The Genus Serratia

More documents

Recommendations

Info

230 F. Grimont and P.A.D. Grimont CHAPTER 3.3.11 The pH is adjusted to 7.2. Autoclave at the same time as solution A. After autoclaving, solutions A and B are mixed aseptically, and the resulting medium is poured in thick layers into sterile, plastic petri dishes (25–30 ml for petri dishes 9–10 cm in diameter). The medium is no longer effective if it is remelted, but plates of CT agar keep well for several weeks at 4°C if contamination and desiccation are prevented. CT agar is very useful for the isolation of Serratia spp. (except S. fonticola) from feces, sputum, and any other polymicrobial clinical sample. Serratia colonies are apparent within 3 days, and further incubation allows colonies grow (2–5 mm). Occasionally, Providencia spp., Acinetobacter spp., or Pseudomonas spp. may develop colonies on this medium. Other bacteria give only pinpoint colonies. When samples are from the natural environment or food, the results are less clear, especially if the sample contains nutrients. Several colonies must then be checked for DNase. Preenrichment in nutrient broth with 4% NaCl, incubated overnight at 20°C, can be used; about 0.1 ml of the enrichment culture is streaked onto CT agar. Broth that has supported overnight bacterial growth usually does not contain enough nutrients to adversely affect the selective isolation of Serratia spp. (P. A. D. Grimont, unpublished observations). Identification The usual methods for the identification of serratiae and other Enterobacteriaceae can be found in Ewing (1986). However, following the pioneering example of Stanier et al. (1966) with the pseudomonads, developments in enterobacterial taxonomy (Bouvet et al., 1985; Grimont and Ageron, 1989; Grimont et al., 1977b; Grimont et al., 1988) have demonstrated the usefulness of carbon source utilization tests. Also, it should be noted that utilization tests are preferable to fermentation tests since strains able to utilize a polyalcohol sometimes fail to produce enough acid products to give a positive reaction in fermentation tests. Although all species can be identified with carbon source utilization tests, some conventional tests can also be used: tetrathionate-reduction and β-xylosidase tests are useful in clinical identification (Le Minor et al., 1970; Brisou et al., 1972). The methodology of carbon source utilization tests and other procedures that are not in general use, but are essential to Serratia identification, are detailed herein. Conditions of Incubation Best results are obtained when Serratia cultures are incubated at 30°C. At 37°C, pigmentation often fails to appear, and the Voges-Proskauer test is often negative. S. plymuthica may even fail to grow at 37°C. Otherwise, there is little difference between the results of tests held at 30°C and those held at 37°C as far as S. marcescens is concerned. Carbon Source Utilization Tests Two methods can be used for carbon utilization tests: a minimal agar medium or API strips (API System, La Balme-les-Grottes, France). The minimal agar medium employs the same M70 medium (Véron, 1975) used in the caprylate-thallous selective agar (see the recipe), but omits the yeast extract, thallous sulfate, and caprylate from solution A. Neutralized carbon sources (the purest brand available) are added to solution A to give a 0.1% final concentration (weight of the anion/vol) in the complete medium (0.2% in the case of carbohydrates). Sterilization of the carbon source in aqueous solution is achieved by filtration throught membrane filters or by heating at 80°C for 20 min. Plates are examined for growth after 4 days (early reading) and 14 days (late reading). The API strip method uses API 50 CH (containing 49 carbohydrates), API 50 AO (49 organic acids), and API 50 AA (49 aminoacids) galleries and a minimal medium containing growth factors provided by the manufacturer. These were described by Gavini et al. (1980). Since about one-third of the carbon sources are useless in the study of the Enterobacteriaceae, we use special galleries containing 99 selected carbon sources (including new compounds not included in the above-mentioned galleries). The inoculated galleries are examined for growth after 2 days (early reading) and 4 days (late reading). These galleries have been used for the description of several bacterial groups (Bouvet et al., 1985; Grimont et al., 1988; Grimont and Ageron, 1989). In our experience, carbon source utilization tests (CSUT) gave reproducible and clear-cut results. Utilization of carbohydrates and polyalcohols often give more useful results than fermentation tests (FT). More than 95% of CSUT and FT using D-xylose, L-arabinose, trehalose, sorbitol, sucrose, and rhamnose gave the same results (P. A. D. Grimont, 1977). However, results with CSUT and FT are not parallel when adonitol, inositol, cellobiose, and lactose are studied. Whereas almost all Serratia strains can grow on inositol, acid from inositol was produced by only 76% of S. marcescens, 36% of S. rubidaea, 92% of S. liquefaciens-S. proteamaculans and 35% of S. plymuthica strains within 2 days. CSUT with adonitol is a very good test for separating S. marcescens from S. liquefaciens (99% vs. 0% positive strains, respectively), but
CHAPTER 3.3.11 The Genus Serratia 231 FT with adonitol is a poor test for separating these two species (32% vs. 0% positive strains in 2 days, respectively). Glucose Oxidation Test The Lysenko test (Lysenko, 1961) was extensively modified by Bouvet et al. 1989. A 1-liter portion of glucose oxidation medium is composed of basal medium containing 8 g of nutrient broth (Difco), 0.02 g of bromcresol purple, and 0.62 g of MgSO 4·7H 2O (2.5 mM). To 9.2 ml of autoclaved (121°C, 15 min) basal medium is added 0.4 ml of a filter-sterilized 1 M D-glucose solution (final concentration, 40 mM). This medium is supplemented with 0.4 ml of a fresh, sterile 25 mM iodoacetate solution (final concentration, 1 mM). The glucose oxidation medium is distributed (0.5 ml portions) into glass tubes (11 by 75 mm), which are plugged with sterile cotton wool. Bacteria grown overnight at 20 or 30°C on tryptocasein soy agar (Diagnostics Pasteur, Marnes-la-Coquette, France) supplemented with 0.2% (wt/vol) D-glucose are collected with a platinum loop, suspended in a sterile 2.5 mM MgSO 4 solution and adjusted to an absorbance (at 600 nm) of about 4. Glucose oxidation medium is then inoculated with 50µ1 of bacteria suspension and vigorously shaken at 270 strokes per minute overnight at 20 or 30°C. The test is positive if a yellow color develop (acid production). Otherwise, the medium remains purple. When negative, the glucose oxidation test is repeated in the presence of 10µM pyrroloquinoline quinone (PQQ). The control strains used are the type strains of S. marcescens (positive without requirement for PQQ) and S. liquefaciens (positive only when PQQ is supplied) (Bouvet et al., 1989). Gluconate and 2-Ketogluconate Dehydrogenase Tests The gluconate dehydrogenase test is performed as follows (Bouvet et al., 1989): Bacteria are grown overnight at 20°C on tryptocasein soy agar supplemented with 0.2% D-gluconate, then collected with a platinum loop and suspended in sterile distilled water to an absorbance (at 600 nm) of about 4. The reaction medium contains 0.2 M acetate buffer (pH5), 1% (wt/vol) Triton X-100, 2.5 mM MgSO 4, 75 mM gluconate, and (added immediately before use) 1 mM iodoacetate. A control medium contains the same ingredients except gluconate. The reaction medium and the control medium are dispensed (100µl portions) into 96-well microtiter plates (Dynatech AG, Denkendorf, Germany). Bacterial suspensions (10µl) are added to the reaction and the control media, and the microtiter plates are incubated at 20°C for 20 min. Then, 10-µl portions of a 0.1 M potassium ferricyanide solution (kept in the dark at room temperature for no longer than 1 week) are added to the wells, and the plates are gently shaken and incubated at 20°C for 40 min. Then 50 µ1 of a reagent containing Fe2(SO 4) 3,0.6 g; sodium dodecyl sulfate, 0.36 g; 85% phosphoric acid, 11.4 ml; distilled water to 100 ml is added to each well. The plates are examined for the development of a green- to-blue color (due to Prussian blue) within 15 min at room temperature. The color in the uninoculated control medium remains yellow. The suggested control strains used are Escherichia coli K-12 (negative) and the type strain of Serratia marcescens (positive). The 2-ketogluconate dehydrogenase test is the same as above, except that the control and reaction media are adjusted to pH 4.0, and 2ketogluconate is used in place of gluconate in the reaction medium. Suggested control strains used are Escherichia coli K-12 (negative) and the type strain of Serratia marcescens (positive at 20°C, not at 30°C) (Bouvet et al., 1989). Tetrathionate Reductase Test The tetrathionate reductase test was found to be very useful for the identification of Serratia spp. The liquid medium of Le Minor et al. (1970) is described below: Tetrathionate Reductase Test for Identifying Serratia spp. (Le Minor et al., 1970) K2S4O6 5 g Bromthymol blue (0.2% aqueous solution) 25 ml Peptone water (peptone [Difco], 10 g; NaCl, 5 g; distilled water, 1 liter) Up to 1 liter Adjust pH to 7.4. Filter-sterilize and dispense 1 ml into 12-x-120-mm tubes. Within 1 or 2 days, reduction of tetrathionate will acidify the medium, indicated by a yellow color. Negative tubes remain green or turn blue. Voges-Proskauer Test Results of the Voges-Proskauer test are variable with some Serratia spp., probably because the end products of the butanediol pathway are metabolized (Grimont et al., 1977b). Many strains of S. plymuthica and S. odorifera give negative results with O’Meara’s method (O’Meara, 1931) and positive results with Richard’s procedure (Richard, 1972). In Richard’s procedure, 0.5-ml aliquots of Clark and Lubs medium (BBL) are inoculated in 16-mm-wide test tubes. After overnight incubation at 30°C, 0.5 ml of αnaphthol (6 g in 100 ml absolute ethyl alcohol) and 0.5 ml of 4 M NaOH are added. Positive tubes will turn red.
Page 1 and 2: Prokaryotes (2006) 6:219-244 DOI: 1
Page 3 and 4: CHAPTER 3.3.11 The Genus Serratia 2
Page 11: CHAPTER 3.3.11 The Genus Serratia 2

The Genus Serratia

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?