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Efficacy of two DNA fingerprinting methods for typing Acinetobacter baumannii isolates
Diagnostic Microbiology and Infectious Disease 39 (2001) 215–223
www.elsevier.com/locate/diagmicrobio
Efficacy of two DNA fingerprinting methods for typing Acinetobacter baumannii isolates Liliana S. Quelle, Mariana Catalano* Departamento de Microbiologı´a, Parasitologı´a e Inmunologı´a, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina Received 8 January 2001; accepted 30 March 2001
Abstract Performance of macrorestriction and repetitive extragenic palindromic DNA sequence-based PCR (REP-PCR) to type Acinetobacter baumannii isolates was quantitatively estimated using a test population of 54 outbreak-related, 29 endemic infection-related and 17 epidemiologically-unrelated isolates. Reproducibility and stability for macrorestriction were 100%, and REP-PCR showed only slightly lower stability. Macrorestriction resolved 18 fingerprints and REP-PCR 10 DNA patterns, forming eight and seven clusters at 75% of similarity level, respectively. Intercluster band variation was ⬎7 bands for both methods. Although, all endemic isolates, except one, were concordantly grouped by both methods, macrorestriction distinguished a greater number of subtypes over one year study. For outbreaks, the epidemiologic concordance for both methods was 88%. The discriminatory index for macrorestriction and REP-PCR was 0.884 and 0.877, respectively. In conclusion, both methods showed similar efficacy as epidemiological markers, and by concordance, this study demonstrated that for REP-PCR typing, a ⱖ7 bands difference seemed an appropriate threshold to identify unrelated strains. © 2001 Elsevier Science Inc. All rights reserved.
1. Introduction Acinetobacter baumannii is frequently involved in hospital-acquired infections (Bergogne-Be´re´zin & Towner, 1996). The ability of A. baumannii to colonize skin and respiratory tract (Cefai et al., 1900; Seifert et al., 1997), to acquire multiple antibiotic resistance (Tankovic et al., 1994) and to survive on inanimate and dry surfaces for prolonged periods of time (Jawad et al., 1998), may contribute to the endemic or epidemic behavior of this nosocomial pathogen. For epidemiological and clinical purposes, accurate methods of strain identification are necessary to monitor A. baumannii infections. On the basis of genomic variability of strains within this species, various molecular techniques have been employed to differentiate clinical isolates, such as ribotyping (Gerner-Smidt, 1992; Biendo et al., 1999); macrorestriction using pulsed-field gel electrophoresis (PFGE) (Gouby et al., 1992; Seifert et al., 1995) or PCR based methods like arbitrary primed PCR (AP-PCR) (Graser et al., 1993; Grundmann et al., 1997); random amplified polymorphic DNA analysis (RAPD) (Grundmann et al., 1997); re-
* Corresponding author. fax: (5411) 4508-3705. E-mail address: [email protected] (M. Catalano).
petitive extragenic palindromic sequence-based PCR (REPPCR) (Reboli et al., 1994; Grundmann et al., 1997); amplified ribosomal DNA restriction analysis (ARDRA) (Koeleman et al., 1998); and amplified fragment length polymorphism (AFLP) analysis (Dijkshoorn et al., 1996; Koeleman et al., 1998). Although several of these techniques have been successfully used to either type members of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex (Ratto et al., 1995; Seifert et al., 1995; Snelling et al., 1996; Liu & Wu, 1997) or to identify A. baumannii outbreak-related strains (Gouby et al., 1992; Dijkshoorn et al., 1993; Catalano et al., 1999), their performance as typing methods at strain level has not been completely and explicitly validated. Furthermore, macrorestriction is viewed by several investigators as the “gold standard” for epidemiological analyses of many bacteria, including A. baumannii (Olive & Bean, 1999). For this purpose, some guidelines were suggested for interpreting PFGE banding patterns as well as for defining the relatedness of isolates recovered over periods of up to 3 months (Tenover et al., 1995). However, there are no criteria for interpreting the banding pattern on long-term studies. In 1996, the European Study Group on Epidemiological Markers (Struelens et al., 1996) proposed guidelines to evaluate the efficacy of typing methods. Updated criteria include, mainly, the study of in vitro
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stability, reproducibility, typeability, and discriminatory power of the typing systems. Another criterion to evaluate typing methods is the epidemiologic concordance (EC) which is defined for each outbreak as the fraction of isolates with the epidemic type. This represents a relative measure of in vivo stability (Struelens et al., 1996). Moreover, it was interesting to compare the identification of clonal groups by independent typing methods (typing methods concordance) because the greater the number of techniques allowing isolates to be concordantly grouped, the more likely such isolates are to be clonally related. Besides, this Study Groups suggested that the DNA banding pattern polymorphism of each species should be taken into account to define strains (Struelens et al., 1996). This study addressed the quantitative evaluation of performance criteria for REP-PCR, and macrorestriction to type A. baumannii isolates. The efficacy of each technique as a typing system was estimated by the quantitative expression of reproducibility, stability, typeability, discriminatory power, and epidemiologic concordance. In addition, typing methods concordance was also evaluated. To this end, the A. baumannii test population included outbreakrelated, sporadic, and epidemiologically unrelated isolates.
2. Materials and methods 2.1. Bacterial strains The 99 A. baumannii isolates studied were obtained from infected patients recovered at 7 different hospitals (H1 to H7) located in Buenos Aires City (Table 1). Fifty-four isolates were recovered during 3 well-characterized outbreaks at hospitals H1 (Ratto et al., 1995), H2 and H4 (Catalano et al., 1999): 29 from endemic infections at hospitals H3 and H5 (isolated during September 1994 –September 1995 and from November 1995 to April 1996, respectively). The remaining sixteen, were epidemiologically unrelated isolates from an A. baumannii collection stored at our laboratory during 1981–1988 and recovered from hospitals H6 and H7. Type strain ATCC 19606 was also included in the study. Isolates were identified at species level using Bouvet and Grimont’s proposed phenotypic scheme (Bouvet and Grimont, 1986). Genospecies 2 was confirmed by EcoRI ribotyping (Gerner-Smidt, 1992). Biotypes were identified as proposed by Bouvet and Grimont (Bouvet & Grimont., 1987). Until used, isolates were frozen at ⫺70°C in Brain Heart Infusion (BHI) (Difco Laboratories, Detroit, USA) and supplemented with 20% glycerol. 2.2. Extraction of genomic DNA For macrorestriction analysis, bacterial concentration was adjusted in TN solution (10 mM Tris-HCl [pH 7.60], 1M NaCl) as previously described (Soares et al., 1993). Then, 150 L of the adjusted suspensions were mixed with
Table 1 Typing results of the 100 Acinetobacter baumannii isolates Isolate source and n° H1 outbreak* (Ratto et al., 1995). 1–3, 5, 6, 8–15, 17, 18 4 7 16 H2 outbreak 1Sa to 16 Sa H3 endemic infections 12, 16 3 9 17, 32 10 4, 6, 11, 29, 36 5, 7 39 18, 20, 22, 24, 26, 40 H4 outbreak (Catalano et al., 1999) 117 116 112 104, 105, 108, 109–111, 113–115, 118, 20, 122, 124, 125 107, 121 119 H5 endemic infections 91, 93, 95, 99 98 90 94 96 Unrelated isolates 19C, 28M 10M 32, 34, 41M 21 64M, 43M 47, 51M 8F 15F 101 50M 48M ATCC 19606
Biotype◊
REP-PCR
PFGE
2 9 2 9
A A B1 C
I I II III
2
A
I
2 2 2 9 9 9 9 9 8
A A A B1 B1 B E E E
I Ia Ib II II d II a IV IV a IV b
2 2 9 9
A A B1 B1
I Ib II b II c
9 8
E1 E1
IV a IV b
2 8 2 9 9
A B1 A E1 F
I I Ia IV b V
2 2 9 2 9 9 8 9 9 9 9 —
A A B1 B C C E E F H F1 J
I Ic II a II c III IIIa IV b IV V VI VII VIII
* H1, H2 and H4 outbreaks occurred during 1992, 1995 and 1996 respectively. H3 and H5 endemic infections occurred during 1995 and 1996 respectively. H6 and H7, unrelated isolates from the stored collection obtained during 1981–1988.
an equal volume of 1.6% low melting point agarose (BioRad, Richmond, CA, USA). Agarose disks were prepared by pippeting 20-L drops onto a plate covered with spacer slides and incubated at ⫺20°C during 5 min. Bacterial cells were lysed at 37°C overnight with gently shaking in lysozyme solution (6 mM Tris-HCl [pH 7,6], 1M NaCl, 100 mM EDTA [pH 7,5], 0.2% deoxycholate, 0.5% sodium lauroyl sarcosine, 0.5% Brij-58, 20 L RNase, and 1 mg lysozyme per mL), followed by a further overnight incubation at 50°C in protelysis buffer (0.5 M EDTA [pH 9], 1%
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sarcosyl, 50 g/mL proteinase K). The disks were washed three times with TE 10/0.1 (10 mM Tris, 0,1 mM EDTA), and were then stored at 4°C. For REP-PCR DNA was extracted as previously described (Ratto et al., 1995). 2.3. PFGE Genomic DNA was digested with ApaI (Promega Corporation, Madison, Wis, USA), according to the procedure described by the manufacturer. Endonuclease-digested genomic DNAs contained in disks were separated by PFGE. Disks were placed into the wells of a 1.2% agarose gel (Bio-Rad) in 0.5 X Tris-borate-EDTA buffer (45 mM TrisHCl-45 mM borate, 1 mM EDTA, pH 8), and separation of the DNA fragment was achieved with Bio-RAD CHEF-DR III system. Running conditions were 24 hrs at 14°C, with an initial switching time of 1 second and final time of 30 seconds, at 6 V/cm. Concatemers of DNA isolated from bacteriophage (New England Biolabs, Beverly, MA, USA) were run next to DNA fragments for size comparison. 2.4. REP-PCR The primer pair REP1R-I (5⬘-IIIICGICGICATCIGGC3⬘) and REP2-I (5⬘-ICGICTTATCIGGCCTAC-3⬘) was used to amplify putative REP-like elements. Amplification reaction was performed in a final volume of 50 L, according to Snelling et al., 1996. REP-PCR amplified DNA fragments were resolved by electrophoresis in a 1.5% agarose gel (GIBCO/BRL Life Technologies, Inc., MO, USA). A molecular weight marker (1 kb ladder, GIBCO/BRL Life Technologies), was loaded onto the agarose gel for size comparison. All amplification reactions were carried out in a Gene Amp PCR System 9600 Perkin Elmer thermal cycler (Perkin Elmer Cetus, Norwalkm, Conn. USA). Fingerprints were considered to be highly similar when all visible bands from two isolates had the same apparent migration distance. Variations in band intensity or shape were not taken into account. 2.5. Analysis of DNA banding patterns DNA banding patterns were analyzed by visual examination, and all loci were scored for the presence or absence of a band. Strains delineation was inferred in terms of percentage similarity of banding patterns (Struelens et al., 1996). Based on our tested population, isolates showing less than 75% of similarity were considered as clonally unrelated and those isolates presenting similarities higher than 75% were classified as subtypes of the same clone. The percent similarity of banding patterns was estimated with the Dice coefficient (Dice, 1945). Cluster analysis was performed by the UPGMA (unweighted pair-group method with arithmetic averages) using the software program NTSyS-PC version 1.40.
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2.6. Performance criteria Reproducibility (R), in vitro stability, typeability (T), and epidemiologic concordance (EC) were calculated suggested by Struelens et al (Struelens et al., 1996). To assess reproducibility, all 100 DNAs were evaluated by three independent digestions or amplifications and electrophoretic runs. To calculate S value, 10 randomly selected isolates were repeatedly passaged on agar culture media. Briefly, the experiment was carried out as follows: isolates from frozen stocks were grown on BHI agar. A single colony of each isolate was transferred to BHI and incubated 18 hrs at 30°C. DNA from each isolate was obtained and designated as FE (first extraction). All 10 isolates were then subjected to 25 serial passages on BHI agar. The FE fingerprint of each isolate was compared with the others obtained using DNA extracted after every 5 subcultures of the 25 total passages (SC5 to SC25, subcultures 5 to 25). DNAs from all isolates (both FE and SC) were analyzed by both REP-PCR and by macrorestriction. As to the outbreaks, EC was calculated on each set of isolates recovered at hospitals H1, H2 and H4. The EC for epidemiologically unrelated isolates was calculated as the fraction of isolates with the most frequent type. Unrelated isolates concordance was estimated with two sets of isolates, 30 endemic infection-related A. baumannii isolates (sporadic cases), recovered at H3 and H5 hospitals, and the other 17 belonging to the epidemiologically unrelated collection. ECs were statistically tested by Fisher’s exact test, with one-tailed P value. Discriminatory power was estimated by the formula of Simpson’s index of diversity (Hunter and Gaston, 1988). To assess DI value, the 16 epidemiologically unrelated isolates from a collection recovered from hospitals H6 and H7 during 1981–1988 and A. baumannii ATCC 19606 type strain were considered.
3. Results Table 1 shows raw data obtained with REP-PCR and macrorestriction, considering the 100 A. baumannii isolates. 3.1. Reproducibility and stability For macrorestriction, and REP-PCR the reproducibility estimation, considering the 100 isolates, yielded a R value of 100%. Macrorestriction classified the 10 isolates randomly selected for stability analysis, in six banding patterns (I, II, III, IV and Ia and IIa). No modifications on banding patterns were observed after 25 serial subculture passages (Table 2). REP-PCR classified the 10 isolates in five fingerprints (A, B, C, E, and B1). Two isolates that predominantly exhibited type A, shifted to type B, at least in one of the serial subcultures (Dice coefficient: 52%). Meanwhile, two further
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Table 2 Macrorestriction using Apa I and REP-PCR in vitro stability results. Isolate
1 2 3 4 5 6 7 8 9 10
PFGE Types
REP-PCR Types
FE*
SC5#
SC10
SC15
SC20
SC25
FE
SC5
SC10
SC15
SC20
SC25
I I I II III I Ia II a IV II a
I I I II III I Ia II a IV II a
I I I II III I Ia II a IV II a
I I I II III I Ia II a IV II a
I I I II III I Ia II a IV II a
I I I II III I Ia II a IV II a
A A A B1 C A A B E B
A A A B1 C A A B E B
A B A B1 C A A B E B
B A A B1 C A A A E A
A A B B1 C A A B E B
A A B B1 C A A B E B
* FE: First extraction, DNA obtained from the first isolate culture. # SC: subculture
isolates that at first exhibited type B shifted to genotype A. The remaining isolates type A preserved this type along culture passaging (Table 2). Furthermore, isolates showing types C, E and subtype B1, remained with an unchanged banding pattern during serial subcultures. The average S value was 90%, ranging from 66.6% to 100%. 3.2. DNA bands resolution Macrorestriction and REP-PCR generated DNA fingerprints for all 100 A. baumannii isolates, thereby, T was 100%. Fingerprints generated by macrorestriction with ApaI showed 11–15 bands per isolate located between 48.5 and 436.6 kbp, and a total of 41 different bands. REP-PCR fingerprints showed 10 to 12 bands per isolate in the 250 – 4,000 bp range, with a total of 26 different bands. Macrorestriction typing of the 99 A. baumannii isolates recovered from infected patients, and the ATCC 19606 strain, generated 18 distinguishable DNA patterns. REP-PCR typing exhibited 10 different fingerprints. 3.3. DNA patterns similarities Fig. 1A shows clustering analysis of all fingerprints generated by macrorestriction, and Fig. 1B several banding profiles. At similarity level (Dice coefficient) of 75%, 8 clusters were distinguished. Similarities within clusters ranged from 25% to 74%, representing fingerprints with 7–17 band difference. Fingerprinting assigned to the same cluster showed Dice coefficients ranging from 74 to 92%. This represented 2– 6 bands difference. Clusters 1 and 5 showed 2 subclusters. Fingerprint Ia, located at one subcluster which was formed at similarity level close to 80% in cluster 1, belonged to one isolate recovered at H3 four months after types I isolation (Table 3), thereby, it seemed appropriate to be considered as a subtype of the parental type I isolates. Subtype Ic, showing 80% of similarity with type I (5 bands difference), belonged to an already known epidemiologically unrelated isolate, therefore, no epidemi-
ological evidence supported related clonality between both fingerprints. Fingerprint IId, located alone at one subcluster linked at 75% of similarity with type II in cluster 5 (6 bands difference), belonged to one isolate recovered at H3 one year after type II isolates (Table 3), and presumably, represented a probably related isolate. Outbreak strain, from H1 and H2 hospitals (Table 1), was located in cluster 1. In spite of the fact that these outbreaks were reported during 1992 and 1995 respectively, they were both caused by the same epidemic strain. In H4 outbreak, the epidemic strain was located in cluster 5, with a similarity coefficient respect to type I close to 40%. Similarities between fingerprints of cross-transmitted strain during H1 and H4 outbreaks and their respective epidemic strains ranged from 24 to 48% showing 10 –19 bands difference. The banding patterns exhibited by endemic infection-related isolates (H3 and H5 hospitals, Table 1) were located in clusters 1, 3, 4 and 5, with similarities ranging from 50% to 75%. Even though the 17 isolates of the epidemiologically unrelated collection were spotted in all clusters, except cluster 3, both clusters 7 and 8, included only fingerprints belonging to this collection. Their similarity range, with respect to the epidemic strains, was 18 – 40%. Dice coefficients of each comparison within the epidemiologically unrelated isolates exhibited by these isolates ranged from 40% to 75%. Fig. 2A shows REP-PCR clustering analysis. At a linkage level of 75%, seven clusters were observed. Similarities between clusters ranged from 24% to 66%, representing fingerprints with 7–14 bands difference. Similarities in fingerprints placed in each cluster were 78 – 82% represented a 3– 4 bands difference. As in macrorestriction clustering analysis, REP-PCR fingerprints of outbreak strains were located in clusters 1 and 5, showing a similarity of 48%. For outbreak-related isolates, the similarity range within crosstransmitted and epidemic strains fingerprints was 48%– 64%. Fingerprints of endemic infection-related isolates were located in clusters 1, 3, 5 and 7. REP-PCR grouping of
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Fig. 1. A. Macrorestriction clustering analysis. The arrow shows cut-off value. Alphanumerical symbols on the right refer to types and subtypes listed in Table 1. B. Macrorestriction fingerprints of 14 A. baumannii strains. Lane 1, type I; lane 2, type Ia; lane 3, type IV; lane 4, type II; lane 5, type IIb; lane 6, type III; lane 7, type Ib; lane 8, type IIa; lane 9, type IId; lane 10, type V; lane 11, type IVb; lane 12, type IIIa; lane 13, type VI; lane 14, type VII. M: lambda phage ladder DNA concatemers (band sizes are expressed on the right in kilobases).
all isolates showing PFGE type II and subtypes IIa, IIb, IIc and IId in one cluster would uphold the relatedness of all these PFGE subtype to type II. Moreover, PFGE fingerprints IId and II exhibited the same REP-PCR subtype (Table 3). For REP-PCR, isolates epidemiologically unrelated collected during 1981–1988 were heterogeneously distributed in all clusters except number 3 (Fig. 2A). As for the ones located in cluster 4 were mainly found within this collection of isolates. Among this collection, the isolate identified as PFGE subtype Ic exhibited REP-PCR type A, similarly to others grouped in cluster 1 by macrorestriction, thereby, it supported the classification of this isolate as clonally related to PFGE type I, mentioned above. The major discrepancy between macrorestriction and REP-PCR clustering analysis lay in the classification of the fingerprint exhibited by a single isolate belonging to the epidemiologically unrelated collection. For macrorestriction
this banding pattern was located alone in cluster 8 (type VII, Fig. 1A); however, by REP-PCR it was located in cluster 7 (subtype F1), showing 78% of similarity (4 bands difference) with type F (Fig. 2B, lanes 5 and 11 respectively). REP-PCR type F exhibited PFGE type V, situated alone in cluster 3, showing 38% of similarity with PFGE type VII (18 bands difference), (Fig. 1B lanes 10 and 14 respectively). 3.4. EC and typing methods concordance All outbreak-related isolates recovered at H2 hospital showed identical fingerprints by either macrorestriction or REP-PCR (EC 100%). For both methods, 16 out of the 18 isolates from H1 outbreak belonged to the epidemic clone (EC 88.8%). The remaining two isolates were identified as two different clones (cross-transmitted strains) (Table 1). Fifteen out of the 20 isolates which was obtained from
Table 3 Concordance of REP-PCR and macrorestriction for typing sporadic cases-related A. baumannii isolates. REP-PCR
Macrorestriction Sep94
Dec94
Mar95
A B B1 E
II a (2)
Apr95
May95
I (1)#
I (1) I b (1)
II a (1)
II a (1)
IV (1)
IV (1)
Jun95
Jul95
Aug95
I a (1) II a (1)
II (2)
# Number of isolates
Sep95
IV a (1)
IV b (3)
IV b (1)
II d (1) IV b (2)
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Fig. 2. A. REP-PCR clustering analysis. The arrow shows the cut-off value. Alphanumerical symbols on the right refer to types and subtypes listed in Table 1. B. REP-PCR fingerprints. Lane 1, subtype B1; lane 2, type A; lane 3, type E; lane 4, type B; lane 5, subtype F1; lane 6, type C; lane 7, type H; lanes 8 and 9, subtype E1; lane 10, ATCC 19606 strain; lane 11, type F. M: 1 Kb DNA ladder.
patients during H4 outbreak, were identified as belonging to the epidemic type by REP-PCR. Fourteen out of these 15 isolates were classified as the epidemic type by macrorestriction while the remaining one as a very closely related subtype (96% of similarity). Thus, EC value was 75% for both methods. Similarly, macrorestriction and REP-PCR classified the remaining 5 isolates in two different types. For outbreak-related isolates, average EC for macrorestriction and REP-PCR was 88%. Hence, by concordance of outbreak-related isolates, all typing methods showed a similar specificity to delineate the epidemic clonal group. For endemic infection-related isolates recovered from H3 hospital, macrorestriction and REP-PCR indicated 9 out of the 21 isolates as belonging to the most frequent type. All 9 isolates exhibited an undistinguished banding pattern by REP-PCR typing (type E, Table 3). For macrorestriction, two of these isolates were identified as type IV, one as subtype IVa, and the remaining 6 subtype IVb. All of these isolates were considered as belonging to the same clone (Table 1). The EC value of each method was 43%. The other 12 isolates were classified into two separate clonal
groups by both methods (Table 1). Macrorestriction identified 5 out of the 8 endemic infection-related isolates recovered from H5 hospital, as belonging to the most frequent type. The others were classified into three different types (EC 63%). REP-PCR classified also these 8 isolates in four types, with an EC value of 50%. Average EC values for macrorestriction, and REP-PCR were 53% and 47%, respectively (P ⫽ 0.5). For epidemiologically unrelated isolates, EC values for macrorestriction and REP-PCR were 24%. Based on the 99 A. baumannii isolates recovered from infected patients, the most frequent type was the one assigned as A/I by REP-PCR and macrorestriction. Actually, isolates showing this type were recovered from all 7 hospitals studied, regardless the year of isolation or epidemiologic condition. A total of 40 out of the 99 A. baumannii isolates showed this type, 39 belonging to biotype 2 and one to biotype 9. However, one isolate showing PFGE type I, exhibited REP-PCR type B1, belonging to biotype 8. All isolates showing PFGE IV, IVa and IVb, were grouped by REP-PCR in cluster 4 as type E or subtype E1. Biotyping
Table 4 Epidemiologic Concordance of the two typing methods. Method
EC* Outbreaks**
Macrorestriction REP-PCR
Endemic infections#
Mean (%)
Range (%)
Mean (%)
Range (%)
Unrelated isolates# (%)
88 88
75–100 75–100
46 53
43–50 43–63
24 24
* EC Epidemiologic concordance. ** Epidemiologic concordance: (frequency of outbreak type/n° of outbreak isolates tested) ⫻ 100. # Epidemiologic concordance for endemic infection-related isolates and epidemiological unrelated isolates: (frequency of the most frequent types/n° of isolates tested) ⫻ 100.
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classified isolates belonging to PFGE IV and IVa as biotype 9, and eight of the 9 isolates PFGE type IV b were biotype 8 (Table 1). Hence, a lack of concordance was observed between biotyping and macrorestriction or REP-PCR typing. 3.5. Discriminatory power and typing of A. baumannii isolates A linkage level lower than 75% of similarity was used as a criterion to classify isolates into different clonal groups. Taking into account the 16 epidemiologically unrelated isolates and A. baumannii ATCC 19606 strains, DI for macrorestriction and REP-PCR were very similar (0.884 vs 0.877).
4. Discussion Strain classification has been achieved by a number of different DNA-based methods, all of which must meet several criteria in order to be widely applicable. In this study, 100 A. baumannii isolates were typed by currently used genotyping methods, to evaluate their performance, particularly their reproducibility, in vitro stability, discriminatory power, and epidemiologic concordance. Macrorestriction and REP-PCR showed the optimal reproducibility value. The analysis of stability under laboratory manipulation demonstrated that macrorestriction was a very stable typing system, since no modifications of banding patterns were observed after 25 serial passaging. However, REP-PCR average S value was close to that showed by macrorestriction. By clustering analysis, macrorestriction classified the 100 A. baumannii isolates into 8 clusters and REP-PCR supported the grouping of PFGE clusters 1, 2, 4, 5, 6, and 7. All fingerprints were linked with similarities exceeding 24%, and the two outbreak types were allocated to clusters 1 and 5 by both methods. DNA patterns exhibited by endemic infection-related isolates were assigned to four different clusters by both techniques. Likewise, epidemiologically unrelated isolates were heterogeneously distributed in all clusters. Two main discrepancies were observed between macrorestriction and REP-PCR clustering analysis results. The first discrepancy was that REP-PCR fingerprints grouped in cluster 7 were located by PFGE in cluster 3 and 8. This finding represents a lack of discrimination by REPPCR rather than the identities of isolates because these fingerprints were exhibited by already known epidemiological unrelated isolates. The second one was the quantity of fingerprints with equal to or greater similarities than 75% identified by macrorestriction within clusters 1, 4 and 5. These discrepancies may be due to the difference in the number of resolution bands observed among these methods, or to the DNA banding pattern polymorphism detected by both methods. In this sense, macrorestriction showed
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greater DNA banding pattern polymorphism than REPPCR. Thus, generation of changes in chromosomal polymorphism, as displayed by PFGE may occur at a faster rate than changes detected by the REP-PCR typing method. This presumption would be stressed by the recover of PFGE subtype IId isolate one year after of the first isolation of type II at H3, showing 6 bands difference and classified as the same type by REP-PCR. In this regard, Arbeit et al., 1990, demonstrated the ability of macrorestriction to resolve recent evolutionary divergence among E. coli isolates which were indistinguishable by other methods. For outbreak-related strains, difference between epidemic strains and cross-transmitted clones were clear cut for both methods, including the H4 outbreak whose span period lasted for 4 months. Thus, for outbreak-related isolates, both typing methods showed similar specificity to delineate the epidemic clonal group (similar EC value). Epidemiologic concordance for outbreak-related isolates may also be considered as a measure of typing method stability under “field” conditions (Struelens et al., 1996). The latter include all the conditions to which strains are subjected during their spread within and among patients, vectors and environmental reservoirs, rather than a single or a few hosts. At this point, macrorestriction and REP-PCR showed an EC value close to the ideal as proposed by Struelens et al (Struelens et al., 1996). Excepting one, all isolates belonging to epidemic strains exhibited indistinguishable banding patterns by both methods, pointing out the highest in vivo stability of these epidemiological markers. With respect to both the epidemiologically unrelated isolates and for the endemic infection-related isolates (sporadic isolates), macrorestriction and REP-PCR showed similar EC to identify the most frequent type. Based on the 17 epidemiological unrelated isolates and a linkage level lower than 75% of similarity to define separate clonal groups, both methods showed similar DI value. Actually, DI value varied according to the criteria used for strain definition. Interpreting criteria for macrorestriction to define the relatedness of isolates were published. However, most of them were mainly directed to identify epidemic strains from isolates recovered over periods of up to 3 months. These general guidelines allow differences of up to six bands (two genetic events) for isolates clonally related (Tenover et al., 1995; Tenover et al., 1997). However, the European Study Group on Epidemiological Markers suggested that the degree of relatedness to define strains should be adjusted to each species because different species may vary in the degree of the DNA banding pattern polymorphism revealed for each typing system (Struelens et al., 1996). In the present study, the known temporary epidemiologically unrelated isolates linked at a similarity lower than 75% (showing 7–17 bands difference), and isolates epidemiological related were linked mainly at similarity equal or greater than 80% (2–5 bands difference). Therefore, taking into account the set of A. baumannii isolates analyzed in the present study, it seemed appropriate to regard differences
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equal or greater than 7 bands to identify separate clonal groups by macrorestriction analysis, even in long-term studies. For PCR-based methods, there are no criteria for interpreting bands size variation or the value of intensity of several bands. The lack of these criteria lay on the fact that there is no restriction site. Hence, bases to standardize strain definition represent an area of active research. In this regard, typing methods concordance is suggested as a good principle to validate the identification of separate clonal groups because natural groups must be identical or very similar when tested by methods that measure independent markers (Struelens et al., 1996). Considering all 100 A. baumannii isolates, by concordance, REP-PCR identified 7 out of the 8 clonal groups recognized by macrorestriction. Hence, a clear cut among related and non-related isolates was observed (more than 82% of similarity) representing 3– 4 bands difference, and 75% of similarity representing more than 7 bands difference. Therefore, an equal or greater number than 7 bands difference seemed appropriate to define separate clonal groups for this genospecies. Using criteria mentioned above to define strains, our results showed a limited numbers of A. baumannii clones involved in nosocomial infections during 1981–1996. During this study, REP-PCR/PFGE type A/I was the most prevalent clone, recognized as epidemic or endemic strain at different hospitals. The limited number of A. baumannii clones causing nosocomial infections was also found by Vaneechoutte et al (Vaneechoutte et al., 1995), using other typing methods. In conclusion, when macrorestriction is regarded as “gold standard” for the molecular typing of A. baumannii isolates, REP-PCR displayed a similar performance. Therefore, when the convenience criteria, such as rapidity, accessibility, and ease of use are considered, REP-PCR is the most suitable method to type A. baumannii isolates for clinical purposes. Relatedness lower than 75% of similarity or 7 bands of difference seemed a good approach to identify separate clonal groups of A. baumannii by this technique. However, given the slightly higher capacity of macrorestriction to recognize recent evolutionary divergence, this method is more accurate when storage of genetic patterns for the creation of reference databases is required.
Acknowledgments This study was supported by grants from Alberto Roemmers Foundation and Universidad de Buenos Aires (UBACYT TM 09).
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L.S. Quelle and M. Catalano / Diagnostic Microbiology and Infectious Disease 39 (2001) 215–223 Liu, P. Y., & Wu, W. (1997). Use of different PCR-based fingerprinting technique and pulsed-field gel electrophoresis to investigate the epidemiology of Acinetobacter calcoaceticus-Acinetobacter baumannii complex. Diag Microbiol Infect Dis, 29, 19 –28. Olive, D. M., & Bean, P. (1999). Principles and applications of methods for DNA-based typing of microbial organisms. J Clin Microbiol, 37, 1661– 1669. Ratto, P., Sordelli, D. O., Abeleira, E., Torrero, M., & Catalano, M. (1995). Molecular typing of Acinetobacter baumannii-Acinetobacter calcoaceticus complex isolates from endemic and epidemic nosocomial infections. Epidemiol Infect, 114, 123–132. Reboli, A. C., Houston, E. D., Monteforte, J. S., Wood, C. A., & Hamill, R. J. (1994). Discrimination of epidemic and sporadic isolates of Acinetobacter baumannii by repetitive element PCR-mediated DNA fingerprinting. J Clin Microbiol, 32, 2635–2640. Seifert, H. L., & Gerner-Smidt, P. (1995). Comparison of ribotyping and pulsed-field gel electrophoresis for molecular typing of Acinetobacter isolates. J Clin Microbiol, 33, 1402–1407. Seifert, H. L., Dijkshoorn, L., Gerner-Smidt, P., Pelzer, N., Tjernberg, I., & Vaneechoutte, M. (1997). Distribution of Acinetobacter species on human skin: comparison of phenotypic and genotypic identification methods. J Clin Microbiol, 35, 2819 –2825. Snelling, A. M., Gerner-Smidt, P., Hawkey, P. M., Heritage, j., Parnell, P., Porter, C., Bodenham, A. R., & Inglis, T. (1996). Validation of use of whole-cell repetitive extragenic palindromic sequence-based PCR (REP-PCR) for typing strains belonging to the Acinetobacter calcoaceticus-Acinetobacter baumannii complex and application of the method to the investigation of a hospital outbreak. J Clin Microbiol, 34, 1193–1202.
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Soares, S., Kristinsson, K. G., Musser, J. M., & Tomasz, A. (1993). Evidence for the introduction of a multiresistant clone of serotype 6B Streptococcus pneumoniae from Spain to Iceland in the late 1980s. J Infect Dis, 168, 158 –163. Struelens, M. J., & the Members of the European Study Group on Epidemiological Markers (ESGEM), of the European Society for Clinical Microbiology and Infectious Diseases (ESCMID). (1996). Consensus guidelines for appropriate use and evaluation of microbial epidemiologic typing systems. Clin Microbiol Infect, 2, 2–11. Tankovic, J., Legrand, P., De Gatines, G., Chemineau, V., Brun-Buisson, C., & Duval, J. (1994). Characterization of a hospital outbreak of imipenem-resistant Acinetobacter baumannii by phenotypic and genotypic methods. J Clin Microbiol, 32, 2677–2681. Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelsen, A., Murray, B. E., Persing, D. H., & Swaminathan, B. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed field electrophoresis: Criteria for bacterial strain typing. J Clin Microbiol, 33, 2233–2239. Tenover, F. C., Arbeit, R. D., & Goering, R. V. (1997). How to select and interpret molecular strain typing methods for epidemiological studies of bacterial infections: A review for healthcare epidemiologists. Infect Control Hosp Epidemiol, 18, 426 – 439. Vaneechoutte, M., Elachouni, A., Maquelin, K., Claeys, G., Van Liedekerke, A., Louagie, H., Verschaegen, G., & Dijshoorn, L. (1995). Comparison of arbitrary primed polymerase chain reaction and cell envelope protein electrophoresis for analysis of Acinetobacter baumannii and A. junii outbreaks. Res Microbiol, 146, 457– 465. Williams, J. G. K., Kubelik, A. R., Livak, K. J., Rafalski, J. A., & Tingey, S. V. (1990). DNA polymorphism amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res, 18, 6531– 6535.
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Efficacy of two DNA fingerprinting methods for typing Acinetobacter baumannii isolates Liliana S. Quelle, Mariana Catalano* Departamento de Microbiologı´a, Parasitologı´a e Inmunologı´a, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina Received 8 January 2001; accepted 30 March 2001
Abstract Performance of macrorestriction and repetitive extragenic palindromic DNA sequence-based PCR (REP-PCR) to type Acinetobacter baumannii isolates was quantitatively estimated using a test population of 54 outbreak-related, 29 endemic infection-related and 17 epidemiologically-unrelated isolates. Reproducibility and stability for macrorestriction were 100%, and REP-PCR showed only slightly lower stability. Macrorestriction resolved 18 fingerprints and REP-PCR 10 DNA patterns, forming eight and seven clusters at 75% of similarity level, respectively. Intercluster band variation was ⬎7 bands for both methods. Although, all endemic isolates, except one, were concordantly grouped by both methods, macrorestriction distinguished a greater number of subtypes over one year study. For outbreaks, the epidemiologic concordance for both methods was 88%. The discriminatory index for macrorestriction and REP-PCR was 0.884 and 0.877, respectively. In conclusion, both methods showed similar efficacy as epidemiological markers, and by concordance, this study demonstrated that for REP-PCR typing, a ⱖ7 bands difference seemed an appropriate threshold to identify unrelated strains. © 2001 Elsevier Science Inc. All rights reserved.
1. Introduction Acinetobacter baumannii is frequently involved in hospital-acquired infections (Bergogne-Be´re´zin & Towner, 1996). The ability of A. baumannii to colonize skin and respiratory tract (Cefai et al., 1900; Seifert et al., 1997), to acquire multiple antibiotic resistance (Tankovic et al., 1994) and to survive on inanimate and dry surfaces for prolonged periods of time (Jawad et al., 1998), may contribute to the endemic or epidemic behavior of this nosocomial pathogen. For epidemiological and clinical purposes, accurate methods of strain identification are necessary to monitor A. baumannii infections. On the basis of genomic variability of strains within this species, various molecular techniques have been employed to differentiate clinical isolates, such as ribotyping (Gerner-Smidt, 1992; Biendo et al., 1999); macrorestriction using pulsed-field gel electrophoresis (PFGE) (Gouby et al., 1992; Seifert et al., 1995) or PCR based methods like arbitrary primed PCR (AP-PCR) (Graser et al., 1993; Grundmann et al., 1997); random amplified polymorphic DNA analysis (RAPD) (Grundmann et al., 1997); re-
* Corresponding author. fax: (5411) 4508-3705. E-mail address: [email protected] (M. Catalano).
petitive extragenic palindromic sequence-based PCR (REPPCR) (Reboli et al., 1994; Grundmann et al., 1997); amplified ribosomal DNA restriction analysis (ARDRA) (Koeleman et al., 1998); and amplified fragment length polymorphism (AFLP) analysis (Dijkshoorn et al., 1996; Koeleman et al., 1998). Although several of these techniques have been successfully used to either type members of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex (Ratto et al., 1995; Seifert et al., 1995; Snelling et al., 1996; Liu & Wu, 1997) or to identify A. baumannii outbreak-related strains (Gouby et al., 1992; Dijkshoorn et al., 1993; Catalano et al., 1999), their performance as typing methods at strain level has not been completely and explicitly validated. Furthermore, macrorestriction is viewed by several investigators as the “gold standard” for epidemiological analyses of many bacteria, including A. baumannii (Olive & Bean, 1999). For this purpose, some guidelines were suggested for interpreting PFGE banding patterns as well as for defining the relatedness of isolates recovered over periods of up to 3 months (Tenover et al., 1995). However, there are no criteria for interpreting the banding pattern on long-term studies. In 1996, the European Study Group on Epidemiological Markers (Struelens et al., 1996) proposed guidelines to evaluate the efficacy of typing methods. Updated criteria include, mainly, the study of in vitro
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stability, reproducibility, typeability, and discriminatory power of the typing systems. Another criterion to evaluate typing methods is the epidemiologic concordance (EC) which is defined for each outbreak as the fraction of isolates with the epidemic type. This represents a relative measure of in vivo stability (Struelens et al., 1996). Moreover, it was interesting to compare the identification of clonal groups by independent typing methods (typing methods concordance) because the greater the number of techniques allowing isolates to be concordantly grouped, the more likely such isolates are to be clonally related. Besides, this Study Groups suggested that the DNA banding pattern polymorphism of each species should be taken into account to define strains (Struelens et al., 1996). This study addressed the quantitative evaluation of performance criteria for REP-PCR, and macrorestriction to type A. baumannii isolates. The efficacy of each technique as a typing system was estimated by the quantitative expression of reproducibility, stability, typeability, discriminatory power, and epidemiologic concordance. In addition, typing methods concordance was also evaluated. To this end, the A. baumannii test population included outbreakrelated, sporadic, and epidemiologically unrelated isolates.
2. Materials and methods 2.1. Bacterial strains The 99 A. baumannii isolates studied were obtained from infected patients recovered at 7 different hospitals (H1 to H7) located in Buenos Aires City (Table 1). Fifty-four isolates were recovered during 3 well-characterized outbreaks at hospitals H1 (Ratto et al., 1995), H2 and H4 (Catalano et al., 1999): 29 from endemic infections at hospitals H3 and H5 (isolated during September 1994 –September 1995 and from November 1995 to April 1996, respectively). The remaining sixteen, were epidemiologically unrelated isolates from an A. baumannii collection stored at our laboratory during 1981–1988 and recovered from hospitals H6 and H7. Type strain ATCC 19606 was also included in the study. Isolates were identified at species level using Bouvet and Grimont’s proposed phenotypic scheme (Bouvet and Grimont, 1986). Genospecies 2 was confirmed by EcoRI ribotyping (Gerner-Smidt, 1992). Biotypes were identified as proposed by Bouvet and Grimont (Bouvet & Grimont., 1987). Until used, isolates were frozen at ⫺70°C in Brain Heart Infusion (BHI) (Difco Laboratories, Detroit, USA) and supplemented with 20% glycerol. 2.2. Extraction of genomic DNA For macrorestriction analysis, bacterial concentration was adjusted in TN solution (10 mM Tris-HCl [pH 7.60], 1M NaCl) as previously described (Soares et al., 1993). Then, 150 L of the adjusted suspensions were mixed with
Table 1 Typing results of the 100 Acinetobacter baumannii isolates Isolate source and n° H1 outbreak* (Ratto et al., 1995). 1–3, 5, 6, 8–15, 17, 18 4 7 16 H2 outbreak 1Sa to 16 Sa H3 endemic infections 12, 16 3 9 17, 32 10 4, 6, 11, 29, 36 5, 7 39 18, 20, 22, 24, 26, 40 H4 outbreak (Catalano et al., 1999) 117 116 112 104, 105, 108, 109–111, 113–115, 118, 20, 122, 124, 125 107, 121 119 H5 endemic infections 91, 93, 95, 99 98 90 94 96 Unrelated isolates 19C, 28M 10M 32, 34, 41M 21 64M, 43M 47, 51M 8F 15F 101 50M 48M ATCC 19606
Biotype◊
REP-PCR
PFGE
2 9 2 9
A A B1 C
I I II III
2
A
I
2 2 2 9 9 9 9 9 8
A A A B1 B1 B E E E
I Ia Ib II II d II a IV IV a IV b
2 2 9 9
A A B1 B1
I Ib II b II c
9 8
E1 E1
IV a IV b
2 8 2 9 9
A B1 A E1 F
I I Ia IV b V
2 2 9 2 9 9 8 9 9 9 9 —
A A B1 B C C E E F H F1 J
I Ic II a II c III IIIa IV b IV V VI VII VIII
* H1, H2 and H4 outbreaks occurred during 1992, 1995 and 1996 respectively. H3 and H5 endemic infections occurred during 1995 and 1996 respectively. H6 and H7, unrelated isolates from the stored collection obtained during 1981–1988.
an equal volume of 1.6% low melting point agarose (BioRad, Richmond, CA, USA). Agarose disks were prepared by pippeting 20-L drops onto a plate covered with spacer slides and incubated at ⫺20°C during 5 min. Bacterial cells were lysed at 37°C overnight with gently shaking in lysozyme solution (6 mM Tris-HCl [pH 7,6], 1M NaCl, 100 mM EDTA [pH 7,5], 0.2% deoxycholate, 0.5% sodium lauroyl sarcosine, 0.5% Brij-58, 20 L RNase, and 1 mg lysozyme per mL), followed by a further overnight incubation at 50°C in protelysis buffer (0.5 M EDTA [pH 9], 1%
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sarcosyl, 50 g/mL proteinase K). The disks were washed three times with TE 10/0.1 (10 mM Tris, 0,1 mM EDTA), and were then stored at 4°C. For REP-PCR DNA was extracted as previously described (Ratto et al., 1995). 2.3. PFGE Genomic DNA was digested with ApaI (Promega Corporation, Madison, Wis, USA), according to the procedure described by the manufacturer. Endonuclease-digested genomic DNAs contained in disks were separated by PFGE. Disks were placed into the wells of a 1.2% agarose gel (Bio-Rad) in 0.5 X Tris-borate-EDTA buffer (45 mM TrisHCl-45 mM borate, 1 mM EDTA, pH 8), and separation of the DNA fragment was achieved with Bio-RAD CHEF-DR III system. Running conditions were 24 hrs at 14°C, with an initial switching time of 1 second and final time of 30 seconds, at 6 V/cm. Concatemers of DNA isolated from bacteriophage (New England Biolabs, Beverly, MA, USA) were run next to DNA fragments for size comparison. 2.4. REP-PCR The primer pair REP1R-I (5⬘-IIIICGICGICATCIGGC3⬘) and REP2-I (5⬘-ICGICTTATCIGGCCTAC-3⬘) was used to amplify putative REP-like elements. Amplification reaction was performed in a final volume of 50 L, according to Snelling et al., 1996. REP-PCR amplified DNA fragments were resolved by electrophoresis in a 1.5% agarose gel (GIBCO/BRL Life Technologies, Inc., MO, USA). A molecular weight marker (1 kb ladder, GIBCO/BRL Life Technologies), was loaded onto the agarose gel for size comparison. All amplification reactions were carried out in a Gene Amp PCR System 9600 Perkin Elmer thermal cycler (Perkin Elmer Cetus, Norwalkm, Conn. USA). Fingerprints were considered to be highly similar when all visible bands from two isolates had the same apparent migration distance. Variations in band intensity or shape were not taken into account. 2.5. Analysis of DNA banding patterns DNA banding patterns were analyzed by visual examination, and all loci were scored for the presence or absence of a band. Strains delineation was inferred in terms of percentage similarity of banding patterns (Struelens et al., 1996). Based on our tested population, isolates showing less than 75% of similarity were considered as clonally unrelated and those isolates presenting similarities higher than 75% were classified as subtypes of the same clone. The percent similarity of banding patterns was estimated with the Dice coefficient (Dice, 1945). Cluster analysis was performed by the UPGMA (unweighted pair-group method with arithmetic averages) using the software program NTSyS-PC version 1.40.
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2.6. Performance criteria Reproducibility (R), in vitro stability, typeability (T), and epidemiologic concordance (EC) were calculated suggested by Struelens et al (Struelens et al., 1996). To assess reproducibility, all 100 DNAs were evaluated by three independent digestions or amplifications and electrophoretic runs. To calculate S value, 10 randomly selected isolates were repeatedly passaged on agar culture media. Briefly, the experiment was carried out as follows: isolates from frozen stocks were grown on BHI agar. A single colony of each isolate was transferred to BHI and incubated 18 hrs at 30°C. DNA from each isolate was obtained and designated as FE (first extraction). All 10 isolates were then subjected to 25 serial passages on BHI agar. The FE fingerprint of each isolate was compared with the others obtained using DNA extracted after every 5 subcultures of the 25 total passages (SC5 to SC25, subcultures 5 to 25). DNAs from all isolates (both FE and SC) were analyzed by both REP-PCR and by macrorestriction. As to the outbreaks, EC was calculated on each set of isolates recovered at hospitals H1, H2 and H4. The EC for epidemiologically unrelated isolates was calculated as the fraction of isolates with the most frequent type. Unrelated isolates concordance was estimated with two sets of isolates, 30 endemic infection-related A. baumannii isolates (sporadic cases), recovered at H3 and H5 hospitals, and the other 17 belonging to the epidemiologically unrelated collection. ECs were statistically tested by Fisher’s exact test, with one-tailed P value. Discriminatory power was estimated by the formula of Simpson’s index of diversity (Hunter and Gaston, 1988). To assess DI value, the 16 epidemiologically unrelated isolates from a collection recovered from hospitals H6 and H7 during 1981–1988 and A. baumannii ATCC 19606 type strain were considered.
3. Results Table 1 shows raw data obtained with REP-PCR and macrorestriction, considering the 100 A. baumannii isolates. 3.1. Reproducibility and stability For macrorestriction, and REP-PCR the reproducibility estimation, considering the 100 isolates, yielded a R value of 100%. Macrorestriction classified the 10 isolates randomly selected for stability analysis, in six banding patterns (I, II, III, IV and Ia and IIa). No modifications on banding patterns were observed after 25 serial subculture passages (Table 2). REP-PCR classified the 10 isolates in five fingerprints (A, B, C, E, and B1). Two isolates that predominantly exhibited type A, shifted to type B, at least in one of the serial subcultures (Dice coefficient: 52%). Meanwhile, two further
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Table 2 Macrorestriction using Apa I and REP-PCR in vitro stability results. Isolate
1 2 3 4 5 6 7 8 9 10
PFGE Types
REP-PCR Types
FE*
SC5#
SC10
SC15
SC20
SC25
FE
SC5
SC10
SC15
SC20
SC25
I I I II III I Ia II a IV II a
I I I II III I Ia II a IV II a
I I I II III I Ia II a IV II a
I I I II III I Ia II a IV II a
I I I II III I Ia II a IV II a
I I I II III I Ia II a IV II a
A A A B1 C A A B E B
A A A B1 C A A B E B
A B A B1 C A A B E B
B A A B1 C A A A E A
A A B B1 C A A B E B
A A B B1 C A A B E B
* FE: First extraction, DNA obtained from the first isolate culture. # SC: subculture
isolates that at first exhibited type B shifted to genotype A. The remaining isolates type A preserved this type along culture passaging (Table 2). Furthermore, isolates showing types C, E and subtype B1, remained with an unchanged banding pattern during serial subcultures. The average S value was 90%, ranging from 66.6% to 100%. 3.2. DNA bands resolution Macrorestriction and REP-PCR generated DNA fingerprints for all 100 A. baumannii isolates, thereby, T was 100%. Fingerprints generated by macrorestriction with ApaI showed 11–15 bands per isolate located between 48.5 and 436.6 kbp, and a total of 41 different bands. REP-PCR fingerprints showed 10 to 12 bands per isolate in the 250 – 4,000 bp range, with a total of 26 different bands. Macrorestriction typing of the 99 A. baumannii isolates recovered from infected patients, and the ATCC 19606 strain, generated 18 distinguishable DNA patterns. REP-PCR typing exhibited 10 different fingerprints. 3.3. DNA patterns similarities Fig. 1A shows clustering analysis of all fingerprints generated by macrorestriction, and Fig. 1B several banding profiles. At similarity level (Dice coefficient) of 75%, 8 clusters were distinguished. Similarities within clusters ranged from 25% to 74%, representing fingerprints with 7–17 band difference. Fingerprinting assigned to the same cluster showed Dice coefficients ranging from 74 to 92%. This represented 2– 6 bands difference. Clusters 1 and 5 showed 2 subclusters. Fingerprint Ia, located at one subcluster which was formed at similarity level close to 80% in cluster 1, belonged to one isolate recovered at H3 four months after types I isolation (Table 3), thereby, it seemed appropriate to be considered as a subtype of the parental type I isolates. Subtype Ic, showing 80% of similarity with type I (5 bands difference), belonged to an already known epidemiologically unrelated isolate, therefore, no epidemi-
ological evidence supported related clonality between both fingerprints. Fingerprint IId, located alone at one subcluster linked at 75% of similarity with type II in cluster 5 (6 bands difference), belonged to one isolate recovered at H3 one year after type II isolates (Table 3), and presumably, represented a probably related isolate. Outbreak strain, from H1 and H2 hospitals (Table 1), was located in cluster 1. In spite of the fact that these outbreaks were reported during 1992 and 1995 respectively, they were both caused by the same epidemic strain. In H4 outbreak, the epidemic strain was located in cluster 5, with a similarity coefficient respect to type I close to 40%. Similarities between fingerprints of cross-transmitted strain during H1 and H4 outbreaks and their respective epidemic strains ranged from 24 to 48% showing 10 –19 bands difference. The banding patterns exhibited by endemic infection-related isolates (H3 and H5 hospitals, Table 1) were located in clusters 1, 3, 4 and 5, with similarities ranging from 50% to 75%. Even though the 17 isolates of the epidemiologically unrelated collection were spotted in all clusters, except cluster 3, both clusters 7 and 8, included only fingerprints belonging to this collection. Their similarity range, with respect to the epidemic strains, was 18 – 40%. Dice coefficients of each comparison within the epidemiologically unrelated isolates exhibited by these isolates ranged from 40% to 75%. Fig. 2A shows REP-PCR clustering analysis. At a linkage level of 75%, seven clusters were observed. Similarities between clusters ranged from 24% to 66%, representing fingerprints with 7–14 bands difference. Similarities in fingerprints placed in each cluster were 78 – 82% represented a 3– 4 bands difference. As in macrorestriction clustering analysis, REP-PCR fingerprints of outbreak strains were located in clusters 1 and 5, showing a similarity of 48%. For outbreak-related isolates, the similarity range within crosstransmitted and epidemic strains fingerprints was 48%– 64%. Fingerprints of endemic infection-related isolates were located in clusters 1, 3, 5 and 7. REP-PCR grouping of
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Fig. 1. A. Macrorestriction clustering analysis. The arrow shows cut-off value. Alphanumerical symbols on the right refer to types and subtypes listed in Table 1. B. Macrorestriction fingerprints of 14 A. baumannii strains. Lane 1, type I; lane 2, type Ia; lane 3, type IV; lane 4, type II; lane 5, type IIb; lane 6, type III; lane 7, type Ib; lane 8, type IIa; lane 9, type IId; lane 10, type V; lane 11, type IVb; lane 12, type IIIa; lane 13, type VI; lane 14, type VII. M: lambda phage ladder DNA concatemers (band sizes are expressed on the right in kilobases).
all isolates showing PFGE type II and subtypes IIa, IIb, IIc and IId in one cluster would uphold the relatedness of all these PFGE subtype to type II. Moreover, PFGE fingerprints IId and II exhibited the same REP-PCR subtype (Table 3). For REP-PCR, isolates epidemiologically unrelated collected during 1981–1988 were heterogeneously distributed in all clusters except number 3 (Fig. 2A). As for the ones located in cluster 4 were mainly found within this collection of isolates. Among this collection, the isolate identified as PFGE subtype Ic exhibited REP-PCR type A, similarly to others grouped in cluster 1 by macrorestriction, thereby, it supported the classification of this isolate as clonally related to PFGE type I, mentioned above. The major discrepancy between macrorestriction and REP-PCR clustering analysis lay in the classification of the fingerprint exhibited by a single isolate belonging to the epidemiologically unrelated collection. For macrorestriction
this banding pattern was located alone in cluster 8 (type VII, Fig. 1A); however, by REP-PCR it was located in cluster 7 (subtype F1), showing 78% of similarity (4 bands difference) with type F (Fig. 2B, lanes 5 and 11 respectively). REP-PCR type F exhibited PFGE type V, situated alone in cluster 3, showing 38% of similarity with PFGE type VII (18 bands difference), (Fig. 1B lanes 10 and 14 respectively). 3.4. EC and typing methods concordance All outbreak-related isolates recovered at H2 hospital showed identical fingerprints by either macrorestriction or REP-PCR (EC 100%). For both methods, 16 out of the 18 isolates from H1 outbreak belonged to the epidemic clone (EC 88.8%). The remaining two isolates were identified as two different clones (cross-transmitted strains) (Table 1). Fifteen out of the 20 isolates which was obtained from
Table 3 Concordance of REP-PCR and macrorestriction for typing sporadic cases-related A. baumannii isolates. REP-PCR
Macrorestriction Sep94
Dec94
Mar95
A B B1 E
II a (2)
Apr95
May95
I (1)#
I (1) I b (1)
II a (1)
II a (1)
IV (1)
IV (1)
Jun95
Jul95
Aug95
I a (1) II a (1)
II (2)
# Number of isolates
Sep95
IV a (1)
IV b (3)
IV b (1)
II d (1) IV b (2)
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Fig. 2. A. REP-PCR clustering analysis. The arrow shows the cut-off value. Alphanumerical symbols on the right refer to types and subtypes listed in Table 1. B. REP-PCR fingerprints. Lane 1, subtype B1; lane 2, type A; lane 3, type E; lane 4, type B; lane 5, subtype F1; lane 6, type C; lane 7, type H; lanes 8 and 9, subtype E1; lane 10, ATCC 19606 strain; lane 11, type F. M: 1 Kb DNA ladder.
patients during H4 outbreak, were identified as belonging to the epidemic type by REP-PCR. Fourteen out of these 15 isolates were classified as the epidemic type by macrorestriction while the remaining one as a very closely related subtype (96% of similarity). Thus, EC value was 75% for both methods. Similarly, macrorestriction and REP-PCR classified the remaining 5 isolates in two different types. For outbreak-related isolates, average EC for macrorestriction and REP-PCR was 88%. Hence, by concordance of outbreak-related isolates, all typing methods showed a similar specificity to delineate the epidemic clonal group. For endemic infection-related isolates recovered from H3 hospital, macrorestriction and REP-PCR indicated 9 out of the 21 isolates as belonging to the most frequent type. All 9 isolates exhibited an undistinguished banding pattern by REP-PCR typing (type E, Table 3). For macrorestriction, two of these isolates were identified as type IV, one as subtype IVa, and the remaining 6 subtype IVb. All of these isolates were considered as belonging to the same clone (Table 1). The EC value of each method was 43%. The other 12 isolates were classified into two separate clonal
groups by both methods (Table 1). Macrorestriction identified 5 out of the 8 endemic infection-related isolates recovered from H5 hospital, as belonging to the most frequent type. The others were classified into three different types (EC 63%). REP-PCR classified also these 8 isolates in four types, with an EC value of 50%. Average EC values for macrorestriction, and REP-PCR were 53% and 47%, respectively (P ⫽ 0.5). For epidemiologically unrelated isolates, EC values for macrorestriction and REP-PCR were 24%. Based on the 99 A. baumannii isolates recovered from infected patients, the most frequent type was the one assigned as A/I by REP-PCR and macrorestriction. Actually, isolates showing this type were recovered from all 7 hospitals studied, regardless the year of isolation or epidemiologic condition. A total of 40 out of the 99 A. baumannii isolates showed this type, 39 belonging to biotype 2 and one to biotype 9. However, one isolate showing PFGE type I, exhibited REP-PCR type B1, belonging to biotype 8. All isolates showing PFGE IV, IVa and IVb, were grouped by REP-PCR in cluster 4 as type E or subtype E1. Biotyping
Table 4 Epidemiologic Concordance of the two typing methods. Method
EC* Outbreaks**
Macrorestriction REP-PCR
Endemic infections#
Mean (%)
Range (%)
Mean (%)
Range (%)
Unrelated isolates# (%)
88 88
75–100 75–100
46 53
43–50 43–63
24 24
* EC Epidemiologic concordance. ** Epidemiologic concordance: (frequency of outbreak type/n° of outbreak isolates tested) ⫻ 100. # Epidemiologic concordance for endemic infection-related isolates and epidemiological unrelated isolates: (frequency of the most frequent types/n° of isolates tested) ⫻ 100.
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classified isolates belonging to PFGE IV and IVa as biotype 9, and eight of the 9 isolates PFGE type IV b were biotype 8 (Table 1). Hence, a lack of concordance was observed between biotyping and macrorestriction or REP-PCR typing. 3.5. Discriminatory power and typing of A. baumannii isolates A linkage level lower than 75% of similarity was used as a criterion to classify isolates into different clonal groups. Taking into account the 16 epidemiologically unrelated isolates and A. baumannii ATCC 19606 strains, DI for macrorestriction and REP-PCR were very similar (0.884 vs 0.877).
4. Discussion Strain classification has been achieved by a number of different DNA-based methods, all of which must meet several criteria in order to be widely applicable. In this study, 100 A. baumannii isolates were typed by currently used genotyping methods, to evaluate their performance, particularly their reproducibility, in vitro stability, discriminatory power, and epidemiologic concordance. Macrorestriction and REP-PCR showed the optimal reproducibility value. The analysis of stability under laboratory manipulation demonstrated that macrorestriction was a very stable typing system, since no modifications of banding patterns were observed after 25 serial passaging. However, REP-PCR average S value was close to that showed by macrorestriction. By clustering analysis, macrorestriction classified the 100 A. baumannii isolates into 8 clusters and REP-PCR supported the grouping of PFGE clusters 1, 2, 4, 5, 6, and 7. All fingerprints were linked with similarities exceeding 24%, and the two outbreak types were allocated to clusters 1 and 5 by both methods. DNA patterns exhibited by endemic infection-related isolates were assigned to four different clusters by both techniques. Likewise, epidemiologically unrelated isolates were heterogeneously distributed in all clusters. Two main discrepancies were observed between macrorestriction and REP-PCR clustering analysis results. The first discrepancy was that REP-PCR fingerprints grouped in cluster 7 were located by PFGE in cluster 3 and 8. This finding represents a lack of discrimination by REPPCR rather than the identities of isolates because these fingerprints were exhibited by already known epidemiological unrelated isolates. The second one was the quantity of fingerprints with equal to or greater similarities than 75% identified by macrorestriction within clusters 1, 4 and 5. These discrepancies may be due to the difference in the number of resolution bands observed among these methods, or to the DNA banding pattern polymorphism detected by both methods. In this sense, macrorestriction showed
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greater DNA banding pattern polymorphism than REPPCR. Thus, generation of changes in chromosomal polymorphism, as displayed by PFGE may occur at a faster rate than changes detected by the REP-PCR typing method. This presumption would be stressed by the recover of PFGE subtype IId isolate one year after of the first isolation of type II at H3, showing 6 bands difference and classified as the same type by REP-PCR. In this regard, Arbeit et al., 1990, demonstrated the ability of macrorestriction to resolve recent evolutionary divergence among E. coli isolates which were indistinguishable by other methods. For outbreak-related strains, difference between epidemic strains and cross-transmitted clones were clear cut for both methods, including the H4 outbreak whose span period lasted for 4 months. Thus, for outbreak-related isolates, both typing methods showed similar specificity to delineate the epidemic clonal group (similar EC value). Epidemiologic concordance for outbreak-related isolates may also be considered as a measure of typing method stability under “field” conditions (Struelens et al., 1996). The latter include all the conditions to which strains are subjected during their spread within and among patients, vectors and environmental reservoirs, rather than a single or a few hosts. At this point, macrorestriction and REP-PCR showed an EC value close to the ideal as proposed by Struelens et al (Struelens et al., 1996). Excepting one, all isolates belonging to epidemic strains exhibited indistinguishable banding patterns by both methods, pointing out the highest in vivo stability of these epidemiological markers. With respect to both the epidemiologically unrelated isolates and for the endemic infection-related isolates (sporadic isolates), macrorestriction and REP-PCR showed similar EC to identify the most frequent type. Based on the 17 epidemiological unrelated isolates and a linkage level lower than 75% of similarity to define separate clonal groups, both methods showed similar DI value. Actually, DI value varied according to the criteria used for strain definition. Interpreting criteria for macrorestriction to define the relatedness of isolates were published. However, most of them were mainly directed to identify epidemic strains from isolates recovered over periods of up to 3 months. These general guidelines allow differences of up to six bands (two genetic events) for isolates clonally related (Tenover et al., 1995; Tenover et al., 1997). However, the European Study Group on Epidemiological Markers suggested that the degree of relatedness to define strains should be adjusted to each species because different species may vary in the degree of the DNA banding pattern polymorphism revealed for each typing system (Struelens et al., 1996). In the present study, the known temporary epidemiologically unrelated isolates linked at a similarity lower than 75% (showing 7–17 bands difference), and isolates epidemiological related were linked mainly at similarity equal or greater than 80% (2–5 bands difference). Therefore, taking into account the set of A. baumannii isolates analyzed in the present study, it seemed appropriate to regard differences
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equal or greater than 7 bands to identify separate clonal groups by macrorestriction analysis, even in long-term studies. For PCR-based methods, there are no criteria for interpreting bands size variation or the value of intensity of several bands. The lack of these criteria lay on the fact that there is no restriction site. Hence, bases to standardize strain definition represent an area of active research. In this regard, typing methods concordance is suggested as a good principle to validate the identification of separate clonal groups because natural groups must be identical or very similar when tested by methods that measure independent markers (Struelens et al., 1996). Considering all 100 A. baumannii isolates, by concordance, REP-PCR identified 7 out of the 8 clonal groups recognized by macrorestriction. Hence, a clear cut among related and non-related isolates was observed (more than 82% of similarity) representing 3– 4 bands difference, and 75% of similarity representing more than 7 bands difference. Therefore, an equal or greater number than 7 bands difference seemed appropriate to define separate clonal groups for this genospecies. Using criteria mentioned above to define strains, our results showed a limited numbers of A. baumannii clones involved in nosocomial infections during 1981–1996. During this study, REP-PCR/PFGE type A/I was the most prevalent clone, recognized as epidemic or endemic strain at different hospitals. The limited number of A. baumannii clones causing nosocomial infections was also found by Vaneechoutte et al (Vaneechoutte et al., 1995), using other typing methods. In conclusion, when macrorestriction is regarded as “gold standard” for the molecular typing of A. baumannii isolates, REP-PCR displayed a similar performance. Therefore, when the convenience criteria, such as rapidity, accessibility, and ease of use are considered, REP-PCR is the most suitable method to type A. baumannii isolates for clinical purposes. Relatedness lower than 75% of similarity or 7 bands of difference seemed a good approach to identify separate clonal groups of A. baumannii by this technique. However, given the slightly higher capacity of macrorestriction to recognize recent evolutionary divergence, this method is more accurate when storage of genetic patterns for the creation of reference databases is required.
Acknowledgments This study was supported by grants from Alberto Roemmers Foundation and Universidad de Buenos Aires (UBACYT TM 09).
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