Bacteroides fragilis

ORIGINAL ARTICLE

A New Selective and Differentiative Medium for the Isolation of Bacteroides fragilis Angeliki Papastathopoulou1,2, Eugenia Bezirtzoglou3 and Nicholas J. Legakis1 From the 1Department of Microbiology, Medical School, University of Athens, 2Microbiological Laboratory, Childrens Hospital ‘Aghia Sofia’, Athens and the 3Department of Microbiology, Medical School, University of Ioannina, Ioannina, Greece Correspondence to: Professor N. J. Legakis, Department of Microbiology, Medical School, University of Athens, M. Assias 75, Goudi, Athens 115 27, Greece. Tel: +30 1 7771139, 7785638; Fax.: +30 1 7709180.

Microbial Ecology in Health and Disease 1998; 10: 110–113 A new selective and differentiative medium for the isolation of Bacteroides fragilis is described. It consists of a Brain Heart Infusion Agar base supplemented with yeast extract, cysteine hydrochloride, haemin, menadione, normal human plasma, bromothymol blue, vancomycin, amikacin, and glucose. If amikacin—resistant aerobic gram — negative bacteria are present, they are colour differentiated due to the presence and metabolism of glucose (0.1%, w/v). Bacteroides fragilis was the only bacterium to form yellow colonies on this medium, whereas a plethora of other aerobic and anaerobic species studied, formed blue colonies. This medium, Bacteroides Amikacin Vancomycin Agar (BAVA) could be useful in Clinical Microbiology laboratories. Key words: Bacteroides fragilis, selective medium, differentiative medium.

Bacteroides fragilis occurs normally in the large bowel of man and animals where it forms part of the predominant anaerobic faecal microbiota (2, 21). It is also found in the normal vagina (16). It is reported to be the commonest cause of all anaerobic infections in humans. ‘Saccharolytic Bacteroides’ have undergone major taxonomic revisions at both the generic and species levels within the last decade. A plethora of media and techniques have been developed for their more rapid and accurate identification (13, 24, 28, 30). Furthermore, multiple selective media have been devised for the specific isolation of B. fragilis (20, 29). Good growth on Bacteroides Bile Esculin (BBE) medium and other 20% bile (2% oxgall) containing media is a general characteristic of the B. fragilis group (28). Morphologic characteristics, special potency disks and some biochemical properties differentiate these species (14). Anaerobic infections are most commonly polymicrobial in nature. Aminoglycosides, well known to inhibit the growth of gram-negative facultatives, have been suggested for some time as additives in selective media for the growth of anaerobes. However, these drugs are becoming less effective due to increased resistance, especially in South European countries (7, 18). The clinical need to rapidly and correctly differentiate B. fragilis from other closely related and similar species stimulated us to develop a new selective and differentiative medium. © Scandinavian University Press 1998. ISSN 0891-060X

MATERIAL AND METHODS Microorganisms The present study utilised 157 aerobic and 120 anaerobic isolates. Aerobes were from the collection of the Department of Microbiology form the Medical School of Athens University and consisted of the following: Escherichia coli (20), Proteus mirabilis (18), P. 6ulgatus (9), Klebsiella spp (15), Pseudomonas spp (11), Citrobacter freundii (2), Serratia liquefaciens (20), Pro6idencia spp (2), Salmonella paratyphi B (2), S. typhimurium (2), S. typhi (5), Shigella flexneri (4), Staphylococcus aureus (8), S. epidermidis (2), Streptococcus pyogenes (4), Enterococcus spp (3). Anaerobic bacteria were provided by four different hospitals (Evangelismos, Laiko, Agia Sofia and Aglaia Kyriakou) situated in Athens, and consisted of the following: Bacteroides fragilis (63), B. thetaiotaomicron (1), Pre6otella melaninogenica (7), Clostridium perfringes (14), Peptococcus spp (26), Peptostreptococcus spp (9). The above microbes were isolated from mixed lesions, including 58 peritonitis, 43 pus abscesses, nine appendicitis, eight bile surgery, four cervicitis, one endocarditis, one umbilical inflammation, one gangrene and one hydatidosis empyema. The presumptive identification of the above anaerobes was made in the Clinical laboratories of the participating hospitals and confirmed in the Department of Microbiology in Athens University Medical School, by conventional biochemical methods and gas Microbial Ecology in Health and Disease

Selecti6e medium for B. fragilis

liquid chromatography (GLC) of metabolic end-products (17, 19). Media and cultural conditions The Bacteroides Amikacin Vancomycin Agar (BAVA) medium has the following composition (grams/liter): Brain Heart Infusion Agar 52.0; yeast extract 5.0; cystein hydrochloride 0.5; haemin 0.02; menadione 0.05; bromothymol blue 0.24; vancomycin 0.075; amikacin 0.075; glycose 1.0; and normal-human plasma 20. The pH of the medium was adjusted to 7.2; the formulation of the medium was evaluated by testing a series of glucose concentrations. The supplementation of the medium at the concentration of 0.1% (w/v) produced the selective and differentiative ability. The above medium without plasma, antibiotics and glucose but enriched with horse blood (5% v/v) referred hereafter as blood-BHIA was used for purity control and as reference medium, tested in parallel with BAVA. For the qualitative evaluation of the BAVA medium, separate overnight Cooked Meat cultures of anaerobes, and analogous Nutrient broth cultures of aerobes were used as original inocula, and tested by surface plating of a standard loopfull (diameter 3 mm). For the quantitative evaluation, parallel counts of B. fragilis and P. melaninogenica were performed on BAVA and blood-BHIA. The original inocula, as above, were diluted in pre-steamed cooled Nutrient broth from 10 − 1 to 10 − 7 and 100 ml of the three last dilutions was seeded over the entire surface of the plates. Those with 50 – 100 colonies were selected for viable counts. In order to ensure the differential capacity of the BAVA, cultures of B. fragilis with P. aeruginosa and K. pneumoniae resistant to amikacin were also tested. All plated cultures were incubated in the same anaerobic conditions (Gas Pak, Oxoid) at 37°C for 48 h.

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Prevotella colonies were yellow pigmented, in the presence of glucose added in the BAVA medium at a final concentration of 0.1% (w/v). The medium’s colour turned from blue to yellow-green in cases of pure culture of B. fragilis, whereas it retained its original blue colour in cases of pure cultures of K. pneumoniae and P. aeruginosa. The usefulness of BAVA — glucose medium for its differential ability was estimated in mixed cultures of B. fragilis —P. aeruginosa and of B. fragilis— K. pneumoniae. The results showed that colonies of aerobes were blue whereas B. fragilis colonies were yellow. The medium at the end of incubation was yellow-green but turned in less than 20 min, after air exposition, to blue. In any case colonies of B. fragilis were consistently of yellow colour and aerobes or facultatives of blue colour. The quantitative evaluation of the medium showed (Table 1) that cultures from the exponential phase of growth (48 h incubation) had comparable colony forming ability on BAVA and blood-BHIA. Also, the size of the colonies was similar on both media. DISCUSSION Over the last years there has been a resurgence of interest in the study of anaerobic infections (9, 11). Most of these infections are fulminating and life-threatening without early diagnosis or proper antibiotic treatment (4, 6, 10, 15, 25). Because of this a plethora of new methods have been developed (3, 26) for their rapid diagnosis. Anaerobes, however, are difficult to identify, in that they are not only slow-growing organisms but also require specialised anaerobic techniques (1, 5, 8, 17). Gas-liquid chromatographic analysis of metabolic end products has been of major importance for the presumptive diagnosis of such infections (19, 23, 27, 28). Significant amounts of either isobutyric, butyric or succinic acids Table 1

RESULTS All the 63 B. fragilis strains tested, grew prolifically on the BAVA medium. This medium consistently gave colonies similar in size when compared to the blood-BHIA medium. The same has been observed with seven strains of P. melaninogenica and the one strain of B. thetaiotamicron examined. On the contrary no growth could be observed with any of the gram-negative anaerobic or aerobic bacteria. However, a substantial proportion of some gram-negative facultatives and aerobic bacteria, namely K. pneumoniae (20%) and P. aeruginosa (30%) did give a relatively small but distinct growth yield in the medium. The above strains when examined for their susceptibility to antimicrobial agents were found to be resistant to amikacin. Interestingly, colonies of P. aeruginosa and K. pneumoniae retained their blue colour, while Bacteroides and

Reco6ery of Bacteroides fragilis strains in exponential phase of growth (a) Strains

1 2 3 4 5 6 7 8 9

Cell number (Log10 CFU/ml) Blood-BHIA

BAVA

8.079 7.973 8.146 8.477 8.079 8.414 7.939 8.000 8.113

7.977 8.033 8.230 8.380 8.146 8.361 7.897 7.903 7.954

Change (%) (b)

79 115 121 80 116 88 91 80 69

(a) 48 h aged cultures. (b) The number of CFU on blood BHIA is taken as 100%.

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detected by GLC in pus specimens is strong evidence of anaerobic infection, notably by B. fragilis (19), but an inconvenience of the above method is that anaerobes are very often implicated in mixed infections with bacteria such as swarming Proteus species (31) and other aerobes that outgrow anaerobes on non-selective media (12, 30). Therefore the use of selective media could produce meaningful results. It is generally known that different selective media can be inhibitory to the organisms for which they have been designed (12). Many selective media proposed (30, 31), contain substances inhibitory to some anaerobic genera or at least to some species within each genus. Mixed cultures of B. fragilis with various aerobic gram-negative rods resistant to amikacin have been performed with our newly developed selective medium for a better understanding of the selectivity and the differential capacity of our medium. Our speculation that B. fragilis would form yellow colonies, but aerobic organisms blue ones, was confirmed. The medium is most likely to be of value where aminoglycoside resistance of gram-negative aerobes is common (7, 18). A combination of this medium with direct immunofluoresence (22) might provide the rapid and presumptive identification of B. fragilis. ACKNOWLEDGEMENTS This work was supported in part from the Greek Ministry of Health and Welfare. We are grateful to Drs. A. Avlami, V. Deligianni, O. Paniara and A. Pagali, Directors of the hospital microbiology laboratories, who are active participants in this work.

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