|
|
|
|
|
|
Antimicrobial Agents and Chemotherapy, August 2004, p . 3130-3132, Vol . 48, No . 8 In Vitro Killing of Mycobacterium ulcerans by Acidified NitriteR . Phillips,1,2,3* S . Kuijper,3 N . Benjamin,4 M . Wansbrough-Jones,2 M . Wilks,5 and A . H . J . Kolk3 Komfo Anokye Teaching Hospital, Kumasi, Ghana,1 St . George's Hospital Medical School,2 Department of Medical Microbiology, St . Bartholomew's Hospital, London,5 Peninsula Medical School, St . Luke's Campus, Exeter, United Kingdom,4 KIT Biomedical Research, Royal Tropical Institute, Amsterdam, The Netherlands3 Received 1 December 2003/ Returned for modification 30 January 2004/ Accepted 26 April 2004
In light of these encouraging clinical results, the present study was designed to investigate the in vitro susceptibility of M . ulcerans to nitrogen oxides . The antimicrobial activity of nitrogen oxides has been clarified to some extent in recent studies, although the exact molecular species responsible for killing is not known (9) . Acidification of nitrite results in production of a complex mixture of nitrogen oxides including nitrous acid, dinitrogen trioxide, nitrogen dioxide, and nitric oxide, all good nitrosating agents (NO+ donors) (20) which diffuse readily across membranes (8) . They react rapidly with reduced thiols to form nitrosothiols, also thought to be important in microbial killing (7) . Nitric oxide can inhibit respiratory chain enzymes through inactivation of iron-sulfur complexes (10) and can disrupt DNA replication by inhibiting ribonucleotide reductase (16) . A clinical isolate of M . ulcerans from Africa, M . ulcerans isolate 1, identified in our laboratory and maintained on Löwenstein-Jensen medium, was cultivated in Middlebrook 7H9 broth (pH 6.8; Difco Laboratories, Detroit, Mich.) supplemented with 10% ADC (albumin, dextrose, catalase; Difco) and incubated at 30°C . The concentration of bacteria was estimated by measuring the optical density in a spectrophotometer at a wavelength of 420 nm, where a reading of 0.15 is equivalent to 108 bacteria per ml (17) . Solutions of anhydrous sodium nitrite (Merck, Darmstadt, Germany) and citric acid monohydrate (BDH, Poole, England) were prepared in deionized water . Freshly prepared 0.4 M (3%), 0.9 M (6%), and 1.7 M (12%) sodium nitrite solutions and 0.2 M (4.5%), 0.4 M (9%), and 0.9 M (18%) citric acid monohydrate solutions were sterilized by passage through a 0.2-µm-pore-size sterile filter (Schleicher & Schuell, Dassel, Germany) . Separate citric acid solutions were made, and the pH was adjusted with 1 M NaOH to that of each of the acidified nitrite solutions (0.2 M [pH 3.4], 0.4 M [pH 3.2], and 0.9 M [pH 3.0]) . Aliquots (0.2 ml) of the bacterial suspension prepared as described above were placed in sterile 2-ml screw-cap tubes (Sarstedt, Nümbrecht, Germany) to which 0.9 ml of nitrite solution and 0.9 ml of citric acid solution were added . Two sets of control tubes contained 0.2 ml of the bacterial suspension, 0.9 ml of sterile water, and 0.9 ml of pH-adjusted citric acid monohydrate solution . After exposures of 10 and 20 min, 0.2 ml of the contents was added to 1.8 ml of Middlebrook 7H9 broth enriched with ADC . Successive 10-fold serial dilutions of these bacterial suspensions were made, and 0.1-ml volumes of the broth mixture were then cultured in duplicate on Middlebrook 7H11 agar (pH 6.6) supplemented with oleic acid, albumin, dextrose, and catalase (OADC; Difco) to further neutralize the effect of the acidified nitrite solution . All cultures were incubated at 30°C in sealed bags, and the resulting CFU were counted after 28 days of incubation . Viable counts were expressed as log10 CFU per milliliter . In human M . ulcerans lesions, the organisms grow in a high-protein environment . Therefore, the effect of acidified nitrite or citric acid on M . ulcerans viability was also tested in Middlebrook 7H9 medium with ADC in which the concentration of bovine serum albumin (BSA; Sigma, St . Louis, Mo.) was increased from 0.5 to 5% (wt/vol) . We first determined the effect of exposure of M . ulcerans to acidified nitrite for 1 and 9 h, since we predicted that prolonged incubation in the presence of acidified nitrite would be necessary to kill M . ulcerans . However, complete killing was found after only 1 h . Table 1 shows the effect of acidified nitrite compared with that of pH-matched citric acid controls after 10- and 20-min incubations . Killing was again rapid, and viable counts were reduced to below detectable limits after only a 10-min exposure to acidified nitrite . Controls showed no reduction in viable counts, suggesting that killing was due to the action of acidified nitrite and not simply to an acid environment . Also, sodium nitrite alone had no effect on the viable counts (data not shown) . Increasing the protein content of the medium to 5% did not inhibit killing by acidified nitrite (Table 1) . In similar experiments, the MIC of acidified nitrite (exposure time, 10 min) for M . ulcerans isolate 1 was determined (Table 2) . The MIC of acidified nitrite for M . ulcerans with an exposure time of 10 min was below 40 mM sodium nitrite and 20 mM citric acid .
In vitro, nitric oxide can kill Escherichia coli (12, 14), Candida spp . (6), Leishmania spp . (13, 18), and M . leprae (1), and it can inhibit Staphylococcus aureus and Propionibacterium acnes (19) . M . ulcerans is an addition to the growing list of susceptible organisms . We have found that Mycobacterium tuberculosis is also susceptible to acidified nitrite at the same concentrations (unpublished data) . These results help to explain the finding that Buruli ulcers caused by M . ulcerans heal more rapidly with topical treatment with acidified nitrite than without it . Further investigations of the actions of this treatment are desirable .
What Is Antibiotic?,
What Is Fermentation?,
What Is Molecular Biology?,
What Is Molecular Microbiology?,
What Is Dna?,
i,
Microbe,
c,
Bacterium,
n,
Microbes,
r,
Microorganism,
n,
Bacteria,
o,
Streptococcal,
e,
Fermentations,
o,
Pseudomonas,
n,
Streptococcal,
s,
Flavobacterium,
a,
Prokaryotes,
n,
Bacillus subtilis,
a,
Schizosaccharomyces,
e,
Lactobacillus,
c,
Escherichia coli,
c,
Thermophile,
s,
Cryptococci,
o,
Microorganisms,
r,
Microorganism,
o,
Cell suspensions,
o,
Cryptococci,
e,
Citrobacter,
n,
Escherichia coli,
i,
Sepsis,
n,
Ps. fluorescens,
a,
Staphylococcus
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
