In addition to the many exoproducts of Pseudomonas aeruginosa which may be associated with virulence, certain secondary metabolites (11) disrupt normal ...
Vol. 25, No. 7
JOUMAL OF CLINICAL MICROBIOLOGY, JUlY 1987, p. 1308-1310 0095-1137/87/071308-03$02.00/0 Copyright © 1987, American Society for Microbiology
Simultaneous Production of Rhamnolipids, 2-Alkyl-4Hydroxyquinolines, and Phenazines by Clinical Isolates of
pseudomonas aeruginosa BRIAN C. SMEAL,' LYNN B3NDER,2 DONALD L. JUNGKIND,2 AND ANNETTE T. FIASTIE3* Jefferson Medical College,1 Clinical Laboratories, Thomas Jefferson University Hospital,2 and Department of Medicine, Jefferson Medical College,3 Thomas Jefferson University, Philadelphia, Pennsylvania 19107 Received 26 November 1986/Acçepted 26 March 1987
Of 72 clinical isolates of Pseudomonas aeruginosa examined for simultaneous production of secondary metabolites, 86% produced 2-alkyl-4-hydroxyquinolines, 75% produced rhamnolipids, and 58% produced phenazines, including pyocyanin. Whereas isolates producing two or one constituted smaller groups, 39% released all three metabolites. Metabolite production did not appear to influence site of infection.
individual) with various sites of infection, including 36 urinary tract, 13 respiratory tract other than cystic fibrosis, 9 blood, 7 wound, and 7 other categories including ear, feces, bile, semen, and peritoneal fluid. Single-colony isolates were grown for 24 h at 37°C with shaking aeration in 20 ml of tryptic soy broth (Scott Laboratories, Inc., Fiskeville, R.I.). Cell-free supernatants were extracted with CHCl3. The residue after evaporation of the CHCl3-soluble phase was suspended in 0.25 ml of methanol. This CHCl3-extracted material was chromatographed on silica gel plates with chloroform-methanol (5:1 or 1:1) as the solvent (6). The blue phenazine pigment, pyocyanin, was observed on the developed plates. The fluorescent 2-alkyl-4-hydroxyquinolines and phenazine derivatives were detected by UV illumination. Ehrlich reagent stained phenazines. Anthrone stained carbohydrates, and charring showed the presence of lipids (6). The presence and migration of materials detected in each CHCl3 extract was recorded. The category of phenazine production included either a phenazine compound tentatively identified as 1-hydroxyphenazine (6), pyocyanin, or both. Usually an isolate produced both, but 10 isolates which produced only one or the other were included in this group. Similarly, isolates producing any combination of the four rhamnolipids (11, 18) were classified together as positive rhamnolipid producers. The 2-alkyl-4-hydroxyquinolines were not individually separated by the chromatography. The levels of sensitivity for the 2-alkyl-4-hydroxyquinolines and phenazines were similar at 50 and 20 ng, respectively. A minimum of 700 ng was necessary for detection of rhamnolipids, which possibly influenced the number of isolates scored positive for these metabolites. P. aeruginosa has been shown to produce 2 mg of rhamnolipid per ml in 24 h (18); production of 100-fold less would still be detectable. Thus, it was concluded that the method was sufficiently sensitive to detect even minimal rhamnolipid production by
In addition to the many exoproducts of Pseudomonas aeruginosa which may be associated with virulence, certain secondary metabolites (11) disrupt normal mammalian cell functions. Phenazine pigments effectively inhibit proliferation of human epidermis (4) and lymphocytes (16), probably by inhibition of electron transport, 1-hydroxyphenazine being more potent than pyocyanin (3). The 2-alkyl-4hydroxyquinolines, first identified as "pyo compounds" (19), in oxidized form also inhibit cytochrome electron transport in heart muscle (12). The rhamnolipids, IBhydroxydecanoyl-p-hydroxydecanoàte with one or two rhamnose molecules, are hemolytic (7, 9, 10, 13), alter polymorphonuclear leukocyte chemotactic responses at subtoxic levels (15), and change electrochemical characteristics of bronchial epithelium (17). These three classes of metabolites impair respiratory ciliated cell function (6, 14). Although rhamnolipid hemolysin production has been linked to virulence (1, 8), pyocyanin or pigment production has not (1, 4). Phenazine pigments were produced by 100% of 11 P. aeruginosa strains in one study (13) and 5 strains in another (21). Production of 2-alkyl-4-hydroxyquinoline has been examined in only one or two strains (18, 19). Production of the rhamnolipid hemolysin was found in 83% of 12 strains (9) to 100% of 27 strains (1). It is unclear to what extent selection of particular P. aeruginosa strains may have occurred in these studies on few isolates and therefore influenced the number reported to produce the metabolite of interest. A study of 198 isolates of P. aeruginosa classified 59 to 99% of the same isolates as hemolysin producers, depending on the source of erythrocytes, but made no distinction between phospholipase c and rhamnolipid hemolysins (20). Two studies on less than 20 isolates (1, 13) reported simultaneous production of both pyocyanin and hemolysin but did not include the 2-alkyl-4-hydroxyquinolines. No study has examined the incidence of clinical isolates simultaneously producing all three metabolites or some combination thereof, which is addressed by this re-
the isolates. Of the 72 isolates, 62 (86%) produced 2-alkyl-4-hydroxyquinolines, 54 (75%) produced rhamnolipids, and 42 (58%) produced phenazine derivatives. On the basis of incidence of metabolite production by the isolates, these secondary metabolites, ranked in descending order, were 2-alkyl-4-
port.
The 72 isolates of P. aeruginosa identified according to standard key characteristics (5) were obtained over a 3month period from different individuals (one isolate per *
hydroxyquinolines
Corresponding author. 1308
>
rhamnolipids
>
phenazines.
NOTES
VOL. 25, 1987 TABLE 1. Pattern of metabolite production by P. aeruginosa
clinical isolates
Metabolite pattern
2-Alkyl-4-
Total ()
Source, no.
(%)l
Rhamnolipids
Phenazines
hydroxyquinolines
+
+
+
28 (39)
Urine, 15 (42) Blood, 5 (55) Respiratory, 5 (38) Wound, 1 (14)
+
-
+
19 (26)
Urine, 9 (25) Blood, 3 (33) Respiratory, 3 (23) Wound, 3 (43)
-
+
+
11 (15)
Urine, 5 (14) Respiratory, 2 (15) Wound, 2 (29)
+
+
-
1(1)
Peritoneal fluid, 1
+
-
-
6 (8)
Urine, 3 (8) Blood, i (11) Respiratory, 2 (15)
-
-
+
4 (6)
Urine, 2 (6) Respiratory, 1 (8)
-
+
-
2 (3)
Urine, 2 (6)
a
no
1(1) Wound, 1(14) %, Number of isolates from a particular site of infection divided by total
for that source.
The largest group, 39% of the isolates, produced all three metabolite classes, whereas groups of isolates producing two of the three classes constituted smaller percentages (26% 2-alkyl-4-hydroxyquinolines and rhamnolipids, 15% 2-alkyl4-hydroxyquinolines and phenazines, and 1% rhamnolipids and phenazines) (Table 1). Groups of isolates producing only one of the classes were smaller still (8% rhamnolipids, 6% 2-alkyl-4-hydroxyquinolines, and 3% phenazines). Only one isolate examined had no detectable amount of the three classes. Groups of isolates demonstrated production of every combination of one or two of the three metabolites. Therefore, it can be concluded that all three are independent biosynthetic products. The distribution based on site of infection generally followed the overall distribution of isolates in the various categories of triple-, double-, or single-metabolite production (Table 1). Exceptions to this generalization, such as isolates from wounds, may be attributed to the small number from that source. Otherwise, metabolite production does not appear to predispose to infection of a particular site. The present report is the first documentation of the high incidence of 2-alkyl-4-hydroxyquinoline production by P. aeruginosa clinical isolates. The 75% incidence of rhamnolipid hemolysin production observed here is somewhat lower than the 83 to 100% in earlier reports (1, 9). A greater difference was noted in the incidence of phenazine pigment production, 58% compared with 100% in earlier reports (13, 21). However, the earlier studies were limited to small numbers of strains and may have selected strains producing the metabolite of interest. In fact, pyocyanin production was
1309
noted in one study (2) as a prerequisite for P. aeruginosa identification. The level of sensitivity and the chromatographic identification of the metabolites in the present report provide confidence in the observed incidence of secondary metabolite production. In summary, the secondary metabolites were ranked, in descending order as follows: 2-alkyl-4-hydroxyquinolines > rhamnolipids > phenazines, based on the incidence of production. The largest group of isolates simultaneously produced all three of these metabolites, although other groups of isolates indicated that the three metabolite classes are independently biosynthesized. Metabolite production does not apparently influence the site of infection. This work was funded in part by a grant from the American Lung Association and by Public Health Service grants T35 HL-07497 and R23 ES-04137 from the National Institutes of Health.
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NOTES
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