Digestion of Algin by Pseudomonas Maltophilia and Pseudomonas Putida V
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1980, p. 92-96 Vol. 39, No. 1 0099-2240/80/01-0092/05$02.00/0 Digestion of Algin by Pseudomonas maltophilia and Pseudomonas putida V. LYLE VON RIESEN Department ofMedical Microbiology, College ofMedicine, University of Nebraska Medical Center, Omaha, Nebraska 68105 Pseudomonas maltophilia and Pseudomonas putida were identified as al- ginolytic species. Two media used for demonstrating alginolytic activity are described. The applied aspects of the ability of these two species to digest algin are discussed. In a search for undescribed biochemical activ- Both media contained yeast extract (0.5%), algin (al- ities of nonfastidious, nonfermentative gram- ginic acid, sodium salt, no. A-7128; Sigma Chemical negative bacilli (NFB) which might be used to Co., St. Louis, Mo.) (1%), and bromothymol blue of these taxonomically (0.002%). Agar (0.5%) was used to form an algin-agar aid in the identification medium and animal charcoal (0.1%) to form an algin- troublesome organisms, a survey of a variety of charcoal medium. The media were prepared in 100- to complex polysaccharides as substrates was made 500-ml amounts in the following manner. Calculated by using a few strains of some well-recognized amounts of yeast extract and bromothymol blue (from species of NFB. When it was discovered that a 1% aqueous solution) were added to an appropriate some of the strains ofPseudomonas maltophilia volume of distilled water. This was then warmed on a showed evidence of an ability to hydrolyze algin magnetic stirrer-heater. When warm (40 to 60°C), (sodium alginate), an available collection of ap- algin and charcoal (algin-charcoal medium) or algin proximately 390 strains of NFB was examined and agar (algin-agar medium) were sprinkled into the of individual strains to digest vigorously stirred solution. As heating and stirring for the ability were continued, obvious lumps were mashed, with a algin. Since many of these organisms do not spatula or glass rod being used to aid the magnetic readily produce acids from carbohydrates, it stirrer. By the time the mixture was close to boiling, seemed that some approach other than the con- it was reasonably free of obvious lumps. Heating was ventional method for carbohydrate metabolism discontinued at this point. The algin-charcoal medium (e.g., 1% carbohydrate in OF base) was neces- was then transferred in 4-ml volumes with a hand- sary. This paper describes the methods used to operated Cornwall pipettor to screw-cap tubes (16 by demonstrate alginolytic activity and identifies 125 mm). The algin-agar medium was left in a flask or two species which can digest algin. fleaker. The media were autoclaved at 121°C for not more than five minutes. The short heating period was to avoid the possible breakdown of algin. Immediately MATERUILS AND METHODS upon removal from the autoclave, the algin-agar was Organisms. The organisms used in this study were distributed in petri dishes to at least half volume. The strains of gram-negative bacilli which had been col- lids were left off the petri dishes to allow steam to lected from human, animal (porcine, bovine, and escape. Before use the plates were exposed in an avian), and environmental sources over a period of 25 incubator at 35°C for further drying. The algin-char- years. They had been maintained through this period coal medium was allowed to cool in an upright position in several ways, but generally by culture in cystine- and the tubes were then refrigerated until used. No Trypticase agar (BBL Microbiology Systems, Cock- settling of the charcoal occurred during storage or eysville, Md.) and storage at room temperature in the during use. The pH of the media by the color of the dark. None of the strains produces acid under anaer- indicator was neutral to very slightly alkaline. obic conditions in OF basal medium (14) with 1% The algin-agar plates were inoculated by spotting glucose, and all are therefore designated as NFB. (patching) cultures, grown on Trypticase soy agar During the course of this study all strains were more (BBL) for 24 h, onto the surface of the medium, 13 critically examined biochemically to obtain as positive strains per plate. (If spreading occurred, the offending an identication of as many strains as was reasonably organisms were tested separately.) The algin-charcoal possible. The methods used to identify the strains deeps were inoculated in a conventional way. Incuba- were identical to or variations of the methods de- tion of all materials was at 35 ± 1°C for 72 h and then scribed by various workers in this field (10, 13, 24, 29, at room temperature (18 to 22°C) to the end of 10 33, 35, 38). A few of the strains remain unidentified, days. Observations were made at 1, 2, 3, 6, and 10 days. since it did not seem possible to identify them with Over a period of several years, a number of formu- recognized species or alphanumerics. lations of algin media and variations of the procedures Algin media. Two forms of algin media were used. described above were examined. The results shown 92 VOL. 39, 1980 ALGIN DIGESTION BY P. MALTOPHILIA AND P. PUTIDA 93 here are based on the procedures described and the TABLE 1. Biochemical characteristics of alginolytic agar and charcoal media were studied concurrently for Pseudomonas species comparison. Positive strains RESULTS Test or medium P. maltophilia P. putida (42 strains) (30 strains) By the criteria used in this study (Table 1) for the identification of the 390 strains of NFB the No. % No. % numbers of strains of species and the alphanu- OF glucose 41 98 30 100 merics identified were: Achromobacter xylosox- Oxidase 0 0 30 100 idans, 10; Acinetobacter anitratum, 24; Acine- Motility 38 91 28 93 tobacter Iwoffii, 17; Alcaligenes denitrificans, 3; H2S 0 0 0 0 Alcaligenes faecalis, 3; Alcaligenes odorans, 7; Tryptophan pyrrolase 40 95 2 7 Bordetella bronchiseptica, 3; Bordetella para- Indole 0 0 0 0 pertussis, 1; Flavobacterium meningosepticum, Starch hydrolysis 0 0 0 0 Deoxyribonuclease 42 100 0 0 1; Flavobacterium odoratum, 2; Moraxella spp. Ribonuclease 42 100 0 0 (undifferentiated), 9; Pseudomonas acidovor- Arginine dihydrolase 0 0 29 97 ans, 7; Pseudomonas aeruginosa, 88; Pseudom- NO3-+ N2 0 0 0 0 onas alcaligenes-pseudoalcaligenes group, 18; N03- N02 18 43 0 0 Pseudomonas cepacia, 13; Pseudomonas dimi- Urease 7 17 2 7 nuta, 6; Pseudomonas fluorescens, 2; Pseudom- Gelatinase 42 100 0 0 onas maltophilia, 42; Pseudomonas paucimo- Esculin hydrolysis 40 95 0 0 bilis, 1; Pseudomonasputida, 30; Pseudomonas Acetamide utilization 0 0 0 0 putrefaciens, 7; Pseudomonas stutzeri, 46; Pseu- Algin hydrolysis 23 55 13 43 domonas testosteroni, 3; Vibrio extorquens, 4; CDC (Center for Disease Control) group lIf, 9; strains produced an appearance of a very weakly CDC group IVc-2, 1; CDC group Vd, 7; CDC developed depression during the first 24 to 72 h; group Ve-1, 1; CDC group Ve-2, 3. Twenty-two however, by day 6 this depression was dissi- strains have not yet been matched to any of the pated. Such an appearance with pectate gels was species or groups. Of these strains, 15 are al- attributed by Hildebrand (12) to possible loss of ginolytic. Efforts to identify these are continu- water from the medium to the developing mass ing. of growth, thus causing shrinkage of the me- Species and alphanumerics not identified in dium. this collection were: Agrobacterium radiobac- With the algin-charcoal medium, alginolytic ter, Pseudomonas mallei; Pseudomonas men- activity was observed as a usually sharply de- docina; Pseudomonas pickettii; Pseudomonas marcated settling of the charcoal. With time, pseudomallei; Pseudomonas vesicularis; and and varying with the individual strains, the line CDC groups IIj, IIk-2, IVe, and Va-i. of settled charcoal rapidly or slowly descended. Table 1 shows the characteristics ofthe strains On each day of observation, the amount of set- identified as P. maltophilia and P. putida. thing (depth of clear zone above the line of char- Twenty-three, or 55%, of the 42 strains of P. coal) was measured and recorded. On day 10, all maltophilia and 13, or 43%, of the 30 strains of tubes showing no evidence of settling, i.e., com- P. putida were alginolytic. All alginolytic strains, parable to the uninoculated control tube, were including the 15 unidentified strains, were posi- recorded as negative. Most of the alginolytic tive on both algin media. strains of both P. maltophilia and P. putida Alginolytic activity was observed with the al- were positive within 24 h of inoculation. A few gin-agar medium, as previously described for strains of both species required up to 72 h to alginolytic activity (9, 42) and for pectinolytic become positive. Table 2 shows the number of activity (36, 37), as a defmite break in the plane strains of P. maltophilia and P. putida produc- of the surface of the medium. With the majority ing 100% settling of the charcoal by days 1, 3, 6, of the positive strains, this was a distinctly dif- and 10. As a further evaluation of the alginolytic ferentiated, bowl-shaped depression containing activity, when the settling of the charcoal was the patch of growth. With some strains a shallow considered 100%, the tubes were manually depression was formed, and in this reaction the shaken to resuspend the charcoal and to obtain growth was frequently contiguous with the pe- an impression of the viscosity of the medium. rimeter of the depression. Most ofthe alginolytic Generally, with P. maltophilia the viscosity was strains of P. maltophilia were positive within 24 as for water (algin presumably completely di- h of inoculation; most strains of P.