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Antimicrobial Agents and Chemotherapy, April 2008, p. 1577-1579, Vol. 52, No. 4
0066-4804/08/$08.00+0     doi:10.1128/AAC.01023-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Killing of Neisseria gonorrhoeae, Streptococcus agalactiae (Group B Streptococcus), Haemophilus ducreyi, and Vaginal Lactobacillus by 3-O-Octyl-sn-Glycerol{triangledown}

B. J. Moncla,1,2* K. Pryke,2 and Charles E. Isaacs3

Department of Obstetrics, Gynecology and Reproductive Sciences,1 Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, Pennsylvania,2 New York State Institute for Basic Research in Developmental Disabilities, Department of Developmental Biochemistry, Staten Island, New York3

Received 3 August 2007/ Returned for modification 21 September 2007/ Accepted 22 January 2008


   ABSTRACT
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The microbicide candidate octylglycerol inactivates sexually transmitted bacterial pathogens at concentrations which spare normal vaginal flora (lactobacillus). Standard minimum microbicidal concentration assays and time-kill assays revealed the drug concentrations and times required for inactivation. Octylglycerol concentrations must exceed the binding capacity of any human serum albumin to be effective.


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Octylglycerol (OG) is an antimicrobial lipid currently being evaluated as a vaginal microbicide to reduce the transmission of human immunodeficiency virus and other sexually transmitted infections (STIs). It is similar in activity to antimicrobial lipids naturally occurring in human milk (5). Its efficacy against human immunodeficiency virus and herpes simplex virus has been demonstrated (3-6). Ideally, vaginal microbicides should not have deleterious effects on the normal vaginal flora such as H2O2-producing Lactobacillus spp. while killing pathogens. We therefore tested the activity of 1-O-octyl-sn-glycerol (1-OG) and 3-OG (Fig. 1) against Neisseria gonorrhoeae, Haemophilus ducreyi, group B streptococci, and Lactobacillus species. 3-OG is much less expensive to synthesize than 1-OG, and therefore most of these studies were done with 3-OG. 3-OG was obtained from Genzyme (Cambridge, MA; lot number 6234). 1-OG was synthesized in C. E. Isaacs's laboratory.


Figure 1
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  		FIG. 1. Structure of 1-OG and 3-OG.

 
The minimum microbicidal concentration (MCC) assay is the current standard to determine the minimum concentration of drug required to kill 99.99% of the test organisms in 30 min. Relevant organisms were studied using the assay exactly as described in the literature (6, 8), with the exception that we used ACES [N-(2-acetamido)-2-aminoethanesulfonic acid] in place of RPMI 1640 as our medium as described previously (8). Bacterial reference strains were obtained from the American Type Culture Collection (ATCC) (Manassas, VA). Field isolates were obtained from human vaginal samples and identified to the species level as described previously (8). Organisms were stored frozen at –70°C in litmus milk until needed and revived by plating onto either blood agar plates (Columbia blood agar base for Lactobacillus and Streptococcus; PML Microbiologicals, Wilsonville, OR) or chocolate agar (PML Microbiologicals or prepared in house) for Neisseria gonorrhoeae. Haemophilus ducreyi isolates, kindly provided by P. Totten, University of Washington, Seattle, were cultured and stored as described for N. gonorrhoeae. All cultures were incubated at 35°C in air enriched to 6% CO2 overnight or until good growth was observed. Pseudomonas aeruginosa was cultured as described for Lactobacillus.

As demonstrated in Table 1, N. gonorrhoeae and H. ducreyi had lower MCCs than the other organisms tested. MCCs of the strains tested with both 1-OG and 3-OG were comparable, but the 1-OG MCCs demonstrated narrower ranges (Table 1). Representative Lactobacillus vaginalis strains were found to be resistant to killing by the highest concentration tested, 50 mM (Table 1).


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  		TABLE 1. MCCs for Lactobacillus jensenii, Lactobacillus crispatus, Lactobacillus iners, and N. gonorrhoeae

 
The presence of serum and whole blood reduced the microbicidal activity of OG and other microbicide candidates screened (data not shown). Albumin, the major serum protein, has well-characterized fatty acid binding sites; therefore, we examined bacterial killing in the presence of human serum albumin (HSA). Killing was reduced partially or completely, depending upon the ratio of HSA to OG. In order to estimate the binding of 3-OG to HSA, MCCs were determined in the presence of different concentrations of HSA. The changes in the apparent MCCs were used to estimate the quantity of free OG. Assays of MCCs without HSA were performed concurrently with each experiment, as outlined above. HSA and "essential-fatty-acid-free" HSA were obtained from Sigma Chemical Co. (1x crystallized and lyophilized; product numbers A9511 and A3782; essentially globulin and fatty acid free). For use, 10% (wt/vol) solutions were prepared in ACES buffer, filter sterilized, and stored at 4°C until used (up to 1 week). ACES buffer was used at a pH of 7.2 with an isotonic strength adjusted to 200 to 300 mosmol/kg (8). The concentration of free OG was estimated assuming different numbers of OG binding sites on the HSA. Using the MCCs for the organisms tested suggested a six-binding-site model (Table 2). Using delipidated HSA resulted in greater protection from killing (Table 3). P. aeruginosa was used to demonstrate that the effect of HSA is a general phenomenon and not just interaction with vaginal organisms.


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  		TABLE 2. Estimation of binding of 3-OG to HSAa

 

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  		TABLE 3. Protection from OG killing of Pseudomonas aeruginosaa by HSA and delipidated HSA

 
An effective microbicide for STI prevention must kill the pathogens in 30 min or less of exposure. In order to demonstrate the rapidity of OG killing, kill curves were used in addition to MCCs (Fig. 2). Organisms cultured on fresh media as described above were suspended in ACES buffer to a McFarland density of 1.0. 3-OG at, below, and above the MCC was prepared as a 10-fold excess solution in the appropriate buffer. Experiments were initiated by the addition of 1/10 volume of 3-OG to the bacterial suspensions and mixing with a vortex mixer. Samples were taken at various times and diluted into 5% HSA containing ACES buffer (HSA binds the OG and stops killing), and serial 10-fold dilutions were prepared and plated in triplicate on the appropriate medium and used to determine the concentration of bacteria at each time point. The number of organisms was determined at time zero from the starting bacterial suspension. The suspension was incubated along with a suspension containing the drug and sampled at 5 min. If the bacterial concentrations differed at the two times, the experiment was rejected. Values of CFU per ml were determined from plates containing between 30 and 300 colonies per plate. The data were used to construct survival curves as log10 CFU/ml versus time of exposure. Decimal reduction times (D values) were calculated using the linear portion of the graph (using linear regression) as D = –1/slope of the graph of log10 CFU/ml versus time (1, 2, 7, 9, 10) and by inspection. 3-OG rapidly kills test organisms at or above the MCC for the organism, with D values typically of about 1 min. Little killing was observed below the MCC. As currently defined, the MCC for an organism and drug represents the concentration where the D value is less than 8 min. Combining both methods easily defines the important parameters of microbicides.


Figure 2
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  		FIG. 2. Survival of microorganisms at the MCCs. Organisms were exposed to 3-OG at the indicated concentration and samples taken and plated to determine the number of CFU surviving. Curves for organisms above their MCCs are not presented. (Top) OG at 2.5 mM (MCC of N. gonorrhoeae ATCC 19424 [{blacksquare}]); (middle), OG at 5 mM (MCC for Lactobacillus jensenii ATCC 25258 [{blacktriangleup}] and Lactobacillus iners 1202 [x]); (bottom) OG at 7.5 mM (MCC for Lactobacillus crispatus ATCC 33197 [{blacklozenge}]).

 
OG inactivated STI pathogens at concentrations lower than those required to kill vaginal lactobacilli, supporting its use as a vaginal microbicide. Use of 1-OG and 3-OG resulted in comparable MCCs. HSA appears to bind the fatty acid moiety of OG, resulting in protection of bacteria until the molar ratio of OG to HSA exceeds 6 plus the MCC, at which point the pathogens are typically inactivated.


   ACKNOWLEDGMENTS
 
This work was supported by grants 6PO1 AI39061 and 5U19 A1051 661-05 from the National Institutes of Health.


   FOOTNOTES
 
* Corresponding author. Mailing address: Room 520, Magee-Womens Research Institute, 204 Craft Avenue, Pittsburgh, PA 15213-3180. Phone: (412) 641-6025. Fax: (412) 641-5290. E-mail: bjm4{at}pitt.edu Back

{triangledown} Published ahead of print on 28 January 2008. Back


   REFERENCES
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 ABSTRACT
 TEXT
 REFERENCES
 

  1. Bruch, M. K. 1991. Methods of testing antiseptics: antimicrobials used topically in humans and procedures for hand scrubs, p. 1028-1068. In S. S. Block (ed.), Disinfection, sterilization, and preservation, 4th ed. Lea & Febiger, Philadelphia, PA.
  2. Canton, E., J. Peman, M. Sastre, M. Romero, and A. Espinel-Ingroff. 2006. Killing kinetics of caspofungin, micafungin, and amphotericin B against Candida guilliermondii. Antimicrob. Agents Chemother. 50:2829-2832.[Abstract/Free Full Text]
  3. Gupta, P., R. Deena, B. K. Patterson, L. Caruso, K. Kathy, L. C. Rohan, M. A. Parniak, C. E. Isaacs, and S. L. Hillier. 2006. Use of frozen-thawed cervical tissues in the organ culture system to measure anti-HIV activities of candidate microbicides. AIDS Res. Hum. Retrovir. 22:419-424.[CrossRef][Medline]
  4. Isaacs, C. E., and M. F. Lampe. 2000. Lactolipids, p. 159-182. In A. S. Naidu (ed.), Natural food antimicrobial systems. CRC Press, Boca Raton, FL.
  5. Isaacs, C. E., J. H. Jia, and W. Xu. 2004. A lipid-peptide microbicide inactivates herpes simplex virus. Antimicrob. Agents Chemother. 48:3182-3184.[Abstract/Free Full Text]
  6. Lampe, M. F., L. M. Ballweber, C. E. Isaacs, D. L. Patton, and W. E. Stamm. 1998. Killing of Chlamydia trachomatis by novel antimicrobial lipids adapted from compounds in human breast milk. Antimicrob. Agents Chemother. 42:1239-1244.[Abstract/Free Full Text]
  7. Mazzola, P. G., T. C. Penna, and A. M. Martins. 2003. Determination of decimal reduction time (D value) of chemical agents used in hospitals for disinfection purposes. BMC Infect. Dis. 3:24.[CrossRef][Medline]
  8. Moncla, B. J., and S. L. Hillier. 2005. Why nonoxynol-9 may have failed to prevent acquisition of Neisseria gonorrhoeae in clinical trials. Sex. Transm. Dis. 32:491-494.[CrossRef][Medline]
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  10. Wickramanayake, G. B., and J. Sproul. 1991. Kinetics of the inactivation of microorganisms, p. 72-84. In S. S. Block (ed.), Disinfection, sterilization, and preservation, 4th ed. Lea & Febieger, Philadelphia, PA.

Antimicrobial Agents and Chemotherapy, April 2008, p. 1577-1579, Vol. 52, No. 4
0066-4804/08/$08.00+0     doi:10.1128/AAC.01023-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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