Induction of Abscess by Non-abscess Producer Staphylococci Using Staphylococcal Fatty Acid Modifying Enzyme (FAME)

Stahpylococcus epidermidis (S. epidermidis), S. warneri and S. aureus subsp. anaerobious were isolated from sheep abscess. Crude FAME was extracted from supernatant fluid of a 24hrs culture of S. aureus subsp. anaerobious grown in Trypticase Soy Broth at 37°C with continuous agitation. Fourteen heads of sheep were used. Three of them were inoculated with S. epidermidis + FAME and other three were inoculated with S. warneri + FAME. Eight animals were divided into four groups, two animals in each, used as control and inoculated with S. epidermidis, S. warneri, S. aureus subsp. anaerobious and FAME. All animals were inoculated subcutaneously at the neck region. After fourteen to eighteen days, sites inoculated with S. epidermidis + FAME, S. warneri + FAME and S. aureus subsp. anaerobious showed abscesses grossly then confirmed bateriologically and histopathologically, while those inoculated with S. epidermidis alone, S. warneri alone and FAME showed no abscess.


Introduction
Abscess disease in sheep has a great economical importance in Sudan.It is caused by S. aureus subsp.anaerobious and characterized by superficial lymph nodes enlargement.For the organism to survive and multiply in the host tissue and causes abscess it must produce an estrifying enzyme known FAME which inactivates the bacteriocidal fatty acids [4,5,7].In natural abscess disease, many Stahpylococcus spp.have been isolated.Some of them were known to produce FAME but unable to form abscess; from these S. epidermidis and S. warneri (coagulase-negative Staphylococci) are examples [6].FAME activity was detected in 54% of CNS and 80% S. aureus strains surveyed but none in E. coli or S. uberis.While some CNS strains produced FAME activity comparable to the lab strain of S. aureus, the pattern of FAME activity varied among strains and across species of staphylococci [9].
S. aureus FAME has a pH optimum between 5.5-6.0 and a temperature optimum of about 40°C.Expression of FAME is controlled genetically by the golbal regulators Agr and Sar [2].FAME has an extracellular activator which can increase FAME expression and activity when added to the FAME-producer Stahpylococcus culture [3].

Preparation of Organisms
Abscesses were collected from sheep for isolation of S. epidermidis, S.warneri and S. aureus subsp.anaerobious.The isolates were identified according to [1,8].S. aureus subsp.anaerobius was grown microaerophilically in ten ml nutrient broth at 37°C overnight while, S. epidermidis and S. warneri were grown aerobically in 10 ml nutrient broth at 37°C overnight.Serial ten-fold dilution from 1 ml of each culture was made and inocula from dilutions 10 -1 (containing about 10 4 bacterial cells) and10 -3 (containing about 10 3 bacterial cells) were used.

Preparation of Crude FAME
Crude FAME was prepared according to the method of Long [6].S. aureus subsp.anaerobius was grown microaerophilically in 100 ml of Trypticase Soy Broth (TSB) at 37°C for 18 hrs.with continuous shaking.The culture was centrifuged at 3000 r.p.m. for 30 minutes using cold centrifuge and the supernatant fluid was sterilized by filtration using 0.22 μm.filter membrane.The culture filtrate was concentrated to half volume at 4°C using dialysis tubes and carbowax.

Inoculation of Experimental Animals (Sheep)
Fourteen healthy fattened sheep were clipped and divided into groups then labeled as follows: Group (1): Three animals were inoculated with 0.5ml of dilution 10 -1 of S. epidermidis + 0.5ml of FAME and 0.5ml of dilution 10 -1 of S. epidermidis + 1.0ml of FAME at each site of the neck region.
Group (2): Three animals were inoculated with 0.5ml of dilution 10 -1 of S. warneri + 0.5ml of FAME and 0.5ml of dilution 10 -1 of S. warneri + 1.0ml of FAME at each site of the neck region.
All animals were inoculated S/C at the neck region, followed up daily and any clinical observations were recorded.After fourteen to eighteen days they were slaughtered and samples were collected for bacteriological examination.Sections from lesions were fixed in 10% neutral formalin for histopathological examination.

Results
Inoculation of sheep with treated bacteria resulted in abscess formation (Figure 1 and Figure 2).Transient increase in body temperature and mild tissue reaction at the inoculation sites were observed in the first three days of inoculation.At day six some swellings developed into abscesses which ruptured by day eight (Figure 3 and Figure 4).Other swellings remained closed until incised at postmortem.The control sites which were injected with S. epidermidis, S. warneri and FAME revealed in no swelling or caseation (Table 1).Results were confirmed histopathologically by the presence of abscess in sites inoculated with S. epidermidis plus FAME and S. warneri plus FAME.A chronic pyogranulomatous reaction was seen; composed of dense collection of mononuclear, lymphocytes and plasma cells with neutrophils in the centre.Most sections showed dermal edema, fibrosis, mononuclear infiltration (observed also perivascularly), subcutaneous muscle degeneration and necrosis with widened and fibrosed intermuscular connective tissues.Pyogranulomas and wide suppuration were seen in deep dermis and subcutaneous tissues.In some sections the granulomatous reaction was not severe and other one showed dermal haemorrahge (Figure 5, Figure 6 and Figure 7).FAME injected sites showed no significant gross change and hitopathologically there was dermal edema.Subcutaneous muscles stained pale and some muscle fibers showed intense eosinophilia (Figure 8).

Discussion
Previous studies about abscess formation showed that, organism to form abscess and causes the disease must possesses two enzymes: FAME and lipase.Those studies explained the role of FAME in abscess formation in vitro.
Studies in Sudan were conducted on different aspects of the disease but there is no work being published on bacterial enzymes specially FAME; the main factor of abscess formation.
As shown in this study, abscess could be generated in animals (sheep) experimentally by non-abscess producer Staphylococci when FAME was added.These results confirmed the findings reported by [4,5,7], who assured that, FAME is an important factor in the process of abscess formation.Other work conducted on extracellular activator of FAME explained that addition of S. aureus activator to S. epidermidis cultures increased FAME production but there was no increase or inhibition in FAME production when S. epidermidis activator was added to S. aureus cultures [3].Also other report showed that, some CNS strains produced FAME activity comparable to the lab strain of S. aureus, while the pattern of FAME activity varied among strains and across species of staphylococci [9].In Sudan also found that injection of S. epidermidis alone (isolated from Sheep abscess) could not produce abscess but when inoculated with other bacteria (S. aureus subsp.anaerobious) abscess was produced (Unpublished data).Three later observations emphasized the present result.
The results of the present study suggest that, S. epidermidis FAME is non-functional and abscess formed due to S. aureus subsp.anaerobious FAME or it was activated by S. aureus activator.

Conclusion and Recommendation
We concluded that FAME is an important factor in abscess formation, and non-abscess producer Staphylococcal FAME can be activated, so as to get rid of the disease must inactivate the FAME.Further researches must be conducted for more information.