Impact of Anaerobic Growth Conditions on Toxic Shock Syndrome Toxin-I Production by Staphylococcus aureus

Objective: The impact of anaerobic growth conditions on the Staphylococcus aureus toxic shock syndrome toxin-1 (TSST-1) production was studied. Methods: Ten strains of S. aureus derived from patients with toxic shock syndrome (TSS), 10 isolates of S. aureus, and documented TSST-1-producing strains recovered from patients with either staphylococcal septicemia or staphylococcal nongenital abscesses were grown under aerobic and anaerobic conditions. The bacterial growth was measured using optical density (OD) determinations at 520 nm. The toxin production was assayed using the TS-RPLA latex agglutination test. Results: Both TSS and non-TSS strains of S. aureus grown under aerobic and anaerobic conditions exhibited comparable OD patterns of growth, and the levels of toxin production remained constant during the logarithmic phase. Toxin titers developed during the logarithmic growth phase and peaked after 24 h of incubation. When stationary-phase isolates grown initially under aerobic conditions were subjected to strict anaerobic conditions, subsequent toxin titers, compared with isolates grown in the continued presence of oxygen, were depressed 2-fold, peaking at a later time. Conclusions: TSST-1 production is diminished under continued anaerobic conditions.

during the logarithmic phase. Toxin titers developed during the logarithmic grovvth phase and peaked after 24 h of incubation. When stationary-phase isolates grown initially under aerobic conditions were subjected to strict anaerobic conditions, subsequent toxin titers, compared with isolates grown in the continued presence of oxygen, were depressed 2-fold, peaking at a later time.
Conclusions: TSST-1 production is diminished under continued anaerobic conditions.

KEY WORDS
Toxic shock syndrome, bacterial growth, tampons oxic shock syndrome (TSS) has been primarily a disease of tampon users at the time of menstruation. 1 '2 Since the withdrawal of selected highly absorbent vaginal tampons and better-informed use of vaginal tampons, the prevalence of TSS has shifted back to clinical situations not associated with menstruation. 3 '4 In obstetrics, the syndrome has been described following vaginal delivery, cesarean delivery, and spontaneous and therapeutic abortions. In the gynecologic patient, TSS has been documented as complicating surgical procedures such as tubal ligation, vaginal and abdominal hysterectomies, urethral and bladder suspensions, explor-atory laparotomy, and therapy of extensive condyloma acuminatum. 5-8 TSS may occur in patients with mastitis, Bartholin's gland abscess, wound infection, or acute salpingitis. What did superabsorbent tampon utilization do to magnify the incidence of disease? Staphylococcus aureus, the causative agent for TSS, and selected strains of S. epidermidis are the only genuses of bacteria with the capacity for being monoetiologic pathogens in both oxygen-rich and oxygen-deprived (abscess) situations. The volumetric expansion achieved by superabsorbent tampons introduced the possibility of the conversion of a

CelI-Densi W Determinations
Ten different cultures of TSS strains of S. aureus were each grown overnight at 37C in T-Y broth under aerobic and anaerobic conditions (gasp, Baltimore Biological Laboratory). The media to be inoculated anaerobically were prereduced in anaerobic jars for 24 h. For each strain, 4 sets of tubes containing 10 ml of T-Y broth were each inoculated with 0.1 ml of the 24-h broth culture of TSS-associated S. aureus. Two sets of the tubes were inoculated with a 24-h aerobic culture. Of these, set was incubated aerobically and the other anaerobically. The remaining 2 sets (prereduced) were inoculated with a 24-h aerobic broth culture. One set was incubated aerobically, and the other set was incubated anaerobically. All tubes were incubated at 37C. The optical density (OD) was measured at 52 nm using a Spectronic 100 spectrophotometer Production of TSST-I Six sets of 8 50-ml culture tubes, each containing 30 ml of T-Y broth, were inoculated with a 0.1ml aliquot of TSST-l-producing S. aureus preculture that had reached a stationary growth phase under aerobic conditions at 37C. Three sets were incubated under aerobic conditions, and 3 sets were incubated under anaerobic conditions at 37C. The media to be inoculated anaerobically were prereduced by boiling for 10 min, dispensed into 50-ml culture tubes, overlaid with nitrogen gas, and autoclaved at 121C for 15 min. The inoculation of the prereduced medium was carried out in the presence of nitrogen gas. The OD (520 nm) was measured at varied time intervals.
The above procedure was repeated using T-Y broth supplemented with 0.06 M phosphate buffer (see above).

Assay of TSST-I
The toxin production was assayed using the TST-RPLA latex agglutination test (Oxoid, Inc., Columbia, MD) in microtiter plates. Briefly, the cell cultures were centrifuged at 17,000 g for 15 min and the supernatant filter was sterilized. Serial 2-fold dilutions of each cell-free filtrate were tested for the presence of TSST-1. The reciprocal of the highest dilution giving a positive agglutination reaction was considered the end point. The toxin titers were expressed in units per milliliter (u/ml).  subjected to anaerobic conditions by placing them in Gas Pack jars. At specific times during aerobic and anaerobic culture conditions, the OD of tube from each set was measured at 520 nm. The contents of each tube were subsequently centrifuged at 17,000 g for 10 min filter-sterilized, measured for pH, and assayed for the presence of toxin.

Enzyme Lysis
At specific times during the culture of TSST-1, S. aureus enzyme lysis was performed. The pellet obtained by centrifugation of broth cultures was mixed with ml of lysozyme and incubated at 37C for h. The supernatant from the above centrifugation was subsequently added to the lysed cells and this mixture was centrifuged at 17,000 g. The resultant preparation was then filter-sterilized and the toxin titers were measured.

RESULTS
The aerobic and anaerobic growth curves of the strains of S. aureus derived from patients with in vivo evidence of TSST-1 production were ana-lyzed. The resulting aerobic and anaerobic curves did not differ in terms of the quantity of bacteria produced during the logarithmic phase of growth or the time frame in which maximum growth was achieved (Fig. 1). While there was no initial difference quantitatively or temporally in the number of S. aureus colonies engendered by aerobic and anaerobic conditions, after 24 h, the curves of the bacteria grown anaerobically began to exhibit significant disparity (Fig. 1). The OD of the cultures of S. aureus grown under aerobic conditions remained relatively level. Those grown under strict anaerobic conditions exhibited a decrease in OD through 168 h. The fall in OD was reversed in cultures grown anaerobically when the culture was reexposed to air (Fig. 1). The quantitative assessment of viability completed by serial dilution demonstrated a l-log reduction in the number of cfu/ml of culture medium for a given strain of S. aureus grown under anaerobic as opposed to aerobic conditions (Fig.   2). However, this change in the number of viable bacteria did not occur until 50-60 h. The changes in the OD observed were not unique to the TSS stains of S. aureus. Comparable changes were observed with the nongenital strains of S. aureus.

TSST-Production
The elaboration of TSST-1 by toxigenic strains of S. aureus occurred during the logarithmic phase of cell growth under aerobic as well as under anaerobic conditions. The peak toxin titers occurred after 48 h of incubation (Fig. 3) and remained constant for 96 h under both aerobic and anaerobic conditions. Despite a fall in the OD of the TSS culture grown anaerobically, the toxin production plateaued and remained at a constant level. The extension of anaerobic conditions resulted in no further increase in toxin production, despite a further fall in OD. In this study, a clear 2-fold-higher toxin titer was obtained under aerobic conditions. An exposure of the stationary-phase aerobic cultures to anaerobic culture conditions revealed that peak toxin titers occurred at 24 h of incubation with no additional toxin production noted under prolonged anaerobic conditions (Fig. 4). The enzyme lysis ofcell cultures resulted in no appreciable increase in toxin titer.
The effect of pH on toxin titer was negligible, as unbuffered media with a pH range of 5.5-7.0 and buffered media with a pH range of 6.7-7.1 exhibited similar peak toxin titers. (Fig. 4).

DISCUSSION
The appearance of a large number of cases of TSS coincided with a significant technological advance in tampon manufacturing. The documentation of the existence of TSS-producing strains of S. aureus antedated the introduction of superabsorbent tampons. In some way, the superabsorbent tampons appeared to produce a change which took the physiologic effects of nonenteric exotoxin from subclinical to clinical levels. Two possible mechanisms were postulated: increased absorbency resulting in augmented substrate availability or vaginal occlusion due to volumetric expansion in tampon size transforming a microaerophilic environment into one capable of sustaining obligatory anaerobic replication. The staphylococci are the only genus of bacteria that is able to function both as class-I anaerobes (bacteria that replicate better in the presence of air than they do in its absence) and class-III anaerobes (bacteria that are destroyed by even transient exposure to molecular oxygen). In this study, the strict anaerobic conditions did not influence the initial quantity of cfu/ml engendered in the logarithmic phase of growth. Even though, after 24 h, the cultures grown anaerobically exhibited a progressive fall in OD, the peak quantity of viable bacteria present in the stationary phase of growth remained constant for 50 h. This report infers that a potential conversion of the endocervical or vaginal area from a microaerophilic environment to an anaerobic environment would not enhance TSST-1 production. Cell rupture did not result in an increase detectable TSSTtiters. TSST-1 is produced primarily by TSS strains of S. aureus during the logarithmic phase of growth, lz The maximum titers are achieved under aerobic growth conditions. Once the stationary phase of growth is reached with prolonged anaerobic conditions, the production of TSST-1 toxin is reduced. This in vitro observation correlates well with in vivo clinical data. Clinically, the majority of TSS cases occur early in the menstrual cycle (within 48 h). With wounds, an infection reflecting the onset of signs and symptoms usually occurs on the fourth day. [13][14][15][16] In those cases of TSS associated with S. aureus wound infections, the TSS usually begins as early as the second day at a time when the local signs of wound infection are minimal or absent.
The determinants of TSS appear to be 1) S. aureus strain selection and 2) augmental bacterial replication for growth that functions in concert. The possibility that superabsorbent tampons promote the probability of TSS by an augmentation of the available substrate for TSS strains of S. aureus requires further experimental analysis.