Efficacy of sodium polyanethol sulfonate on herpes simplex virus-1 infection in vitro.

Objective: To investigate the effect of sodium polyanethol sulfonate (SPS) on herpes simplex virus type 1 (HSV-1) infection in vitro. Methods: Human corneal epithelial (HCE-T) cells and Vero cells were infected with HSV-1 [HSV-1 f strain, HSV-1f; HSV-1-H129 with green fluorescent protein (GFP) knock-in, HSV-1g]. SPS was added to the culture medium at various concentrations in time-of-addition assay. Experiments including photography of fluorescence in HSV-1g or plaque formation by HSV-1f, western blot assays, real-time RT-PCR assays, cytopathic effect inhibition assays, cytotoxicity assays, and viral absorption and penetration assays were performed to explore the antiviral effect and mechanism of the compounds. Results: We identified that SPS reduced the replication of HSV-1 in HCE-T and Vero cells in a dose-dependent manner. HSV-1g fluorescence was reduced by 66.3% and 65.4% in HCE-T and Vero cells, respectively, after treatment with 0.4 µg/ml SPS. Furthermore, the viral fluorescence intensities were inhibited by SPS in a dose-dependent manner when the viruses or cells were preincubated with SPS. Relative levels of the ICP4 protein and VP16 mRNA were decreased by SPS in a dose-dependent manner. Moreover, the IC50 values of SPS for HSV-1g and HSV-1f in HCE-T cells were 0.69±0.09 µg/ml and 1.63±0.44 µg/ml, respectively. Even 10,000 µg/ml SPS had no obvious cytotoxicity toward HCE-T and Vero cells. Importantly, viral absorption and penetration assays showed that the relative fluorescence intensity of HSV-1g was significantly reduced by SPS in a dose-dependent manner in the absorption test, but no change was observed in the penetration test. Conclusions: SPS inhibits HSV-1 replication in HCE-T and Vero cells, indicating that SPS has the potential for treating HSV-1 infection, particularly HSV-1 keratitis.

SPS-neutralization in tissue samples for efficacy testing of antimicrobial peptides.

BACKGROUND: Accurate determination of the efficacy of antimicrobial agents requires neutralization of residual antimicrobial activity in the samples before microbiological assessment of the number of surviving bacteria. Sodium polyanethol sulfonate (SPS) is a known neutralizer for the antimicrobial activity of aminoglycosides and polymyxins. In this study, we evaluated the ability of SPS to neutralize residual antimicrobial activity of antimicrobial peptides [SAAP-148 and pexiganan; 1% (wt/v) in PBS], antibiotics [mupirocin (Bactroban) and fusidic acid (Fucidin) in ointments; 2% (wt/wt))] and disinfectants [2% (wt/wt) silver sulfadiazine cream (SSD) and 0.5% (v/v) chlorhexidine in 70% alcohol]. METHODS: Homogenates of human skin models that had been exposed to various antimicrobial agents for 1 h were pipetted on top of Methicillin-resistant Staphylococcus aureus (MRSA) on agar plates to determine whether the antimicrobial agents display residual activity. To determine the optimal concentration of SPS for neutralization, antimicrobial agents were mixed with PBS or increasing doses of SPS in PBS (0.05-1% wt/v) and then 105 colony forming units (CFU)/mL MRSA were added. After 30 min incubation, the number of viable bacteria was assessed. Next, the in vitro efficacy of SAAP-148 against various gram-positive and gram-negative bacteria was determined using PBS or 0.05% (wt/v) SPS immediately after 30 min incubation of the mixture. Additionally, ex vivo excision wound models were inoculated with 105 CFU MRSA for 1 h and exposed to SAAP-148, pexiganan, chlorhexidine or PBS for 1 h. Subsequently, samples were homogenized in PBS or 0.05% (wt/v) SPS and the number of viable bacteria was assessed. RESULTS: All tested antimicrobials displayed residual activity in tissue samples, resulting in a lower recovery of surviving bacteria on agar. SPS concentrations at ≥0.05% (wt/v) were able to neutralize the antimicrobial activity of SAAP-148, pexiganan and chlorhexidine, but not of SSD, Bactroban and Fucidin. Finally, SPS-neutralization in in vitro and ex vivo efficacy tests of SAAP-148, pexiganan and chlorhexidine against gram-positive and gram-negative bacteria resulted in significantly higher numbers of CFU compared to control samples without SPS-neutralization. CONCLUSIONS: SPS was successfully used to neutralize residual activity of SAAP-148, pexiganan and chlorhexidine and this prevented an overestimation of their efficacy.

Sodium Polyanethol Sulfonate Modulates Natural Transformation of SigH-Expressing Staphylococcus aureus.

Expression of genes required for natural genetic competence in Staphylococcus aureus is controlled by an alternative transcription sigma factor, SigH. However, even in the SigH-expressing cells, the DNA transformation efficiency varies depending on culture conditions. We report here that cells grown in the competence-inducing medium (CS2 medium) exhibit enlarged morphology with disintegrated cell walls. Notably, an autolysis inhibitor, Sodium Polyanethol Sulfonate (SPS), facilitated transformation in CS2 medium in a dose-dependent manner, suggesting the involvement of the cell wall metabolism in transformation. However, the transformation efficiency of cells grown in TSB was not improved by physical or enzymatic damage on the cell walls.

Expanding the diagnostic use of PCR in leptospirosis: improved method for DNA extraction from blood cultures.

BACKGROUND: Leptospirosis is a neglected zoonosis of ubiquitous distribution. Symptoms are often non-specific and may range from flu-like symptoms to multi-organ failure. Diagnosis can only be made by specific diagnostic tests like serology and PCR. In non-endemic countries, leptospirosis is often not suspected before antibiotic treatment has been initiated and consequently, relevant samples for diagnostic PCR are difficult to obtain. Blood cultures are obtained from most hospitalized patients before antibiotic therapy and incubated for at least five days, thus providing an important source of blood for PCR diagnosis. However, blood cultures contain inhibitors of PCR that are not readily removed by most DNA-extraction methods, primarily sodium polyanetholesulfonate (SPS). METHODOLOGY/PRINCIPAL FINDINGS: In this study, two improved DNA extraction methods for use with blood cultures are presented and found to be superior in recovering DNA of Leptospira interrogans when compared with three previously described methods. The improved methods were easy and robust in use with all tested brands of blood culture media. Applied to 96 blood cultures obtained from 36 patients suspected of leptospirosis, all seven patients with positive convalescence serology were found positive by PCR if at least one anaerobic and one aerobic blood culture, sampled before antibiotic therapy were tested. CONCLUSIONS/SIGNIFICANCE: This study suggests that a specific and early diagnosis can be obtained in most cases of severe leptospirosis for up to five days after initiation of antimicrobial therapy, if PCR is applied to blood cultures already sampled as a routine procedure in most septic patients.

Sodium polyanethole sulfonate as an inhibitor of activation of complement function in blood culture systems.

Sodium polyanethole sulfonate (SPS; trade name, Liquoid) is a constituent in culture media used to grow bacteria from blood samples from patients suspected of bacteremia. SPS prevents the killing of bacteria by innate cellular and humoral factors. We analyzed the effect of SPS on the three complement activation pathways: the classical, alternative, and lectin pathways, respectively. Inhibition of complement activity by SPS is caused by a blocking of complement activation and is not a result of complement consumption. The classical pathway is inhibited at SPS concentrations greater than 0.1 mg/ml, and complete inhibition is seen at 0.4 mg/ml. An SPS concentration of 0.5 mg/ml completely inhibits the binding of C1q and subsequent incorporation of C3, C4, and C9. The same was observed for the alternative pathway with an inhibition at SPS concentrations from 0.1 mg/ml and a complete inhibition from 0.4 mg/ml. Here, properdin binding was completely absent, and no incorporation of C3 and C9 was observed. In contrast, the lectin complement pathway remains unaffected at these SPS concentrations, and inhibition is first observed from 0.7 mg/ml. A complete inhibition required concentrations greater than 1 mg/ml. SPS is used in growth media (e.g., BACTEC and BacT/Alert) at concentrations from 0.3 to 0.5 mg/ml. The well-known finding that certain bacteria are growth inhibited by blood factors could therefore be a consequence of the lectin pathway, which is not inhibited at these concentrations. In addition, our findings also open up the possibility of a new assay for the assessment of the functional capacity of the lectin complement pathway.

[Negative effect of the components of the lysis-centrifugation system in the growth of mycobacteria in MGIT and Septi-Chek AFB liquid media]. / Efecto negativo de los componentes del sistema lisis-centrifugación en el crecimiento de micobacterias en los medios líquidos MGIT y Septi-Chek AFB.

BACKGROUND: The lysis-centrifugation system (Isolator system) is a technique with excellent results in the recovery of mycobacteria from blood specimens. This system consists mainly of saponin (SAP), polypropylenglycol (PPG), and sodium polianthol sulfonate (SPS). The objective of this work was to determine the effect of SAP, PPG, and SPS on the growth of Mycobacterium avium, M. kansasii, M. tuberculosis, and M. xenopi in fluid culture media MGIT and Septi-Chek AFB. METHODS: Two concentrations each of SAP, PPG, and SPS were prepared, and were added in 0.1 ml amounts (alone, in pairs and in combination) to fluid media MGIT and Septi-Chek AFB. Fluid culture media were then in individually inoculated with two different concentrations (10(3) and 10(5) CFU/ml) of each of the four mycobacterial strains used in this study. Culture media were incubated at 37 degrees C and were checked for growth daily. RESULTS: SAP, PPG, and SPS did not inhibit growth of mycobacteria but growth of these strains was indeed retarded (a lengthier time was required for detection of bacterial growth compared with the positive control). Final concentrations of SAP, PPG, and SPS which retarded mycobacterial growth varied, depending upon species, mycobacterial inoculum size, and fluid culture media used. CONCLUSIONS: Components included in the lysy-centrifugation system (SAP, PPG, and SPS), either alone or in combination retarded growth of M. avium, M. kansasii, M. tuberculosis, and M. xenopi in 10(3) and 10(5) CFU/ml concentrations in fluid culture media MGIT and Septi-Chek AFB. These results suggest that strategies should be adopted to decrease the concentrations of these three components, present in the sediment of the processed blood by the Isolator System, which eventually are going to be added to fluid media MGIT and Septi-Check AFB.

Age-associated differences in neutrophil oxidative burst (chemiluminescence).

Phagocytic defensive functions consist of a sequence of events, including migration, phagocytosis, secretion, and the release of reactive oxygen species (ROS). The last of these (also called "oxidative burst") has not received due attention in the elderly, even though it can be considered the most important event in the process of killing an invading microorganism. The aim of the present study was to investigate the oxidative burst activity of polymorphonuclear neutrophil leukocytes (PMNs) in relation to age, using a technique that specifically identifies ROS production: luminol-amplified chemiluminescence (LACL). Besides the use of LACL, a particular feature of the study was the use of five rather than just one or two different stimulants: two particulate (Candida albicans and zymosan) and three soluble ones [N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol 12 myristate 13 acetate (PMA), and polyanetholesulfonate (liquoid)]. This approach allowed us to observe a dichotomy between the effects of Candida and zymosan (particulates), which were not significantly different in the elderly subjects compared to the young controls, and those of fMLP, PMA, and liquoid (solubles), which showed a significant reduction in LACL in the elderly group. Considering the different results obtained with the various stimulants adopted that are all believed to have NADPH oxidase as a common final target of oxidative burst, it may be postulated that aging can influence the different transductional pathways in different ways.

Adherence of Mycoplasma pneumoniae to human alveolar macrophages.

The human pathogen Mycoplasma pneumoniac causes primary atypical-cold agglutinin-positive pneumonia. Since alveolar macrophages internalize mycoplasma as part of their immune defense, we studied characteristics of the human macrophage receptor for opsonized and nonopsonized M. pneumoniae. The glass-adhering subpopulation of M. pneumoniae attached more than the non-adherent subpopulation. The attachment was dose-dependent and enhanced by opsonization in the presence of human serum. It is inhibited by sulfated compounds such as dextran-sulfate and polyanetholsulfonic acid, but not by dextran or several monosaccharides, suggesting that sulfated glycolipids on the macrophage surface may act as receptors for M. pneumoniae binding. In addition, sialylated compounds, such as fetuin and alpha 1-acid glycoprotein, were found to be potent inhibitors of the attachment, also indicating the role of sialic acid residue in recognition and attachment of M. pneumoniae to human alveolar macrophages.

Salt-induced cell lysis of Staphylococcus aureus.

Cell lysis was efficiently induced in Staphylococcus aureus by the addition of 0.3 M NaCl to exponentially growing cultures at 30 degrees C. When cells harvested at the exponential phase were incubated in buffer with NaCl, autolysis occurred. Treatment with chloramphenicol failed to induce cell lysis by NaCl. The effects of NaCl on growing cells and harvested cells were inhibited by the addition of sodium polyanethole sulfonate, subtilisin, cardiolipin, and lipoteichoic acid. These agents diminished the activity of a cell wall-lytic enzyme liberated from the cells in the presence of NaCl. Lysis induced by salt appears to be catalyzed by a similar lytic enzyme in growing and harvested cells.

Salt-induced L-forms of Staphylococcus aureus.

When growing cultures of a salt-sensitive strain of Staphylococcus aureus were inoculated on nutrient agar containing 0.8 M NaCl and 0.5% bovine serum albumin, typical colonies of L-form developed extensively after 2 days of incubation at 30 C. Incubation of growing cultures with lipoteichoic acid, sodium polyanethole sulfonate and subtilisin resulted in inhibition of L-form induction.

Assessment of gelatin-supplemented BACTEC blood culture medium in a pediatric hospital.

Sodium polyanetheolesulfonate (SPS), an anticoagulant used in blood culture media, adversely affects the isolation of Neisseria meningitidis. The addition of gelatin appears to counteract this effect. Studies using the radiometric BACTEC system, however, have noted a lower isolation rate of other bacteria from gelatin-supplemented media. We wished to evaluate the effect of the addition of gelatin (1.2%) to a nonradiometric BACTEC aerobic medium (NR6A) on the recovery of N. meningitidis and other pathogens. The NR6A medium with gelatin (NR6A analogue) also contained a lower concentration of SPS (0.025% vs 0.035%). We did 6045 paired comparisons of blood cultured in routine NR6A medium and the NR6A analogue. Eight isolates of N. meningitidis were recovered, five only from the gelatin-supplemented medium and three from both bottles. There was no statistically significant difference in total recovery of aerobic and facultative bacteria or Candida species from both bottles. Haemophilus influenzae was detected earlier in the nonsupplemented NR6A medium. We conclude that the use of the NR6A analogue medium appeared to increase the yield of N. meningitidis without adversely affecting the recovery of other common pathogens, although the recovery of H. influenzae was slightly delayed.

Use of the RapID-ANA system and sodium polyanetholesulfonate disk susceptibility testing in identifying Haemophilus ducreyi.

Haemophilus ducreyi has traditionally been difficult to identify. We have utilized simple test methods to identify 19 fresh isolates obtained during a recent outbreak of chancroid in Houston and six strains of H. ducreyi from other outbreaks. Tests were performed from growth on chocolate agar after 48 h of incubation at 35 degrees C with increased humidity and CO2. All isolates exhibited typical colonial morphology and Gram stain. Isolates were catalase negative and oxidase and nitrate positive (in enriched broth). The RapID NH system failed to identify these strains because of negative reactions with alkaline phosphatase and nitrate reductase. However, by using the RapID-ANA system, all strains were positive for alkaline phosphatase and arginine, glycine, and serine aminopeptidases. Their biochemical profiles were distinct from those obtained with 66 strains representing 13 species similar to H. ducreyi. We also investigated the use of sodium polyanetholesulfonate (SPS) disk susceptibility to identify and differentiate H. ducreyi from other species. All H. ducreyi isolates were susceptible, as evidenced by the presence of a zone of inhibition with an average size of 15 mm around the SPS disk. With the exceptions of Neisseria gonorrhoeae, Gardnerella vaginalis, and Capnocytophaga spp., no other strain showed any evidence of inhibition. The latter three organisms can be easily differentiated from H. ducreyi by various features including reactions in the RapID-ANA. We conclude that, by considering simple growth and biochemical characteristics, SPS susceptibilities, and reactions in RapID-ANA, it is possible for more clinical laboratories to definitively identify this organism.

Controlled evaluation of trypticase soy broth with and without gelatin and yeast extract in the detection of bacteremia and fungemia.

The addition of gelatin to blood culture media has been suggested to prevent the inhibition of Neisseria meningitidis, Neisseria gonorrhoeae, Gardnerella vaginalis, and Peptostreptococcus anaerobius that is caused by sodium polyanetholsulfonate. To determine the effect of such supplementation on the overall yield of microorganisms, we compared the yield and speed of detection of clinically important microorganisms from 5422 paired 10-ml samples of blood cultured in Trypticase soy broth (TSB) containing 0.03% sodium polyanetholesulfonate (SPS) and TSB/SPS containing 1.2% gelatin and 1.0% yeast extract (mTSB). The atmosphere of incubation (open venting unit) and ratio of blood to broth (1:5) were the same for both samples. Only cultures with adequate blood sample (greater than or equal to 80% of stated volume) were compared statistically. Addition of gelatin and yeast extract resulted in inhibited growth of Enterobacteriaceae (p less than 0.001), Pseudomonas aeruginosa (p less than 0.01), fungi (p less than 0.05), and the overall set of microorganisms encountered (p less than 0.001). It delayed growth of Enterobacteriaceae (p less than 0.001) but reduced the time to recover staphylococci (p less than 0.02). Of 12 isolates of species usually inhibited by SPS, seven grew only with the addition of gelatin and yeast extract, none grew only without supplementation, and five grew in both media. Although gelatin and yeast extract may improve the yield of some specific bacteria, the routine use of these additives cannot be recommended for all blood culture media.

Selective inhibition of human natural killing and antibody-dependent cellular cytotoxicity by a polyanion.

A high molecular polyanion, Liquoid, was found to inhibit at nontoxic concentrations (12-50 micrograms/ml) the natural killing (NK) and the antibody-dependent cellular cytotoxic (ADCC) activity of human peripheral blood mononuclear cells selectively. Whereas NK of the K 562 target cell was slightly or not at all affected, the spontaneous lysis of PDe-B-1, an EBV-transformed B-cell line, was strongly inhibited or even completely abolished. ADCC activity could only be inhibited by Liquoid if the target cells were mycoplasma-free, while the polyanion had no effect when mycoplasma-contaminated target cells were used. Liquoid did not alter the target binding capacity of the NK effector cells and did not activate monocytes or induce other suppressive cells. Alpha interferon, but neither beta nor gamma interferon, was able to neutralize the NK reduction. These results suggest that Liquoid inhibits a target cell-related, selective process in the post-binding stage of NK cell lysis.

Inhibition of fibrinolytic system by liquoid (polyanetholesulfonate). A contributing factor in the liquoid-induced renal cortical necrosis.

Liquoid induces microvascular thrombosis and renal cortical necrosis in experimental animals. We hypothesized that thrombosis and renal cortical necrosis may, at least in part, result from the inhibition of the fibrinolytic system by liquoid. Effects of liquoid on plasminogen activation by rat kidney, purified human tissue plasminogen activator (TPA), urokinase, streptokinase, and on the amidolytic activities of TPA, urokinase, and plasmin were studied using chromogenic substrates and clot lysis. Liquoid had a strong inhibitory effect on the fibrinolytic system in vivo and in vitro. The inhibition was most effective at the plasminogen activation level, with activation by streptokinase being most susceptible. The demonstrated stoichiometric binding between liquoid and plasminogen, and to a lesser degree the direct inactivation of plasminogen activators and plasmin, is probably responsible for the reduction of plasminogen activation in circulation and in the kidneys.

Disseminated intravascular coagulation (DIC) induced by liquoid (polyanetholsulfonate) in the rat. V. Effects on circulating fibronectin.

Levels of plasma fibronectin (Fn) were 63% lower than normal 15 min after the intravenous injection of liquoid (P less than 0.01); 3 h later they were still low but rebounded to 35% above normal (P less than 0.01) by 24 h. Concurrently microthrombi containing fibrinogen, Fn and Factor VIII related-antigens (VIII:Ag) were detected in the kidneys and lungs by immunohistopathological studies. Ultrastructurally, thrombi were composed of dense granular and occasional fibrillar non-striated material. In liquoid-injected rats 125I-fibrinogen mainly localized in kidneys and lungs, especially in the latter (P less than 0.01), and the lungs had a higher wet-to-dry weight ratios than did controls (P less than 0.01). It is concluded that the polyanion (liquoid)-induced intravascular coagulation-like reaction sequestered Fn concomitantly with the precipitation of fibrinogen and VII:Ag in the microclots. The reduced concentration of plasma Fn may have impaired the disposal of coagulation products thus enhancing the expression of the coagulopathy-mediated renal and pulmonary histopathology. It is suggested that the liquoid-related coagulopathy may have resulted in enzymatic lysis of Fn.

Inhibition of wall autolysis of staphylococci by sodium polyanethole sulfonate "liquoid".

Liquoid (polyanethole sulfonate) was neither capable of influencing the growth nor the viability of staphylococci. But liquoid induced a suppression of the activity of different autolytic wall systems of normally growing staphylococci, i.e., autolysins which participate in cross wall separation as well as autolysins which are responsible for cell wall turnover. Additionally, the lysostaphin-induced wall disintegration of staphylococci was inhibited by liquoid. However, no indication could be found for a direct inhibition of lytic wall enzymes by liquoid; rather an interaction of liquoid with the target structure for the autolytic wall enzymes, the cell wall itself, was postulated. On the basis of the experimental data with the teichoic acid- mutant S. aureus 52A5 the sites of wall teichoic acid were supposed to be an important target for the binding of liquoid to the staphylococcal cell wall.

Synergistic effects of synthetic adjuvants on the humoral immune response.

The effect of combinations of adjuvants on the humoral immune response to sheep red blood cells (SRBC) as antigen was investigated. Adjuvants belonging to two categories differing in physicochemical properties were used: surfactants (N,N-dioctadecyl-N',N'-bis-(2-hydroyethyl)propanediamine (CP-20,961), dimethyldioctadecylammonium bromide (DDA), neutrally charged liposomes, polyol (L 101 and L 121) and polyanions [dextran sulfate (DXS), liquoid and suramine]. All adjuvants but suramine augmented humoral responses to 2 X 10(7) SRBC measured by the number of direct anti-SRBC plaque-forming cells (PFC) in the spleen. The response to 2 X 10(6) SRBC was enhanced considerably by L 121 and DXS but hardly or not at all by the other adjuvants. Combinations of two adjuvants were made at distinct ratios (1:3, 2:2, and 3:1) and injected intraperitoneally with 2 X 10(6) SRBC. Low responses (5 X 10(3) PFC per spleen) were induced by combinations of liquoid or suramine with DDA or DXS, and by combinations of CP-20,961, liposomes, L 101 or L 121 with DDA. Combinations of the surfactants DDA, CP-20,961, liposomes, L 101 or L 121 with DXS evoked responses which were significantly higher than the sum of responses supported by the single adjuvants. Ratios of 1:3 or 2:2 (surfactant: DXS) resulted in the most effective combinations. The data obtained suggest that only adjuvants derived from two different physicochemical groups are able to act synergistically.

[The influence of carrageenan, colchicine, 2,4-dinitrophenol, ethanol, galactose, indomethacin and liquoid on hemolytic complement activity (CH50E)]. / Die Beeinflussung der hämolytischen Komplementaktivität (CH50E) durch Carrageenan, Colchicin, 2,4-Dinitrophenol, Ethanol, Galactose, Indomethacin und Liquoid.

The influence of Liquoid (0.00005, 0.00001%), Carrageenan (0.001% up to 0.000001%), Colchicine (0.01% up to 0.0025%), Indomethacin (1 X 10(-4) up to 2.5 X 10(-5) mol/1), Ethanol (10% up to 1.25%), Galactose (1 X 10(-1) up to 1 X 10(-2) mol/l) and 2.4-Dinitrophenol (5 X 10(-4) up to 1 X 10(-5) mol/l) on the total hemolytic activity of complement was tested. By Liquoid, Carrageenan, Colchicine and Ethanol in all studied concentrations an remarkable inhibition of the CH50E could be estimated. Indomethacin up to an concentration of 3.75 X 10(-5) mol/l reduced the activity partially. In contrast, Galactose and 2.4-Dinitrophenol have not influenced the complement titers.