Evaluation of Media for Differentiating Nonfermenting Gram-negative Bacteria of Medical Significance

Transcription

1 APPLIED MICROBIOLOGY, Sept. 1969, p Copyright 1969 American Society for Microbiology Vol. 18, No. 3 Printed in U.S.A. Evaluation of Media for Differentiating Nonfermenting Gram-negative Bacteria of Medical Significance GERALD L. GILARDI Microbiology Division, Department of Laboratories, Hospital for Joint Diseases and Medical Center, New York, New York Received for publication 27 June 1969 An evaluation was made of media and tests used for differentiating nonfermenting gram-negative bacteria encountered in medical bacteriology in order to determine those diagnostic procedures most useful in identifying these bacteria. The organisms examined included Alcaligenes faecalis, A. odorans var. viridans, Moraxella duplex (Mima polymorpha var. oxidans), Acinetobacter anitratum (Herellea vaginicola), A. Iwoffi (Mima polymorpha), Pseudomonas fluorescens, P. putida, P. maltophilia, P. pseudomallei, P. stutzeri, P. alcaligenes, and atypical strains of P. aeruginosa. The media and tests evaluated included Sellers' medium; Hugh and Leifson's OF medium; acid production from 10% lactose infusion agar; gluconate oxidation; starch, aesculin, and Tween 80 hydrolysis; lysine decarboxylase, arginine dihydrolase, deoxyribonuclease, and tyrosinase activity; tolerance to triphenyl tetrazolium chloride, cetrimide, cadmium sulfate, 2.5% and 6.5% sodium chloride, and ph 5.6; utilization of glucose, acetamide, and malonate. An evaluation was made of diagnostic procedures for examining nonfermenting gramnegative bacteria in order to determine those methods most useful in the identification of these bacteria. The organisms examined included representative species of Pseudomonas, Alcaligenes, Moraxella, and Acinetobacter. This paper reports on those findings. MATERIALS AND METHODS Cultures. The strains of Pseudomonas pseudomallei were obtained from the American Type Culture Collection (ATCC 11668), R. Hugh, George Washington University, Washington, D.C. (NCTC 1691, B-ill, RH 2108), and Major M. R. Smith, 406th Medical Laboratory, U.S. Army ( and ). Two strains of Moraxella duplex (Mima polymorpha var. oxidans) were obtained from E. Bottone, Mt. Sinai Hospital, New York City. All other isolates, including Alcaligenes faecalis, A. odorans var. viridans, M. duplex, Acinetobacter anitratum (Herellea vaginicola), A. Iwoffi (Mima polymorpha), P. fluorescens, P. putida, P. maltophilia, P. stutzeri, P. alcaligenes, and atypical strains of P. aeruginosa, were recovered from clinical materials in this laboratory from May 1965 to May The 35 strains of P. aeruginosa examined were atypical in that they were apocyanogenic and failed to produce the odor of trimethylamine. The diagnostic criteria used to identify these bacteria have previously been described (9-11). Studies performed. The tests, media, and references to procedures employed included: acid production 355 from 1% glucose, OF Basal Medium [Difco, (16)]; acid production from 10% lactose, Purple Agar Base [Difco, (1)]; acid production from glucose, fluorescein production, nitrogen gas production, and anaerobic growth in the presence of nitrate, Sellers' Differential Agar [Difco, (23)]; dihydrolase and decarboxylase activity, Decarboxylase Base Moller [Difco, (20)]; deoxyribonuclease activity, DNase Test Agar [Difco, (6)]; gluconate oxidation, Gluconate Substrate [Key Scientific Products, (8)]; malonate utilization, Malonate Broth [Difco 0395, (17)]; tyrosinase activity, Trypticase Soy Agar (TSA; BBL) with 1% 1-tyrosine (4); starch hydrolysis, TSA with 1% starch (19); lipase activity, Sierra medium with 1% polyethylene sorbitan monooleate [Tween 80, (24)]; aesculin hydrolysis, TSA with 0.1% aesculin and 0.05% ferric citrate (4); growth on cetrimide, Pseudosel Agar [BBL, (3)]; growth on triphenyl tetrazolium chloride (TTC), TSA with 1% TTC (3); growth on cadmium, TSA with 0.04% cadmium sulfate (28); growth at ph 5.6, Sabouraud Dextrose Agar (Difco); sodium chloride tolerance, TSA adjusted to NaCl concentrations of 2.5% and 6.5%; and utilization of glucose and acetamide as sole sources of carbon and energy in a basal mineral medium previously described (11). RESULTS AND DISCUSSION The reactions obtained with the aerobic gramnegative bacteria on the different media are given in Table 1. Sellers' medium. All strains of A. anitratum oxidized glucose in Sellers' medium as indicated

2 356 GILARDI APPL. MICROBIOL. by the yellow band at the junction of the slant some of the apyocyanogenic strains of P. aeruand butt. In all cases, the band was distinct and ginosa, previous studies (7, 23) report that pyoreproducible on repeat examination. Among the cyanogenic strains fail to produce a yellow pseudomonads, P. pseudomallei, P. stutzeri, the band. Formation of a yellow band has been simple fluorescent pseudomonads (P. fluorescens used (23) to distinguish A. anitratum from A. and P. putida), and the apyocyanogenic strains Iwoffi which is nonglucolytic, but, since some of of P. aeruginosa produced variable oxidation of the pseudomonads produced a yellow band, glucose. With many of these strains the yellow this character cannot be considered specific band was weak or indefinite and not always for A. anitratum. uniformly reproducible on repeat examination. Nitrogen gas was produced by all strains of P. Although band formation was detected with pseudomallei, P. stutzeri, and A. odorans var. Test or substrate TABLE 1. Reactions of the aerobic gram-negative bacteria on different mediaa _ i 'X" Sellers' medium Alkaline slant Acid band Alkaline butt Nitrogen gas Fluorescence Oxidation of 1% Glucose (OFBM) 10% Lactose (PAB) Gluconate (GS) Tyrosinase activity Arginine dihydrolase Lysine decarboxylase Deoxyribonuclease activity Starch hydrolysis Aesculin hydrolysis Tween 80 hydrolysis Growth in ph % NaCl % NaCl TTC Cetrimide Cadmium Utilization of Malonate (MB) Acetamide (BMM) Oc Glucose (BMM) c a Figures in parentheses represent number of strains examined; other figures represent number of strains positive for the test. Abbreviations: OFBM, OF Basal Medium; PAB, Purple Agar Base; GS, Gluconate Substrate; TTC, triphenyl tetrazolium chloride; MB, Malonate Broth; BMM, basal mineral medium. All tests were incubated at 37 C. The tests for gluconate oxidation, deoxyribonuclease activity, and starch hydrolysis were performed after 24 hr of incubation. Tyrosinase activity and oxidation of glucose in OF medium by P. maltophilia generally were detected only after 48 to 72 hr of incubation. All other reactions were interpreted after 24 to 48 hr of incubation. b Synonyms, respectively: Herellea vaginicola, Mima polymorpha, M. polymorpha var. oxidans. BMM supplemented with methionine. ZV

3 VOL. 18, 1969 NONFERMENTING GRAM-NEGATIVE BACTERIA 357 viridans, as indicated by the gas bubbles in the condensate and the cracked agar butt. The capacity to denitrify is generally considered (26) a universal property of P. aeruginosa, but it has been reported (12) to be a variable character as demonstrated in this study. The strains of P. aeruginosa were also tested in Trypticase Nitrate Broth Medium (BBL) with the same variable results (unpublished observations). All strains of P. aeruginosa, P. pseudomallei, P. stutzeri, and A. odorans var. viridans grew anaerobically in the presence of nitrates as indicated by the blue color of the alkaline butt. Fluorescence was detected on exposure to ultraviolet light with all strains of P. aeruginosa, P. fluorescens, and P. putida. The absence of anaerobic growth, glucose oxidation, fluorescence, and nitrogen gas production was characterized by P. alcaligenes, A. iwoffi, M. duplex, and A. faecalis. Strains of A. faecalis which produce gas and grow anaerobically in Sellers' medium have been examined (23); such strains have also been designated A. denitrificans (18). Oxidation of carbohydrates. Acid was produced only in the unsealed tubes of OF medium containing glucose by A. anitratum and by all pseudomonads except P. alcaligenes; such a phenomenon is indicative of obligate aerobes. An alkaline reaction was produced by all other strains. The advantage of using Hugh and Leifson's OF medium (16) in place of conventional carbohydrate-peptone media to demonstrate oxidative ability has been pointed out (9, 12). All strains of A. anitratum and 16 of the 108 strains of glucolytic pseudomonads produced acid from 10% lactose in infusion base. Oxidation of 10% lactose has been employed (1) to distinguish A. anitratum from A. iwoffi, but since some of the pseudomonads gave a positive reaction, this test can not be considered specific for A. anitratum. The usefulness of the oxidation of gluconate to keto-gluconate in differentiating P. aeruginosa from other pseudomonads has been established (8). Positive (14) and variable (28) gluconate oxidation has been reported for P. fluorescens. In a recent study (21), the test was 93% accurate for P. aeruginosa, but an accuracy of only 63% was demonstrated in the present study using apyocyanogenic strains of P. aeruginosa. Of the 21 strains of simple fluorescent pseudomonads, 13 gave a positive test. Extracellular enzyme activity. Previous studies show that aesculin hydrolysis is a characteristic of xanthomonads (5) and strains of P. pseudomallei which oxidize sucrose (29). Three of the strains of P. pseudomallei examined in this study oxidized sucrose in OF medium (unpublished data), but only two of these three strains hydrolyzed aesculin. In addition to P. pseudomallei, all strains of P. maltophilia gave a positive test. Starch hydrolysis was an exclusive character of P. stutzeri. Seven of eight strains of P. stutzeri were starch-positive, and the same seven strains were lipase-positive. One starch- and lipasenegative strain of P. stutzeri was deoxyribonuclease-positive. Previous studies report positive (22) and negative (19, 27) starch hydrolysis for P. pseudomallei, but none of the strains were positive in the present study. The test for deoxyribonuclease production is used primarily for the identification of pathogenic staphylococci and Serratia marcescens, but other bacteria are known to produce this enzyme (6). In the present study, all but one strain of P. maltophilia produced deoxyribonuclease. Except for P. maltophilia, one strain of P. stutzeri, and four strains of P. aeruginosa, all other strains gave negative reactions. Lipolytic activity as indicated by the hydrolysis of Tween 80 has been used to identify P. aeruginosa (26) and Acinetobacter (2). In this study, only 77% of the apyocyanogenic strains of P. aeruginosa hydrolyzed Tween 80. Nearly all strains of P. maltophilia, P. pseudomallei, P. stutzeri, Acinetobacter, and A. faecalis were lipase-positive. The only species that were uniformly negative included M. duplex and A. odorans var. viridans. Although A. faecalis was lipase-positive, the species has been reported (7) lipase-negative. Arginine dihydrolase and lysine decarboxylase. The anaerobic breakdown of arginine by arginine dihydrolase by all fluorescent pseudomonads and P. pseudomallei was demonstrated by M0ller's method. Arginine dihydrolase activity is reported variable (26) and negative (15) for the alcaligenes group of pseudomonads, and all strains of P. alcaligenes were negative in the present study. All strains of P. maltophilia were positive for lysine decarboxylase as determined by M0ller's method. Tyrosinase activity. P. maltophilia produced a diffusable light-brown pigment as a result of the formation of melanine from tyrosine. This character is not limited to P. maltophilia since other pseudomonads (12, 19) and xanthomonads (5) are known to produce melanine. Asimilation of carbon compounds. Malonate utilization in Leifson's malonate broth (17) is used to differentiate members of the enterobacteriaceae and has been applied to the identification of pseudomonads (14, 15). In the present study, all strains of P. aeruginosa, P. maltophilia, P. pseudomallei, P. stutzeri, and A. odorans var.

4 358 GILARDI APPL. MICROBIOL. viridans utilized malonate, whereas utilization was variable among the simple fluorescent pseudomonads and A. anitratum. P. maltophilia strains produced a positive reaction only when a heavy inoculum was added to the medium due to the requirement of the organism for methionine as growth factor (26). Acetamide is reported to serve as the sole carbon source in a basal medium for P. aeruginosa (13, 26) and some strains of P. putida (26). In the present study, all the strains of A. odorans var. viridans, two of 19 strains of P. putida, but only 57% of the apyocyanogenic strains of P. aeruginosa utilized acetamide. Glucose was assimilated by all pseudomonads except P. alcaligenes, but not by any other genera. Although A. anitratum was capable of oxidizing glucose in a complex medium, it could not utilize glucose as the sole carbon source. Tolerance to chemical and physical agents. Tolerance to various chemical and physical agents has been used (3, 25, 27, 30) as an aid in the isolation and identification of Pseudomonas and Acinetobacter. In the present study, P. pseudomallei and A. faecalis were the only species that could not grow in 2.5% NaCl. P. stutzeri and A. odorans var. viridans were the only species uniformly capable of tolerating 6.5% NaCl. P. stutzeri and M. duplex were the only species unable to grow at ph 5.6. Variable growth of A. iwoffi at ph 5.5 has been reported (25). Growth in 0.03 % cetrimide occurred with 95% of the fluorescent pseudomonads and all strains of A. odorans var. viridans. P. aeruginosa is reported (3, 28) capable of growing in 1% TTC; in this study, growth occurred with 91% of the simple fluorescent pseudomonads, but with only 69% of the apyocyanogenic strains of P. aeruginosa. Growth of P. aeruginosa on 0.2% cadmium sulfate agar has been reported (28), but all bacteria examined in the present study were inhibited by this concentration. Eighty-nine percent of the strains of P. aeruginosa did grow in 0.04% cadmium, as did P. pseudomallei, A. faecalis, and 73% of the simple fluorescent pseudomonads. LITERATURE CITED 1. Aiken, M. A., M. K. Ward, and E. 0. King A study of a group of gram-negative bacteria resembling the tribe Mimeae (DeBord). Public Health Lab. Bull. Conf. State Prov. Public Health Dir. 14: Baumann, P., M. Doudoroff, and R. Y. Stanier A study of the Moraxella group. II. Oxidative-negative species (genus Acinetobacter). J. Bacteriol. 95: Brown, V. I., and J. L. Lowbury Use of an improved cetrimide agar medium and other culture methods for Pseudomonas aeruginosa. J. Clin. Pathol. 18: Cowan, S. T., and K. J. Steel Manual for the identification of medical bacteria. Cambridge University Press. 5. Dye, D. 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