Biotypes, Serovars and Antimicrobial Resistance Patterns of Acinetobacter baumannii Clinical Isolates

Zbl. Bakt. 284, 550-558 (1996) © Gustav Fischer Verlag, Stuttgart· Jena . New York

Biotypes, Serovars and Antimicrobial Resistance Patterns of Acinetobacter baumannii Clinical Isolates MURILO GOMES OLIVEIRAl, KINUE IRIN02, TANIA MARA IBELLI VAZ2, CELIA RODRIGUES GON<::ALVES2, and CARLOS EMILIO LEVy3 Department of Microbiology, Instituto de Ciencias Biol6gicas e Geociencias, Universidade Federal de Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil 2 Department of Bacteriology, Instituto Adolfo Lutz, Sao Paulo, SP, Brazil 3 Laboratory of Microbiology, Faculdade de Medicina de Ribeirao Preto, USP, Ribeirao Preto, Sao Paulo, Brazil 1

Received November 18, 1994 . Revision received November 16, 1995 . Accepted January 11,1996

Summary 255 Acinetobacter strains, from clinical specimens of inpatients and outpatients, were identified phenotypically according to the new taxonomy proposed by Bouvet and Grimont. A. baumannii was the most frequent species (80.8%). This species underwent biotyping and serotyping according to the scheme of Bouvet and Grimont, and that of Traub, respectively. 81.2 % of samples belonged to biotypes 2, 6 and 9 with a predominance of biotype 2. 86.6% of the strains could be serotyped; 2 new serotypes were encountered. The new serotype 29, being the most frequently isolated, was related to biotype 2 (86.6%), whereas serotype 13 was related to biotype 6 (84.8%). These clones presented marked multiple resistance patterns and were widespread in different wards. No outbreak was reported during the period studied. These phenotypical methods proved to be useful in differentiating strains of A. baumannii and, if used together, they showed a high discriminatory power.

Introduction Since the 1970's, the genus Acinetobacter, despite its low virulence, has been recognized as an emerging opportunist pathogen frequently involved in severe nosocomial outbreaks in several countries. These bacteria usually infect patients on antibiotic or immunosupressive therapy, those with malignancies, and those under tracheal or intravascular intervention or under prolonged treatment in intensive care units. These infections are a therapeutic challenge partly because of a significant increase in incidence and partly because of a spread of multiple resistance to antibiotics (1,2, 16, 17, 18, 21,22).

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Bouvet and Grimont (3) have recently introduced an extensive change in the taxonomy of the genus Acinetobacter. Through DNA-DNA hybridization, 12 genomic groups (genospecies) were discriminated, 11 of which could be biochemically differentiated using 28 phenotypical tests. Four genomic groups (2, 4, 5 and 7) were recognized as new species and named A. baumannii, A. haemolyticus, A. junii and A. johnsonii. Species formerly known as A. calcoaceticus and A.lwoffii were redefined and allocated to groups 1 and 8, respectively. Tjernberg and Ursing (24) presented three additional DNA groups, coded 13 through 15, and Bouvet and Jeanjean (5) described five DNA groups of proteolytic Acinetobacter strains which were numbered 13 through 17. Due to these taxonomic changes, phenotypic typing methods such as serotyping (25), biotyping (4), phage-typing (6), and analysis of outer membrane protein (8) or enzyme profiles (23) have been reassessed and genotypic methods, such as plasmid analysis (30), restriction patterns of chromosomal DNA (12), peR (13) and ribotyping (7, 10,23) have been proposed for epidemiological studies of infections by A. baumannii, the most commonly isolated species. In 1989, Traub (25), introduced a tube agglutination test for serotyping of A. baumannii. At first, 20 serovars were identified, 8 further serovars were subsequently found; meanwhile, serotyping has been extended to isolates comprising genospecies 3 (26,27). This study reports the serological analysis of A. baumannii isolates from Brazilian inpatients and outpatients and its correlation with Bouvet and Grimont's biotyping system.

Materials and Methods

Bacterial strains. 202 clinical isolates of A. baumannii were studied. They were isolated from inpatients (142 strains), outpatients (53 strains), and from unknown sources (7 strains), during the years 1990 and 1991 in different departments of two hospitals situated in the city of Ribeirao Preto, state of Sao Paulo, in southeastern Brazil. The isolates had been identified as A. baumannii according to Bouvet and Grimont (3). From 202 A. baumannii strains, 74 had been isolated from respiratory tract, 40 from wounds, 33 from urine, 19 from catheters, 4 from blood, 4 from surgical wounds, 2 from cerebrospinal fluid, and 26 from other clinical specimens. Reference bacterial strains. These strains were the same described elsewhere (20, 21). Strains corresponding to serotypes 1 to 28 of A. baumannii were kindly provided by Dr. W. H. Traub for the preparation of antisera. Biotyping of A. haumannii strains. Strains classified as A. baumannii were biotyped as described by Bouvet and Grimont (4), using 6 different carbon sources (levulinate, citraconate, L-phenylalanine, phenylacetare, 4-hydroxybenzoate and L-tartarate). Sera typing of A. baumannii strains. Antigen preparation corresponding to the 28 serovars of A. baumannii already determined and to the strains to be analyzed was carried out as previously described by Traub (25). The antisera against the 28 serovars were obtained after immunization of rabbits with antigenic suspension in accordance with Traub and Fukushima (28). All the strains were serotyped by tube agglutination test according to Traub (25). Two strains (345/91 and 774/91), selected from those which did not agglutinate with the 28 antisera, were inoculated IOto rabbits, and the resultant antisera were titrated against homologous and heterologous strains. Each antiserum was then twice absorbed with homologous and heterologous cells, including strains from genospecies 3 as described by

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Traub (25). The remaining agglutinating activity of each antiserum was assessed by titration against homologous and heterologous cells and comparison with the titres of non-absorbed antisera. Strains not agglutinating with any of the 28 antisera were tested against antisera 345/91 and 774/91, following the same procedures as for the tube agglutination reaction. Antimicrobial susceptibility test. Susceptibility testing was performed by using a previously described agar diffusion method and in accordance with the guidelines established by the National Committee for Clinical Laboratory Standards (19). Discriminatory power. Discriminatory power was determined with the aid of the Hunter and Gaston (14) discrimination index.

Results Of 202 strains of A. baumannii, Bouvet and Grimont's scheme identified 13 out of 20 biotypes previously established for this species (4, 6, 21). Biotypes 2, 6 and 9 were most frequently found (164 of 202 strains = 81.2 %), with a predominance of biotype 2 (43.6%). The initial serological study involving 202 strains of A. baumannii led to the classification of 102 strains distributed among 28 serovars determined by Traub (25,27). Serovars 13, 15 and 4 were the most frequent ones. However, a high proportion (49.5%) of strains could not be typed. Checkerboard tube agglutination tests with polyclonal rabbit immune sera against two non-typable strains and selective cross-absorption studies permitted identification of 2 new serovars of A. baumannii designated serovars 29 and 30, sequentially in Traub's scheme (Table 1). Employing these 2 new antisera against the 100 untypable isolates, 73 strains proved to be typable; 60 corresponded to serovar 29, and 13 proved to be serovar 30. Table 2 shows the distribution of serotypes according to the origin of the strains. It was noted that serovars 29, 13, and 15 predominated among inpatients, whereas serovars 29, 13, and 4 were preponderant among outpatients. 13.4% of strains remained serologically non-typahle. Table 1. Absorption of rabbit immune sera(RIS) 345/91 and 774/91 with homologous and heterologous strains of A. baumannii RIS

Treatment"

Titrated against

345/91

none 2X Abs. Serovars 1-28 2X Abs. Strain 345/91 2X Abs. Strain 774/91

Strain 345/91

none 2X Abs. Serovars 1-28 2X Abs. Strain 345/91 2X Abs. Strain 774/91

Strain 774/91

774/91

a

160 160

o

160

160 160 160

o

RIS were twice absorbed as described in Materials and Methods. RIS were employed at a starting dilution of 1 : 5 (= 1: 10 final dilution); titres recorded are final titres.

b The

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Table 2. Serovar distribution of A. baumannii clinical isolates according to occurrence of strains Occurrence" among

Serovar

1 2 4 6 9 10 11 12 13 14 15 17 18 19 20 21 22 23 24 28 29 30 NTb Total

Inpatients

Outpatients

2 3 5 (3.5)

1 1 6 (11.3) 2

Total

2 2 38 (26.7) 10 (7.0) 23 (16.2)

18 (33.9) 3 4

3 4 11 (5.4) 2 3 2 5 1 33 (16.3) 1 17(8.4) 3 3 4 2 1 1 1 3 2 60 (29.7) 13 (6.4) 27 (13.3)

142

53

202 e

3 2 5

1

26 (18.3) 1 12 (8.4) 2 3 1

7 (13.2) 4 1 3 2 1 1

1

1

The percentages are given in parenthesis and are relative to inpatients, outpatients and total. b Nontypable strain. e 7 strains were of unknown origin. a

The association of 14 biotypes with the 22 serovars and non-typable (NT) strains of A. baumannii is shown in Table 3. The predominant serovars proved to be more homogeneous in their associations. Except for NT strains distributed among biotypes 2,6 and 9, serovar 29 strains were found to be associated with biotype 2 in 86.6% (52 of 60 strains). The same was true with regard to the combination of serovar 13 and biotype 6 (84.8%), serovar 30 and biotype 2 (92.3%), and serovar 15 and biotype 9 (58.8%). The distribution of these more frequent associations of serovars and biotypes, according to the origin of the strains (Table 4) shows the predominance of the combinations between serovar 29 and biotype 2, and serovar 13 and biotype 6 both among outpatients and inpatients. These predominant clones were equally distributed in the majority of the wards studied. As seen in Table 5, multiple resistance patterns to antimicrobial drugs were associated with the main clones, except for the clone biotype 9/serovar 15.

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Table 3. Association between serovars and biotypes of A. baumannii strains Biotype

Serovar 2

7

8

9

10

11

12

4

13

16

19 20

1

1

3

3 4

11

7

1 1

3 1

2 3 2 5 1 33 1 17 3 3 4 2

1 3

1 I I

12

Total

6

1

1 2 4 6 9 10 11 13 14 15 17 18 19 20 21 22 23 24 28 29 30 NT"

5

Total

5 I I

28

2

1 1

10 1 1

1

1

1 1

1 3

1

1

1

1

1 1 3 2 60 13 27

1 1

5

8

52 12 5

3

6

88

3

40

2 1

4

2 1 1 5 36

1

1

1

1 7

4

1

2

1

6

1

202

* NT = Nontypable strains.

Table 4. Correlation between serovars and biotypes of A. baumannii according to the source of strains Biotype/Serovar

2/29 2/30

2INTd 6/13 61NT 9/15

9INT

9/4

Source Inpatients

Outpatients

33(23.2)" 9(6.3) 6(4.2) 22(15.5) 5(3.5) 7(4.9) 4(2.8) 2(1.4)

15(28.3)b 3(5.6) 3(5.6) 6(11.3) 1(1.9) 2(3.8) 1(1.9) 5(9.4)

" % relative to 142 strains. % relative to 53 strains.

b

Total

C% relative to 202 strains. d NT = nontypable strains.

52(25.7)C 12(5.9) 9(4.4) 28(13.8) 6(3.0) 10(4.9) 5(2.5) 7(3.4)

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Table 5. Antimicrobial resistance patterns of main A. baumannii clones Pattern

Antimicrobial drugs

Biotype/Serovar 2/30

2/29

(52)

6113 (28)

9115 (10)

4 5 3

13 24 7

3 18 7

5 4

(12)

1 2 3 4

Beta-lactams Amp Cef Ct Cfx Cazs Amp Cef Ct R Amp Cef Ct CfxR Amp Cef Ct Cfx Caz R

5 6 7 8 9

Aminoglycosides Gen Am Net Tob s Gen Am Net R Gen Am Tob R Gen Net Tob R Gen Am Net Tob R Fluoroquinolones Nor Cip PefS NorR Nor pefR Nor CipR Nor Cip PefR

10 11

12 13 14

1 1 3

9

4

3 5 4 29

3 23

3 3 1

12 4

2 2 2

7

2

25

17

1

( ) Total of strains. Amp = ampicillin; Cef = cefalotin; Ct = cefoxitin; Cfx = cefotaxime; , Caz = ceftazidime; Gen = gentamicin; Am = amikacin; Net = netilmicin, Tob = tobramycin; Nor = norfloxacin; Pef = pefloxacin; Cip = ciprofloxacin. S = susceptibility. R = Resistance.

Analysis of the discriminatory power of the 2 typing methods showed a higher discriminatory index (0.850) for serotyping compared with that of biotyping (0.739). The association between the 2 phenotypic markers was excellent (0.909). Discussion

In Brazil, as previously reported (20, 21, 29), A. baumannii is the prevalent species among the members of the genus Acinetobacter, in clinical materials, which point to the importance of this species in nosocomial infections. Traditional or modern typing methods will better support studies about the ecology and epidemiology of this species. Thus, Bouvet and Grimont's biotyping scheme allowed us to demonstrate the prevalence of biotypes 2,6 and 9, highlighting biotype 2. Bibliographic data show biotype 9 as the most frequently isolated one, also in other parts of Brazil (4, 6, 7, 9, 15,23,29). Serological studies published prior to Bouvet and Grimont's reclassification were inconclusive due to a lack of accuracy and consistent methods for identification. 36

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Some researchers (5, 6, 11,24) have noticed a phenotypic as well as genotypic similarity among genospecies 1,2,3 and 13, with antigenic reactions among them being likely. Recent serological studies demonstrated cross reactions, many of them reciprocal, among serovars of A. baumannii and A. genospecies 3 (26,27). The systematic use of the quantitative agglutination reaction with absorbed antisera contributes to a more reliable definition of A. baumannii serovars whose phenotypic differentiation from genospecies 3 is only made by growth at 44°C. It is therefore possible that application of the serological study to other species of the so-called "A. calcoaceticus-A. baumannii complex" may lead to a better differentiation of the strains in this group. During the study period, we noticed the prevalence of the new serovar 29 and serovar 13 among the strains from in- and outpatients. Traub (25), when introducing his serotyping method, described a higher incidence of serovar 4 in 3 outbreaks in intensive care and surgery units and 1 outbreak due to serovar 10 in a external paediatric hospital. The predominance of particular clones among our A. baumannii strains became evident from the association between biotypes and serovars. The high frequency of association between the new serovar 29 and biotype 2 (86.6%), and between serovar 13 and biotype 6 (84.8%), has demonstrated the stability of these markers. The presence of these clones in different wards as well as in outpatients suggests their wide spread and persistence in the area and in the studied period. A common source of contamination was difficult to establish, but the high level of resistance could suggest their possible nosocomial origin. Although we had seen the prevalence of these clones, epidemiological data could not evidence any outbreak. Otherwise, the prevalence of these clones with marked multiple resistance to antimicrobial drugs is a problem of concern and outbreaks involving these microorganisms could represent a serious risk for the hospitalized patients. The evidence of a distinct profile of serovars in a geographic area incorporating 2 new serovars proves the effectiveness of serotyping in the differentiation of A. baumannii strains. In spite of the difficulties to obtain the antisera, the method has proved to be easy and reproducible, with a high discriminatory index when associated to biotyping, which makes it a useful tool for epidemiological investigations. Addendum: Traub, in a recent paper (Med. Microbiol. Lett. 3 (1994) 120-127) has presented an update of the A. baumannii serotyping schema, extending it to 34 serovars in which serovars 29 and 30 were included. Acknowledgement. We thank Dr. W. H. Traub for providing the A. baumannii type strains and for his helpful suggestions.

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