Serological Typing ofPseudomonas aeruginosa - Journal of Clinical ...

JOURNAL OF CLINICAL MICROBIOLOGY, June 1977, p. 640-649 Copyright © 1977 American Society for Microbiology

Vol. 5, No. 6 Printed in U.S.A.

Serological Typing of Pseudomonas aeruginosa: Use of Commercial Antisera and Live Antigens III* Department ofParasitology and Laboratory Practice, School of Public Health, University of North Carolina, Chapel Hill, North Carolina 27514; Department of Microbiology and Public Health, School of Medicine, Zaragoza, Spain; and Bacteriophage-Bacteriocin Laboratory, Enteric Section, Center for Disease Control, CHARLES D. BROKOPP,1 RAFAEL GOMEZ-LUS,

AND

J. J. FARMER

Atlanta, Georgia 30333* Received for publication 7 February 1977

A practical slide agglutination test, with commercial antisera (Difco) and live antigens (antigens of live bacteria) taken directly from 24-h antimicrobial susceptibility plates, has been established for serotyping Pseudomonas aeruginosa. Until recently, the lack of both a standard antigenic scheme and a source of commercial antisera has made serological typing ofthis organism impractical. A simplified procedure with 17 unabsorbed antisera and live antigens prepared from materials readily available in most clinical microbiology laboratories makes epidemiological typing of this organism possible in hospital laboratories. The distribution of each serotype examined in this study was determined by using 425 consecutive patient isolates from six different hospitals. The distribution of 0 antigen groups (live antigen) was as follows: 01, 11.5%; 02, 1.6%; 03, 3.8%; 04, 7.8%; 05, 4.2% 06, 27.1%; 07,8, 5.9%; 09, 6.8%; 010, 2.4%; 011, 8.2%; 012 through 017, each less than 1%. Ten and six-tenths percent of the above agglutinated in two antisera, 3.3% agglutinated in more than two antisera, and 5.2% did not agglutinate in any antisera. A comparison of live and heated antigens shows that 93.2% were typable with the live antigen, and 94.5% were typable with the heated antigen. When both antigens were used, we typed 96.3% of 725 isolates. The reproducibility and specificity of the serological procedure were examined. We recommend using the live antigen for routine serological typing in clinical microbiology laboratories for "in house" epidemiology and reserving the heated antigen for reference and research typing (and for those few cases where results cannot be obtained using the live antigen). The application of serotyping in the study of outbreaks of P. aeruginosa is also presented.

Infections caused by Pseudomonas aeruginosa have become increasingly prevalent in the hospital. About 7 of every 1,000 hospitalized patients develop an infection with P. aeruginosa (2). Although effective antibiotics are available, infection with this organism is a major cause of morbidity and mortality among patients with altered immune defenses. The use of antibiotics, immunosuppressive agents, antimetabolites, and radiation therapy, as well as numerous supportive procedures (urethral catherization, tracheostomy, lumbar puncture, and intravenous infusion), increases the susceptibility of patients to infection by this organism. This is especially true in very young and very old patients, and in those with burns or cancer.

Our knowledge of the epidemiology and con1 Present address: Enteric Section, Center for Disease Control, Atlanta, GA 30333.

trol of pseudomonas infections depends on the availability of accurate typing procedures. Numerous typing schemes have been used to differentiate strains of P. aeruginosa. These include bacteriophage typing (3, 20, 26, 29), pyocin production (10, 14, 32), pyocin sensitivity (10), serological typing (6, 12, 15, 16, 19, 22, 24, 27, 30), and the use of antibiograms (7) and gross phenotypic characteristics (5). Since no single typing method has gained widespread acceptance, two or more methods are often used for comparison typing of isolates. During the past 60 years, the antigenic structure and immune response produced by P. aeruginosa have been widely studied. The species was first reported to be serologically uniform, since attempts to prepare specific antisera were unsuccessful (23); however, the species was later shown to possess specific antigens which could be used to prepare typing sera. Several serological typing schemes have been 640

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SEROLOGICAL TYPING OF P. AERUGINOSA

described for P. aeruginosa (6, 12, 15, 16, 19, 22, 24, 27, 30), but only a few have been widely used (12, 15, 30). Until recently, the lack ofboth a well-defined set of antigens and a commercial source of antisera prevented the standardization of serotyping procedures. The purpose of this study is to describe a rapid, practical slide agglutination technique with antigens of live bacteria (hereafter referred to as live antigens) and commercially available antisera. Serotyping can now be used to study pseudomonas infections even in small hospitals. In addition to a simplified procedure, we describe a similar slide agglutination technique with heated antigens which might be used for reference and research. MATERIALS AND METHODS Media. Mueller-Hinton (MH) agar (Bioquest, Cockeysville, Md.) and veal infusion agar were used to prepare the live and heated antigens, respectively. MH agar was prepared as for susceptibility testing according to the manufacturer's instructions. Veal infusion agar contained 25 g of veal infusion broth (Difco Laboratories, Detroit, Mich.), 15 g of agar (Difco), and 1,000 ml of distilled water. Sixty milliliters of MH agar was dispensed into plastic petri plates (150 by 20 mm), and 20 ml of VI agar was placed in plastic petri plates (100 by 15 mm). Tubes containing 3 ml of Trypticase soy broth (TSB; Bioquest) were used to prepare the inoculum for both the heated and live antigens. TSB/100 contained 0.3 g of TSB in 1,000 ml of distilled water. All media were stored at 40C before use, and all incubations were at 36 + 1°C. Strains of Pseudomonas. P. aeruginosa used in this study included 249 isolates from 24 outbreaks throughout the United States; the 24 pyocin producer strains (NIH A-NIH X) and 27 pyocin indicator strains (NIH 1-NIH 27) described by Farmer and Herman (10); and 425 patient isolates from six different hospitals. These isolates include: 94 consecutive isolates from patients at the Clinical Center, National Institutes of Health, Bethesda, Md. (hospital 1); 53 isolates from patients at Druid City Hospital, a 500-bed community hospital in Tuscaloosa, Ala. (hospital 2); 103 isolates from patients at North Carolina Memorial Hospital, a 550-bed university hospital in Chapel Hill, N.C. (hospital 3); and 38 isolates from patients at Freeport Memorial Hospital, a 250-bed community hospital in Freeport, Ill. (hospital 4). In addition, 45 isolates from patients at Riberirfo Preto Hospital, a 359-bed general hospital in Sao Paulo, Brazil (hospital 5); and 92 isolates from patients at University Hospital, School of Medicine, Zaragoza, Spain (hospital 6) were examined. Twenty-four outbreaks of P. aeruginosa infections were studied after isolates were received at the Center for Disease Control (CDC) for epidemiological typing. Of the 249 isolates from these outbreaks, 227 were from hospitals; the remaining 22 were from two outbreaks of skin rash associated with contaminated swimming pools and whirlpools. Sixteen standard

641

antigenic strains (01-016) were obtained from P. V. Liu, University of Louisville, Louisville, Ky. In addition, 11 P. aeruginosa, 19 Pseudomonas putida, and 15 Pseudomonas fluorescens from the American Type Culture Collection were studied. All isolates of P. aeruginosa were identified by the clinical microbiologists at the hospitals or laboratories from which the isolates were obtained. We did not perform further identification of oxidasepositive isolates with typical colonial morphology, characteristic blue-green pigment, and typical odor. Isolates that did not possess these characteristics were confirmed by the Enteric Section of CDC on the bases of their oxidative metabolism of 1-glucose and D-xylose, oxidation of D-gluconate to 2-keto-D-gluconate, production of arginine dihydrolase, and growth in TSB at 42°C. Isolates that agglutinated in more than two antisera or did not agglutinate in any of the antisera were included only after being confirmed as P. aeruginosa. Throughout this study, each isolate was stored at room temperature in TSB/ 100. P. aeruginosa antisera. Seventeen unabsorbed P. aeruginosa antisera, which are now commercially available, were obtained from A. E. Bunner (Difco). The origin of the strains used to make the antisera is shown in Table 1. These 0-groups have been proposed as the standards for serotyping P. aeruginosa by the Subcommittee on Pseudomonadaceae and Related Organisms of the International Committee of Systematic Bacteriology of the International Association of Microbiological Societies. The lyophilized antisera were rehydrated with 1.0 ml of merthiolated saline (1:10,000 thimersal; Merthiolate, Eli Lilly & Co., Indianapolis, Ind.) and diluted 1:10 by adding 9.0 ml of merthiolated saline. Antisera at this dilution were used in the slide agglutination test. Three antisera pools, each containing five antisera, were also prepared. Pool A contained antisera 1, 3, 4, 6, and 10; pool B contained antisera 2, 5, 7, 8, and 15; and pool C contained antisera 9, 11, 12, 13, and 14.

TABLE 1. Description of the 17 0-antigen groups being studied by the Subcommittee on Pseudomonadaceae and Related Organisms 0-antigen 1 2 3 4 5 6 7 8 9 10 11

12 13 14 15 16 17

Previous nomenclature

Reference

Habs 0:1 Habs 0:2 Habs 0:3 Habs 0:4 Habs 0:5 Habs 0:6 Habs 0:7 Habs 0:8 Habs 0:9 Habs 0:10 Habs 0:11 Habs 0:12 Sandvik 0:11 Verder and Evans 0:5 Lanyi 0:12 Homma T:13 Meitert X

(15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (27) (30) (19) (16)

(25)

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BROKOPP, GOMEZ-LUS, AND FARMER

Antisera 16 and 17 were not included in any of the three pools. Each antiserum in a pool was at a final dilution of 1:10; the dilution used to test the sera individually. Preparation of heated antigens. Four to five wellisolated colonies of the same morphological type were inoculated into a tube containing 3 ml of TSB. This tube was incubated at 36°C until the suspension was as turbid as that used to standardize the inoculum for disk sensitivity tests (1). This turbidity is visually equivalent to a standard prepared by adding 0.5 ml of 0.048 M BaCl, to 99.5 ml of 0.36 N H2SO4. Alternatively, an overnight culture may be diluted with saline (0.85% NaCl) to the proper turbidity. A sterile cotton swab was dipped into the standardized suspension, and any excess broth was removed by rotating the swab firmly against the side of the tube. A plate of VI agar was streaked evenly to obtain a uniform inoculum. After overnight incubation, the surface of the VI agar was flooded with 10 ml of saline. The growth from the entire surface was suspended in the saline, and then FIG. 1. Slide agglutination with 17 monovalent transferred to a screw-cap tube (16 by 125 mm) with a pipette. The cell suspension was autoclaved for 30 antisera and serum control showing positive agglutimin at 12100, allowed to cool, and then centrifuged nation in antiserum 4. (1,000 x g) for 10 min to sediment the bacteria. The supernatant fluid was discarded, and the bacterial mass was resuspended in 0.8 ml of saline. After this was mixed, the dense suspension was used as the I heated antigen. Seventeen control antigens are available commercially (Difco), but we did not test these. Preparation of live antigen. The live antigen is a saline suspension of cells removed from a plate of II t MH agar. The live antigen is conveniently prepared --.II directly from a susceptibility plate which should be available for most isolates of P. aeruginosa obtained in clinical laboratories. Organisms from a circular area 25 mm in diameter (the size of a quarter) were transferred with a cotton swab to a tube containing 1 ml of saline. This suspension was mixed well to disperse the bacteria and then used directly as the I p antigen. A 1:30 dilution ofthis live antigen (0.1 ml of i live antigen and 2.9 ml of saline) has an optical ,6 k,a;} density (OD) of 0.25 + 0.1 when read in glass tubes (13 by 100 mm) at a wavelength of 650 nm. Slide agglutination procedure. The slide aggluti0j nation procedure has been simplified by using a disposable plastic petri plate as a surface on which to test the organism. Although conventional glass slides marked off with a wax pencil may be used, small or large plastic petri dishes are more convenFIG. 2. Antiserum applicator with syringes conient (Fig. 1). The antisera and normal rabbit serum taining monovalent antisera. control were dispensed with a multisyringe dropper. The device designed by Farmer et al. (11) allows uniform drops of all antisera (approximately 0.01 examined with indirect lighting from a Quebec colony counter (model C 100, New Brunswick Scienml) to be dispensed in a single operation (Fig. 2). The antigen was added to 1 drop (0.01 ml) of tific, New Brunswick, N.J.). The strength of the each antiserum with a disposable tuberculin syringe agglutination reaction after 1 min was recorded as fitted with a 25-gauge needle (Sherwood Medical shown in Table 2. Only reactions with a strength of 2 Industries, Deland, Fla.). One drop (0.006 ml) of the or more (at least 25% of the antigen agglutinated) heated antigen or 2 drops (0.012 ml) of the live were considered positive (Fig. 3). Misleading results antigen was added to each drop of antiserum and were obtained when the reactions were interpreted control and quickly mixed with a single wooden after more than 1 min. Agglutinations in two antiapplicator stick to form a uniform suspension. The sera (P2), three antisera (P3), or more than three entire plate was rocked gently for 1 min and then antisera (P > 3) were recorded. Agglutinations in f

,

f

VOL. 5, 1977


three pairs of antisera (7 and 8, 9 and 10, 5 and 16) were so commonly noted that such reactions were recorded separately. Evaluation of pooled antisera. Two hundred sixty-nine P. aeruginosa were typed by using both the three antisera pools and the 17 monovalent antisera prepared as described above. Positive reactions were recorded by listing the results in the pooled antisera, followed by the results in the monovalent antisera. For example, "A:1" means that agglutination occurred in pool A and monovalent 1, whereas "A,C:9,10" means that agglutination occurred in pools A and C, and in monovalent 9 and 10. Three types of reactions (I to III) were noted (see Table 6). Reproducibility of serological procedures. Serological typing of 125 P. aeruginosa was repeated after cultures had been stored for at least 2 months in TSB/100. Each organism was selected and coded by a person other than the one doing the serotyping to prevent possible bias. The code was broken only after each isolate had been serotyped the second time. Serological typing of P. fluorescens and P. putida. Since P. fluorescens and P. putida are commonly confused with P. aeruginosa because of their ability to produce the fluorescent pigment, pyoverdin (fluorescein), we attempted to serotype 19 isolates of P. putida and 15 of P. fluorescens.

RESULTS Comparison of heated and live antigens. Table 3 shows the results obtained when 725 P. aeruginosa were typed with both live and heated antigens. With the heated antigen, 685 (94.5%) of the isolates were typable, whereas 676 (93.2%) were typable with the live antigen. Combining both techniques, that is, by using the live antigen and then the heated antigen to TABLE 2. Notation of slide agglutination reactions Notation

Degree of agglutination

0 1 2 3 4

No agglutination Less than 25% of antigen agglutinated 25-50% of antigen agglutinated 50-75% of antigen agglutinated More than 75% of antigen agglutinated

UIG.3.

643

type those which were not typable with the live antigen, 698 (96.3%) of the isolates could be typed. The same number were typable when the heated antigen was used and then the live antigen for those isolates which could not be typed with the heated antigen. Only 27 (3.7%) of the isolates were not typable when both antigens were used. Since little difference was noted between the results obtained with the heated and live antigens, all results reported in the remainder of this paper are based on live antigens, which are more suited to routine use in clinical laboratories. Distribution of 0 antigen groups. Important epidemiological information is obtained by determining the distribution of the 0 antigens in a hospital or other area. For example, if 25% of the isolates from a particular population belong to serotype A, and only 2% belong to serotype B, the findings of two isolates with serotype B would be epidemiologically more significant than the finding of two isolates of serotype A. The distribution of the 0 antigen groups in each of the six hospitals studied is shown in Table 4. These percentages altered only slightly when heated antigen was used. Although the distribution of the 0 antigens varied from hospital to hospital, serogroup 6 was the most common in each of the six hospitals. Serogroups 1, 4, 6, 9, and 11 accounted for more than 61% of all isolates. Over 19% of the isolates agglutinated in two or more antisera. Three combinations (7 and 8, 9 and 10, 5 and 16) accounted for most of these multiple reactions. No isolates were found to be serogroup 7, 8, 13, 15, or 17. Only 5.2% of all isolates did not agglutinate in any of the antisera when the live antigen was used. With the heated antigen, only 4% could not be typed with this antisera. Use of antisera pools. The ingredients of each ofthe three antisera pools were selected on the basis of the cross-reactions of the antisera with the standard antigenic strains. The crossreactions observed are shown in Table 5. When

Typical agglutination reactions (left to right) 4+, 3+, 2+, I +, O. FIG. 3. Typical agglutination reactions (left to right) 4 +, 3 +, 2 +, 1 +, 0.

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J. CLIN. MICROBIOL.

TABLE 3. Comparison of agglutinations with heated and live antigens for 725 strains of P. aeruginosa Live anti-

T

617,8

Heated antigen: | 1 1 | 16 1 9 15 16 1 9 10 111 112J13J 0 0 0 0 0 0 0 0 0

gen 1 [ 2 j 4 5 1 61 b 0 0 0 0 0 0 2 0 10 0 0 0 0 0 0 0 0 0 3 0 0 23 0 0 0 0 0 0 0 0 4 0 0 0 57 0 0 0 0 0 0 0 5 0 0 0 0 14 0 0 0 0 0 0 6 0 0 0 0 0 152 0 0 0 0 0 7,8 0 0 0 0 0 0 31 0 0 0 0 9 0 0 0 0 0 0 0 36 0 0 0 10 0 0 0 0 1 0 0 0 22 0 0 11 0 0 0 0 0 0 0 0 0 125 0 12 0 0 0 0 0 0 0 0 0 0 27 13 0 0 0 0 0 0 0 0 0 0 0 14 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0 0 0 0 0 0 0 0 0 0 0 16 0 0 0 0 0 0 17 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 9,10 0 0 1 0 0 0

oI

23

0 0 0 0 0 0 0 0 0 0oI 1 0 0 01 0 0

5,16 0 0 0 0 2 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 2 P2 0 1 0 0 0 0 0 0 1 0 0 P3 0 o P>3 0 0 0 0 1 0 1 0 0 0 0 0 2 o 3 1 1 0 0 NA Total 61 10 57 21 11601 311 42 2311281 27 a NA, No agglutination. bNumber of strains that agglutinated (2+ or greater) antigens.

0l

21

possible, cross-reacting strains were placed in the same pool. The concentration of each of the five antisera in the three pools was the same as the monovalent antisera. Pooled antisera have been used previously to serotype P. aeruginosa (13, 31), and we believe their use can reduce the time required to serotype this organism. This is especially true if the antisera are dispensed individually. We noted three reaction types (I to III) when using pooled antisera and have listed them in Table 6. Two hundred and fortynine of 269 (92.6%) of the isolates tested agglutinated in the pool antisera and in one of the monovalent antisera represented in that pool. Only 19 isolates agglutinated in pooled antisera but not in the monovlaent. Although antisera 16 and 17 were not in any of the three pools, isolates of 0 antigen group 16 most often agglu-

1

1

0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1

9,1015,16 0

0 0 0 1 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 00

2. 0 8

0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

0 0

0 0

Ojl 0

0 2

0

0

3 0 0 0 0 12

P21 0 0 0 0 0 01 0 0 0 11 0 1 0 .0

0

0 0 0 0 0 0 2 0 0 0 0 0 0 0 0

00

0 0 0 0 0 0

01 1

3 1

oI

0 0

9 01 0 0 0

0

0

0 0 0

3

281 31

0

0 0 0 1 0 0 0 0 1 0 0 0 0 0 00

0 0 0 0 0

0

0 18

0 0 0

11 0 0

N T P3IP>3JNAaLoa 61 0 0 0 10 0 0 0

0

1 0

1 4

3 3

0 1

4

1

13

0 0 0 0 0

00 0 1 1 2 5

24

1 0 4 2 0 2 1 0

57 20 156 31 41 25 125

27

0 0

1 1

01 0 0

2 8

0

0 0

2 1 0 27 40

20 36

10 14

7 49 725

in the antisera when tested as heated and live

tinated in pool B. This was expected, since serotype 16 cross-reacts with serotype 5, which is in pool B. Reproducibility of serological procedure. The serotyping of 125 isolates was repeated after the organisms had been stored for at least 2 months. A comparison of the results of the two runs shows that 115 of 125 (92%) isolates were typed the same both times. The 10 isolates that differed in their serotype are shown in Table 7. The numbers in parentheses represent the strength of the reaction as previously defined. Each of the changes involved either the loss of or addition of one or more positive reactions of strength 3 or less. One isolate, number 246, was found to be serotype 1 on the first run and serotype 7,8 on the second. Since each of these reactions had a strength of 4, we sus-

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TABLE 4. Percent distribution of 0-groups in six different hospitals % Isolates in 0-group in hospital no: 0-groupa 0-groupa 1 2 3 4 5 6 (94)b (53) (103) (38) (45) (92) 1 5 9 14 5 7 21 2 4 4 0 3 0 0 3 2 2 5 7 0 5 4 9 8 7 7 23 1 5 2 2 6 0 2 9 6 26 30 28 32 27 24 7,8 4 2 4 11 2 12 9 7 9 7 4 5 8 10 3 0 4 0 4 1 11 14 6 10 11 9 0 12 4 0 0 0 0 0 13 0 0 0 0 0 0 14 0 0 1 0 0 0 15 0 0 0 0 0 0 16 1 0 1 0 4 0 17 0 0 0 0 0 0 4 6 4 9,10 0 0 3 7 9 5 7 7 3 5,16 P2 0 2 1 3 2 2 P3 2 8 1 3 0 2 P>3 3 0 0 3 0 0 NAd 4 0 5 3 9 9

~~~~~~~~~Totalc (425) 11.5 1.6 3.8 7.8 4.2 27.1 5.9

6.8 2.4 8.2 0.5 0.0

0.2 0.0 0.9 0.0 3.3 5.9 1.4 2.4 0.9 5.2

9 and 10, and one agglutinated in antiserum 4. The remaining 12 strains of P. putida did not agglutinate in any of the 17 monovalent antisera. Application of serological typing for the study of epidemiology and hospital-acquired infections. Between January 1974 and September 1976, 26 outbreaks of P. aeruginosa were studied with our serotyping procedures. Twenty-four of these outbreaks occurred in hospitals; the other two outbreaks were associated with contaminated swimming pools or whirlpools. We identified epidemic strains of P. aeruginosa and further divided isolates with identical antibiograms. Table 8 contains three examples of serotyping used to trace infections TABLE 5. Cross-reactions observed using commercial antisera (final dilution of 1:10) and standard antigen strains Antise-

Degree of agglutination a with standard antigenic strain (live antigen):

rum

lb21 3 4I 5 6 7 8 9 10 11 12 13 14 15 16

2

Based on live antigen. Number of isolates from each hospital. c Percent of all 425 isolates that belong to 0-

4

4

4

5

group.

agglutination.

--

1 _

2

3

b

pected an error in our coding system and studied this organism further. The original stock culture was streaked for isolation, and two colony types were observed. The 0 antigen of each colony type was determined, and we found that the larger, more pigmented colonies were serotype 1, and the smaller, less pigmented colonies were serotype 7,8. We concluded that the original stock culture contained two distinct serotypes and that a change in the 0 antigen had not occurred. Serological typing of fluorescent pseudomonads other than P. aeruginosa. The fluorescent pseudomonads, P. aeruginosa, P. fluorescens, and P. putida, might easily be confused by the clinical microbiologist who performs only a limited number of tests for identification. Only 10 of 15 ATCC strains of P. fluorescens grew on MH agar at 36°C. Live antigens prepared from five of these isolates agglutinated in antisera 13 and 14, a combination not found with P. aeruginosa; one isolate agglutinated in antisera 7 and 8; and four isolates did not agglutinate in any of the 17 antisera. Sixteen of 19 ATCC strains of P. putida grew on MH agar at 36°C. Two isolates agglutinated in antisera 13 and 14, one agglutinated in antisera

31

1

a

d NA, No

645

4

2

6 7

3 3

8

2 4

9

3

10

1

11

1 3

4

12

4

13

3 1

14

1 4

15

1 4

16

3

1

We did not have the standard strain for 0-group 17. bStandard antigenic strains (live antigens).

a

TABLE 6. Comparison of agglutination reactions in pools with individual sera (live antigen) Reaction No. Description type

I II III

Agglutination in pools same as in individual sera Agglutination in pool, but not in individual sera comprising pool Agglutination in individual sera but not in pool

249 19 1

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TABLE 7. Changes in serotype of 10 isolates after storage for at least 2 months

J. CLIN. MICROBIOL.

single, antibiotic-resistant strain, and this assumption was supported when we found all isolates to be serotype 11. Antibiotic-resistant Serogroup of isolate Isolate no. strains may be differentiated further by seroFirst run Second run typing as is shown for hospital C. 2 NA a 6(3) b DISCUSSION 14A NA 6(2),10(2) 14B 6(4) 6(2),10(2) P. aeruginosa has become a leading cause of 23 9(3) 9(4),10(2) hospital-acquired infections because the num40 5(3) 5(3),10(2) ber of susceptible patients in hospitals has in84B 9(4),10(3) 1(3),9(4),10(4) creased. Prolonged treatment with immuno85 9(3),10(2) 1(3),9(4),10(4) suppressive agents, antimetabolites, antibiot90 5(3) 5(4),16(3) ics, and radiation therapy have decreased the 93 5(3),10(2) NA ability of hospital patients, especially those 469 9(3),10(2) 9(3) with severe burns or malignant diseases, to NA, No agglutination. combat this organism effectively. Although our b Numbers in parentheses indicate degree of agknowledge of the ecology and epidemiology of glutination (Table 2). these infections has increased, control of pseudomonas infections depends on the availability TABLE 8. Use of serotyping in three different of accurate typing methods. epidemiological situations In addition to serological typing, other epideOutbreak miological typing procedures have been used to or hospiSource Resistanta to 0-group differentiate strains ofP. aeruginosa. The simtal code plest of these include the use of gross phenoTracheal aspi11 A typic properties (5) and the use of antibiotic rate Tracheal aspiCB Polyagglu- susceptibility data (5, 7). Although these methods are rarely specific enough to distinguish rate tinating between strains unless unusual characteristics Tracheal aspi11 rate are present, they may be the only typing methTracheal aspi10 ods available in smaller hospitals. Pyocin prorate duction (10, 14, 32), pyocin sensitivity (10), and Tracheal aspi11 bacteriophage typing (3, 20, 26, 29) have also rate been used to differentiate strains of this orgaTracheal aspi11 nism. These methods provide more sensitive rate differentiation, since they depend on the finer Tracheal aspi4 genetic characteristics of the organism. Howrate ever, they are also technically more difficult to B 11 Urine CB,GM perform, and therefore have been reserved pri11 Urine CB,GM marily for reference and research. A combina11 Urine CB,GM tion of two or more methods is commonly used 11 Urine CB,GM to study outbreaks. For example, serological 11 Urine CB,GM followed by either pyocin or bacteriotyping, 11 Urine CB,GM phage typing, has been suggested (9, 17). 11 Urine CB,GM The serotype of P. aeruginosa is generally considered as a stable characteristic, although 6 C Urine CB,GM investigators have reported changes in it after 9 Urine CB,GM 6 Urine CB,GM repeated subculture, storage at room tempera6 Urine CB,GM ture, or exposure to bacteriophage (4, 6, 18, 21, Urine CB,GM 9,10 22). The organism may undergo dissociation 6 Urine CB,GM and result in colonies with different character6 Urine CB,GM istics, but the serotype of the various colonial a Resistance is listed only for carbenicillin (CB) forms rarely changes (13, 28). Liu (21) examined 100 colonies that had been exposed to and gentamicin (GM). -, Not resistant to either. phages, and found that 20% had changes in their serotype. However, he also observed no caused by P. aeruginosa. Outbreak A was changes in somatic antigens associated with thought to be a single-strain outbreak; how- the loss of a prophages. Phage-induced seroconever, four different serotypes were identified. version has been demonstrated in rats infected Outbreak B was thought to be caused by a with a single strain of pseudomonas (4), but

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similar seroconversion has not been seen in human infections. Until recently, serological typing of P. aeruginosa was not usually done in the United States. Although numerous schemes have been proposed (6, 12, 15, 16, 19, 22, 24, 27, 30), the lack of a well-defined set of standard strains and commercial antiserum has made serotyping of this organism impractical. The International Subcommittee on Pseudomonadaceae and Related Organisms has proposed a set of standard antigenic strains for preparing typing sera. Commercially prepared typing serum is now available (Difco, Pasteur Institute), and we suggest that serological typing of P. aeruginosa be the first step in any typing procedure for this organism. More definitive typing procedures, such as pyocin or bacteriophage typing, can then be used as necessary to further characterize the organisms within any serogroup. We designed the slide agglutination tests in this study to be as simple and practical as possible, so that they can even be used in most small hospital laboratories. We developed a typing procedure that is reproducible, requires materials readily available in most microbiology laboratories, and is simple to perform. The reproducibility of the test depends on careful standardization of the test conditions. The inoculum for both the live and heated antigens is prepared as described for sensitivity testing (1). We chose this inoculum so that the serotype could be determined directly from growth taken from a 24-h susceptibility plate. The amount of antigen and antiserum used and the timing of the agglutination test have been adjusted to obtain reproducible results. Our comparison of both live and heated antigens shows that a live antigen can be used to serotype P. aeruginosa, an observation also made by Lanyi (19). The combination of both antigens allowed us to type over 96% of our isolates, comparing favorably with a range of 62 to 100% noted by investigators using different serological schemes. Those able to type all of their isolates used both somatic and flagellar antiserum (30). Since the antiserum we used was prepared by immunizing rabbits with heated cell preparations, it should be free of agglutinating antibodies to thermolabile antigens. Some typing schemes have made use of thermolabile antigens (13, 30). Unlike the flagellar antigens of Salmonella, the heatlabile components ofP. aeruginosa may include surface antigens other than the single polar flagellum. These antigens, whatever their source, have been used to supplement 0-grouping. However, since researchers have had great difficulty in making specific flagellar anti-

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serum, these typing schemes have not been widely accepted. Less than 20% of the isolates that we typed with live antigens and unabsorbed commercial antisera agglutinated in two or more antisera. Three pairs of antisera (7 and 8, 9 and 10, 5 and 16) accounted for most (76%) of the multiple reactions. Although absorbed antisera may be used to reduce the number of cross-reactions (8), absorption of antisera is not a practical procedure for clinical microbiology laboratories, and our results show that absorption of commercial antiserum is unnecessary. Since slide agglutination tests are less specific than tube agglutination tests, low levels of crossreacting antibodies do not interfere with slide agglutination. Cross-reactions obtained when typing P. aeruginosa with live antigens must be considered when differentiating between two or more isolates. For example, if isolate A agglutinated in antisera 9 (3) and 10 (2), and isolate B agglutinated in antisera 9 (3) and 10 (1), in which the numbers in parentheses indicate the strength of the reaction, isolate A would be considered type 9,10 and isolate B, type 9. If isolates A and B were serotyped again, both isolates might be either serotype 9 or 9,10 depending on the interpretation given to the weak reaction in antiserum 10. Such discrepancies are part of any typing procedure used to answer specific epidemiological questions. We have not found isolates of P. aeruginosa that agglutinate only in antiserum 7 or only in antiserum 8; however, isolates that agglutinate in both 7 and 8 are quite common. We suspect that the combination of 7,8 represents a single serotype, and prefer to consider the 7,8 combination a single serotype until specific antiserum is available to distinguish between the two types. Pooled antisera can shorten the time required to serotype an isolate, especially if the antiserum is dispensed without the use of a mechanical device. Changing the monovalent antiserum in each pool might result in more efficient use of pooled antisera. Since serotypes 12, 13, 14, and 15 are rarely found, antisera representing these serotypes might be omitted from the pools. Alternatively, a pool containing those serotypes rarely found could be used along with the monovalent antisera. Isolates could first be examined using monovalent antisera to the most common types found among hospital patients, and then be screened with one or two pools composed of those types less commonly found. This would reduce the number of monovalent antisera and the number of pools needed to type an isolate. If certain sero-

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types were more prevalent in a particular location, appropriate substitutions in the pools could be made. Our recommendations for serological typing of P. aeruginosa with commercial antisera are as follows. For routine serological typing, which includes the study of intrahospital strains, use a live antigen taken from a 24-h sensitivity plate. We have described a simplified procedure which can be used and have shown that it compares very favorably with a more difficult, time-consuming procedure requiring a heated antigen. The additional media, equipment, and personnel time do not warrant the routine use of the heated antigen; nor do our results show that its use is necessary to obtain information that is epidemiologically useful. Similarly, absorbed antisera are quite useful in a reference laboratory, but are not required in a clinical laboratory. The practical serological typing procedures that we have described are applicable to the investigation of infections caused by P. aeruginosa. Studies that will evaluate the practical application of these procedures in the investigation of more than 25 outbreaks are in progress, and comparison of serological typing with other typing procedures will be published soon.

J. CLIN. MICROBIOL.

7. Dayton, S. L., D. Blasi, D. D. Chipps, and R. F. Smith. 1974. Epidemiological tracing of Pseudomonas aeruginosa: antibiogram and serotyping. Appl. Microbiol. 27:1167-1169. 8. Duncan, N. H., N. A. Hilton, J. L. Penner, and I. B. R. Duncan. 1976. Preparation of typing antisera specific for 0 antigens of Pseudomonas aeruginosa. J. Clin. Microbiol. 4:124-128. 9. Edmonds, P., R. R. Suskind, B. G. MacMillan, and I. A. Holder. 1972. Epidemiology of Pseudomonas aeruginosa in a burn hospital: evaluation of serological, bacteriophage, and pyocin typing methods. Appl. Microbiol. 24:213-218. 10. Farmer, J. J., III, and L. G. Herman. 1969. Epidemiological fingerprinting ofPseudomonas aeruginosa by production of and sensitivity to pyocin and bacteriophage. Appl. Microbiol. 18:760-765. 11. Fanner, J. J., III, F. W. Hickman, and J. V. Sikes. 1975. Automation of Salmonella typhi phage typing. Lancet ii:787-790. 12. Fisher, M. W., H. B. Devlin, and F. J. Gnabasik. 1969. New immunotype schema for Pseudomonas aeruginosa based on protective antigens. J. Bacteriol. 98:835-836. 13. Gaby, W. L. 1946. A study of the dissociative behavior of Pseudomonas aeruginosa. J. Bacteriol. 51:217-234. 14. Gillies, R. R., and J. R. W. Govan. 1966. Typing of Pseudomonas pyocyanea by pyocyine production. J. Pathol. Bacteriol. 91:339-345. 15. Habs, I. 1957. Untersuchungen uber die 0-antigine von Pseudomonas aeruginosa. Z. Hyg. Infektionskr. 144:218-228. 16. Homma, J. Y., H. Shionoya, H. Yamada, and Y. Kawabe. 1971. Production of antibody against Pseudomonas aeruginosa and its serological typing. Jpn. J. Exp. Med. 41:89-94. ACKNOWLEDGMENTS 17. Jones, L. F., J. P. Zakanycz, E. T. Thomas, and J. J. This research was performed as part of the Laboratory Farmer, III. 1974. Pyocin typing of Pseudomonas Practice Training Program, School of Public Health, Uniaeruginosa: a simplified method. Appl. Microbiol. versity of North Carolina, in cooperation with the Bureau of 27:400-406. Laboratories, Center for Disease Control, Atlanta, Ga., and 18. Kawaharajo, K. 1971. Changes in serotype ofPseudomwas supported by a General Purpose Traineeship and a onas aeruginosa. Jpn. J. Exp. Med. 43:225-226. Public Health Special Purpose Traineeship, grant 19. Lanyi, B. 1966. Serological properties of Pseudomonas A04AH00008, from the Division of Allied Health Manaeruginosa. I. Group specific somatic antigens. Acta power, Bureau of Health Resource Development, Health Microbiol. Acad. Sci. Hung. 13:295-318. Resource Administration, Public Health Service. 20. Lindberg, R. B., and R. L. Latta. 1974. Phage typing of We thank L. R. McCarthy, North Carolina Memorial Pseudomonas aeruginosa: clinical and epidemiologic Hospital, and T. J. Bacher, Freeport Memorial Hospital, for considerations. J. Infect. Dis. 130:S33-S42. isolates used in this study. 21. Liu, P. V. 1969. Changes in somatic antigens of Pseudomonas aeruginosa induced by bacteriophages. J. LITERATURE CITED Infect. Dis. 119:237-246. 1. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. 22. Mayr-Harting, A. 1948. The serology of Pseudomonas Turck. 1966. Antibiotic susceptibility testing by a pyocanea. J. Gen. Microbiol. 2:31-39. standardized single disk method. Am. J. Clin. Pa- 23. Meader, P. D., G. H. Robinson, and V. Leonard. 1925. Pyorubin, a red water soluble pigment characteristic thol. 45:493-496. 2. Bennett, J. V. 1974. Nosocomial infections due to Pseuof Bacillus pyocyaneus. Am. J. Hyg. 5:682-708. 24. Meitert, T. 1964. Contribution a l'etude de la structure domonas. J. Infect. Dis. 130:54-57. 3. Bergan, T. 1972. A new bacteriophage typing set for antig6nique des B. pyocyaniques (Pseudomonas Pseudomonas aeruginosa. 1. Selection procedure. aeruginosa). II. Individualisation des groupes serologiques au moyen des antigenes 0. Arch. Roum. PaActa Pathol. Microbiol. Scand. Sect. B 80:177-188. 4. Bergan, T., and T. Midtvedt. 1975. Epidemiological thol. Exp. Microbiol. 23:679-688. markers for Pseudomonas aeruginosa. 4. Change of 25. Meitert, T., and E. Mietert. Utilisation combinee du serotypage et de la lysotypie des souches de Pseudom0-antigen and phage sensitivity after phage infection in vitro and in vivo ofPseudomonas aeruginosa. Acta onas aeruginosa en vue d'approfondir les investigations epidemiologiques. Arch. Roum. Pathol. Exp. Pathol. Microbiol. Scand. (B) 83:1-9. 5. Bobo, R. A., E. J. Newton, L. F. Jones, L. H. Farmer, Microbiol. 25:427-434. and J. J. Farmer III. 1973. Nursery outbreak of Pseu- 26. Postic, B., and M. Finland. 1961. Observations on bacdomonas aeruginosa: epidemiological conclusions teriophage typing of Pseudomonas aeruginosa. J. from five different typing methods. Appl. Microbiol. Clin. Invest. 40:2064-2075. 27. Sandvik, 0. 1960. Serological comparison between 25:414-420. strains of Pseudomonas aeruginosa from human and 6. Christie, R. 1948. Observations on the biochemical and animal sources. Acta Pathol. 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28. Shionoya, H., and J. Y. Homma. 1968. Dissociation in Pseudomonas aeruginosa. Jpn. J. Exp. Med. 38:8194. 29. Sutter, R. L., V. Hurst, and J. Fennell. 1965. A standardized system for phage typing Pseudomonas aeruginosa. Health Lab. Sci. 2:7-16. 30. Verder, E., and J. Evans. 1961. A proposed antigenic schema for the identification of strains ofPseudomo-

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