SELECTION AND RAPID IDENTIFICATION OF PSEUDOMONAS PSEUDOMALLEI FROM OTHER GRAM-NEGATIVE BACTERIA

Transcription

1 THE AMERICAN JOURNAL OF CLINICAL PATHOLOGY Vol. 9, No. G Copyright 068 by The Williams & Wilkins Co. Printed in U.S.A. SELECTION AND RAPID IDENTIFICATION OF PSEUDOMONAS PSEUDOMALLEI FROM OTHER GRAMNEGATIVE BACTERIA HANNAH FARKASHIMSLEY, M.Sc, PH.D. WITH THE TECHNICAL ASSISTANCE OF SALLY BEASTALL, B.S. Department of Microbiology, School of Hygiene, University of Toronto, Toronto 5, Ontario, Canada Recently a casualty report of melioidosis in Vietnam 8 was released by the United States Armed Forces. The disease is known to produce pulmonary and multiple articular lesions and, in fatal cases, the spleen, the liver, and sometimes the kidneys have abscesses and caseous deposits. Since the discovery of melioidosis by Whitmore in 93 3 most of the cases have been reported in the Far East. Therefore, it is likely that doctors trained in the United States may not easily recognize cases of melioidosis in returned military or civilian population. Likewise medical personnel trained in the United States and stationed at present in Vietnam may not have previously seen a clinical case of this disease. Despite this unfamiliarity and the consequent difficulty in recognizing the disease and the causative organism Bacillus whitmori, which is now called Pseudomonas pseudomallei, this organism was isolated recently by the United States Army Surgeon General's Office from 3 servicemen, of whom the disease was fatal to nine. 8 The bacteria have been reported to be present in moist soil, perhaps because of the presence of excreta from domestic animals which were shown to harbor them. 3 ' ' 3 Human infection could thus occur via abrasions in the skin and via the respiratory system, provided that an aerosol of contaminated soil particles were inhaled. Indeed, experimental infections in mice and hamsters indicated the ease of infection by an aerosol. 6 Considering the above, one would expect to isolate Ps. pseudomallei from: skin lesions or wounds; the respiratory tract, i.e., nasopharynx, sputum, pleural fluid, or pulmonary lesions; or soil contaminated with urine and fecal excretions. Furthermore, the organisms can be encountered in tissue and Received July, 967. exudates of affected organs and, in the case of an acute systemic infection, in blood, cerebrospinal fluid, and urine. Therefore, any Gramnegative bacteria which could be associated with the above mentioned sources or specimens would have to be considered for differentiation purposes from Ps. pseudomallei. The purpose of the present communication is to describe a rapid selection and identification procedure for Ps. pseudomallei which distinguishes it from other Gramnegative bacilli likely to be present in any of the above mentioned specimens. 850 MATERIALS AND METHODS Selection Procedures It is recommended that the specimen be heavily inoculated on the selective medium, so that half of the plate would yield confluent growth, whereas the other half would have discrete colonies. The plate should be incubated for and 8 hr. at 37 C. Selective Medium. MacConkey's agar (Difco Laboratories, Inc., Detroit, Mich.) to which 0 jug per ml. ColyMycin (colistins*) were added was found to be an excellent selective medium, even when the inoculum consisted of only a small number of cells of Ps. pseudomallei. Freshly prepared plates had to be dried for at least 30 min. at 37 C. in order to prevent spreading of Proteus species. Identification Tests and Media Modified King's medium? This consisted of King's medium A to which were added sucrose at a final concentration of % and phenol red as indicator. This medium was contained in Petri plates. Organisms which * Supplied by WarnerLambert Canada, Ltd., Toronto, Canada. Downloaded from on 0 January 08

2 June 968 IDENTIFICATION OF PS. PSEUDOMALLEI S5 TABLE GUAMNEGATIVE BACTERIA WHICH MAY ACCOMPANY PS. PSEUDOMALLBI Family Genus Species Number Strains Pseudomonadaeeae Spirillaceae Achro mobuc Icrnccae Brucellaceae Enterobaeteriaceae? Pseudomoiias Vibrio A Indigenes Achromobacter Piisteurellii Bordetella Brucella Actinobacillus Moraxclla Salmonella Shigella Escherichia Proteus Providence B. anitratum r."ottil 5 (6?) attacked sucrose and formed acid were easily identified by the yellow color of the medium. Pigmentproducing bacteria such as Ps. aeruginosa appeared with a metallic sheen, and fluorescence was marked when illuminated under an ultraviolet light provided with a Wood's glass filter.* Mannose medium. This consisted of nutrient agar (Difco) to which mannose at a final concentration of % and phenol red as indicator were added. This medium was contained in 'Petri plates. Organisms which attacked mannose formed acid and turned the medium yellow. 'Desoxycholate citrate agar. This agar (Difco) was also contained in Petri plates. Oxidase test. 9 Oxidase tests were performed at once on all colonies to be tested by pouring the reagent 9 onto filter paper laid down in a Petri plate and replicating the colonies onto it. The appearance of a purple color within 0 to GO sec. was regarded as an oxidasepositive test. Confirmation tests and media. Other media recommended for confirmation of the presence of Ps. pseudomallei were arginine di * Ultraviolet lamp, Hanovia 'Division, Engelhard Industries, Inc., Newark, N. J. hydrolase, 7 the oxidative fermentative (0/ F) 7 medium (Difco), malonate broth, 0 lipase medium 6 (Difco 0950), and Sellers medium 6 (Difco 0S95). Replica plating. 0 A velveteen pad was used to pick up at once an inoculum from all of the colonies grown on the primary selective medium to replicate in rapid succession to the identification test media described above. RESULTS The Gramnegative bacilli tested which could be associated with specimens obtained for identification of Ps. pseudomallei are listed in Table. Thus, a total of 3 species and 73 strains of Gramnegative bacteria were examined on MacConkey agar and Mac Conkey agar supplemented with various concentrations of penicillin or ColyMycin. MacConkey medium inhibited a number of Gramnegative bacteria but not Ps. pseudomallei. Next, MacConkey media supplemented with ColyMycin 00 down to 5 Mg. per ml. were tested. ColyMycin at 5 to 0 fig. per ml. selected Ps. pseudomallei (eight strains) but inhibited most of the examined Gramnegative strains. However, as shown in Table, growth occurred with Ps. aerugi Downloaded from on 0 January 08

3 S5 FARKASHIMSLEY Vol. 9 TABLE GRAMNEGATIVE SPECIES SURVIVING COLYMYCIN AND THEIR SENSITIVITY TO PENICILLIN Bacterial Species P. pseudomallei P. aeruginosa V. comma; V. ElTor A. faecalis P. pseudotuberculosis B. bronchiseptica B. anitratum S. sonnei S. scholtmulleri Proteus Providence MacConkey Agar jcolyjviycin (0 g./ml.) * ( ) () () () () () ( ) G fpenicillin G (30 U./ml.) () or () *, Good growth;, fair growth; (), poor growth;, no growth. nosa, Vibrio comma and ElTor, Alcalige7ies faecalis, Pasteurella pseudotuberculosis,* and Bordetella bronchiseptica* as well as with Bacterium anitratum, Shigella sonnei, Salmonella schottmulleri, Proteus, and Providence, provided the inoculum was high (0 7 to 0 8 bacteria). Proteus mirabilis and occasionally Proteus vulgaris were found to spread on this medium, unless the medium was predried as described above in "Materials and Methods." The above findings indicated that a good selective medium was at our disposal, one which allowed the use of a heavy inoculum in the first isolation step. On the other hand, MacConkeypencillin media from 00 down to 5 ng. per ml. of penicillin G (30 to 8 units per ml., respectively) were also found to inhibit a number of Gramnegative bacteria. Table shows the effect of penicillin G (30 units per ml.). However, as for the eight strains of Ps. pseudomallei tested, an either complete or partial inhibition was noted with even the lowest concentrations of antibiotic. Thus the disadvantage of using penicillin G as a selective antibiotic for Ps. pseudomallei became apparent. The sensitiv * Two strains of B. bronchiseptica and seven strains of P. pseudotuberculosis among eight strains tested for each species were sensitive even to 5 jug per ml. of ColyMycin. ity of Ps. pseudomallei to penicillin was also noticed by Green and Tuffnell. 6 Effect of size of inoculum. The effect of ColyMycin and penicillin G at various concentrations was tested on eight strains of Ps. pseudomallei, using variable bacterial numbers as inoculum. The advantage of Coly Mycin over penicillin as a selective antibiotic for Ps. pseudomallei even in the presence of small inocula was clearly demonstrated. The following results were obtained for ColyMycin. At 50 ^g per ml. of medium the eighs strains of Ps. pseudomallei tested were not inhibited for hr., provided that an inoculum of 0 3 bacteria or greater was tested. With approximately 00 cells per inoculum or less, a slight inhibitory effect was observed transiently, resulting in good growth after incubation for S hr. Concentrations of ColyMycin below 50 ng. per ml. of medium did not inhibit any of the eight strains of Ps. psexulomallei tested, even when only approximately 0 cells per inoculum were tested, and distinct colonies to mm. in diameter were seen after 8hr. incubation at 37 C. Such results are shown in Figure for ColyMycin at 0 ng. per ml. Contrary to ColyMycin, penicillin G was found to inhibit Ps. pseudomallei even when as many bacteria as 0 s per inoculum were used. Penicillin G at 00 jug per nil. of medium (30 units per ml.) either completely or partially inhibited for hr. incubation seven out of eight strains of Ps. pseudomallei tested. After incubation for S hr. only four strains showed good growth, whereas three strains were still partially inhibited and one strain remained completely inhibited. Penicillin G, even at 5 ng. per ml. of medium, inhibited one strain (known to be virulent 6 ) when tested at such a high inoculum as 0 8 bacteria and incubated for S hr. As shown in Figure, penicillin G at 00 Mg per ml. of medium (60 U. per ml.) inhibited all of the eight strains tested with inocula up to 0 5 bacteria during the first hr. of incubation. After incubation fors hr., three strains were still unable to grow, two strains were partially inhibited, and only three strains out of eight grew well. However, when the inoculum consisted of 0 bacteria, all eight strains were inhibited by 00 ng. per Downloaded from on 0 January 08

4 June 968 IDENTIFICATION OF PS. PSEUDOMALLEI S53 Hrs. <rt 37 C. 8 Hrs. ot 37 C. Inoculum Size (number of bacteria) FIG.. Effect of ColyMycin and penicillin G on eight strains of Pseudomonas pseudomallei. Test conditions:, MacConkeyColyMycin at 0 Mg per ml.;, MacConkeypenicillin G medium at 00 Mg. per ml. (60 units per ml.);, no growth; ±, slight growth;, good growth. Oxidasenegative P. pseudotuberculosis B. anlitralum Enterobactcriaeeae A B C Fluorescence Sucrose JIannose Dcsoxycholate citrate Oxidasepositive P. pseudomallei P. aeruginosa V. comma; V. ElTor A. faecalis B. bronchiseplica * () or () or *, negative reaction or growth;, positive reaction or growth; (), slow reaction or growth. ml. of penicillin G of medium, even when inhibited completely the respective inocula incubated for 8 hr. at 37 C. Contrary to of 0,0 3, and 0 bacteria, results with ColyMycin at 0 ^g per ml., In view of the sensitivity of Ps. pseudothe smaller the bacterial inoculum the less mallei to penicillin G, it is recommended penicillin G was required to inhibit the that the use of penicillin G be discontinued growth of all eight strains of Ps. pseudomallei. as a selective agent for the isolation of Ps. Penicillin G at 00, 50, and 0 /ug. per ml. pseudomallei, as is the practice at present. 9 Downloaded from on 0 January 08

5 85 FAUKASHIMSLEY Vol. 9 Se/ective Mec/i< Master Plate /ettnfijit Mec/i'a ation FIG.. Identification of P. pseudomallei from other Gramnegative bacteria It is recommended that ColyMycin at 0 tig. per ml. of medium be used instead. At this concentration even the smallest number of Ps. pseudomallei cells in the specimen would not be suppressed and could be detected. In order to identify Ps. pseudomallei and to differentiate this species from other Gramnegative organisms which might be encountered in specimens suspected of containing Ps. pseudomallei and which might grow to some extent on the MacConkey ColyMycin (0 /xg. per ml.) medium, replica plating was used as described in "Methods and Materials" and shown in Figure. Thus, the selective MacConkeyColyMycin plate and the colonies thereon served as the master plate. The test media onto which the colonies were replicated were King's modified medium, mannoseagar, and desoxycholate citrate medium, followed by the oxidase test. The media and the oxidase reagentsoaked filter paper were all contained in Petri plates to facilitate and hasten replica plating. The oxidase test enabled the differentiation within 0 to 60 sec. of the oxidasepositive from Downloaded from on 0 January 08

6 June 968 IDENTIFICATION OF PS. PSEUDOMALLEI S55 TABLE 3 CHARACTERISTIC REACTIONS EOR PS. PSEUDOMALLEI AND SOME OTHER GRAMNEGATIVE OXIDASEPOSITIVE BACILLI Bacterial Species Media or Tests Ps. pseudomallei Ps. aeruginosa V. comma or V. ElTor A. faecalis and B. bronchiseptica Oxidalive/fermentutive Arginincdi hydrolase ( hr.) Indole' Malonafe ( hr.) Li pase Sellers: SLint Glucoseoxidized (yellow bund) Fluorescence Butt Na gas produced Alkaline Variable /* () _ or () No change or alkaline Variable, chiefly / (or false ) Alkaline Rarely Alkaline Acid NT / Acid or () / _ No change or alkaline No change Variable *, strong positive reaction;, positive reaction; (), slow or negligible reaction;, negative reaction; NT, not testable. the oxidasenegative bacteria, such as P. pseudotuberculosis and B. anitratum, and the enteric bacteria such as S. sonnet, Salmonella, Proteus, and.providence. The oxidasepositive species other than Ps. pseudomallei could be Ps. aeruginosa, V. comma, V. ElTor, A. faecalis, and B. bronchiseptica. These species could be differentiated and recognized easily on the three identification media used. It may be seen in Figure that Ps. pseudomallei did not fluoresce, attacked sucrose but not mannose, and did not grow on desoxycholate citrate agar. This inhibition was confirmed with eight different strains of Ps. pseudomallei and inoculum of 0 8 bacteria. Ps. aeruginosa, on the other hand, usually fluoresced on King's modified medium, did not produce acid from sucrose or mannose, and grew well on desoxycholate citrate agar. A. faecalis and B. bronchiseplica, unlike Ps. pseudomallei, were inert to sucrose and mannose and grew well on desoxycholate citrate medium. V. comma and V. ElTor produced acid from sucrose and therefore turned the medium bright yellow. Complete or partial inhibition was found on desoxycholate citrate agar, depending on, the strain tested. Thus the differentiation from Ps. 'pseudomallei was found difficult. However, mannose oxidation aided in their differentiation since the 0 strains of V. comma and four strains of V. ElTor tested attacked mannose and formed acid, whereas the eight strains of Ps. pseudomallei did not attack mannose. Final confirmation tests for Ps. pseudomallei are summarized in Table 3. For comparative purposes, Table 3 also includes reactions obtained for cultures of Gramnegative bacteria which grew on MacConkey ColyMycin at 0 pg. per ml. medium and were oxidasepositive and hence appeared similar to Ps. pseudomallei. SUMMAKY The prevalence of melioidosis among the United States Armed Forces in Vietnam and the difficulty of the identification procedures of the etiologic agent Pseudomonas pseudomallei encouraged us to develop a selective medium and a rapid diagnostic procedure for its differentiation from other Gramnegative bacilli which may be associated in clinical specimens. The selective medium consisted of MacConkey's medium reinforced with ColyMycin, 0 jug per ml. medium. At this Downloaded from on 0 January 08

7 856 PARKASHIMSLEY Vol. 9 concentration, an inoculum of Ps. pseudomallei of less than 0 bacteria was not inhibited, whereas many other Gramnegative bacilli which could possibly accompany Ps. pseudomallei were inhibited when even a heavy inoclum of 0 s bacteria was used. Exceptions were Ps. aeruginosa, Vibrio comma and Vibrio ElTor, Alcaligenes faecalis, Pasteurella pseudotuberculosis, Shigella sonnet, Salmonella scholtmiuleri, Proteus species and Providence, all of which occasionally grew in the presence of colymycin, 0 jug. per ml., provided 0 7 and 0 8 bacteria per inoculum were tested. For the final identification of Ps. pseudomallei the replica plating method was used in order to test many colonies at once by inoculating several media in rapid succession. A modified King's medium, mannoseagar, desoxycholate citrate agar, and the oxidase test were found adequate to differentiate Ps. pseudomallei from all other Gramnegative bacilli which were not inhibited on the selective medium containing ColyMycin. Several additional tests are tabulated for the final confirmative identification of Ps. pseudomallei. REFERENCES. Breed, R. S., Murray, E. G. D., and Smith, N. R.: Bergey's Manual of Determinative Bacteriology, Ed. 7,p. 00. Baltimore:The Williams & Wilkins Company, Chambon. L.: Isolement du bacille de Whitmore a partir du milieu exterieur. Ann. Just. Pasteur, 89: 935, Cottew, G. S.: Melioidosis in sheep in Queensland. A description of the causal organism. Australian J. Exper. Biol. & M. Sci., 8: , Cowan, S. T., and Steel, K. J.: Manual for the Identification of Medical Bacteria. Cambridge: Cambridge University Press, 965, p Dannenberg, A. M., Jr., and Scott, E. M.: Melioidosis: pathogenesis and immunity in mice and hamsters. I. Studies with virulent strains of Malleomyces pseudomallei. J. Exper. Med., 07: 5366, Green, R. N., and Tuffnell, P. G.: A case of laboratory acquired melioidosis. Am. J. Med., U: (April) Hugh, R., and Leifson, E.: The taxononiic significance of fermentative versus oxidative metabolism of carbohydrates by various Gram negative bacteria. J. Bad;., 66: 6, King, E. O., Ward, M. K., and Raney, D. E.: Two simple media for the demonstration of pyocyanin and fluorescin. J. Lab. & Clin. Med., U: 30307, Kovacs, N.: Identification of Pseudomonas pyocyanea by the oxidase reaction. Nature, London, 78: 703, Lederberg, J., and Lederberg, E. M.: Replica plating and indirect selection of bacterial mutants. J. Bad,., 63: 39906, 95.. Lewis, F. A., and Olds, R. J.: Melioidosis in sheep and a goat in North Queensland. Australian Vet. J., 8: 550, 95.. Nigg, C, and Johnston, M. M.: Complement fixation test in experimental clinical and subclinical melioidosis. J. Bact., 8: 59 68, Olds, R. J., and Lewis, F. A.: Melioidosis in a pig. Australian Vet. J., SI: 737, Rimington, R. A.: Melioidosis in North Queensland. M. J. Australia, : 5053, Sellers, W.: Medium for differentiating the Gramnegative nonfermenting bacilli of medical interest. J. Bact., 87: 68, Starr, M. P.: Spirit blue agar: A medium for the detection of lipolytic microorganisms. Science, 93: 33333, Thornley, M. J.: The differentiation of Pseudomonas from other Gramnegative bacteria on the basis of arginine metabolism. J. Appl. Bact., 3: 375, Time (Magazine): Viet Nam's "time bomb." Medicine Section, 89: 50, U. S. Army Medical Center, Washington, D. C. Personal communication. 0. U. S. Public Health Service: Enterobacteriaceae, Biochemical Methods for Group Differentiation. Washington, D. C: U. S. Government Printing Office, 966, p. 9. Downloaded from on 0 January 08