J. Med. Microbiol. - Vol. 46 (1997), 39-44 0 1997 The Pathological Society of Great Britain and Ireland IDENTIFICATION AND TYPING OF BACTERIA Media and tests to simplify the recognition and identification of members of the Proteeae B. W. SENIOR Department of Medical Microbiology, University of Dundee Medical School, Ninewells Hospital, Dundee DDI 9SY Several important and diverse human pathogens are found in the tribe Proteeae. By identifying and concentrating on key biochemical reactions, it has been possible to devise six simple media that permit the identification of all the important members of the tribe with ease, speed and accuracy. This was confirmed by optional additional confirmatory media and tests. Introduction antibiotic susceptibility. For example, Prov. stuartii isolates are some of the most antibiotic resistant of the The tribe Proteeae comprises three genera, Proteus, Enterobacteriaceae, whereas problems of antibiotic Morganella and Providencia, and 10 species. Its resistance are unusual in Prov. alcalifaciens and members have diverse characteristics. Some, like P Proteus spp. The antibiotic susceptibility of M. myxofaciens - which was isolated from larvae of the morganii strains is very different from that of Proteus gypsy moth (Porthetria dispar) [l] and Prov. heimba- and Providencia spp. chae [2] which has been isolated from penguin faeces - have as yet never been associated with infections in Therefore, accurate and rapid identification of mem- man. On the other hand, I? mirabilis is one of the most bers of the Proteeae is essential. This should not be frequently encountered organisms in the diagnostic difficult, although it frequently does not happen. Prov. medical microbiology laboratory. stuartii is frequently misidentified, particularly when some commercial test kits are used, possibly because Members of the tribe are associated with a wide it may reveal traits of the plasmid-borne genes isolates variety of infections. After Escherichia coli, I? often carry. Moreover, the description ‘indole-positive mirabilis is probably the commonest cause of urinary Proteus’ which is often seen in the literature may be a tract infection, particularly in elderly patients of both misnomer for any lactose negative, indole and urease sexes [3]. Unlike E. coli, it has a predilection for the forming member of the Enterobacteriaceae and as upper urinary tract, where it can cause stone formation such could be M. morganii, Prov. rettgeri, Prov. and pyelonephritis. Prov. stuartii is also a major cause stuartii or P vulgaris. The latter organism is the only of urinary tract infection particularly in long-term true ‘indole-positive Proteus’. catheterised elderly patients [4, 51. However, Prov. alcallfaciens is rarely associated with urinary tract The purpose of this study was to evaluate the key infections but is now a recognised cause of diarrhoea biochemical reactions useful in the identification of [6,7]. M. morganii, which can be isolated from members of the Proteeae and combine them in an diarrhoea1 stools in the absence of other known appropriate way so that an accurate, speedy and bacterial enteric pathogens, has often been suspected unambiguous identification of each species within the to cause diarrhoea but this has yet to be proved. tribe could be made with the minimal number of tests Excepting the two organisms that are not associated and volume of media. with human infection, all other members of the tribe are also encountered frequently in infections of wounds, blood and sputum. Materials and methods Bacterial strains Members of the Proteeae also differ widely in their A collection of 198 strains was examined. This Received 9 Jan. 1996; accepted 19 June 1996. comprised I? mirabilis (24 strains), P vulgaris (24), Corresponding author: Dr B. W. Senior. I? penneri (15), M. morganii subsp. morganii (26), M. 40 B. W. SENIOR morganii subsp. sibonii (25), Prov. rettgeri (17), Prov. colour a few seconds after the addition of a drop of stuartii (14), Prov. alcalifaciens (24), Prov. rustigianii aqueous ferric chloride 10% w/v to the PAD medium (17) and Prov. heimbachae (12). The strains were indicated phenylanine deaminase activity and the selected at random from a large collection of isolates formation of phenylpyruvic acid. The formation of a from around the world. They had been identified by pink colour in the urea-indole medium indicated extensive biochemical tests and all gave reactions urease formation. The subsequent development of a characteristic for the species. The collection included pink colour at the surface of this medium after the NCTC and ATCC reference strains and excepting addition of Ehrlich's or Kovac's reagent indicated isolates of Prov. heimbachae (kindly donated by Dr H. indole formation. Growth and the development of E. Miiller) from penguin faeces, all were clinical alkalinity (blue colouration) in the ornithine decarboxy- isolates from man. lase medium indicated formation of this enzyme. Fermentation of sugars resulted in a pH change to acid which was indicated by a colour change from Media and tests blue to yellow. Phenylalanine deaminase (PAD) medium contained Tryptone water (Oxoid CM 87) 1.5 g, L-phenylalanine 1 g and agar 1.3 g in 100 ml of distilled water. After sterilisation at 121°C for 15 min, it was dispensed in Results and discussion either 200-pl amounts into microwell plates or 2-ml amounts into sterile tubes which were sloped. The results indicate that accurate identification of members of the Proteeae can be made satisfactorily Urea-indole medium was prepared by supplementing, with only a few tests if selection is made of key when cool, tryptone water (1.5 g in 100 ml of distilled reactions unique to the tribe or a given species within water) which had been sterilised at 121°C for 15 min, it. with filtered sterile urea (40% w/v in water) to 2% w/v and with a 1 in 200 dilution of phenolphthalein Excepting the recently defined rare organisms Tatu- 1% in isopropanol. The medium was dispensed asep- mella ptyseos [8] and Rahnella aqztatilis [9], the tically in 1-ml volumes into sterile tubes. ability to oxidatively deaminate certain amino acids, usually phenylalanine or tryptophan, to the corre- Ornithine decarboxylase medium contained tryptone sponding keto acid and ammonia, is a feature among water 0.5 g, L-ornithine.HC1 1 g and 2.5 ml of the Enterobacteriaceae found only in strains belonging bromocresol purple dye 0.08% in 100ml of distilled to the Proteeae. Upon the addition of aqueous ferric water and was sterilised at 121°C for 15 min. When chloride ( 10%) the deaminated product of phenylala- cool, the medium was supplemented aseptically with nine becomes dark green and later fades, whereas that sterile glucose 10% w/v in water to 0.1% w/v and of tryptophan remains red/brown. Members of the dispensed aseptically in either 200-pl amounts into Proteeae are also readily recognised by their forma- microwell plates or 2.5-ml amounts into screw-capped tion of a similar coloured product, but this time of a bottles. diffusible melanin-like pigment, when grown under aerobic conditions on media containing iron and Peptone water sugars were made by supplementing, certain L aromatic amino acids including tryptophan aseptically, when cold, Peptone water (Oxoid CM 9) [lo, 111. This reaction is unique to Proteeae within (1.5 g and 2.5 ml of bromocresol purple 0.08% in the Enterobacteriaceae, but the same or a similar 100 ml of distilled water) which had been sterilised at reaction and the formation of a product of a similar 121°C for 15 min, with a sugar or sugars, each to a colour is also given by some other bacteria including final concentration of I% w/v, from a sterile (steamed species of Pseudomonas [ 121, Aeromonas and Acine- for 1 h) stock solution of each sugar, 10% w/v in tobacter [ 131 and Legionella [ 141. water. They were dispensed in either 200-pl amounts into microwell plates or 1-ml amounts into sterile Because of the importance of the test, and the possible tubes. confusion of the brown colour with that of the medium, the phenylalanine deaminase test with its All media were inoculated with one drop (c. 25 pl) of distinctive coloured reaction product was used here in either a nutrient broth culture of the strain incubated preference to that of tryptophan deaminase and red- overnight at 37"C, or a suspension of a colony in brown pigment production. saline, and incubated at 37°C. For tests in microwell plates, after inoculation of the media, drops of sterile All strains tested formed phenylalanine deaminase. liquid paraffin were added to wells containing However, with one isolate of Prov. heimhuchae the ornithine decarboxylase medium and the plates were PAD reaction was weak after incubation for 24 h. It is sealed with tape. This was punctured over wells important that the test is done under aerobic conditions. containing PAD medium. Tests were read after Although PAD-negative isolates of Proteeae may exist, incubation for 16-24 h. Development of a dark green they are rare. In general, therefore, any isolate which LDENTIFICATION OF THE PROTEEAE 41 fails to deaminate phenylalanine should be considered not to belong to the tribe. A combination of this test, with a sugar fermentation test, was examined by incorporation of a sugar to 0.5% and bromophenol blue indicator dye into the medium. Provided the sugar was not fermented, the phenylalanine deaminase reaction was unaffected by the presence of the sugar and the indicator. Fermentation of the sugar interfered with the formation or detection or both of phenylpyru- vic acid. Thus addition of lactose to the phenylalanine deaminase medium would give, if not already known, I++l IS1 the additional information that the PAD positive isolate was also lactose negative (as are all Proteeae) and, therefore, most likely to belong to the tribe, preventing any possible confusion with isolates of R.