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1.  Bactericidal Activity of N-Chlorotaurine against Biofilm-Forming Bacteria Grown on Metal Disks 
Many orthopedic surgeons consider surgical irrigation and debridement with prosthesis retention as a treatment option for postoperative infections. Usually, saline solution with no added antimicrobial agent is used for irrigation. We investigated the activity of N-chlorotaurine (NCT) against various biofilm-forming bacteria in vitro and thereby gained significant information on its usability as a soluble and well-tolerated active chlorine compound in orthopedic surgery. Biofilms of Staphylococcus aureus were grown on metal alloy disks and in polystyrene dishes for 48 h. Subsequently, they were incubated for 15 min to 7 h in buffered solutions containing therapeutically applicable concentrations of NCT (1%, 0.5%, and 0.1%; 5.5 to 55 mM) at 37°C. NCT inactivated the biofilm in a time- and dose-dependent manner. Scanning electron microscopy revealed disturbance of the biofilm architecture by rupture of the extracellular matrix. Assays with reduction of carboxanilide (XTT) showed inhibition of the metabolism of the bacteria in biofilms. Quantitative cultures confirmed killing of S. aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa biofilms on metal alloy disks by NCT. Clinical isolates were slightly more resistant than ATCC type strains, but counts of CFU were reduced at least 10-fold by 1% NCT within 15 min in all cases. NCT showed microbicidal activity against various bacterial strains in biofilms. Whether this can be transferred to the clinical situation should be the aim of future studies.
doi:10.1128/AAC.02700-13
PMCID: PMC4023792  PMID: 24492358
2.  New Insight into Amphotericin B Resistance in Aspergillus terreus 
Amphotericin B (AMB) is the predominant antifungal drug, but the mechanism of resistance is not well understood. We compared the in vivo virulence of an AMB-resistant Aspergillus terreus (ATR) isolate with that of an AMB-susceptible A. terreus isolate (ATS) using a murine model for disseminated aspergillosis. Furthermore, we analyzed the molecular basis of intrinsic AMB resistance in vitro by comparing the ergosterol content, cell-associated AMB levels, AMB-induced intracellular efflux, and prooxidant effects between ATR and ATS. Infection of immunosuppressed mice with ATS or ATR showed that the ATS strain was more lethal than the ATR strain. However, AMB treatment improved the outcome in ATS-infected mice while having no positive effect on the animals infected with ATR. The in vitro data demonstrated that ergosterol content is not the molecular basis for AMB resistance. ATR absorbed less AMB, discharged more intracellular compounds, and had better protection against oxidative damage than the susceptible strain. Our experiments showed that ergosterol content plays a minor role in intrinsic AMB resistance and is not directly associated with intracellular cell-associated AMB content. AMB might exert its antifungal activity by oxidative injury rather than by an increase in membrane permeation.
doi:10.1128/AAC.01283-12
PMCID: PMC3623369  PMID: 23318794
3.  N-Chloramines, a Promising Class of Well-Tolerated Topical Anti-Infectives 
Antibiotic resistance is a growing public health crisis. To address the development of bacterial resistance, the use of antibiotics has to be minimized for nonsystemic applications in humans, as well as in animals and plants. Possible substitutes with low potential for developing resistance are active chlorine compounds that have been in clinical use for over 180 years. These agents are characterized by pronounced differences in their chlorinating and/or oxidizing activity, with hypochlorous acid (HOCl) as the strongest and organic chloramines as the weakest members. Bacterial killing in clinical practice is often associated with unwanted side effects such as chlorine consumption, tissue irritation, and pain, increasing proportionally with the chlorinating/oxidizing potency. Since the chloramines are able to effectively kill pathogens (bacteria, fungi, viruses, protozoa), their application as anti-infectives is advisable, all the more so as they exhibit additional beneficial properties such as destruction of toxins, degradation of biofilms, and anticoagulative and anti-inflammatory activities. Within the ample field of chloramines, the stable N-chloro derivatives of ß-aminosulfonic acids are most therapeutically advanced. Being available as sodium salts, they distinguish themselves by good solubility and absence of smell. Important representatives are N-chlorotaurine, a natural compound occurring in the human immune system, and novel mono- and dichloro derivatives of dimethyltaurine, which feature improved stability.
doi:10.1128/AAC.02132-12
PMCID: PMC3591902  PMID: 23295936
4.  NVC-422 Inactivates Staphylococcus aureus Toxins 
Bacterial pathogens have specific virulence factors (e.g., toxins) that contribute significantly to the virulence and infectivity of microorganisms within the human hosts. Virulence factors are molecules expressed by pathogens that enable colonization, immunoevasion, and immunosuppression, obtaining nutrients from the host or gaining entry into host cells. They can cause pathogenesis by inhibiting or stimulating certain host functions. For example, in systemic Staphylococcus aureus infections, virulence factors such as toxic shock syndrome toxin 1 (TSST-1), staphylococcal enterotoxin A (SEA), and staphylococcal enterotoxin B (SEB) cause sepsis or toxic shock by uncontrolled stimulation of T lymphocytes and by triggering a cytokine storm. In vitro, these superantigens stimulate the proliferation of human peripheral blood mononuclear cells (PBMC) and the release of many cytokines. NVC-422 (N,N-dichloro-2,2-dimethyltaurine) is a broad-spectrum, fast-acting topical anti-infective agent against microbial pathogens, including antibiotic-resistant microbes. Using mass spectrometry, we demonstrate here that NVC-422 oxidizes methionine residues of TSST-1, SEA, SEB, and exfoliative toxin A (ETA). Exposure of virulence factors to 0.1% NVC-422 for 1 h prevented TSST-1-, SEA-, SEB-, and ETA-induced cell proliferation and cytokine release. Moreover, NVC-422 also delayed and reduced the protein A- and clumping factor-associated agglutination of S. aureus cultures. These results show that, in addition to its well-described direct microbicidal activity, NVC-422 can inactivate S. aureus virulence factors through rapid oxidation of methionines.
doi:10.1128/AAC.01945-12
PMCID: PMC3553688  PMID: 23208720
5.  N-Chlorotaurine, a Long-Lived Oxidant Produced by Human Leukocytes, Inactivates Shiga Toxin of Enterohemorrhagic Escherichia coli 
PLoS ONE  2012;7(11):e47105.
N-chlorotaurine (NCT), the main representative of long-lived oxidants produced by granulocytes and monocytes, is known to exert broad-spectrum microbicidal activity. Here we show that NCT directly inactivates Shiga toxin 2 (Stx2), used as a model toxin secreted by enterohemorrhagic Escherichia coli (EHEC). Bacterial growth and Stx2 production were both inhibited by 2 mM NCT. The cytotoxic effect of Stx2 on Vero cells was removed by ≥5.5 mM NCT. Confocal microscopy and FACS analyses showed that the binding of Stx2 to human kidney glomerular endothelial cells was inhibited, and no NCT-treated Stx2 entered the cytosol. Mass spectrometry displayed oxidation of thio groups and aromatic amino acids of Stx2 by NCT. Therefore, long-lived oxidants may act as powerful tools of innate immunity against soluble virulence factors of pathogens. Moreover, inactivation of virulence factors may contribute to therapeutic success of NCT and novel analogs, which are in development as topical antiinfectives.
doi:10.1371/journal.pone.0047105
PMCID: PMC3491008  PMID: 23139739
6.  Molecular analysis of volatile metabolites released specifically by staphylococcus aureus and pseudomonas aeruginosa 
BMC Microbiology  2012;12:113.
Background
The routinely used microbiological diagnosis of ventilator associated pneumonia (VAP) is time consuming and often requires invasive methods for collection of human specimens (e.g. bronchoscopy). Therefore, it is of utmost interest to develop a non-invasive method for the early detection of bacterial infection in ventilated patients, preferably allowing the identification of the specific pathogens. The present work is an attempt to identify pathogen-derived volatile biomarkers in breath that can be used for early and non- invasive diagnosis of ventilator associated pneumonia (VAP). For this purpose, in vitro experiments with bacteria most frequently found in VAP patients, i.e. Staphylococcus aureus and Pseudomonas aeruginosa, were performed to investigate the release or consumption of volatile organic compounds (VOCs).
Results
Headspace samples were collected and preconcentrated on multibed sorption tubes at different time points and subsequently analyzed with gas chromatography mass spectrometry (GC-MS). As many as 32 and 37 volatile metabolites were released by S. aureus and P. aeruginosa, respectively. Distinct differences in the bacteria-specific VOC profiles were found, especially with regard to aldehydes (e.g. acetaldehyde, 3-methylbutanal), which were taken up only by P. aeruginosa but released by S. aureus. Differences in concentration profiles were also found for acids (e.g. isovaleric acid), ketones (e.g. acetoin, 2-nonanone), hydrocarbons (e.g. 2-butene, 1,10-undecadiene), alcohols (e.g. 2-methyl-1-propanol, 2-butanol), esters (e.g. ethyl formate, methyl 2-methylbutyrate), volatile sulfur compounds (VSCs, e.g. dimethylsulfide) and volatile nitrogen compounds (VNCs, e.g. 3-methylpyrrole).
Importantly, a significant VOC release was found already 1.5 hours after culture start, corresponding to cell numbers of ~8*106 [CFUs/ml].
Conclusions
The results obtained provide strong evidence that the detection and perhaps even identification of bacteria could be achieved by determination of characteristic volatile metabolites, supporting the clinical use of breath-gas analysis as non-invasive method for early detection of bacterial lung infections.
doi:10.1186/1471-2180-12-113
PMCID: PMC3444334  PMID: 22716902
Volatile organic compounds (VOCs); Gas chromatography mass spectrometry (GCMS); Breath analysis; In vitro headspace sampling; Adsorptive enrichment; Multibed sorption tubes; Volatile metabolites; Staphylococcus aureus; Pseudomonas aeruginosa
7.  Tolerability of inhaled N-chlorotaurine in an acute pig streptococcal lower airway inflammation model 
BMC Infectious Diseases  2011;11:231.
Background
Inhalation of N-chlorotaurine (NCT), an endogenous new broad spectrum non-antibiotic anti-infective, has been shown to be very well tolerated in the pig model recently. In the present study, inhaled NCT was tested for tolerability and efficacy in the infected bronchopulmonary system using the same model.
Methods
Anesthetized pigs were inoculated with 20 ml of a solution containing approximately 108 CFU/ml Streptococcus pyogenes strain d68 via a duodenal tube placed through the tracheal tube down to the carina. Two hours later, 5 ml of 1% NCT aqueous solution (test group, n = 15) or 5 ml of 0.9% NaCl (control group, n = 16) was inhaled via the tracheal tube connected to a nebulizer. Inhalation was repeated every hour, four times in total. Lung function and haemodynamics were monitored. Bronchoalveolar lavage samples were removed for determination of colony forming units (CFU), and lung samples for histology.
Results
Arterial pressure of oxygen (PaO2) decreased rapidly after instillation of the bacteria in all animals and showed only a slight further decrease at the end of the experiment without a difference between both groups. Pulmonary artery pressure increased to a peak 1-1.5 h after application of the bacteria, decreased in the following hour and remained constant during treatment, again similarly in both groups. Histology demonstrated granulocytic infiltration in the central parts of the lung, while this was absent in the periphery. Expression of TNF-alpha, IL-8, and haemoxygenase-1 in lung biopsies was similar in both groups. CFU counts in bronchoalveolar lavage came to 170 (10; 1388) CFU/ml (median and 25 and 75 percentiles) for the NCT treated pigs, and to 250 (10; 5.5 × 105) CFU/ml for NaCl treated pigs (p = 0.4159).
Conclusions
Inhaled NCT at a concentration of 1% proved to be very well tolerated also in the infected bronchopulmonary system. This study confirms the tolerability in this delicate body region, which has been proven in healthy pigs previously. Regarding efficacy, no conclusions can be drawn, mainly because of the limited test period of the model.
doi:10.1186/1471-2334-11-231
PMCID: PMC3178512  PMID: 21875435
8.  In vitro activity of N-chlorotaurine (NCT) in combination with NH4Cl against Trichomonas vaginalis 
Trichomoniasis, caused by the protozoan Trichomonas vaginalis, is usually treated with metronidazole, however resistance is on the rise. In this study, N-chlorotaurine (NCT), a new endogenous mild active chlorine compound for topical use, killed T. vaginalis in vitro within 15 min of treatment at a concentration of 55 mM (1%), which is well tolerated by human tissue. The activity of NCT was further enhanced by addition of ammonium chloride (NH4Cl). A combination of 5.5 mM (0.1%) NCT plus 19 mM (0.1%) NH4Cl killed 100% of trichomonads within 5 min.
doi:10.1016/j.ijantimicag.2010.09.006
PMCID: PMC3030749  PMID: 21074373
Trichomonas vaginalis; Susceptible; N-Chlorotaurine; Oxidant; In vitro
9.  Tolerability of inhaled N-chlorotaurine in the pig model 
Background
N-chlorotaurine, a long-lived oxidant produced by human leukocytes, can be applied in human medicine as an endogenous antiseptic. Its antimicrobial activity can be enhanced by ammonium chloride. This study was designed to evaluate the tolerability of inhaled N-chlorotaurine (NCT) in the pig model.
Methods
Anesthetized pigs inhaled test solutions of 1% (55 mM) NCT (n = 7), 5% NCT (n = 6), or 1% NCT plus 1% ammonium chloride (NH4Cl) (n = 6), and 0.9% saline solution as a control (n = 7), respectively. Applications with 5 ml each were performed hourly within four hours. Lung function, haemodynamics, and pharmacokinetics were monitored. Bronchial lavage samples for captive bubble surfactometry and lung samples for histology and electron microscopy were removed.
Results
Arterial pressure of oxygen (PaO2) decreased significantly over the observation period of 4 hours in all animals. Compared to saline, 1% NCT + 1% NH4Cl led to significantly lower PaO2 values at the endpoint after 4 hours (62 ± 9.6 mmHg vs. 76 ± 9.2 mmHg, p = 0.014) with a corresponding increase in alveolo-arterial difference of oxygen partial pressure (AaDO2) (p = 0.004). Interestingly, AaDO2 was lowest with 1% NCT, even lower than with saline (p = 0.016). The increase of pulmonary artery pressure (PAP) over the observation period was smallest with 1% NCT without difference to controls (p = 0.91), and higher with 5% NCT (p = 0.02), and NCT + NH4Cl (p = 0.05).
Histological and ultrastructural investigations revealed no differences between the test and control groups. The surfactant function remained intact. There was no systemic resorption of NCT detectable, and its local inactivation took place within 30 min. The concentration of NCT tolerated by A549 lung epithelial cells in vitro was similar to that known from other body cells (0.25–0.5 mM).
Conclusion
The endogenous antiseptic NCT was well tolerated at a concentration of 1% upon inhalation in the pig model. Addition of ammonium chloride in high concentration provokes a statistically significant impact on blood oxygenation.
doi:10.1186/1471-2466-9-33
PMCID: PMC2722574  PMID: 19602222
10.  Reassessment of the microbicidal activity of reactive oxygen species and hypochlorous acid with reference to the phagocytic vacuole of the neutrophil granulocyte 
Journal of medical microbiology  2003;52(Pt 8):643-651.
During phagocytosis, neutrophils undergo a burst of respiration in which oxygen is reduced to superoxide (O2−), which dismutates to form H2O2. Myeloperoxidase (MPO) is discharged from the cytoplasmic granules into the phagosome following particle ingestion. It is thought to utilize H2O2 to oxidize halides, which then react with and kill ingested microbes. Recent studies have provided new information as to the concentration of O2− and proteins, and the pH, within the vacuole. This study was conducted to examine the antimicrobial effect of O2−, H2O2 and hypochlorous acid under these conditions and it was found that the previously described bactericidal effect of these agents was reversed in the presence of granule proteins or MPO. To establish which cellular proteins were iodinated by MPO, cellular proteins and bacterial proteins, iodinated in neutrophils phagocytosing bacteria in the presence of 125I, were separated by 2D gel electrophoresis. Iodinated spots were detected by autoradiography and the oxidized proteins were identified by MS. The targets of these iodination reactions were largely those of the host cell rather than those of the engulfed microbe.
PMCID: PMC2635949  PMID: 12867557
11.  Cytotoxic Activity of N-Chlorotaurine on Acanthamoeba spp.▿  
Acanthamoeba spp. are the causative agents of Acanthamoeba keratitis (AK), which mainly occurs in contact lens wearers, and of skin lesions, granulomatous amoebic encephalitis (GAE), and disseminating diseases in the immunocompromised host. AK therapy is complex and irritating for the eye, skin lesions are difficult to treat, and there is no effective treatment for GAE. Therefore, new anti-Acanthamoeba drugs are needed. We investigated the anti-Acanthamoeba activity of N-chlorotaurine (NCT), an endogenous mild antiseptic. It was shown that NCT has amoebicidal qualities, both in phosphate-buffered saline (PBS) and in amoebic culture medium. After 6 h of treatment with 10 mM NCT in PBS, the levels of trophozoites of all strains investigated already showed at least a 2-log reduction. When the trophozoites were treated with 20 mM NCT in culture medium, they showed a 2-log reduction after 24 h. The addition of NH4Cl to NCT led to a faster decrease in the numbers of living cells, if tests were carried out in PBS. A delay of excystation was observed when cysts were treated with 55 mM (1%) NCT in culture medium. A complete failure of excystment was the result of treatment with 1% NCT plus 1% NH4Cl in PBS. Altogether, NCT clearly demonstrated amoebicidal activity at concentrations well tolerated by human tissues and might be useful as a topical drug for the treatment of Acanthamoeba infections. The addition of ammonium chloride can be considered to enhance the activity.
doi:10.1128/AAC.00715-07
PMCID: PMC2224745  PMID: 18039920
12.  Phagocytosis and Killing of Bacteria by Professional Phagocytes and Dendritic Cells 
Dendritic cells (DC) represent a class of professional antigen-presenting cells whose primary function is to alert the immune system, not to clear invading microorganisms. The objective of our study was to compare the abilities of polymorphonuclear neutrophilic leukocytes (PMN), monocytes, monocyte-derived macrophages (MDM), monocyte-derived immature DC (imDC), and mature DC (maDC) to ingest and destroy Staphylococcus aureus and Escherichia coli. Acridine orange staining and fluorescence microscopy demonstrated that MDM, followed by monocytes, imDC, and PMN, internalized bacteria well but that maDC exhibited less pronounced phagocytic activity. PMN, monocytes, and MDM exhibited a much higher capacity to kill ingested bacteria than both imDC and maDC. In summary, these data are in agreement with the generally accepted idea that different types of leukocytes fulfill specialized tasks in antigen presentation and killing of pathogens.
doi:10.1128/CDLI.9.6.1165-1168.2002
PMCID: PMC130096  PMID: 12414745
13.  Impact of N-Chlorotaurine on Viability and Production of Secreted Aspartyl Proteinases of Candida spp. 
N-Chlorotaurine, an endogenous long-lived oxidant, demonstrated fungicidal activity against Candida spp. and a postantifungal effect. Secreted aspartyl proteinases, important fungal virulence factors, proved to be a first target of impact. These results provide support for the topical application of N-chlorotaurine as an antimicrobial agent in yeast infections.
doi:10.1128/AAC.46.6.1996-1999.2002
PMCID: PMC127226  PMID: 12019124
14.  Studies of In Vitro Activities of Voriconazole and Itraconazole against Aspergillus Hyphae Using Viability Staining 
The minimal fungicidal concentrations (MFCs) of voriconazole and itraconazole for five clinical isolates each of Aspergillus terreus, Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger were determined by a broth macrodilution method. Conidial suspensions as inocula were compared to hyphae as inocula since the invasive form of aspergillosis is manifested by the appearance of hyphal structures. In addition, cell viability staining with the dye FUN-1 was performed to assess time-dependent damage of hyphae exposed to various concentrations of the antifungal agents. With conidial inocula the MFC ranges of voriconazole were 0.5 to 4 μg/ml and those of itraconazole were 0.25 to 2 μg/ml, whereas the MFCs (2 to >16 μg/ml) with hyphal inocula were substantially higher (P < 0.01) for both itraconazole and voriconazole. Only minor differences between the tested antifungals were observed since 16 of 20 and 17 of 20 of the isolates of Aspergillus spp. tested appeared to be killed by voriconazole and itraconazole, respectively. The results of FUN-1 viability staining correlated closely to colony counts, but various time- and dose-dependent levels of viability of hyphae were also observed. In conclusion, our study demonstrates the importance of the type of inoculum used to test antifungals and the applicability of FUN-1 staining as a rapid and sensitive method for assaying the viability of hyphae.
doi:10.1128/AAC.45.1.124-128.2001
PMCID: PMC90249  PMID: 11120954
15.  Bactericidal Activity of Micromolar N-Chlorotaurine: Evidence for Its Antimicrobial Function in the Human Defense System 
N-Chlorotaurine, the main representative of long-lived oxidants found in the supernatant of stimulated granulocytes, has been investigated systematically with regard to its antibacterial activity at different physiological concentrations for the first time. N-Chlorotaurine (12.5 to 50 μM) demonstrated a bactericidal effect i.e., a 2 to 4 log10 reduction in viable counts, after incubation at 37°C for 6 to 9 h at pH 7.0, which effect was significantly enhanced in an acidic milieu (at pH 5.0), with a 3 to 4 log10 reduction after 2 to 3 h. Moreover, bacteria were attenuated after being incubated in N-chlorotaurine for a sublethal time, as demonstrated with the mouse peritonitis model. The supernatant of stimulated granulocytes exhibited similar activity. Transmission electron microscopy revealed changes in the bacterial cell membrane and cytoplasmic disintegration with both reacting systems, even in the case of a mere attenuation. The results of this study suggest a significant bactericidal function of N-chlorotaurine and other chloramines during inflammation.
PMCID: PMC90093  PMID: 10952603
16.  Bactericidal Activity of Vancomycin in Cerebrospinal Fluid 
Intraventricular application of vancomycin is an effective therapeutic regimen for the treatment of shunt-associated staphylococcal ventriculitis. We examined the in vitro activity of vancomycin at high concentrations against Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis ATCC 12228 in human cerebrospinal fluid samples. Time-kill curves revealed equal efficacies for concentrations of 10, 100, and 300 μg/ml, and incubation times of 24 to 48 h were needed to achieve a 3 log10 reduction of viable bacteria. A concentration of 5 μg/ml showed a slightly lower activity, but this difference was not significant. In an infant who was successfully treated for shunt-associated ventriculitis due to S. epidermidis by once-daily local administration of vancomycin (3 mg for 2 days and 5 mg for 4 days [0.5 to 0.8 mg/kg of body weight]) the in vivo kill kinetics were similar to those for the in vitro results. These results support time-dose regimens that provide trough vancomycin levels of 5 to 10 μg/ml.
PMCID: PMC89393  PMID: 10428915
17.  Acanthamoeba castellanii : growth on human cell layers reactivates attenuated properties after prolonged axenic culture 
Fems Microbiology Letters  2009;299(2):121-127.
The free-living, but potentially pathogenic, bacteriovorous amoebae of the genus Acanthamoeba can be easily grown axenically in a laboratory culture. This, however, often leads to considerable losses in virulence, and encystment capacity, and to changes in drug susceptibility. We evaluated potential options for a reactivation of a number of physiological properties, attenuated by prolonged axenic laboratory culture, including encystment potential, protease activity, heat resistance, growth rates and drug susceptibility against N-chlorotaurine (NCT). Toward this end, a strain that had been grown axenically for 10 years was repeatedly passaged on human HEp-2 cell monolayers or treated with 5′-azacytidine (AzaC), a methyltransferase inhibitor, and trichostatin A (TSA), a histone deacetylase inhibitor, in order to uplift epigenetic gene regulation. Culture on human cell monolayers resulted in significantly enhanced encystment potentials and protease activities, and higher susceptibility against NCT, whereas the resistance against heat shock was not altered. Treatment with AzaC/TSA resulted in increased encystment rates and protease activities, indicating the participation of epigenetic mechanisms. However, lowered resistances against heat shock indicate that possible stress responses to AzaC/TSA have to be taken into account. Repeated growth on human cell monolayers appears to be a potential method to reactivate attenuated characteristics in Acanthamoeba.
doi:10.1111/j.1574-6968.2009.01680.x
PMCID: PMC2810444  PMID: 19732153
Acanthamoeba; encystment; protease; axenic culture; N-chlorotaurine

Results 1-17 (17)