The Genus Serratia

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228 F. Grimont and P.A.D. Grimont CHAPTER 3.3.11 Enzymes The most probable potential use of chitinase is in the treatment of the chitin-containing wastes produced by the seafood packing industry, biocontrol agents against plant pathogenic fungi, production of adhesives and wound dressings, heavy metal recovery from waters, delayedrelease agrochemicals or drugs, and dialysis membranes (Joshi et al., 1989). S. marcescens produces five proteins (molecular masses of 57, 52, 48, 36, and 21 kDa) with chitinolytic activity (Fuchs et al., 1986) whereas S. liquefaciens produces three proteins (molecular masses of 56, 51, and 36 kDa) with such activity (Joshi et al., 1989). The sequences of two S. marcescens chitinase genes, chiA encoding the 57 kD enzyme and chiB encoding the 52 kD enzyme, have been published (Jones et al., 1986; Harpster and Dunsmuir, 1989). Biotechnology Serratia strains have been used in whole-cell bioconversion processes. Resting-cell suspensions of S. marcescens are able to convert vanillin to vanillic acid. At high substrate concentration (0.3%), 75% vanillin is converted (Perestello et al., 1989). 2,5-Diketogluconic acid, a key intermediate in the synthesis of ascorbic acid, can be produced from glucose at 20°C by S. marcescens (without requirement for a cofactor) and by S. liquefaciens and S. grimesii when supplied with the cofactor, pyrroloquinoline quinone (Bouvet et al., 1989). Some genetic tools are available to construct S. marcescens strains of biotechnological interest. These tools include general transduction (Matsumoto et al., 1973), transformation (Reid et al., 1982), and use of nuclease-deficient, antibioticsensitive, and restrictionless mutants (reference is not an exact match Takagi and Kisumi, 1985). Recombinant strains obtained were histidaseless regulatory mutants producing L-histidine (Kisumi et al., 1977), L-arginine-producing mutants (Kisumi et al., 1978), an isoleucine-producing strain (Komatsubara et al., 1980), and a threonine-hyperproducing strain (Komatsubara et al., 1983). Isolation Underlying Principles Pigmented species and biotypes of Serratia often exhibit pink or red colonies on nutrient agar. Use of low-phosphate agar without glucose, such as peptone-glycerol agar (Difco peptone, 5 g; glycerol, 10 ml; Difco agar, 20 g; distilled water, 1 liter), is best in order to demonstrate pigmentation (Williams and Hearn, 1967). Although the genus Serratia includes all the redpigmented enterobacteria, identification of a pink or red colony should be confirmed by biochemical tests because a few nonenterobacteria may also produce prodigiosin (see “Introduction”). On nutrient agar, nonpigmented species or biotypes of Serratia give opaque-whitish, mucoid, or transparent smooth colonies. None of these traits is specific for the isolation of Serratia from a mixture of enteric bacteria. Colonies of S. odorifera and S. ficaria and occasionally S. rubidaea give off a specific, potato-like odor. Detection of this odor may suggest the presence of at least one colony of that species on the plate. The salt tolerance and relatively low minimal growth temperature of all Serratia species may help devise a means of enriching them. A nutrient broth containing 4% NaCl, incubated at 15– 20°C, will allow multiplication of Serratia but not of Enterobacter cloacae or Aeromonas hydrophila (P. A. D. Grimont, unpublished observations). This procedure has been used by us, but no systematic study of its effectiveness has been conducted. Strains of the genus Serratia do not normally require addition of growth factors to a minimal medium. Thus, an enrichment medium for Serratia can be a minimal medium with a carbon source that is commonly used by Serratia spp. but not by non-Serratia spp. Production of extracellular gelatinase, lecithinase, and DNase are essential characteristics of the genus Serratia (Ewing, 1986; Grimont et al., 1977b), S. fonticola excepted. Agar media that show the presence of one or a combination of extracellular enzymes can be used to differentiate Serratia colonies. Finally, Serratia spp. are resistant to several compounds, including colistimethate, cephalothin (Greenup and Blazevic, 1971), and thallium salts (Starr et al., 1976), and one of these compounds can be used to make enrichment or differential media selective for Serratia. However, some S. plymuthica or S. rubidaea strains may be more susceptible to colistimethate, cephalothin, or ampicillin than is S. marcescens. No medium has yet been devised to selectively isolate one particular Serratia species (other than S. marcescens), although specific carbon sources or conditions are listed in taxonomic papers (Grimont et al., 1977b). Occasionally, a selective medium may be devised according to a specific pattern of antibiotic resistance displayed by a nosocomial strain of S. marcescens (Denis and Blanchard, 1975), but such media cannot be of general utility.
CHAPTER 3.3.11 The Genus Serratia 229 Selective Media Based on DNase Production and Antibiotic Resistance Farmer et al. 1973 proposed the following deoxyribonuclease-toluidine blue-cephalothin (DTC) agar. DTC Agar for Isolating Serratia (Farmer et al., 1973) Deoxyribonuclease test agar (BBL) 21 g Agar (Difco) 2.5 g Toluidine blue O 0.05 g Distilled water 500 ml Mix on a mechanical stirrer until the dye dissolves, autoclave with a Teflon stirring bar at 121°C for 15 min, cool to 50°C, add 5 ml (500 mg) of cephalothin (Keflin injectible, Eli Lilly), stir and dispense in sterile petri dishes. Typically, Serratia spp. grow on this blue DTC medium producing a red halo extending several millimeters around the colonies. “False negatives” (no growth or no halo) are very rare among S. marcescens isolates, but several strains of S. rubidaea and S. plymuthica failed to give the proper reaction (Starr et al., 1976). A medium with DNase test agar, toluidine blue, cephalothin (30 µg/ml and colistimethate (30µ/ml) has been proposed by Cate (1972); and a medium with DNase test agar, toluidine blue, egg yolk, and cephalothin (100µ/ml) has been proposed by Goldin et al. 1969. The latter combines DNase and lecithinase detection. Berkowitz and Lee (1973) devised the following medium for the isolation of S. marcescens: DNase Medium for Isolating Serratia marcescens (Berkowitz and Lee, 1973) Deoxyribonuclease test agar with methyl green (Difco) 42 g L-Arabinose 10 g Phenol red 0.05 g Methyl green, 1% 4 ml Distilled water up to 1 liter After autoclaving, add ampicillin (5 µg/ml), colistimethate (5 µg/ml), cephalothin (10 µg/ml), and amphotericin B (2.5 µg/ml). Serratia colonies hydrolyze DNA, and the green component of the medium’s dark color disappears around the colonies. S. marcescens (or S. entomophila), unable to ferment L-arabinose, will give colonies surrounded by a red halo, whereas other Serratia species will give a yellow halo. The other Serratia species, however, may be inhibited by the antibiotic mixture. Wright et al. 1976 used this medium to isolate and enumerate S. marcescens from vegetable salads. Selective Media Based on Carbon-Source Utilization A minimal medium with meso-erythritol as the sole carbon source was devised by Slotnick and Dougherty (1972). A modification of this medium that included the antiseptic “Irgasan” (4′,2′,4′-trichloro-2-hydroxydiphenylether), has been proposed (Lynch and Kenealy, 1976). Unfortunately, Serratia liquefaciens, S. plymuthica, S. entomophila, S. proteamaculans biotypes Clc and RB, S. odorifera biotype 1, and the nosocomial biotypes A5, A8a, A8b, A8c and TCT of S. marcescens cannot grow with erythritol as sole carbon source (Grimont et al., 1977b; Grimont et al., 1978a; Starr et al., 1976). Therefore, these media should not be used in epidemiological or ecological surveys unless the study is knowingly limited to erythritol-positive serratiae. A minimal medium with meso-inositol as sole carbon source has been proposed for the selective isolation of Klebsiella pneumoniae and Serratia spp. (Legakis et al., 1976). However, only a few genera and species have been studied by these authors, and it has been shown (Grimont et al., 1977b) that Enterobacter aerogenes, E. cloacae, Erwinia herbicola, and pectinolytic Erwinia spp. can also grow on a minimal medium with inositol as sole carbon source. A caprylate-thallous (CT) agar medium has been devised for the selective isolation of all Serratia species and biotypes. This CT agar, derived from M70 minimal medium (Véron, 1975), is made up as follows (Starr et al., 1976): CT Agar for Selective Isolation of Serratia (Starr et al., 1976) First, a trace element solution (Véron, 1975) is prepared: H3PO4 1.96 g FeSO4·7H2O 0.0556 g ZnSO4·4H2O 0.0287 g MnSO4·4H2O 0.0223 g CuSO4·5H2O 0.025 g Co(NO3)2·6H2O 0.003 g H3BO3 0.0062 g Distilled water 1 liter Store at 4°C (keeps well for at least 1 year). Then the following solutions (solution A and solution B) are prepared: Solution A: CaCl2·2H2O 0.0147 g MgSO4·7H2O 0.123 g KH2PO4 0.680 g K2HPO4 2.610 g Trace element solution (see above) 10 ml Caprylic acid Yeast extract (Difco) 1.1 ml (5% wt/vol solution) 2 ml Thallous sulfate 0.25 g Distilled water up to 500 ml The pH is adjusted to 7.2 with NaOH. Autoclave at 110°C (or 120°) for 20 min. Solution B: NaCl 7 g (NH4)2SO4 1 g Agar (Difco) 15 g Distilled water 500 g
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