Vol. 8, No. 1

JOURNAL OF CLINICAL MICROBIOLOGY, July 1978, p. 73-3

0095-1137/78/0008-0073$02.00/0 Copyright © 1978 American Society for Microbiology

Printed in U.S.A.

Biotyping of Serratia marcescens and Its Use in Epidemiological Studies PATRICK A. D. GRIMONTt* AND FRANCINE GRIMONTt Laboratoire de Bactériologie, Université de Bordeaux II, and Laboratoire de Bactériologie, Hôpital Pellegrin, Bordeaux, France Received for publication 23 May 1978

A Serratia marcescens biotyping system using eight carbon sources (benzoate, DL-carnùtùne, m-erythritol, 3-hydroxybenzoate, 4-hydroxybenzoate, lactose, Dquinate, and trigonelline), a tetrathionate reduction test, production of prodigiosin, and horse blood hemolysis was derived from a recent numerical taxonomic study (Grimont et al., J. Gen. Microbiol. 98:39-66, 1977). A total of 98.6% of 2,210 isolates from various sources could be assigned to 1 of 19 biotypes. Distribution and spread of 1,088 S. marcescens isolates throughout 13 clinical departments of Pellegrin Hospital (Bordeaux, France) were studied from 1968 through 1975. Except for one that colonized the intestinal tract of newborns, the six pigmented biotypes were seldom isolated. Each of the 13 nonpigmented biotypes showed a particular pattern of distribution and spread. The usefulness of S. marcescens biotyping was shown by relating several isolates recovered from patients and their inanimate environment and by pointing out the possible existence of infections or colonizations by two unrelated biotypes. S. marcescens strains isolated from the natural environment (water) are usually pigmented, and their biotypes are uncommon in hospitals. Biotyping can, therefore, be of help in epidemiological and ecological surveys. cens, and more H antigens have recently been proposed (17, 28). Although antisera are now commercially available in the U.S.A. (Lee Laboratories, Grayson, Ga.), the technique of agglutination of S. marcescens can be tedious because of cross-reactions (19, 28). What else can a hospital laboratory use? In despair, some clinical bacteriologists have attempted to use the API 20E (Analytab Products, Inc., New York, N.Y.) numerical profile (called by the manufacturer "biotype") as an epidemiological tool (21, 22). Unfortunately, although API 20E is a satisfactory system for species identification, reproducibility testing of the numerical profile for enterics showed that only 56% of 110 strains gave identical "biotypes" when repeated (2). The most uncertain readings of S. marcescens biochemical characters (with API 20E) are for inositol fermentation and urea hydrolysis (22). In a recent numnerical taxonomic study (12), the S. marcescens phenon was shown to be composed of several subphenons, each characterized by a specific pattern of carbon source utilization, tetrathionate reduction, and red pigmentation. Moreover, agar gel electrophoresis of Serratia proteinases showed that most subphenons could also be differentiated by their proteinase pattern (11).

Many hospital bacteriologists, epidemiologists, and clinicians are now faced with nosocomial infections caused by members of the genus Serratia (10, 23, 32). Although we consider this genus to be composed of four species [S. marcescens, S. liquefaciens, S. plymuthica, and S. marinorubra (rubidaea) (12)], only S. marcescens (the single Serratia species recognized by Bergey's Manual [1]) is involved in hospital outbreaks (10, 15). Methods for selective isolation and identification of Serratia have recently been improved (12, 24), and there is now a need for an easy, unambiguous and reproducible typing method. Phage typing (9, 13) and typing by production of (7) or susceptibility to bacteriocins (6, 30) are very sensitive methods. They give excellent results in local studies (26, 27), but they have not yet been able to give an overall view of the distribution of S. marcescens strains among different wards, hospitals, towns, or countries because any single change in one reaction is likely to give a different pattern number (6). Serotyping (3, 4) might provide this general view of the worldwide distribution and dispersion of Serratia strains. There are 15 somatic (O) and 13 flagellar (H) antigens (4) known in S. marcest Present address: Service des Entérobactéries, Institut Pasteur, 75724 Paris, Cedex 15, France.

73

74

GRIMONT AND GRIMONT

J. CLIN. MICROBIOL.

Since July 1974, we have been using these subphenon characteristics to characterize nosocomial strains isolated in Pellegrin Hospital, Bordeaux, France, and the original subphenons (12) have evolved into a biotyping system. The purpose of this article is to describe this new biotyping system for S. marcescens and to show its general utility in (i) splitting a large collection of isolates into a number of types, (ii) providing an overall view of nosocomial S. marcescens infections in a large general hospital during 8 years, (iii) relating environmental intrahospital strains and clinical strains, and (iv) providing a means to compare nosocomial strains with strains occurring in the natural environnent.

MATERIALS AND METHODS Bacterial strains. A total of 2,210 S. marcescens isolates were studied. Origins of these isolates are summarized in Table 1. Strains from the hospital environment were isolated on caprylate-thallous selective medium (24). Carbon source utilization tests. The M70 inorganic mixture of Véron (31) was used for carbon source utilization tests. The trace elements solution used in this medium is composed of: distilled water, 1,000 ml; H3PO4, 1.96 g; FeSO4 7 H20, 0.056 g; ZnSO4 7 H20, 0.029 g; MnSO4 4 H20, 0.022 g; CuS04*5 H20, 0.002 g; Co(NO3)2 6 H20, 0.003 g; and H3B03, 0.007 g. This solution was kept unsterilized at 4°C. The M70 medium is composed of two solutions-A and B-autoclaved separately and mixed aseptically to give 1 liter of the final medium. Solution A contained: CaCI2 2 H20, 0.015 g; MgSO47 H20, 0.123 g; KH2PO4, 0.680 g; K2HPO4, 2.610 g; trace elements solution, 10 ml; and distilled water, up to 500 ml. The pH was adjusted to 7.2 with NaOH by using a pH meter. This solution was autoclaved at 1100C (or 1210C) for 20 min. Solution B contained: NaCl, 7 g; (NH4)2SO4, 1 g; agar (Difco), 15 g; distilled water, 500 ml. The pH was adjusted to 7.2, and this agar base was autoclaved TABLE 1. Source of S. marcescens isolates Source

No. ofisolates

Pellegrin Hospital, Bordeaux, France ... 1,375 64 3 other hospitals in Bordeaux ..... ..... 285 ....... 6 other hospitals in France ...... 96 J. Van de Pitte, Leuven, Belgium ....... 21 C. Kressmann, Bonn, Germany ..... .... 35 .. S. Kominos, Pittsburg, Pa 18 ....... J. Brisou, Poitiers, France ...... 125 L. Le Minor, Paris, France ...... ....... B. Davis, Center for Disease Control, At120 lanta, Ga ............................ 39 Culture collections .......... .......... 9 Other individuals ........... .......... 23 Water isolates (from southwest France) Total ..............................

2,210

under the same conditions as for solution A. After autoclaving, solutions A and B were mixed aseptically, and 5 ml of a 20% carbon source solution were added. Carbon source solutions contained 1 g (in 5 ml of distilled water) of one of the following: adonitol, benzoic acid (Fluka, Buchs, Switzerland), DL-carnitine (General Biochemicals, Chagrin Falls, Ohio), mesoerythritol, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, D-quinic acid, D-sorbitol (Fluka), trigonelline (K & K Laboratories, Inc., Plainview, N.J.), or 2 g of Larabinose, lactose, or L-rhamnose (Fluka). The pH of these solutions was adjusted to 7.2 with NaOH, and these solutions were filtered through nitrocellulose filters (Millipore Corp., Bedford, Mass.) (0.45 jim) or heated at 800C for 20 min. An 80-ml amount of M70 medium with carbon source was poured into sterile plastic petri dishes (14-cm diameter). The dishes were stored at 4°C until used. Plates of M70 medium with carbon source were dried before use by placing them in a 37°C incubator for a few hours. Tetrathionate reductase test. The liquid medium of Le Minor et al. (16) contained: K2S406, 5 g; bromothymol blue 0.2% (wt/vol) aqueous solution, 25 ml; peptone water (peptone [Difco]), 10 g; NaCl, 5 g; and distilled water, up to 1 liter (pH 7.4). This medium was filter sterilized and dispensed in wells of sterile microculture plates (0.1 ml per well). Horse blood agar. Mueller-Hinton agar (Difco) supplemented with 5% (vol/vol) defibrinated, sterile horse blood was dispensed in petri dishes (50 ml per 14-cm diameter petri dish). Peptone-glycerol agar. Peptone-glycerol agar contained: peptone (Difco), 5 g; glycerol, 10 ml; agar (Difco), 20 g; and distilled water, 1 liter. This medium enhances production of prodigiosin (35). Inoculation methods and reading. We use the Denley automatic multipoint inoculator (Denley Instruments Ltd., Bolney, Sussex, England), modified to inoculate, simultaneously, 48 cultures on petri dishes (14-cm diameter). The inoculum consisted of 24-h-old Trypticase soy broth (Baltimore Biological Laboratory, Cockeysville, Md.) cultures dispensed in 48 wells (out of 96) of a microculture plate (ca. 0.1 ml/well). About 2 x 107 bacterial cells per drop (0.02 ml) were applied onto the media. Media were inoculated in this order: carbon source plates, horse blood agar, peptoneglycerol agar, and tetrathionate reductase medium. Ail media were incubated at 300C. Horse blood hemolysis and tetrathionate reduction (acidification) were read after 24 h. M70 media with carbon source were scored for growth every other day for up to 4 days (growth on lactose) or 14 days (all other carbon sources). Growth was considered positive when unambiguous. Pinpoint colonies were considered negative. Any doubt was resolved by restreaking the doubtful growths on fresh medium. Doubtful results with tetrathionate reductase tests were checked by inoculating the same medium in tubes (1 ml) under a layer of paraffin oil. Epidemiological study. All strains of Serratia isolated at Pellegrin Hospital, a teaching hospital with 1,250 beds, were kept in our collection, together with general information about the patients (name, source, date of isolation, and ward) from which the strains were isolated. When needed, more clinical data were obtained from the patients' charts. No attempt will be

VOL. 8, 1978

S. MARCESCENS BIOTYPING

made here at differentiating between significant infection, asymptomatic infection, or colonization of the patients, or between bacteremia and septicemia. In interpreting the results, one should keep in mind that, as for any other infrasubspecific division (serotype, phage type, etc.), several isolates that belong to a given biotype may or may not be of the same strain. RESULTS

Identification check. Utilization of adonitol, L-arabinose, D-sorbitol, and L-rhamnose was included in the biotyping system to screen for any misidentification of S. marcescens (12). Growth on adonitol and D-sorbitol occurred for 99.5 and 99.9%, respectively, of our 2,210 isolates. No strain of S. marcescens could grow on L-arabinose or L-rhamnose. The 10 isolates that failed to grow on adonitol and the 5 others that did not grow on D-sorbitol were considered to be S. marcescens strains after a complete set of identification tests was run (12). Definition of biotypes. A recent numerical taxonomic study of the genus Serratia (12) has indicated seven significant subphenons (groups): Ai, A2, A3, A4, A5, A6, and A8. To avoid confusion, we are using this nomenclature. As more strains were studied with respect to the original subphenon characteristics, the previous groups were further subdivided, and three undescribed biochemical patterns (labeled with the initials of positive reactions) were found. Table 2 gives the definition of 19 biotypes. Although lactose-positive strains occur in other biotypes, lactose-positive strains of group A8 were considered a separate biotype (A8c) since their distribution in Bordeaux and their antibiotic resistance ap-

75

peared quite different from those of lactose-negative A8a strains. Biochemical characteristics of the 19 biotypes are given in Table 3. Among 2,210 S. marcescens isolates studied, 2,179 (98.6%) could be assigned to 1 of 19 biotypes (Table 3). Six isolates (not included in Table 3) differed from the described biotypes by one character: three isolates resembled Ala, A2b, and A5, but without tetrathionate reductase; one resembled A4, but could grow on trigonelline; and one resembled A5, but could grow on erythritol. Auxotrophic isolates (i.e., those unable to grow on minimal medium) accounted for 15 isolates (0.68%). Ten isolates (0.45%) had biochemical patterns different from those presented in Table 2. The positive features of these isolates (not mentioning growth on adonitol and D-sorbitol) were: growth on carnitine and trigonelline (three isolates), growth on quinate and 4-hydroxybenzoate with positive tetrathionate reduction (three isolates) and the same pattern with red pigmentation (one isolate), growth on trigonelline (two isolates), and tetrathionate reduction as the sole positive test (one isolate). Such rare biochemical patterns are not explicitly labeled here. Hemolytic activity of S. marcesoens biotypes on horse blood agar. We observed four types of change occurring around S. marcescens colonies on horse blood agar. Type 1 hemolysis is characterized by a clear zone with fuzzy limits after 24 h of incubation. This zone is clearer near the colony. Reproducibility of this hemolytic type is very good. Type 1 hemolysis is associated with group A4 (80% of A4 strains as opposed to 0.7% of strains of other biotypes). Type 2 he-

TABLE 2. Definition of the biotypesa Biotype designations Characteristic

AI

A2

A4

A3

A6

A8

A5 a

b

a

b

a

b

c

d

Growth on: + + + + m-Erythritol + + + - + + - + Trigonelline Quinate and/or . . . . . . . . 4-hydrosyben-

a

b

+

+

+

+ +

a

b

+ +

a

b

c

+ +

+

+

+

+ +

+

+

TCT

TT

+

+

TC

zoateb

3-Hydroxybenzoate Benzoate

-

-

+

+

-

-

+

+.+

- + DL-Carnitine + + _ + Lactose + + + - - + + + Tetrathionate re+ + + + + + + + + + + + duction Red pigment + + a Only the reactions that define biotypes are given. Symbols: +, Positive at 14 days (carbon sources) or at 1 day (tetrathionate reduction); -, negative at 14 days (carbon sources) or at 1 day (tetrathionate reduction); blank, character not part of the definition of the biotype. b Quinate and 4-hydroxybenzoate are likely to be dissimilated through the same pathway in S. marcescens (P. A. D. Grimont and F. Grimont, work in progress).

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76

Clearing beless reproducible. is much molysis and progressively spreads. the colony gins under After 24 h, the clear zone is often a little larger o than the colony itself. This hemolytic zone is O8o Co80080~8 always perfectly translucent with clear-cut limits. Upon repetition of this procedure, this type o88 00082 hemolysis may sometimes be unnoticed if the is smaller than the colony. This hemolytic zone _~~~~~~~~~ zone was given by 28% of the strains of all biotypes. Type 3 hemolysis is characterized by a double zone. Around the colony there is a zone o similar to type 2 hemolysis, then a ring of intact erythrocytes and, around this ring, a poorly limited clearer zone. This aspectitsis not always noticeable in 24 h, which affects reproducibility. a Cr 088S00008 OMg. 0 Only a few pigmented strains showed this type e: 3 hemolytic pattern (10 to 16% of Ai, A2, and A6a strains). Type 4 hemolysis was given only S by some pigmented strains (5 to 6% of Ala and 0 A2a strains). It is a barely visible zone, a bit CfO Cf Q o clearer than the rest of the blood agar plate, extending around the colony. It can be observed by careful examination of blood agar plates by transillumination. Only one hemolytic type seems of interest: type 1, which can help identify o o o Oo nO OO OO biotypes A4a and A4b. 8 Distribution and spread of S. marcescens in Pellegrin Hospital from 1968 biotypes 0 . c.e z L--( CD U through 1975. To study the distribution and of S. marcescens biotypes in our hospital, 0 .f spread isolate per patient was retained in the o one only 0 ao o 8 e study, except when several biotypes were re=C .0 covered from the same patient (then one isolate oC was studied). We were e ;. of each biotype found O ~8 0080081,088 isolates. Table 4 ,D4 concerned, then, with S o gives the number of isolates per year for each of isolates among bio.t -: biotype. The 13distribution 0 clinical the in departments is very un= types : : even (Table 5). En Ga.). . Biotype A3a has been almost regularly isoG>G>E _= 0; E @ ç e O lated from Pellegrin Hospital patients in the c, 'O m O- Z w o o Internal Medicine Department since April 1972. Three patients were infected with this biotype o

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S. MARCESCENS BIOTYPING

VOL. 8, 1978

77

TABLE 4. S. marcescens biotypes isolated per year (1968 to 1975) in Pellegrin Hospital No. of isolates during following year:

Biotype

Alaa Alba A2aa A2ba A3a A3b A3c A3d A4a A5 A6aa A8a A8b A8c TCT TT

1968 O

1969 O

1970 O

1971 O

1972 0

1973 1

1974 0

1975 0

Total 1

0 0 0 0 0 0 0 20 0

0 1 0 0 1 0 0 44 0

0 0 0 0 0 0 0 23 1

0 0 0 1 0 0 1 33 0

0 0 0 4 0 0 0 41 5

0 2 0 6 2 2 0 34 7

0 0 42 13 2 2 0 68 83

1 1 31 17 14 1 18 14 37

1

a

0.1

0.1 0.4 6.7 3.8 1.7 0.5 1.7 25.5 12.2 0.1 10.5 3.1 20.3 12.3 0.5 0.5

4 73 41 19 5 19 277 133

o

o

o

o

o

o

1

o

1

4 1 0 0 0 0

4 3 0 9 0 0

5 6 0 3 0 0

14 5 7 5 0 0

9 7 9 12 0 0

7 0 38 15 0 0

23 6 76 13 1 0

48 6 91 77 4 6

114 34 221 134 5 6

25 Total Pigmented biotypes.

62

38

66

87

114

330

366

1,088

Auxotrophs

%

100

TABLE 5. S. marcescens biotypes isolated for 8 years from 13 clinical departments of Pellegrin Hospitala No. of isolates from following dept: Biotype

Alab Albb A2ab A2bb A3a A3b A3c A3d A4a A5 A6ab A8a

A8b A8c TCT TT Auxotrophs

Urol O O 2

Medi 1 O 1

0

0 13 7 2

1

0 1 il 169 16 1 40 3

160 88 4 5

1

12 28

Surg 0 O

0 0 4 1 0 1 41

35

Neph

Trau 0 O

Neur 0 O

Otor 0 0

0 1

0 0 3 3 0 0 14 3

0 0 6 5 1 6 10 4

0 0 2 0 0 0 2 13

0 0 0 0 0 0 10 9

Card 0

Reha 0

0 1

0

Derm 0 0

Neon 0 0

Obst 0 0

Burn o 0

0 0 6 0 0 0

0 0

0 0 0 0 0

0 0 3 0 1 0 0 9

0 0 7 9

0 0 1 2 0 0 8 3

4 3

0 73 0 0 0 0 0 1

0 1 1

1

0

0

0

0

0

0

o

o

o

o

o

o

11 9 4 19 0 0

21 2 4 4 0 0

15 11 8 2 0 0

6 1 5 6 1 0

6 8 0 2 0 0

3 0 28 0 0 0

4

3 0 5 5 0 0

2 0 4 1 0 0

0 0 1 1 0 0

0 0 0 0 0 0

3 0 0 0 0 1

0 2 6 0 0

501 108 113 59 51 31 Total 33 51 27 20 76 1 17 a Abbreviations: Urol, Urology; Medi, Internal Medicine; Surg, General Surgery; Trau, Traumatology; Neur,

Neurosurgery; Otor, Otorhinolaryngology; Neph, Nephrology; Card, Cardiology; Reha, Rehabilitation; Derm, Dermatology; Neon, Neonatology; Obst, Obstetrics; Burn, Burn Unit. b Pigmented biotypes.

in the trauma unit (Traumatology) (July to September 1973), three others in the Burn Unit (July, August, and October 1975), and six in Neurosurgery (May and June 1975). Other cases were scattered throughout the hospital. Biotype A3b was recovered from six cases in Internal Medicine (April, May, June 1975), five

in Neurosurgery (January to April 1975), and a few other scattered cases. On the other hand, the five cases of biotype A3c infection were not apparently related. Biotype A3d, which had been isolated once in 7 years, was involved in an outbreak in Neurosurgery (six cases from January to June 1975)

78

,~ ~

J. CLIN. MICROBIOL.

GRIMONT AND GRIMONT

and in Urology (10 cases from September to December 1975). Biotype A4a was certainly the most persistent biotype in Pellegrin Hospital. Figure 1 shows its occurrence in almost all wards from 1968 through 1975, with some clustering of cases in some wards at certain times. Biotype A4a was isolated from the following cases: 19 septicemias or bacteremias, 201 urinary tract infections or colonisations, 32 surgical wounds, 3 serious fluids, BU DE RE CA NE 0T NS

_

2). Only one isolate of this biotype had been isolated before May 1972. Then this biotype was found in different departments in our hospital, the peak year being 1974 when biotype A5 was the predominant S. marcescens biotype in Internal Medicine, Surgery, Ear-Nose-Throat (Otorhinolaryngology), and Cardiology. This

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s.

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TR

su

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1 cerebrospinal fluid, and 7 tracheal exudates (263 out of 270 known origins). Biotype A5 had a different distribution (Fig.

e'ig|

ME UR

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e

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j

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1975 1970 1972 1974 1971 1973 '68 1969 FIG. 1. Distribution of S. marcescens biotype A4a among the several clinical departments of Pellegrin Hospital from July 1968 to December 1975. Abbreviations for clinical departments: BU, Burn Unit; DE, Dermatology; RE, Rehabilitation; CA, Cardiology; NE, Nephrology; OT, Otorhinolaryngology; NS, Neurosurgery; TR, Traumatology; SU, General Surgery; ME, Internal Medicine; UR, Urology. Each symbol (-) represents a patient. BU DE RE CA NE OT NS TR

IA4AAti A

A A

T

1

A

A

LA

~

~

#

t

t

A

A&

à

T4

A&

A

A

t 4A

Su

ME UR II 1 1à

A,

1

A"

.."à

44

1975 1974 1973 1971 1972 '68 1969 1970 FIG. 2. Distribution of S. marcescens biotype A5 among the several clinical departments of Pellegrin Hospital from July 1968 to December 1975. Abbreviations for clinical departments are the same as in Fig. 1. Each symbol (A) represents a patient.

S. MARCESCENS BIOTYPING

VOL. 8, 1978

biotype has been recovered from 8 septicemias or bacteremias, 43 urinary infections, 45 surgical wounds, 8 serous fluids, and 27 tracheal-exudates (out of 148 known origins). Biotype A8a was limited to Urology in 1968 and 1969. From 1970 through 1975, biotype A8a was recovered from almost all clinical departments. No clear clustering of cases was detected on a background of quasi-regular occurrence, with the exception of 21 cases from May to November 1975 in Urology. This biotype was involved in 8 septicemias or bacteremias, 57 urinary tract infections or colonizations, and 27 wound infections and was isolated from 15 tracheal exudates (out of 117 known origins). Biotype A8b had a more limited distribution in the hospital (Fig. 3), although it was persistently isolated from 1968 to 1972 in the Traumatology and from August 1971 to October 1972 in the Ear-Nose-Throat Unit. Several cases of infections due to this biotype also occurred in Internal Medicine from mid-1974 to mid-1975. This biotype was recovered from 4 septicemias or bacteremias, 10 urinary tract infections or colonisations, 9 wound infections, and 8 tracheal exudates (out of 35 clinically documented isolates). Biotype A8c had never been isolated in Bordeaux prior to October 1971 (Fig. 4). Biotype A8c could then be recovered from almost every clinical department, but its main epidemiological feature was an explosive spread among patients in Urology, where it became the dominant S. marcescens biotype. This is also reflected in the kidney unit (Nephrology) (including the artifi-

79

cial kidney ward), which exchanges patients with Urology. Biotype A8c was involved in 20 septicemias or bacteremias, 179 urinary tract infections, 28 surgical wound infections, and 2 serious fluids (out of 237 clinically documented isolates). Biotype TCT was occasionally isolated from almost all departments with some clustering of cases, until 1975 (Fig. 5). In 1975, an outbreak of S. marcescens biotype TCT infections occurred in Urology. This biotype was recovered from 3 septicemias or bacteremias, 120 urinary tract infections or colonisations, 11 surgical wound infections, 1 serious fluid infection, and 2 tracheal exudate colonisations (out of 141 clinically documented isolates). Biotype TT, a rare one, was isolated in Urology (four isolates recovered from December 1974 to June 1975) and in Neurosurgery (one strain in December 1975). All six auxotrophic S. marcescens strains were isolated in 1975; five were from Urology and one from the Burn Unit. Obviously, each biotype in Pellegrin Hospital showed a particular pattern of diffusion and distribution. Selected clinical cases showing the usefulness ofS. marcescens biotyping. Different isolates from the same patient or from the patient's environment can be compared according to their biotype. In the infant ward, a systematic search of Serratia in feces of 24 hospitalized newborns (mostly premature) from 16-26 March 1976 showed 5 newborns with red-pigmented S. marcescens biotype A2b. A search in the ward dis-

BU DE RE CA NE OT NS TR Su

ME

a*

:

à

0

==

w

--

*-

a

a

a

Mz'

a

a *à a

a.m.M:

UR 1975 1974 '68 1969 1973 1972 1971 1970 FIG. 3. Distribution of S. marcescens biotype A8b among the several clinical departments of Pellegrin Hospital from July 1968 to December 1975. Abbreviations for clinical departments are the same as in Fig. 1. Each symbol (.) represents a patient.

80

J. CLIN. MICROBIOL.

GRIMONT AND GRIMONT

BU DE RE CA NE OT NS TR

=~~~~~~~~~~~~~o

=s

e .3.

*

=

*

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* * *s-- $:~~~~~~~~~~ O

0~~~~~~~~~~~~~~~~~~~~.3.

Su

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'00

1YT

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luid

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FIG. 4. Distribution of S. marcescens biotype A8c among the several clinical departments of Pellegrin Hospital from July 1968 to December 1975. Abbreviations for clinical departments are the same as in Fig. 1. Each symbol (O) represents a patient. Bu DE RE CA NE OT NS TR

Ise

Su

ME

00

UR

0

0 0

0

00

0

0

0

.vi iliffi '68 1969 1971 1975 1970 1974 1972 1973 FIG. 5. Distribution of S. marcescens biotype TCT among the several clinical departments of Pellegrin Hospital from July 1968 to December 1975. Abbreviations for clinical departments are the same as in Fig. 1. Each symbol (O) represents a patient. f.

lm m

Imm

mi

1111.111

§il..

m

covered this biotype in 8 out of 12 plastic bottles Hodgkin's disease, had an endotracheal tube filled with "sterile saline," which was also used connected to a mechanical respirator. S. marfor baby cleaning. Subsequently, biotype A2b cescens biotype A5 was isolated from purulent was recovered from the facial skin of six babies tracheal secretions on 4 April 1974. The same and from four hand towels. Earlier, in 1974 and day, biotype A5 was recovered from the respira1975, the same biotype had been isolated from tor, the sink in the patient's room, and a sponge two sinks and two sponges adjacent to the sinks. adjacent to the sink. On 11 April 1974, a blood No other biotype of S. marcescens and no other culture from this patient yielded the same biospecies of Serratia has yet been isolated from type A5. the newborns' environment in the Neonatology In Urology, the urinary tract of patient 2 was Ward. colonized by S. marcescens biotype A8c from 21 In Internal Medicine, patient 1, affected with January to 19 June 1975, as was shown by re-

S. MARCESCENS BIOTYPING

VOL. 8, 1978

peated urine cultures. In the same ward, patient 3, whose urine carried S. marcescens biotype A8a on 2 September 1975, developed a bacteremia with biotype A8a on 5 September. Another example of chronic urinary tract infection with S. marcescens was provided by patient 4, whose urine repeatedly yielded an auxotrophic strain from 19 March to 2 December 1975. Infection by two biotypes of S. marcescens may occur and, as a matter of fact, up to 10% of Serratia-infected patients in urology wards in 1975 were infected or colonized by two biotypes (G. Romet, M.D. thesis, University of Bordeaux II, Bordeaux, France, 1976). The following are examples of probable double infection. In Urology, two colonial types of S. marcescens were isolated from urine of patient 5 on 14 May 1974. The large (3 mm), white, opaque colonial type was nonhemolytic on horse blood agar, resistant to gentamicin (minimal inhibitory concentration [MIC] = 32 ,ug/ml), and identified as biotype A&; whereas the smaller, translucent colonial type was hemolytic on horse blood, susceptible to gentamicin (MIC = < 1 ,ug/ml), and identified as biotype A4a. In the same department, two S. marcescens biotypes were isolated from urine of patient 6 on 9 April 1975 (biotype A8c and TCT) and again on 15 April. In both instances, biotype A8c colonies were resistant to streptomycin (MIC = > 256 ug/ml) and susceptible to tobramycin (MIC = 1 gag/ml), whereas biotype TCT colonies were more susceptible to streptomycin (MIC = 16 ,ug/ml) and resistant to tobramycin (MIC = 16 gg/ml). Biotyping may also help to differentiate a new infection from a relapse. Patient 7 in Internal Medicine had an endocarditis due to S. marcescens biotype A5 (positive blood cultures from 31 January to 17 February 1976). Antibiotherapy combined with bacteriophage ingestions and injections turned the blood cultures negative, and the patient became afebrile. On 5 March and on 9 March 1976, two strains of S. marcescens were isolated, respectively, from a skin lesion and from urine while the patient was again febrile. These two strains happened to belong to biotype TCT (a biotype that infected other patients in the same ward at that time). This clinical case was reported more completely elsewhere (submitted for publication). Usefulness of biotyping S. marocens isolates in ecological studies. Biotypes of 47 strains isolated in Pellegrin Hospital (patients' environment) and of 29 strains isolated from water (natural environment) are listed, respectively, in Tables 6 and 7. It seems clear that S. marcescens biotypes recovered in the hospital correspond to the biotypes known to infect or colonize patients in that hospital. In water, 22

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TABLE 6. Biotypes of 47 S. marcescens strains isolated in the hospital (patients' environment) Source (no. of isolates) "Sterile" saline (8), sinks (2), sponges (2), hand towels (3) 2 Sponge, scouring pad A3a 4 Sinks (2), brushes (2) A3d 2 Sink (1), bathroom door knob (1) A4a 14 Toilet bowl in nurses' restroom (1), hand A5 towel (1), brushes (3), sinks (2), sponges (3), respirator (1), suction tubes (3) 6 "Bidet" (1), brush (1), sponges (2), sink A8a (1), scouring pad (1) Bench 1 A8b 1 A8c Soap holder TT 1 Sponge 1 Sink Auxotroph a Pigmented biotype (Neonatology Ward). No. 15

Biotype A2bV

TABLE 7. Biotypes of 29 S. marcescens strains isolated from water Bio-

No.

Source (no. of isolates)

Alaa

6

Alba

A2aa

2 11

Water pumping plant, drinking water (2), lake, river, well River, seawater

A3b

1 6

type

A4a

Seawater (3), river (2), well (3), spring, effluent from a distillery, lake Lake Lake (3), well, seawater, unknown water

Lake, river, spring Pigmented biotypes.

A6aa a

3

out of 29 strains belong to pigmented biotypes Ala, Alb, A2a, and A6a-biotypes which are very rare among patients in Bordeaux. Although more studies are needed for adequate comparison of S. marcescens strains from these different ecological niches, biotyping appears to be a useful tool for effecting this comparison.

DISCUSSION A reliable typing system for S. marcescens has been needed for ecological and epidemiological studies. Although our system is not more rapid than serotyping, tetrathionate reduction and hemolysis may provide presumptive evidence of biotype A4a or A4b within 24 h, and growth on meso-erythritol, trigonelline, quinate, and lactose are most often observable within 48 h. The rest of the results are available within 5 days. Observation for 14 days provides sufficient time to resolve any ambiguity in reading. By then, positive growth is a patch of culture about 5 to 7 mm large, whereas negative cultures show, at most, pinpoint colonies. However, an estimate of the results may be obtained in less than 5 days. Although reproducibility was not statisti-

82

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cally studied (e.g., with strains taken at random), more than 100 strains were biotyped twice, and in no case did the second result differ from the first. The trace element solution (see Materials and Methods) used in making the minimal medium needs to be made once a year, and solutions A and B are quickly prepared. A number of carbon source minimal media may be stored for months in a refrigerator, provided they are poured under sterile conditions and protected from desiccation. Any multi-inoculation apparatus, such as the Denley Automatic Multipoint Inoculator, Steers mechanical point inoculator (25), or any homemade inoculating device may be used to deposit about 0.02 ml each of a number of cultures on the plates. Overcrowding of plates should be avoided, and we recommend not exceeding 20 to 24 strains on a 10-cm petri dish and 50 to 55 strains on a 14-cm petri dish. This biotyping system can be applied to strains isolated in different parts of Europe and the United States and to strains isolated long ago, such as Fortineau's Erythrobacilluspyosepticus, Koch's S. indica, or S. anolium (12). About 0.7% of all studied strains could not grow on minimal medium (without growth factors), but this auxotrophic property, plus the ability to reduce tetrathionate and lyse horse blood, can also be used as an epidemiological marker. The retrospective study of S. marcescens nosocomial infections and colonizations in Pellegrin Hospital showed a striking increase in the number of isolates of S. marcescens from 1968 to 1975. Two major reasons may have contributed to this increase: (i) an actual increase in the Serratia infection and colonization rate, and (ii) an increased concern among clinicians about nosocomial infections, and, consequently, an increase in sampling asymptomatic patients. Other reasons for an apparent increase in the number of isolates (increased interest in Serratia, progress in identification, and use of a selective medium) did not play a significant role because the authors were interested in Serratia since 1968, the deoxyribonuclease test was routinely used since 1968, and the caprylate-thallous selective medium was not introduced in routine work on patient specimens during the period of study. This study of nosocomial S. marcescens showed how different the behavior of a Serratia biotype can be. Strain or biotype A4a seems to be present in an even background of infections and colonizations. Strains or biotypes such as A8c and A5 were first isolated in the hospital in 1971 and 1972 and became endemic in certain wards. Once the strains were introduced into a given hospital area, they were soon isolated from

J. CLIN. MICROBIOL.

a large number of patients, a finding similar to the Nashville outbreak due to S. marcescens (23). Other biotypes, such as A8b, were limited to a few wards, and the most limited was pigmented biotype A2b (infant ward). It is not known why pigmented biotypes can multiply in the intestinal tract of newborns, often with few pathological consequences (15, 18, 33), nor why adults are much more likely to be infected or colonized by nonpigmented biotypes (10, 34). Our findings confirm that pigmented and nonpigmented Serratia strains often differ in their biochemical properties (12). The exceptions are biotypes A2a and A2b, which resemble biotypes A3c and A3d, respectively, when pigmentation is not considered. However, there is work in progress that shows that biotypes A2a and A3c, as well as A2b and A3d, are antigenically distinct. Our biotyping system was sufficiently precise to point out the existence of infection by two unrelated biotypes or to distinguish between a relapse and an infection by another biotype. The strains isolated from water were usually pigmented, and their biotypes are uncommon in hospitals. Biotyping thus provides a way to distinguish-in the course of an epidemiological survey-between biotypes that can be found in hospitals and biotypes that are related to the natural environment. More work needs to be done in this direction, and biotyping with our method may help. Intensive surveys of existing biotypes in hospitals located in different states and countries and in different nonhospital habitats are now in progress. ACKNOWLEDGMENTS Our work, carried out at the bacteriological laboratories of Pellegrin Hospital and Université de Bordeaux Il, was supported by Contrat d'Action Thématique 75.1.440.50 from the Institut National de la Santé et de la Recherche Médicale. We acknowledge the hospitality and advice of the late H. L. C. Dulong de Rosnay. Thanks are due to M. P. Starr, A. Balows, and P. H. A. Sneath for their helpful suggestions, to the Service d'Hygiène Hospitalière (M. Serisé) for help in sampling the patients' environment, and to the many individuals who sent us strains. LITERATURE CITED 1. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Bergey's manual of determinative bacteriology, 8th ed. The Williams & Wilkins Co., Baltimore. 2. Butler, D. A., C. M. Lobregat, and T. L. Gavan. 1975. Reproducibility of the Analytab (API 20E) system. J. Clin. Microbiol. 2:322-326. 3. Davis, B. R., and J. M. Woodward. 1957. Some relationships of the somatic antigens of a group of Serratia marcescens cultures. Can. J. Microbiol. 3:591-597. 4. Edwards, P. R., and W. H. Ewing. 1972. Identification of Enterobacteriaceae, 3rd ed. Burgess Publishing Co.,

Minneapolis.

5. Ewing, W. H., B. R. Davis, and R. W. Reavia. 1959. Studies on the Serratia group. U.S. Department of

VOL. 8, 1978 Health, Education, and Welfare, Atlanta. 6. Farmer, J. J. HI. 1970. Mnemonic for reporting bacteriocin and bacteriophage types. Lancet ii:96. 7. Farmer, J. J. III. 1972. Epidemiological differentiation of Serratia marcescens: typing by bacteriocin production. Apple. Microbiol. 23:218-225. 8. Farmer, J. J. m. 1972. Epidemiological differentiation of Serratia marcescens: typing by bacteriocin sensitivity. Apple. Microbiol. 23:226-231. 9. Farmer, J. J. EU. 1975. Lysotypie de Serratia marcescens. Arch. Roum. Pathol. Exp. Microbiol. 34:189. 10. Farmer, J. J. III, B. R. Davis, F. W. Hickman, D. B. Presely, G. P. Bodey, M. Negut, and R. A. Bobo. 1976. Detection of Serratia outbreaks in hospital. Lancet ii:455-458. 11. Grimont, P. A. D., F. Grimont, and H. L C. Dulong de Rosnay. 1977. Characterization of Serratia marcescens, S. liquefaciens, S. plymuthica and S. marinorubra by electrophoresis of their proteinases. J. Gen. Microbiol. 99:301-310. 12. Grimont, P. A. D., F. Grimont, H. L. C. Dulong de Rosnay, and P. H. A. Sneath. 1977. Taxonomy of the genus Serratia. J. Gen. Microbiol. 98:39-66. 13. Hamilton, L. R., and W. J. Brown. 1972. Bacteriophage typing of clinically isolated Serratia marcescens. Appl. Microbiol. 24:899-906. 14. Hernandez Marchant, R., P. O. Rojas, and O. Arcaya. 1960. Sindrome del panal rojo. Rev. Chu. Pediatr. 31:336-339. 15. Johnson, E., and P. D. Ellner. 1974. Distribution of Serratia species in clinical specimens. Apple. Microbiol. 28:513-514. 16. Le Minor, L., M. Chippaux, F. Pichinoty, C. Coynault, and M. Piéchaud. 1970. Méthodes simples permettant de rechercher la tétrathionate réductase en cultures liquides ou sur colonies isolées. Ann. Inst. Pasteur (Paris) 119:733-737. 17. Le Minor, S., and F. Pigache. 1977. Etude antigénique de souches de Serratia marcescens isolées en France. I. Antigènes H: individualisation de six nouveaux facteurs H. Ann. Microbiol. (Paris) 128B:207-214. 18. McCormack, R. C., and C. M. Kunin. 1966. Control of a single source nursery epidemic due to Serratia marcescens. Pediatrics 37:750-755. 19. Negut, M., B. R. Davis, and J. J. Farmer II. 1975. Différenciation épidémiologique de Serratia marcescens: comparaison entre lysotypie et sérotypie. Arch. Roum. Pathol. Exp. Microbiol. 34:189. 20. Negut, M., B. R. Davis, and J. A. Washington Il. 1975. Biochemical and serological characteristics of Serratia marcescens isolated from various clinical and environmental sources. Arch. Roum. Pathol. Exp. Microbiol. 34:33-39. 21. Roemisch, E., and F. E. Kocka. 1976. Comparison of methods for differentiating among Serratia marcescens isolated from clinical specimens. Am. J. Clin. Pathol.

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66:96-100. 22. Rubin, S. J., S. Brock, M. Chamberland, and R. W. Lyons. 1976. Combined serotyping and biotyping of Serratia marcescens. J. Clin. Microbiol. 3:582-585. 23. Schaberg, D. R., R. H. Alford, R. Anderson, J. J. Farmer HI, M. A. Melly, and W. Schaffner. 1976. An outbreak of nosocomial infection due to multiply resistant Serratia marcescens: evidence of interhospital spread. J. Infect. Dis. 134:181-188. 24. Starr, M. P., P. A. D. Grimont, F. Grimont, and P. B. Starr. 1976. Caprylate-thallous agar medium for selectively isolating Serratia and its utility in the clinical laboratory. J. Clin. Microbiol. 4:270-276. 25. Steers, E., E. L Foltz, and B. S. Graves. 1959. Inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9:307-311. 26. Traub, W. H. 1972. Continued surveillance of Serratia marcescens infections by bacteriocin typing: investigation of two outbreaks of cross-infection in an intensive care unit. Apple. Microbiol. 23:982-985. 27. Traub, W. H., and I. Kleber. 1974. Continued epidemiological surveillance of Serratia marcescens infections by bacteriocin typing, with particular reference to strains isolated at Erlangen. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 229:372-382. 28. Traub, W. H., and I. Kleber. 1977. Serotyping of Serratia marcescens: evaluation of Le Minor's H-immobilization test and description of three new flagellar H antigens. J. Clin. Microbiol. 5:115-121. 29. Traub, W. H., and E. A. Raymond. 1971. Epidemiological surveillance of Serratia marcescens infections by bacteriocin typing. Appl. Microbiol. 22:1058-1063. 30. Traub, W. H., E. A. Raymond, and T. S. Startsman. 1971. Bacteriocin (marcescin) typing of clinical isolates of Serratia marcescens. Apple. Microbiol. 21:837440. 31. Veron, M. 1975. Nutrition et taxonomie des Enterobacteriaceae et bactéries voisines. I. Méthodes d'études des auxanogrammes. Ann. Microbiol. (Paris) 126A:267274. 32. von Graevenitz, A. 1977. The role of opportunistic bacteria in human disease. Annu. Rev. Microbiol. 31:447-471. 33. Waisman, H. A., and W. H. Stone. 1958. The presence of S. marcescens as the predominating organism in the intestinal tract of the newborn. Pediatrics 21:8-12. 34. Wilfert, J. N., F. F. Barrett, W. H. Ewing, M. Finland, and E. H. Kass. 1970. Serratia marcescens: biochemical, serological, and epidemiological characteristics and antibiotic susceptibility of strains isolated at Boston City Hospital. Apple. Microbiol. 19:345-352. 35. Williams, R. P., and W. R. Hearn. 1967. Prodigiosin, p. 410-432 and 449-451. In D. Gottlieb, and P. D. Shaw (ed.), Antibiotics. Il. Biosynthesis. Springer-Verlag New York, Inc., New York.

Biotyping of Serratia marcescens and its use in epidemiological studies.

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