibiotic‐susceptible S. pneumoniae, and S. pyogenes being 0.3, 1.0, 1.0, 30, 0.5, 50, 50, 30, 0.3–1.0, 0.5, and 0.3 μg/mL, respectively. Additionally, with the exception of P. gingivalis, hinokitiol exerted bactericidal effects against all bacterial strains 1 hr after exposure. Hinokitiol did not display any significant cytotoxicity toward the human gingival epithelial cell line Ca9‐22, pharyngeal epithelial cell line Detroit 562, human umbilical vein endothelial cells, or human gingival fibroblasts, with the exception of treatment with 500 μg/mL hinokitiol, which decreased numbers of viable Ca9‐22 cells and gingival fibroblasts by 13% and 12%, respectively. These results suggest that hinokitiol exhibits antibacterial activity against a broad spectrum of pathogenic bacteria and has low cytotoxicity towards human epithelial cells.     

Enhanced staphylolytic activity of the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 HydH5 virion-associated peptidoglycan hydrolase: fusions, deletions, and synergy with LysH5

Virion-associated peptidoglycan hydrolases have potential as antimicrobial agents due to their ability to lyse Gram-positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion-associated peptidoglycan hydrolase from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88. Full-length HydH5 and two truncated derivatives containing only the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain exhibited high lytic activity against live S. aureus cells. In addition, three different fusion proteins were created between lysostaphin and HydH5, each of which showed higher staphylolytic activity than the parental enzyme or its deletion construct. Both parental and fusion proteins lysed S. aureus cells in zymograms and plate lysis and turbidity reduction assays. In plate lysis assays, HydH5 and its derivative fusions lysed bovine and human S. aureus strains, the methicillin-resistant S. aureus (MRSA) strain N315, and human Staphylococcus epidermidis strains. Several nonstaphylococcal bacteria were not affected. HydH5 and its derivative fusion proteins displayed antimicrobial synergy with the endolysin LysH5 in vitro, suggesting that the two enzymes have distinct cut sites and, thus, may be more efficient in combination for the elimination of staphylococcal infections.     

Intracellular localization of Staphylococcus aureus within primary cultured mouse kidney cells.

Staphylococcus aureus Cowan I was incubated with monolayers of cells derived from several portions of mouse kidney, and found to be ingested by all types of the renal cells. Intracellular localization of S. aureus was determined by resistance of intracellular cocci against lysostaphin digestion and confirmed by electron microscopy. From renal medulla, three morphological variants of the hyperosmolarity-tolerant (HOT) cells were obtained. The rate of cocci-ingesting cells varied from 16.9% to 93.4% among these of the HOT cells at the end of 3-hr incubation. From renal cortex, three morphological variants of epithelial cells grew in medium RK-1. Among them, only the cells on the edge of colony ingested Cowan I, while the epithelial cells on the center of colony ingested few cocci. Transferred from medium RK-1 to MEM supplemented with 10% FBS, part of the cortical cells changed into fibroblast-like appearance and obtained the capacity to ingest Cowan I. This result may indicate the correlation between ingesting capacity and cellular morphology. From a glomerulus, epithelial (GE) cells and fibroblast-like (GF) cells were obtained. The GE cells ingested not only S. aureus Cowan I but Staphylococcus epidermidis and Staphylococcus saprophyticus after 30-min incubation, while the GF cells, like both of the HOT cells and the cortical cells, ingested only S. aureus. These results suggest a possibility that S. aureus is located within nonprofessional phagocytes during its infection and intracellular coccus plays an important role in its pathogenicity.     

Effect of tumor necrosis factor-α on intracellular Staphylococcus aureus in vascular endothelial cells

Although tumor necrosis factor-α (TNF-α) is an important host factor against intracellular bacteria, little is known about the effect of TNF-α on the persistence of intracellular Staphylococcus aureus in vascular endothelial cells. It was investigated whether recombinant human TNF-α influences the survival of intracellular S. aureus (ATCC 29213) in human umbilical vein endothelial cells (HUVEC) under a condition with an antistaphylococcal agent, and its mechanism. The HUVECs were incubated with TNF-α, oxacillin, or both in 24-well plates for up to 48 h following internalization of S. aureus (10⁶ CFU well⁻¹) into HUVECs for 1 h. TNF-α (1 ng mL⁻¹) significantly reduced the number of intracellular S. aureus in HUVECs, and TNF-α plus oxacillin eliminated more intracellular S. aureus in HUVEC than oxacillin alone. The LDH viability assay and quantification of apoptosis using photometric enzyme-immunoassay showed that TNF-α preferentially induced cell death and apoptosis of HUVECs infected with S. aureus compared with noninfected HUVECs. These results indicate that TNF-α helps antistaphylococcal antibiotics to eliminate intracellular S. aureus in vascular endothelial cells, partly because TNF-α preferentially induces apoptosis of endothelial cells infected by S. aureus .