Vol. 29, No. 5 Printed in U.SA.
APPLD MICROBIOLOGY, May 1975, p. 615-620 Copyright 0 1975 American Society for Microbiology
Inhibition of Microbial Growth by Fatty Amine Catalysts from Polyurethane Foam Test Tube Plugs JOHN A. BACH,* RICHARD J. WNUK, AND DELANO G. MARTIN Research and Development and Physical and Analytical Chemistry, The Upjohn Company, Control Analytical Kalamazoo, Michigan 49001 Received for publication 9 December 1974
When polyurethane foam test tube plugs are autoclaved, they release volatile fatty amines that inhibit the growth of some microorganisms. The chemical structures of these amines were determined by the use of a gas chromatographmass spectrometer. They are catalysts used to produce the foam. The problem of contaminating growth media with toxic substances released from polymeric materials is discussed.
While attempting to develop an optimal isolation medium for some pink-pigmented bacteria identified as strains of Vibrio extorquens (8), we observed frequent and unexplainable failure of the organisms to grow. Eventually, we began to suspect that the growth medium was being contaminated by an inhibitory substance from polyurethane foam test tube plugs. The following article contains experimental proof for this hypothesis and a description of the isolation and identification of the inhibitory substances.
MATERIALS AND METHODS General. Except where specifically mentioned, all tubes, vials, flasks, and other equipment used to prepare or analyze the inhibitory substances from polyurethane foam were made of borosilicate glass and were closed with either ground glass stoppers or plastic screw caps with white rubber liners. Reagents. Analytical grade absolute methanol (acetone-free), hydrochloric acid, and sodium hydroxide (50% aqueous solution) were all purchased from Mallinckrodt Chemical Works, St. Louis, Mo. Glassdistilled, non-spectro grade hexane was purchased from Burdick and Jackson Laboratories, Muskegon, Mich. Regisil, a mixture of 99% N,O-bis(trimethylsilyl)-trifluoroacetamide and 1% trimethylchlorosilane, was purchased from Regis Chemical, Morton Grove, Ill. Samples of Armeen DM16D (a mixture of the tetradecyl, hexadecyl, and octadecyl derivatives of dimethylamine) and Armeen NCMD (a mixture of the N-octyl, N-decyl, N-dodecyl, N-tetradecyl, Nhexadecyl, and N-octadecyl derivatives of morpholine) were a gift from the Armour Industrial Chemical Co., Chicago, Ill. Culture media. Dehydrated culture media were obtained from the following sources: Trypticase soy broth was purchased from BBL, Cockeysville, Md., tryptone glucose extract agar was purchased from Difco, Detroit, Mich. We prepared tryptone glucose
extract broth of the same composition as the agar medium from tryptone (Difco), beef extract, and dextrose, and we prepared Sabouraud dextrose broth from peptone and dextrose obtained from Roussel UCLAF, Paris, France. The media were prepared for use according to the manufacturer's recommendations. Flasks were closed with aluminum foil and test tubes were closed with polypropylene caps. Peptone and dextrose concentrations in the Sabouraud dextrose broth were 1 and 2%, respectively. Microbial cultures. All of the microbial cultures, with three exceptions, were obtained from Alma Dietz, curator of the Upjohn Culture Collection. The exceptions were some pink-pigmented gram-negative organisms that were isolated from water at The Upjohn Co. They were very similar in colonial and microscopic appearance and in nutritional require-
ments to the strains of Vibrio extorquens described by Stocks and McCleskey (8). All cultures were grown in Trypticase soy broth, stored in the gas phase of a liquid nitrogen tank, and recultured in Trypticase soy broth for 1 to 2 h before using as inocula for experiments.
Test tube closures. The polyurethane test tube plugs were (0.75 x 1.5 inch [ca. 1.89 x 3.81 cm]) Series T 1380 Dispo plugs sold by Scientific Products. The company supplying Scientific Products with these plugs is the T. E. Forrest Co., Baltimore. The polypropylene test tube caps were Kap-uts sold by Bellco Glass Co. Inc., Vineland, N. J. Preparative extraction of antimicrobial substances from polyurethane foam plugs. Two methods of extraction were used. In the first method, the plugs were placed directly into distilled water in an aluminum foil covered flask and autoclaved at 121 C. After cooling, the plugs were removed from the flask with a sterile stainless-steel spatula. Water absorbed by the plugs was returned to the flask by squeezing each plug with the spatula as it was withdrawn. The resulting solution was called an immersed plug extract. A modified cell culture jar (Bellco Catalogue no. 1961) was used for the second extraction method. A 615
616
BACH, WNUK, AND MARTIN
APPL. MICROBIOL.
50-ml graduated cylinder was inverted in the jar to had to be protected from contamination by environprovide a platform on which to place a 50-ml beaker mental organisms. Generally, these solutions were containing 35 ml of 0.1 N HCl. Fifty polyurethane heat sterilized at pH 1 to 2 and adjusted to pH 7 with plugs were packed around the cylinder. The two side 50% (wt/vol) NaOH under sterile conditions. Sterile arms were closed loosely with glass stoppers and 100 preparations could also be obtained by evaporation of ml of distilled water was poured over the polyurethane hexane or methanol solutions with a stream of filterplugs. The beaker of HCl was placed on the cylinder sterilized nitrogen. base, the top was placed on the jar, and the entire Gas chromatography-mass spectrometry. Samapparatus was autoclaved at 121 C. After cooling, the ples were dissolved in chloroform and analyzed using jar was opened and the hydrochloric acid solution was a Varian MAT CH-7 gas chromatograph-mass specpoured into a sterile screw-capped bottle. The result- trometer. The Pyrex glass chromatographic column ing solution was called a steam-distilled extract (1.22 m by 3 mm) was packed with 1% OV-17 on Gas (SDE). Chrom Q (80/100 mesh). The column temperature Thin-layer chromatography. Commercially pre- was programmed from 130 to 230 C at 8 C/min. pared 250-Mm-thick silica gel plates (Analtech, Inc., Injector temperature was 235 C and helium (40 Newark, Del.) were used without heat activation. ml/min) was used as a carrier gas. The mass spectra Samples were applied with glass pipettes and dried were recorded at 70 eV ionizing voltage. Ion source thoroughly at room temperature before developing. temperature was 250 C. High resolution measurePlates were developed with methanol in closed glass ments were performed on a Varian CH-5 DF mass containers lined with filter paper. spectrometer using the direct introduction probe and Separated components were revealed by absorption using the peak-matching technique. Perfluorokeroof iodine vapor or by fluorescence under short wave or sene or acetone was used as the internal standlong wave ultraviolet light (UVSL.25, Mineralight, ard. Ultraviolet Products, Inc., San Gabriel, Calif.). The RESULTS antimicrobial activity of separated components was determined by scraping an area of silica gel correof the cause of growth Determination sponding to the R, of each component into 15-ml inhibition. Two sets of 19 test tubes each were conical centrifuge tubes and eluting the components with three 5-ml portions of methanol. The methanol washed with detergent, thoroughly rinsed with washes were combined and evaporated to dryness distilled water, and filled with 10 ml of Sabouwith a stream of sterile nitrogen and the residue was raud dextrose broth. One set was closed with tested for antimicrobial activity by the tube dilution polyurethane foam plugs and the other with method described below using V. extorquens strain polypropylene caps. Both sets of tubes were A. A portion of each residue was always re-chromato- autoclaved at 121 C for 90 min, cooled to room graphed to prove that the proper components had temperature, inoculated with 400 viable cells of been eluted. V. extorquens strain A per tube, and incubated Estimation of antimicrobial activity. Antimi- at 25 C. Growth was observed in all tubes closed crobial potency was most satisfactorily determined by a serial twofold tube dilution method using tryptone with polypropylene caps within 4 days. Turbidglucose extract broth as the growth medium. The test ity developed in only eight tubes closed with organisms were Mima polymorpha UC 3183 and a polyurethane plugs within 14 days and only 17 culture of V. extorquens designated strain A. V. tubes were positive within 53 days. extorquens strain A is at least 125 times more sensiSince the bag of plugs had been open for at tive than M. polymorpha to the antimicrobial sub- least a month, we considered the possibility stance, but requires 3 to 4 days incubation at 28 C to that they had become contaminated in storage. grow to visible turbidity from a 100- to 1,000-cell However, attempts to clean them prior to use by inoculum. M. polymorpha tube dilution assays could in distilled water and rinsing thoroughly be read after 24 h of incubation at 37 C. Routinely, boiling in deionized water failed to prevent inhibition. 0.1-ml aliquots of inoculum containing about 106 viable cells each were stored in the gas phase of a liquid Thus, we concluded that the inhibitory subnitrogen tank. Prior to use, the inocula were rapidly stance was probably a natural constituent of the thawed, diluted with 10 ml of tryptone glucose extract foam. broth, and incubated for 1 to 2 h to allow repair of Antimicrobial spectrum of the growth infreeze-thaw damage. Then they were diluted further hibitory substance. An immersed plug extract and used for inoculating tube dilution assays. The cell was prepared according to the procedure in population of the final inoculum was determined by Materials and Methods by using 40 polyurespreading 0.1 ml on a layer of tryptone glucose extract in 200 ml of distilled water and agar in a polystyrene petri dish, incubating the dish thane plugs for 30 min. The relative inhibitory autoclaving for 2 to 4 days, and counting colonies. for 19 species of bacteria of this solution activity Solutions of antimicrobial substance above pH 4 could not be sterilized by heat or membrane filtration from the Upjohn Culture Collection and three without losing antimicrobial activity. Therefore, solu- strains of V. extorquens isolated from water was tions intended for estimation of antimicrobial potency determined by serial dilution assay. Four of the
VOL. 29, 1975
617
INHIBITION OF MICROBIAL GROWTH
Upjohn Culture Collection species and all three strains of V. extorquens were inhibited. Inhibitory activity is expressed in Table 1 in terms of the maximal dilution that will prevent turbid growth from a small inoculum. The fact that the inhibitor is extremely active against the V. extorquens strains explains why we began to notice growth inhibition by these plugs only when we started working with these organisms. Identification of the antimicrobial substance. Further experimentation with the foam plugs revealed that the antimicrobial substance was a volatile basic material that could be trapped in dilute hydrochloric acid solution as it is released from steam-heated foam. This steam distillation method produced an extract that was free of most of the extraneous ultraviolet absorbing substances present in immersed plug extracts. Fifty milliliters of SDE were evaporated at 80 C under vacuum and the slight brownish residue was analyzed by combined gas chromatography-mass spectrometry (GC-MS). The gas chromatograph of SDE is shown in Fig. 1 and indicates the presence of five major components labeled A through E. The mass spectra of peaks A, C, D, and E are very similar. The spectrum of peak C is shown in Fig. 2. These compounds show an intense ion at mass 100.0762 which corresponds to C5HiONO+ (calculated, 100.0762). In addition, the corresponding molecular ions are odd molecular weights at m/e 227 for A, 255 for C, 283 for D, and 311 for compound E. An accurate mass determination on the molecular ion of peak C showed that its elemental composition corresponded to C 16H,SNO (calculated, 255.2562, found: 255.2556). Since the m/e 100 ion contains the nitrogen and oxygen atoms present in this
molecule, the remaining portion of the molecule contains the remaining carbon and hydrogen atoms present. Subtracting CXHNO from C1,H8,NO leaves C,1H27, a saturated long chain hydrocarbon. The features of the mass spectra of compounds A, C, D, and E show very little fragmentation between their molecular ions and the m/e 100 ion, indicating that the hydrocarbon chain is probably linear and not branched since branching in a hydrocarbon chain produces ions of significant intensity at the point of branching. The mass spectra of compounds A, C, D, and E indicate that these materials belong to a (CsHloNO) CnH2n + 1 series and differ by 28 mass units (C2HJ. The loss of CnHml, + 1 from these compounds results in the base peak at
m/e 100.
Compound B (Fig. 3) differed in its mass spectral pattern from A, C, D, and E in that it did contain a large ion at mass 100. The largest ion present in this spectrum is at mass 58.0653 and corresponds to CSHSN+ (calculated 58.0657). Although the mass spectrum shows an ion of higher intensity at mass 270, the molecular weight of this material was established as being 269. The larger 270 ion is typical for nitrogen-containing materials and is also caused by ion molecule reactions due to high sample pressure. The 58 ion C HSN+ is usually a strong ion in tertiary amines (4) and is due to cleavage of the carbon-carbon bond adjacent to the nitrogen atom (beta cleavage) giving rise to CH2an ion of the type (CH.)2N+ The mass spectrum of compound B also shows very little fragmentation between the molecular ion at 269 and the lower part of the spectrum. Although the empirical composition of the molecular ion was notdefinitely established, the tentative structure assigned to this =
1. Maximal inhibitory dilution of immersed plug extract for several bacterial speciesa Maximal inhibitory dilution Inoculum Upjohn Culture Organismb (MID) (viable cells) Collection no.
TABL
57 3183 3035 128
Klebsiella pneumoniae Mimapolymorpha Pseudomonas barkerii Sarcina lutea Vibrio extorquens, strain A Vibrio extorquens, strain B Vibrio extorquens, strain C
610 4,170
1,030 4,620 1,012 150 170
Undiluted Undiluted Twofold diluted Twofold diluted 125-fold diluted 32-fold diluted Greater than 64-fold
a The following organisms were not inhibited by the immersed plug extract: Aerobacter aerogens (UC 3), Aeromonas hydrophila (UC 3054), Alcaligenes faecalis (UC 3032), Bacillus cereus (UC 8), Bordetella bronchiseptica (UC 3287), Escherichia coli (UC 48), Erwinia atroseptica (UC 631), Herellea sp. (UC 3200), Neisseria catarrhalis (UC 3021), Proteus vulgaris (UC 93), Pseudomonas sp. (UC 3369), Serratia marcescens (UC 132), Shigella dysenteriae (UC 135), Streptococcus faecalis (UC 157), and Vibrio comma (UC 813). "The Vibrio extroquens tests were incubated for 12 days. The rest were incubated for 2 days. The growth medium was tryptone glucose extract broth made from Difco ingredients.
618
APPL. MICROBIOL. BACH, WNUK, AND MARTIN for compounds A, C, D, and E, the ones that appeared attractive to us were long chain nitro-. gen-substituted morpholines of the type
R N
CO) ce
z
0
a.
C
=
LII
0 C., miI
0
A
B
\ l E I
0
2
l 4
lI
l 6
l
, 8
10
12
TIME (MINUTES) FIG. 1. Gas chromatogram of SDE.
was (CH,)2N(C1HX,,), NN-dimethyl hex .adecyl amine. 1o determine if the components present in SD]E contained any active protons, the sample was reacted with Regisil. The derivatized sample was analyzed on a Varian MAT CH-7 GC;-MS under conditions similar to the under ivatized material. The mass spectra and the rete ntion times of the peaks present in silylated SD]E were identical to their unsilylated counterpar ts, indicating that no exchangeable protons are present in any of these compounds. ALfter considering several possible structures cor apound
The morpholine structure was attractive because we knew that morpholines were used as catalysts in the preparation of polyurethane foam (1, 2). Because the SDE residue contained a mixture of compounds, we could not be certain which ones were responsible for antimicrobial activity. Therefore, we attempted to purify the components of the residue so that antimicrobial activity could be correlated with one or more structures. Thirty-five milliliters of SDE were adjusted to pH 9 with sodium hydroxide and extracted twice with equal volumes of n-hexane. The hexane solution was concentrated to 0.5 ml by evaporation under a stream of nitrogen. Five microliters were spotted on a thin-layer plate and the plate was developed with methanol. Irradiation by short and long wave ultraviolet light and exposure to iodine vapor revealed only two well-separated iodine-abs6rbing spots. The R. of each spot was noted and chromatography was repeated with the remainder of the hexane solution. The two components were eluted from the silica gel with methanol and concentrated by evaporation. An area of silica gel between the two components was also sampled and used as a control. Both components had high antimicrobial activity, whereas the residue from the control area was totally inactive. Both components were analyzed by GC-MS. The slowest component contained a single peak with a fragmentation pattern similar to peak B in Fig. 1. The fastest component contained three peaks with fragmentation patterns similar to peaks C,
D, and E.
A search for information on alkyl amines and
morpholines turned up the following additional indirect evidence to support the general structures proposed for these compounds. Rosen and Goldsmith (7) listed the manufacturer and trade names of tertiary amines and morpholines with structures that fit the mass spectral data obtained from our extracts. A price list (14 October 1968) from the manufacturer, Armour Industrial Chemical Co., stated that these tertiary aliphatic amines (Armeens) were used as catalysts for isocyanate foams. A publication by Hueck et al. (5) showed that many fatty amine and fatty morpholine compounds inhibit the
INHIBITION OF MICROBIAL GROWTH
VOL. 29, 1975
619
100-
75-
.-100 CS H1O NO +
-IISM
50-
0-
-I
25x
0
L1 .
.,I,
I. I
i
I,"I,..
....
L
IL.
100
D
C1. H33 No +
10 W
150
Is
200
l
.255(M+)
' r~l 'ITS Im]I
250
300
m/e FIG. 2. Mass spectrum of compound C. 100-
75-
-
)I-
-I
58
C3HsN+ 50225x
0
1A
A
50
10
C1, H39 N + 269 (M+)
.
I
I
100
*0I
...
150
M'e
200
250 250
300 300
FIG. 3. Mass spectrum of compound B.
growth of bacteria, fungi, and algae. Analysis of Armeen DM16D and Armeen NCMD by GC-MS showed that they contain compounds with fragmentation patterns indentical to those found in steam distilled extracts of polyurethane foam stoppers. Thus, it is highly probable that the antimicrobial substances released from polyurethane foam stoppers are fatty amine catalysts used to prepare the foam. DISCUSSION A literature search revealed no published reports on the release of antimicrobial agents from polyurethane foam stoppers. However, two verbal reports of inhibition of algal cultures by
these stoppers have been received. These reports are consistent with the high activity of fatty amine compounds against algae reported by Hueck et al. (5). At least two other reports of growth inhibition by polymeric materials have appeared in the literature. For example, Rightsel et al. (6) reported that vinyl plastic containers are toxic for monkey kidney cells in culture unless the containers are washed with 95% denatured alcohol prior to use. They also found that polyester and were toxic for these cells. Dutka et al. (3) reported reduced recoveries of Escherichia coli deposited on membrane filters compared to control samples deposited directly on
acetate films
620
BACH, WNUK, AND MARTIN
agar. Recovery efficiencies depended on several factors such as the source of the membranes, the method of sterilizing the membrane, and the incubation temperature for growth. These reports and our own experience with polyurethane foam stoppers clearly indicate that one should not assume any polymeric material (or any other material) to be biologically inert until it has been shown to be inert under the conditions of actual use. ACKNOWLEDGMENTS We appreciate the assistance of Richard S. Hanson in identifying our strains of pink-pigmented organisms. We are also grateful to Linda K. Thompson for typing the manuscript and to Eugene Beals and Frank Meints for preparation of the figures.
LITERATURE CITED 1. Agabeg, R. C. 1964. Intermediates for polyurethane foams, p. 8. In T. T. Healy (ed.), Polyurethane foams. Iliffe
APPL. MICROBIOL.
Books, Ltd., London. 2. Dombrow, G. A. 1954. Polyurethanes, p. 21. Rembold Publishing Corp., New York. 3. Dutka, B. J., M. J. Jackson, and J. B. Bell. 1974. Comparison of autoclave and ethylene oxide-sterilized membrane filters used in water quality studies. Appl. Microbiol. 28:474-480. 4. Gohlke, R. S., and F. W. McLafferty. 1962. Mass spectrometric analysis of aliphatic amines. Anal. Chem. 34:1281-1287. 5. Hueck, H. J., D. M. M. Adema, and J. R. Wiegmann. 1966. Bacteriostatic, fungistatic, and algistatic activity of fatty nitrogen compounds. Appl. Microbiol. 14:308-319. 6. Rightsel, W. A., P. Schultz, D. Muething, and I. W. McLean, Jr. 1956. Use of vinyl plastic containers in tissue cultures for virus assays. J. Immunol. 76:464-474. 7. Rosen, M. J., and H. Goldsmith. 1972. Systematic analysis of surface active agents, p. 518-19. John Wiley and Sons, Inc., New York. 8. Stocks, P. K., and C. S. McCleskey. 1964. Identity of the pink-pigmented methanol-oxidizing bacteria as Vibrio extorquens. J. Bacteriol. 88:1065-1070.
APPLD MICROBIOLOGY, May 1975, p. 615-620 Copyright 0 1975 American Society for Microbiology
Inhibition of Microbial Growth by Fatty Amine Catalysts from Polyurethane Foam Test Tube Plugs JOHN A. BACH,* RICHARD J. WNUK, AND DELANO G. MARTIN Research and Development and Physical and Analytical Chemistry, The Upjohn Company, Control Analytical Kalamazoo, Michigan 49001 Received for publication 9 December 1974
When polyurethane foam test tube plugs are autoclaved, they release volatile fatty amines that inhibit the growth of some microorganisms. The chemical structures of these amines were determined by the use of a gas chromatographmass spectrometer. They are catalysts used to produce the foam. The problem of contaminating growth media with toxic substances released from polymeric materials is discussed.
While attempting to develop an optimal isolation medium for some pink-pigmented bacteria identified as strains of Vibrio extorquens (8), we observed frequent and unexplainable failure of the organisms to grow. Eventually, we began to suspect that the growth medium was being contaminated by an inhibitory substance from polyurethane foam test tube plugs. The following article contains experimental proof for this hypothesis and a description of the isolation and identification of the inhibitory substances.
MATERIALS AND METHODS General. Except where specifically mentioned, all tubes, vials, flasks, and other equipment used to prepare or analyze the inhibitory substances from polyurethane foam were made of borosilicate glass and were closed with either ground glass stoppers or plastic screw caps with white rubber liners. Reagents. Analytical grade absolute methanol (acetone-free), hydrochloric acid, and sodium hydroxide (50% aqueous solution) were all purchased from Mallinckrodt Chemical Works, St. Louis, Mo. Glassdistilled, non-spectro grade hexane was purchased from Burdick and Jackson Laboratories, Muskegon, Mich. Regisil, a mixture of 99% N,O-bis(trimethylsilyl)-trifluoroacetamide and 1% trimethylchlorosilane, was purchased from Regis Chemical, Morton Grove, Ill. Samples of Armeen DM16D (a mixture of the tetradecyl, hexadecyl, and octadecyl derivatives of dimethylamine) and Armeen NCMD (a mixture of the N-octyl, N-decyl, N-dodecyl, N-tetradecyl, Nhexadecyl, and N-octadecyl derivatives of morpholine) were a gift from the Armour Industrial Chemical Co., Chicago, Ill. Culture media. Dehydrated culture media were obtained from the following sources: Trypticase soy broth was purchased from BBL, Cockeysville, Md., tryptone glucose extract agar was purchased from Difco, Detroit, Mich. We prepared tryptone glucose
extract broth of the same composition as the agar medium from tryptone (Difco), beef extract, and dextrose, and we prepared Sabouraud dextrose broth from peptone and dextrose obtained from Roussel UCLAF, Paris, France. The media were prepared for use according to the manufacturer's recommendations. Flasks were closed with aluminum foil and test tubes were closed with polypropylene caps. Peptone and dextrose concentrations in the Sabouraud dextrose broth were 1 and 2%, respectively. Microbial cultures. All of the microbial cultures, with three exceptions, were obtained from Alma Dietz, curator of the Upjohn Culture Collection. The exceptions were some pink-pigmented gram-negative organisms that were isolated from water at The Upjohn Co. They were very similar in colonial and microscopic appearance and in nutritional require-
ments to the strains of Vibrio extorquens described by Stocks and McCleskey (8). All cultures were grown in Trypticase soy broth, stored in the gas phase of a liquid nitrogen tank, and recultured in Trypticase soy broth for 1 to 2 h before using as inocula for experiments.
Test tube closures. The polyurethane test tube plugs were (0.75 x 1.5 inch [ca. 1.89 x 3.81 cm]) Series T 1380 Dispo plugs sold by Scientific Products. The company supplying Scientific Products with these plugs is the T. E. Forrest Co., Baltimore. The polypropylene test tube caps were Kap-uts sold by Bellco Glass Co. Inc., Vineland, N. J. Preparative extraction of antimicrobial substances from polyurethane foam plugs. Two methods of extraction were used. In the first method, the plugs were placed directly into distilled water in an aluminum foil covered flask and autoclaved at 121 C. After cooling, the plugs were removed from the flask with a sterile stainless-steel spatula. Water absorbed by the plugs was returned to the flask by squeezing each plug with the spatula as it was withdrawn. The resulting solution was called an immersed plug extract. A modified cell culture jar (Bellco Catalogue no. 1961) was used for the second extraction method. A 615
616
BACH, WNUK, AND MARTIN
APPL. MICROBIOL.
50-ml graduated cylinder was inverted in the jar to had to be protected from contamination by environprovide a platform on which to place a 50-ml beaker mental organisms. Generally, these solutions were containing 35 ml of 0.1 N HCl. Fifty polyurethane heat sterilized at pH 1 to 2 and adjusted to pH 7 with plugs were packed around the cylinder. The two side 50% (wt/vol) NaOH under sterile conditions. Sterile arms were closed loosely with glass stoppers and 100 preparations could also be obtained by evaporation of ml of distilled water was poured over the polyurethane hexane or methanol solutions with a stream of filterplugs. The beaker of HCl was placed on the cylinder sterilized nitrogen. base, the top was placed on the jar, and the entire Gas chromatography-mass spectrometry. Samapparatus was autoclaved at 121 C. After cooling, the ples were dissolved in chloroform and analyzed using jar was opened and the hydrochloric acid solution was a Varian MAT CH-7 gas chromatograph-mass specpoured into a sterile screw-capped bottle. The result- trometer. The Pyrex glass chromatographic column ing solution was called a steam-distilled extract (1.22 m by 3 mm) was packed with 1% OV-17 on Gas (SDE). Chrom Q (80/100 mesh). The column temperature Thin-layer chromatography. Commercially pre- was programmed from 130 to 230 C at 8 C/min. pared 250-Mm-thick silica gel plates (Analtech, Inc., Injector temperature was 235 C and helium (40 Newark, Del.) were used without heat activation. ml/min) was used as a carrier gas. The mass spectra Samples were applied with glass pipettes and dried were recorded at 70 eV ionizing voltage. Ion source thoroughly at room temperature before developing. temperature was 250 C. High resolution measurePlates were developed with methanol in closed glass ments were performed on a Varian CH-5 DF mass containers lined with filter paper. spectrometer using the direct introduction probe and Separated components were revealed by absorption using the peak-matching technique. Perfluorokeroof iodine vapor or by fluorescence under short wave or sene or acetone was used as the internal standlong wave ultraviolet light (UVSL.25, Mineralight, ard. Ultraviolet Products, Inc., San Gabriel, Calif.). The RESULTS antimicrobial activity of separated components was determined by scraping an area of silica gel correof the cause of growth Determination sponding to the R, of each component into 15-ml inhibition. Two sets of 19 test tubes each were conical centrifuge tubes and eluting the components with three 5-ml portions of methanol. The methanol washed with detergent, thoroughly rinsed with washes were combined and evaporated to dryness distilled water, and filled with 10 ml of Sabouwith a stream of sterile nitrogen and the residue was raud dextrose broth. One set was closed with tested for antimicrobial activity by the tube dilution polyurethane foam plugs and the other with method described below using V. extorquens strain polypropylene caps. Both sets of tubes were A. A portion of each residue was always re-chromato- autoclaved at 121 C for 90 min, cooled to room graphed to prove that the proper components had temperature, inoculated with 400 viable cells of been eluted. V. extorquens strain A per tube, and incubated Estimation of antimicrobial activity. Antimi- at 25 C. Growth was observed in all tubes closed crobial potency was most satisfactorily determined by a serial twofold tube dilution method using tryptone with polypropylene caps within 4 days. Turbidglucose extract broth as the growth medium. The test ity developed in only eight tubes closed with organisms were Mima polymorpha UC 3183 and a polyurethane plugs within 14 days and only 17 culture of V. extorquens designated strain A. V. tubes were positive within 53 days. extorquens strain A is at least 125 times more sensiSince the bag of plugs had been open for at tive than M. polymorpha to the antimicrobial sub- least a month, we considered the possibility stance, but requires 3 to 4 days incubation at 28 C to that they had become contaminated in storage. grow to visible turbidity from a 100- to 1,000-cell However, attempts to clean them prior to use by inoculum. M. polymorpha tube dilution assays could in distilled water and rinsing thoroughly be read after 24 h of incubation at 37 C. Routinely, boiling in deionized water failed to prevent inhibition. 0.1-ml aliquots of inoculum containing about 106 viable cells each were stored in the gas phase of a liquid Thus, we concluded that the inhibitory subnitrogen tank. Prior to use, the inocula were rapidly stance was probably a natural constituent of the thawed, diluted with 10 ml of tryptone glucose extract foam. broth, and incubated for 1 to 2 h to allow repair of Antimicrobial spectrum of the growth infreeze-thaw damage. Then they were diluted further hibitory substance. An immersed plug extract and used for inoculating tube dilution assays. The cell was prepared according to the procedure in population of the final inoculum was determined by Materials and Methods by using 40 polyurespreading 0.1 ml on a layer of tryptone glucose extract in 200 ml of distilled water and agar in a polystyrene petri dish, incubating the dish thane plugs for 30 min. The relative inhibitory autoclaving for 2 to 4 days, and counting colonies. for 19 species of bacteria of this solution activity Solutions of antimicrobial substance above pH 4 could not be sterilized by heat or membrane filtration from the Upjohn Culture Collection and three without losing antimicrobial activity. Therefore, solu- strains of V. extorquens isolated from water was tions intended for estimation of antimicrobial potency determined by serial dilution assay. Four of the
VOL. 29, 1975
617
INHIBITION OF MICROBIAL GROWTH
Upjohn Culture Collection species and all three strains of V. extorquens were inhibited. Inhibitory activity is expressed in Table 1 in terms of the maximal dilution that will prevent turbid growth from a small inoculum. The fact that the inhibitor is extremely active against the V. extorquens strains explains why we began to notice growth inhibition by these plugs only when we started working with these organisms. Identification of the antimicrobial substance. Further experimentation with the foam plugs revealed that the antimicrobial substance was a volatile basic material that could be trapped in dilute hydrochloric acid solution as it is released from steam-heated foam. This steam distillation method produced an extract that was free of most of the extraneous ultraviolet absorbing substances present in immersed plug extracts. Fifty milliliters of SDE were evaporated at 80 C under vacuum and the slight brownish residue was analyzed by combined gas chromatography-mass spectrometry (GC-MS). The gas chromatograph of SDE is shown in Fig. 1 and indicates the presence of five major components labeled A through E. The mass spectra of peaks A, C, D, and E are very similar. The spectrum of peak C is shown in Fig. 2. These compounds show an intense ion at mass 100.0762 which corresponds to C5HiONO+ (calculated, 100.0762). In addition, the corresponding molecular ions are odd molecular weights at m/e 227 for A, 255 for C, 283 for D, and 311 for compound E. An accurate mass determination on the molecular ion of peak C showed that its elemental composition corresponded to C 16H,SNO (calculated, 255.2562, found: 255.2556). Since the m/e 100 ion contains the nitrogen and oxygen atoms present in this
molecule, the remaining portion of the molecule contains the remaining carbon and hydrogen atoms present. Subtracting CXHNO from C1,H8,NO leaves C,1H27, a saturated long chain hydrocarbon. The features of the mass spectra of compounds A, C, D, and E show very little fragmentation between their molecular ions and the m/e 100 ion, indicating that the hydrocarbon chain is probably linear and not branched since branching in a hydrocarbon chain produces ions of significant intensity at the point of branching. The mass spectra of compounds A, C, D, and E indicate that these materials belong to a (CsHloNO) CnH2n + 1 series and differ by 28 mass units (C2HJ. The loss of CnHml, + 1 from these compounds results in the base peak at
m/e 100.
Compound B (Fig. 3) differed in its mass spectral pattern from A, C, D, and E in that it did contain a large ion at mass 100. The largest ion present in this spectrum is at mass 58.0653 and corresponds to CSHSN+ (calculated 58.0657). Although the mass spectrum shows an ion of higher intensity at mass 270, the molecular weight of this material was established as being 269. The larger 270 ion is typical for nitrogen-containing materials and is also caused by ion molecule reactions due to high sample pressure. The 58 ion C HSN+ is usually a strong ion in tertiary amines (4) and is due to cleavage of the carbon-carbon bond adjacent to the nitrogen atom (beta cleavage) giving rise to CH2an ion of the type (CH.)2N+ The mass spectrum of compound B also shows very little fragmentation between the molecular ion at 269 and the lower part of the spectrum. Although the empirical composition of the molecular ion was notdefinitely established, the tentative structure assigned to this =
1. Maximal inhibitory dilution of immersed plug extract for several bacterial speciesa Maximal inhibitory dilution Inoculum Upjohn Culture Organismb (MID) (viable cells) Collection no.
TABL
57 3183 3035 128
Klebsiella pneumoniae Mimapolymorpha Pseudomonas barkerii Sarcina lutea Vibrio extorquens, strain A Vibrio extorquens, strain B Vibrio extorquens, strain C
610 4,170
1,030 4,620 1,012 150 170
Undiluted Undiluted Twofold diluted Twofold diluted 125-fold diluted 32-fold diluted Greater than 64-fold
a The following organisms were not inhibited by the immersed plug extract: Aerobacter aerogens (UC 3), Aeromonas hydrophila (UC 3054), Alcaligenes faecalis (UC 3032), Bacillus cereus (UC 8), Bordetella bronchiseptica (UC 3287), Escherichia coli (UC 48), Erwinia atroseptica (UC 631), Herellea sp. (UC 3200), Neisseria catarrhalis (UC 3021), Proteus vulgaris (UC 93), Pseudomonas sp. (UC 3369), Serratia marcescens (UC 132), Shigella dysenteriae (UC 135), Streptococcus faecalis (UC 157), and Vibrio comma (UC 813). "The Vibrio extroquens tests were incubated for 12 days. The rest were incubated for 2 days. The growth medium was tryptone glucose extract broth made from Difco ingredients.
618
APPL. MICROBIOL. BACH, WNUK, AND MARTIN for compounds A, C, D, and E, the ones that appeared attractive to us were long chain nitro-. gen-substituted morpholines of the type
R N
CO) ce
z
0
a.
C
=
LII
0 C., miI
0
A
B
\ l E I
0
2
l 4
lI
l 6
l
, 8
10
12
TIME (MINUTES) FIG. 1. Gas chromatogram of SDE.
was (CH,)2N(C1HX,,), NN-dimethyl hex .adecyl amine. 1o determine if the components present in SD]E contained any active protons, the sample was reacted with Regisil. The derivatized sample was analyzed on a Varian MAT CH-7 GC;-MS under conditions similar to the under ivatized material. The mass spectra and the rete ntion times of the peaks present in silylated SD]E were identical to their unsilylated counterpar ts, indicating that no exchangeable protons are present in any of these compounds. ALfter considering several possible structures cor apound
The morpholine structure was attractive because we knew that morpholines were used as catalysts in the preparation of polyurethane foam (1, 2). Because the SDE residue contained a mixture of compounds, we could not be certain which ones were responsible for antimicrobial activity. Therefore, we attempted to purify the components of the residue so that antimicrobial activity could be correlated with one or more structures. Thirty-five milliliters of SDE were adjusted to pH 9 with sodium hydroxide and extracted twice with equal volumes of n-hexane. The hexane solution was concentrated to 0.5 ml by evaporation under a stream of nitrogen. Five microliters were spotted on a thin-layer plate and the plate was developed with methanol. Irradiation by short and long wave ultraviolet light and exposure to iodine vapor revealed only two well-separated iodine-abs6rbing spots. The R. of each spot was noted and chromatography was repeated with the remainder of the hexane solution. The two components were eluted from the silica gel with methanol and concentrated by evaporation. An area of silica gel between the two components was also sampled and used as a control. Both components had high antimicrobial activity, whereas the residue from the control area was totally inactive. Both components were analyzed by GC-MS. The slowest component contained a single peak with a fragmentation pattern similar to peak B in Fig. 1. The fastest component contained three peaks with fragmentation patterns similar to peaks C,
D, and E.
A search for information on alkyl amines and
morpholines turned up the following additional indirect evidence to support the general structures proposed for these compounds. Rosen and Goldsmith (7) listed the manufacturer and trade names of tertiary amines and morpholines with structures that fit the mass spectral data obtained from our extracts. A price list (14 October 1968) from the manufacturer, Armour Industrial Chemical Co., stated that these tertiary aliphatic amines (Armeens) were used as catalysts for isocyanate foams. A publication by Hueck et al. (5) showed that many fatty amine and fatty morpholine compounds inhibit the
INHIBITION OF MICROBIAL GROWTH
VOL. 29, 1975
619
100-
75-
.-100 CS H1O NO +
-IISM
50-
0-
-I
25x
0
L1 .
.,I,
I. I
i
I,"I,..
....
L
IL.
100
D
C1. H33 No +
10 W
150
Is
200
l
.255(M+)
' r~l 'ITS Im]I
250
300
m/e FIG. 2. Mass spectrum of compound C. 100-
75-
-
)I-
-I
58
C3HsN+ 50225x
0
1A
A
50
10
C1, H39 N + 269 (M+)
.
I
I
100
*0I
...
150
M'e
200
250 250
300 300
FIG. 3. Mass spectrum of compound B.
growth of bacteria, fungi, and algae. Analysis of Armeen DM16D and Armeen NCMD by GC-MS showed that they contain compounds with fragmentation patterns indentical to those found in steam distilled extracts of polyurethane foam stoppers. Thus, it is highly probable that the antimicrobial substances released from polyurethane foam stoppers are fatty amine catalysts used to prepare the foam. DISCUSSION A literature search revealed no published reports on the release of antimicrobial agents from polyurethane foam stoppers. However, two verbal reports of inhibition of algal cultures by
these stoppers have been received. These reports are consistent with the high activity of fatty amine compounds against algae reported by Hueck et al. (5). At least two other reports of growth inhibition by polymeric materials have appeared in the literature. For example, Rightsel et al. (6) reported that vinyl plastic containers are toxic for monkey kidney cells in culture unless the containers are washed with 95% denatured alcohol prior to use. They also found that polyester and were toxic for these cells. Dutka et al. (3) reported reduced recoveries of Escherichia coli deposited on membrane filters compared to control samples deposited directly on
acetate films
620
BACH, WNUK, AND MARTIN
agar. Recovery efficiencies depended on several factors such as the source of the membranes, the method of sterilizing the membrane, and the incubation temperature for growth. These reports and our own experience with polyurethane foam stoppers clearly indicate that one should not assume any polymeric material (or any other material) to be biologically inert until it has been shown to be inert under the conditions of actual use. ACKNOWLEDGMENTS We appreciate the assistance of Richard S. Hanson in identifying our strains of pink-pigmented organisms. We are also grateful to Linda K. Thompson for typing the manuscript and to Eugene Beals and Frank Meints for preparation of the figures.
LITERATURE CITED 1. Agabeg, R. C. 1964. Intermediates for polyurethane foams, p. 8. In T. T. Healy (ed.), Polyurethane foams. Iliffe
APPL. MICROBIOL.
Books, Ltd., London. 2. Dombrow, G. A. 1954. Polyurethanes, p. 21. Rembold Publishing Corp., New York. 3. Dutka, B. J., M. J. Jackson, and J. B. Bell. 1974. Comparison of autoclave and ethylene oxide-sterilized membrane filters used in water quality studies. Appl. Microbiol. 28:474-480. 4. Gohlke, R. S., and F. W. McLafferty. 1962. Mass spectrometric analysis of aliphatic amines. Anal. Chem. 34:1281-1287. 5. Hueck, H. J., D. M. M. Adema, and J. R. Wiegmann. 1966. Bacteriostatic, fungistatic, and algistatic activity of fatty nitrogen compounds. Appl. Microbiol. 14:308-319. 6. Rightsel, W. A., P. Schultz, D. Muething, and I. W. McLean, Jr. 1956. Use of vinyl plastic containers in tissue cultures for virus assays. J. Immunol. 76:464-474. 7. Rosen, M. J., and H. Goldsmith. 1972. Systematic analysis of surface active agents, p. 518-19. John Wiley and Sons, Inc., New York. 8. Stocks, P. K., and C. S. McCleskey. 1964. Identity of the pink-pigmented methanol-oxidizing bacteria as Vibrio extorquens. J. Bacteriol. 88:1065-1070.
