The anaerobic and aerobic bacterial flora of leg ulcers in patients with sickle-cell disease

Journal of Infection (I988) I7, I I5--I20

The anaerobic and aerobic bacterial flora of leg ulcers in patients with sickle-cell disease S. A. A d e m i l u y i , * V. O. Rotirni,t~ A. O. C o k e r t T. O. B a n j o t a n d O. Akinyanju~

*Department of Surgery, tDepartment of Medical Microbiology and Parasitology and ~Department of Medicine, College of Medicine, Lagos University Teaching Hospital, Idi-Araba, Lagos, Nigeria Accepted for publication 25 October I987 Summary Leg ulcers in 26 patients with sickle-cell disease (SCD) were studied bacteriologically over a period of 6 months. The average age of the patients was 2o'92 years and the mean duration of the ulcers was 3'43 years. In order of frequency, Staphylococcus aureus, Pseudomonasaeruginosa,and Bacteroidesmelaninogenicuswere the predominant organisms. Anaerobes were isolated from I4 (54 %) of 26 patients and represent 2x % of the total 77 isolates. The presence of anaerobes correlated well with odorous ulcers. Isolation of anaerobes from leg ulcers of patients with SCD has added to knowledge of bacterial infection in SCD.

Introduction L e g ulcers m a y be due to various factors including venous hypertension, arterial insufficiency, malignant lesions, vasculitis of a u t o - i m m u n e disease and oedema of the legs in severe heart failure. T h e y may complicate haemolytic disorders such as h o m o z y g o u s sickle-cell disease ( S C D ) , hereditary spherocytosis, Felty s y n d r o m e and thalassaemia. T h e types c o m m o n l y described in tropical Africa include Buruli ulcers, tropical phagadenic ulcers, decubitus ulcers and S C D ulcers. T h e S C D ulcer, although considered u n c o m m o n in Africa in the past, is a major manifestation of S C D and m a y sometimes be the only initial presenting symptom. T h e s e ulcers are often chronic, disabling and difficult to treat. T h e y usually present in the second or third decade of life) S C D ulcer is a continuous source of misery and frustration to every S C D patient in w h o m the ulcer develops. 2 It often leads to psychological disturbances that result in retardation of educational progress and diminished prospects for employment. T h e r e are few reports on w e l l - c o n d u c t e d bacteriological studies of S C D ulcers. 3'4 T h e p r e d o m i n a n t organisms m e n t i o n e d in these reports are mainly of the resident skin flora, opportunistic G r a m - n e g a t i v e organisms and a few G r a m - p o s i t i v e facultative bacteria. H o w all these influence the healing processes and the m a n a g e m e n t of the ulcers has not been fully elucidated. M a n y of the S C D patients seen in our clinic sometimes present with foulsmelling ulcers. F o r this reason and because of the fact that the bacterial flora of m a n y infections vary from hospital to hospital and from one geographical :~ Corresponding author. oi63-4453/88/o5oi I5 +06 $o2.oo/o

© I988 The British Society for the Study of Infection

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location to another, we investigated the ulcers of SCD patients for both anaerobic and aerobic bacteria. Materials and methods

A total of 26 consecutive SCD patients with leg ulcers, attending the haematology clinic of the Lagos University Teaching Hospital ( L U T H ) , were studied. Haematological profiles of each patient performed by haemoglobin electrophoresis showed that they were homozygous SCD patients. T h e general characteristics of each patient, such as age, sex, duration of the ulcer, laterality of the ulcers (one or both legs), size and shape, anatomical site, odour and the general state of the patients' health, were all fully recorded. Ulcers were cleaned with sterile saline before sampling. Specimens were obtained in duplicate from the most active site and from the edge of the ulcer, away from the normal skin, by rolling sterile cottonwool swabs (Sterilin) over the surface of the sampling sites. These swabs were then broken into Amies transport m e d i u m and taken to our Research Laboratory for immediate processing. In the laboratory, a smear was made of one of each pair of swabs and Gram-stained while the other was streaked on the surface of a set of selective and non-selective culture media. These included plain blood agar, anaerobically pre-reduced blood agar, MacConkey agar (Oxoid), 'chocolate' agar, crystal violet agar (blood agar supplemented with i : 5 o o o o o o crystal violet), mannitol-salt agar (Oxoid), pre-reduced neomycin blood agar (containing Ioo rag/1 neomycin) and pre-reduced B M K agar (an anaerobic m e d i u m described by Holbrook e t a l . 5 and to which was added 75 mg/1 kanamycin). After inoculating these media each was placed in freshly-prepared cookedmeat broth and incubated for 2 4 h as an extra culture for anaerobic bacteriological study. T h e plain blood agar, the mannitol-salt agar and MacConkey agar plates were incubated in air at 37 °C for 24 h, while the chocolate agar and the crystal violet agar plates were all incubated in air plus 5 - r o % carbon dioxide in a candle extinction jar for 2 4 h ; incubation was prolonged for 4 8 h whenever necessary. T h e pre-reduced blood agar, neomycin blood agar and the B M K agar plates were incubated anaerobically in anaerobic jars (Oxoid) containing gas generating kits (Oxoid). Each jar was then placed in an ordinary 37 °C incubator for an initial period of 48 h and then re-incubated for an extended period of up to 5 days as a standard procedure. At the end of the appropriate incubation periods, representative colonies on the aerobic plates were identified by the methods of Cowan 6 and the API20E System (API System, SA, La Balme les Grottes, Vercieu, France). Colonies on the anaerobic plates were presumptively identified by their initial sensitivity to a 5/zg metronidazole disc and resistance to IO/zg genatamicin disc, placed on the primary agar plates as well as lack of growth in air plus Io % carbon dioxide. Isolates were further identified by methods previously described. 7,s Results

T h e general characteristics of the patients are shown in Table I. Males outn u m b e r e d females by a ratio of 3 : I (2o males and 6 females); ages ranged from

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T a b l e I General characteristics of the patients with sickle-cell disease Characteristic features

No. of patients (%)

Age (years) O'-IO I I-I 9 20-29 30-39

4o and above Sex Female Male Laterality of ulcer Left leg Right leg Both legs Duration of ulcer 0-4 weeks > I month-1 year > I year-5 years > 5 years

0 I3 (5 O) I I (42"3) 2 (7"7)

o 6 (23'I) 20 (76"9) 17 (65"4) 6 (23"I) 3 (I 1'5) 5 (I9"2) 6 (23-I) 13 (5o) 2 (7"7)

I2-36 years with a m e a n of 20"92 years and a modal age group of 2I-25 years that included 3I % of the 26 patients. T h e cumulative frequency of age distribution showed that 50 % of the patients were between 2I and 25 years of age, 80"7 % were below 25 years and 9 r 5 % were 30 years old or less. T h e duration of the ulcers ranged from 2 weeks to I9 years with a m e a n of 3"43 years. T h r e e of the 26 patients had ulcers on both legs but were treated as single patients because of the similarity in the flora of the two ulcers. T h e left legs (I7 of 23) were more affected than the right legs (6 of 23) by a ratio of almost 3 : I. T h e overall p r e d o m i n a n t bacterial flora were Staphylococcus aureus. Pseudomonas aeruginosa and Bacteroides melaninogenicus which were isolated from I2, I2 and nine of the 26 patients respectively. Other p r o m i n e n t isolates were Escherichia coli isolated from eight patients, Streptococcus pyogenes from seven, Proteus mirabilis from six and Bacteroides fragilis from four. T h e isolation rates were reflected by the same proportions in the overall total isolates (see Table II). I n a11, anaerobes were isolated from I4 (53"8 %) of the 26 patients and accounted for i6 ( 2 I ' 8 % ) of the total 77 isolates. O f these i6 isolates, nine (56"3%) were B. melaninogenicus, and four ( 2 5 % ) were B. fragilis. T h e other anaerobes, Bacteroides intermedius, Fusobacterium sp. and Peptostreptococcus sp., were each single isolates. An anaerobe was not isolated f r o m any ulcer of < 4 weeks duration. As shown in Table I I I , all the anaerobes were isolated in mixed cultures with facultative bacteria f r o m almost all the ulcers. Only two patients were each infected by two anaerobes, but, in the overall analysis, each patient was invariably infected by two or m o r e organisms (anaerobes and aerobes) at a time. Assessment of the remaining I2 of the 26 patients showed that they were

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Table II Bacteria isolated from leg ulcers of patients with sickle-cell disease No. of patients colonised (%), n = 26

Organisms

Staphylococcus aureus Streptococcus pyogenes Escherichia coli Staphylococcus albus Pseudomonas aeruginosa Proteus mirabilis Candida albicans Viridans streptococci Bacteroides fragilis Bacteroides intermedius Bacteroides melaninogenicus Fusobacterium sp. Peptostreptococcus sp. Eikenella corrodens Coryneforms

Percent of the total isolates, n = 77

I2 (46) 7 (26"9) 8 (30'7) 3 (I I'5) I2 (46) 6 (23) 3 (I I'5) 2 (7'6) 4 (15"4) I (3'8) 9 (34'6) I (3"8) I (3"8) 2 (7"6) 3 (I I'5)

I5"6 9 IO'3 4 I5"6 7"8 4 2"6 5"2 I'3 I I'7 I'3 I'3 2'6 4

Table III Prevalence of anaerobes in leg ulcers No. of patients infected (%), n~26

Bacterial group Aerobes only Anaerobes only Anaerobes + aerobes

I2 (46) o I4 (53'6)

infected by aerobes only. T h e y were those with ulcers of < 4 weeks and I m o n t h - I year's duration. Discussion

Results of this study include two important observations. First, we have shown that, in addition to the complex polymicrobial flora of these ulcers, over half were infected by anaerobes in mixed cultures. This is the first report of a high isolation rate of anaerobes from the leg ulcers of SCD patients. Second, although the general clinical features of the patients agree essentially with those already reported, the relative rarity of bilateral ulcers in this series is striking when compared with reports from elsewhere. I° T h e predominant aerobic bacteria isolated were S. aureus and Ps. aeruginosa as in some earlier reports, i. 4.9 However, we also found relatively more patients infected by S. pyogenes, P. mirabilis and E. coll. Most ulcers infected by these pathogens also harboured one or two species derived from the normal resident skin flora such as Staphylococcus albus, viridans streptococci and coryneforms. Compared with the figures reported in the only previous study to demonstrate anaerobes in SCD ulcers, 4 the isolation of anaerobes from a relatively large

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n u m b e r of leg ulcers in our patients is very significant, particularly with respect to our empirical antibiotic t r e a t m e n t of these ulcers. Isolation of anaerobes in m a n y mixed cultures agrees with reports of investigations c o n d u c t e d on other types of infected peripheral ulcers without concomitant systemic disease. 11-13 F i n d i n g m a n y cases with mixed flora is not surprising since facultative or aerobic organisms often accompany anaerobes in m a n y mixed infectious processes, especially infections superimposed on areas of devitalised tissues due to ischaemic changes such as m a y be the case in these SCD ulcers. Cultures of specimens obtained from the bilateral ulcers of three patients yielded identical flora. T h i s aptly demonstrates similarity in the organisms that colonise individual SSD patients. 4 As m i g h t be expected, the isolation of anaerobes in this study correlated well with every malodorus leg ulcer in our patients, although the only Peptostreptococcus sp. isolated was in one patient with a non-offensive ulcer. T h i s confirms our previous experience with infected ulcers. 13 Also, the isolation of anaerobes was related to the chronicity of the leg ulcers since anaerobes were not isolated from any ulcer of < 4 weeks' duration. T h e s e were the only predictive indices for any bacterial category in this study. N e i t h e r age, sex nor the size of the ulcers related to any particular bacterial flora. We suggest that both anaerobes and aerobes, in particular B. melaninogenicus, S. aureus and Ps. aeruginosa, may be involved in the pathogenesis of the ulcers. T h e healing of these ulcers m a y therefore improve if these opportunistic pathogens are eliminated. Because of poor blood supply, systemic antibiotics may not be effective and their use precluded. Topical therapy such as debridement, the application of granulated sugar, peroxide or iodine m a y assist. T h e e n h a n c e m e n t of i m m u n i t y in these S C D patients m a y also be a useful adjunct to the healing of their u l c e r s ) Our bacteriological findings should help to guide empirical use of antimicrobial agents for treating ulcers considered clinically to be infected.

i. 2. 3. 4. 5. 6. 7. 8. 9.

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