S U M M A R Y :Spermine was found t o inhibit the growth of various micro-organisms. The most susceptible were Staphylococcus aureus and Staph. albus, NeisseTia gonorrhoeae and N . meningitidis, and to a lesser degree Bacillus anthracis. The Enterobacteriaceae were relatively resistant. The action of spermine was enhanced by an increase in pH of the medium. The antibacterial effect was bactericidal ; it was slowed but not prevented by a decrease in temperature from 37 to 6'.
The inhibitory activity of human semen on the growth of Staphylococcus aureus has been reported previously (Rozansky, Gurevitch, Brzezinsky, and Eckerling, 1949).The Function of Human Semen The investigation has now been extended using 12 addi.ional freshly isolated strains of Staph. aureus.Materials and Methods.-Seventy-five specimens of semen from 53 patients (of the male sterility clinic) between the ages of 23 and 51 years were examined. Six of these were azoospermic, 25 oligospermic (less than 60 million spermatozoa per ml.), and 22 were normospermic. Ten specimens of human blood serum, 10 of cerebrospinal fluids, 10 of fluids from ovarian cysts, eight of amniotic fluids, four of pleural exudate, three of tears, and three of ox semen* were also tested in a similar manner. Materials were only taken from patients who had not received antibiotic treatment. The specimens of human semen were kept at room temperature for four hours before examination, then tested immediately, or after 72 hours at 80 C. or 14 days' refrigeration at 80 C. Twelve specimens were also examined after heating at 900 C. for 30 minutes. In addition to the three strains of Staph. aureus used in the previous study, 12 freshly isolated strains from surgical cases or pyogenic skin infections were tested. All strains were actively haemolytic, coagulase-positive, and mannitol-positive. The method described in the previous study (Rozansky et al., 1949)
SUMMARY: Spermine and spermidine inhibited the growth of Staphylococcus aureus under both aerobic and anaerobic conditions. The activity of spermine was decreased by sera of different species; sheep and bovine serum showed most effect. This antagonistic effect was destroyed by heating the sera at 100' for 30 min. Sodium and potassium also decreased the antibacterial effect of spermine and spermidine. Spermine exerted a bactericidal effect on staphylococci in broth at 37" and at 6 O , but not on washed organisms suspended in buffer at 6'. The dependence of the bactericidal effect of the bases seems to depend on some metabolic activity of the cocci.The antibacterial action of spermine and spermidine was described previously (Gurevitch, Rozansky, Weber, Brzezinsky & Eckerling, 1951 ; Rozansky, Bachrach & Grossowicz, 1954; Rozansky, Razin & Grossowicz, 1954) ; Hirsch & Dubos (1952) studied the antimycobacterial effect of these substances. According to Hirsch (1953 a, b ) spermine itself was not antimycobacterial but a derivative of it, obtained by the action of an oxidase present in sheep and bovine sera, was the active agent. I n the present paper evidence is presented that spermine itself is bactericidal against staphylococci. METHODSThe technique described previously was used (Rozansky, Bachrach & Grossowicz, 1954). Spermine tetrahydrochloride and spermidine phosphate (HoffmanLa Roche Inc.) were used. The antistaphylococcal tests were made in Difco nutrient broth. The pH value of the medium was adjusted by adding sodium hydroxide or hydrochloric acid and was measured by a Beckman pH meter. Nutrient broth containing increasing twofold dilutions of spermine or spermidine was distributed into 2 ml. quantities in 100 x 12 mm. test tubes. The tubes were plugged with cotton-wool and autoclaved for 20 min. at 15 lb./sq.in.StaphyZococcus aureus strain 23 was the main test organism; 0.1 ml. of a 1/104 dilution of a 24 hr. broth culture was used as inoculum. The tubes were incubated at 37O, unless otherwise specified. The antibacterial activity was expressed as the minimal concentration of spermine or spermidine which caused complete inhibition of growth visible to the naked eye. In experiments to test the bactericidal effect of spermine, a plate count technique was used.Anaerobic tests were performed in a jar from which the air was evacuated and the remaining oxygen removed by pyrogallol and NaOH; reduced methylene blue was used as indicator of anaerobiosis.
Several workers have ascribed antibacterial properties to human cervical mucus. Barton and Wiesner ( 1 ) found that cervical mucus sometimes produced zones of growth inhibition of staphylococci in agar plates. Pommerenke( 2 ) reported the inhibitory action of cervical mucus on Streptococcus heiiiolyticus and Staphylococcus aurem and stated that hemolytic streptococci and staphylococci incubated for 2 hours with cervical mucus and subsequently plated out on solid media yielded 70-8056 fewer colonies than untreated control cultures. No other reports on this subject could be traced in the available English literature.Our aim in this study was to reinvestigate this reported antibacterial activity and if possible to isolate the substance or substances responsible for it. The bacterial flora of the mucus were also investigated.Material and wethods. The cervical mucus from 110 healthy women between 18 and 40 years of age was examined. Jlucus was taken between the 10th and 17th day of the menstrual period, i.e., near the time of ovulation when mucus is more copious, clearer and less viscous.The cervis was cleaned with gauze, and the mucus aspirated by a sterile tuberculin syringe introduced into the cervical canal. Only women with a healthy cervix, with no erosion or purulent discharge, were selected.The bacterial flora were examined in 85 spximens by inoculating part of the mucus into Difco Thioglycollate broth. Positive cultures were subinoculated on blood agar and chocolate agar and incubated aerobically and anaerobically.Tests for antibacterial activity. All samples were preserved at 6'(' until esamined. The selection of a siiitahle technique was difficult due to the viscous nature of the mucus. Even the most clear and "fluid" samples were hardly miscible in fluids and could not be diluted for exact quantitative tests. Various attempts to liquefy the mucus were unsuccessful. Treatment with hyaluronidase or with hyaluronidase together with 0.05 NaOH as described by Pommerenke was of no avail. Liquefaction could be obtained by adding 1N SaOH or 1N H2S04, but since these reagents might themselves destroy the antibacterial substance this treatment was not considered desirable. Another drawback of this method is the necessity of neutralizing the mucus after liquefaction, so that the substance looked for is unduly diluted. The following technic was finally adopted: Circular holes, 5 mm in diameter, were punched in agar plates inoculated with the organisms examined. The holes were filled with mucus and the plates incubated at 37°C. In the latter part of the study 2-3 drops of a 1 mg/ml solution of lysozyme chloride (crystalline, egg diite. product of L. Light & Co.) were introduced into an additional similar hole.After 24 hours incubation the plates were inspected for the appearance of zones of inhibi t ion.The following micro-organisms were tested: Staphylococcus aureus (coagulase positive) , .'taphylococcus albus (coagulase negative) , Strcptococcus heinolyticus, Streptococcus fecalis, Escherichia coli, Micrococcus lys...
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