Characterization of oligonucleotide probes for the identification of Acinetobacter spp., A. baumannii and Acinetobacter genomic species 3

© INST1TUTPASTEUR/ELSEVIER Paris 1998

Res. Microbiol. 1998, 149, 557-566

Characterization of oligonucleofide probes for the identification of Acinetobacter spp., A. baumannii and Acine¢obacter genomic spedes 3 C. Lagatolla (*), A. Imvenia, E. Tonin, C. Monti-Bragadin and L. Dolzani

Dipartimento di Scienze Biomediche, Universith degli Studi, Via Fleming 22, 34127 Trieste (Italy)

SUMMARY

The 16S-23S intergenic spacer regions of four Acinetobacter genomic species belonging to the A. calcoaceticus-A, baumannii (Acb) complex, i.e. genomic species 1 (11. calcoaceticus), genomic species 2 (A. baumannii), genomic species 3 and ~emberg and Ursing (TU) genomic species 13, have been cloned and sequenced. Sequence analysis led to the discovery of a single copy of lie and Ala tRNA genes within each spacer. Sequence comparison allowed the identification of a 192-base-pair long highly conserved sequence between the 3' end of the 16S rRNA and the 5" end of the tRNAA~a genes. Moreover, two short regions, which were specific to, respectively, genomic species 2 and 3, could be identified. Oligonucleotides corresponding to these sequences were constructed and tested for the ability to hybridize with chromosomal DNA extracted from Acinetobacter belonging to different genomic species and with chromosomal DNA of other bacterial genera. One of these oligonucleotides was demonstrated to be useful as a sensitive and specific probe for A. baumannii. A less sensitive probe for Acinetobacter genomic species 3 was also developed.

Key-words: Oligonucleotide probe, Acinetobacter, Acinetobacter baumannfi; Identification.

INTRODUCTION Bacteria belonging to the genus Acinetobacter are divided into DNA groups or genomic species, on the basis of DNA/DNA relatedness (Bouvet and Grimont, 1986; Nishimura et al., 1987; Bouvet and Jeanjean, 1989 ; Tjernberg and Ursing, 1989; Gemer-Smidt and Tjemberg, 1993). Genomic species are designated by numbers, and seven have been given a species name, i.e. genomic species 1, 2, 4, 5, 7, 8/9 and 12 which correspond to A. cal-

Submitted March 6, 1998, accepted June 23, 1998. (*) Correspondingauthor.

coaceticus, A. baumannii, A. haemolyticus, A. junii, A. johnsonii, A. lwoffii and A. radioresistens, respectively. Due to discrepancies in the numbering systems adopted by different laboratories, the designation of genomic species 13-15 is equivocal (Bouvet and Jeanjean, 1989; Tjernberg and Ursing, 1989). We will refer to genomic species 13 sensu Tjernberg and Ursing (TU) throughout this work. Acinetobacter belonging to different genomic species are recovered with different frequencies from clinical specimens (Bouvet and Grimont,

558

C. LAGATOLLA ET AL.

1987; Tjemberg and Ursing, 1989; Gemer-Smidt and Tjemberg, 1993; Joly-Guillou and Brun-Buisson, 1996). Clinical isolates associated with nosocomial outbreaks, particularly from respiratory infections, blood c u l ~ s and wounds, are mostly identified as A. baumannii. This genomic species alone is res~nsible for more than 80% of all Acinetobacter infections in the hospital environment (Bouvet and Grimont, 1987; Bergogne-B&tzin and Towner, 1996). Genomic species 3 is also frequently reported as a cause of infection in hospitalized patients, while other genomic species (i.e. A. lwofffi, A. johnsonii and A. junii) are found with variable frequency in pathological specimens, particularly from general practice (Gemer-Smidt and Frederiksen, 1993). A. calcoaceticus is considered mostly an environmental species (Bouvet and Grimont, 1987; Gerner-Smidt and Tjernberg, 1993). As a matter of fact, however, elucidation of the ecology and the epidemiology of the different genomic species has long been hindered by the absence of simple and reliable identification methods. This problem principally concerns the genomic species belonging to the so-called Acb complex, i.e. genomic species 1, 2, 3 and 13TU which are phenotypically very similar (GernerSmidt et al., 1991). Two other genomic species, indicated as "Close to 13TU" and "Between 1 and 3", have been described and assigned to the Acb complex (Gerner-Smidt and Tjemberg, 1993), but are probably rare. Apart from DNA/DNA hybridization, which is considered the gold standard, a reliable identification of the genomic species in the Acb complex can be achieved by ribotyping (Gerner-Smidt, 1992), by restriction analysis of the rDNAItS genes (Vaneechoutte et al., 1995), of the 16S-23S rDNA internal transcribed spacers (Dolzarfi et al., 1995) or of the entire rRNA transcription units (Garcia-Arata et al., 1997) and by AFLP fingerprinting (Janssen et al., 1997). These methods are quite complex or may be time-consuming to set up, so identifications in the clinical laboratories often do not reach the species level, notwith-

Acb

=

A. calcoaceticus-A, baumannii (complex).

DIG LB

= =

digoxigenin. Luria-Bertani (medium).

standing the fact that the complex includes both the more frequent pathogens and ,an environmental genomic species. While the importance of complete identification for the management of a single patient can be questionable, for epidemiological purposes it is mandatory. For this reason, the development of new identification methods would be very desirable. The 16S-23S rDNA intergenic spacer regions have been successfully used as markers for bacterial identification, both at the species and the subspecies level (Barry et al., 1991 ; Kostman et al., 1992; Jensen et al., 1993; Dolzani et al., 1994; Gtirtler and Stanisich, 1996; Lagatolla et al., 1996). In a previous work, we applied restriction analysis of these regions to the identification of Acinetobacter in the Acb complex (Dolzani et al., 1995). In the present work, the complete sequences of the 16S-23S rDNA intergenic spacer regions of four type or reference strains of the Acb complex were obtained and analysed, with the aim of finding sequences of potential use for designing molecular probes.

MATERIALS AND METHODS

Bacterial strains

The 16S-23S intergenic spacers were cloned from the reference or type strains of four genomic species belonging to the Acb complex (Bouvet and Grimont, 1986; Tjernberg and Ursing, 1989), respectively: ATCC 23055 x (A. calcoaceticus), ATCC 19606T (A. baumannii), ATCC 19004 (genomic species 3) and ATCC 17903 (genomic species 13TU). Oligonucleotide probes were tested for hybridization on 28 Acinetobacter strains well characterized in previous works (table D and on 108 clinical isolates, both of Acinetobacter and of other Gram-negative bacteria (tables I, II), which were collected at the Trieste City Hospital (Ospedale di Cattinam). Species identification of isolates different from Acinetobacter was achieved by conventional methods. Acinetobacter genomic species were determined by restriction analysis of both the

SDS

=

Taq TU

=

Thermus aquaticus.

=

Tjemberg and Ursing (genomic species).

sodium dodecyl sulphate.

DNA P R O B E S FOR IDENTIFYING ACINETOBACTER

16S-23S rDNA spacers (Dolzani et al., 1995) and the 16S rRNA genes (Vaneechoutte et al., 1995). All strains were grown in Luria-Bertani (LB) medium (Sambrook et al., 1989). Long-term storage was obtahled by freezing in LB medium additioned of 10 % dimethylsulphoxide.

DNA extraction Bacterial DNA was extracted as previously described (Dolzani et al., 1995). Briefly, bacteria of an overnight culture were washed once in Tris-HCl 10 mM, pH 8.0, EDTA 5 mM, and then suspended in the same buffer. Lysozyme (Sigma, USA) 1 mg/ml was added and cells were incubated at 37°C for 3060 min. SDS 0.5% and proteinase K (Sigma, USA) 100 ILtg/ml were then added and incubation was continued at 55°C until the solution became clear. Proteins were then removed by phenol extraction and nucleic acids were recovered by ethanol precipitation. Pellets were dissolved in TE buffer (Tris-HCl 10 raM, pH 8.0, EDTA 1 mM) and treated with T1 RNase (Boehringer GmbH, Mannheim, Germany). DNA quantification was performed by measuring the absorption of the solution at 260 nm and/or the fluorescence intensity after exposure to the intercalating dye Hoechst 33258 (Labarca and Paigen, 1980).

Amplification of the 16S-23S intergenic spacer regions Spacer regions were amplified by using primers complementary to conserved regions at the 3' end of the 16S gene and at the 5' end of the 23S region (Jensen et al., 1993). A recognition site for EcoRl (primer E) or for BamHl (primer B) was added at the 5' end of the oligonucleotide sequences to facilitate subsequent cloning. The modified primer sequences were: primer E: 5'-GGCTCGAATTCGAAGTCGTAACAAGG-3'; primer B: 5'TCAGTGGATCC.CAAGGCATCCACCGT-3' (recognition sites are underlined). Amplification reaction mixtures consisted of Tris-HCl 10 mM, pH 8.3, KCI 50 mM, MgCI 2 3.0 mM, dNTP 200 ~tM each, primers 0.5 l.tM each, 2.5 U Taq polymerase (AmpliTaq, PE Applied Biosystems, USA) and 100 ng template DNA, for a total reaction volume of 100 lxl. Samples were submitted to 35 cycles of 1 min at 95°C, 1 min at 55°C and 1 min at 72°C. Amplification products were separated by electrophoresis in 2 % agarose gels.

Cloning of the 16S-23S intergenic spacer regions Amplified DNA fragments corresponding to the spacer regions of the four Acinetobacter strains were

559

passed through a "Microspin S400HR" (Pharmacia Biotech, Bruxelles) column to remove unpolymerized nucleotides and excess primers. They were subsequently digested with EcoRI and BamHI restriction e n d o n u c l e a s e s , purified again by phenol extraction and ethanol-precipitated. They were finally dephosphorylated and ligated to the polylinker site of pUC19 (Yanisch-Perron et al., 1985). Escherichia coli DH5o~ was used as a recipient of recombinant plasmids. Dephosphorylation, ligation and transformation procedures were performed as described by Sambrook et al. (1989).

DNA sequencing and analysis One clone for each genomic species was sequenced using the Silver Sequence DNA Sequencing System (Promega, USA). Each sequencing reaction consisted of four samples containing: Tris-HCl 50 mM, pH 9, MgCI23.0 raM, dNTP 10 l.tM each, plus one of the ddNTP (ddGTP 15 l.tM, ddATP 175 l.tM, ddCTP 100 I.tM, ddTTP 300 l.tM), primer 2 l.tM, 1.2 U sequencing grade Taq polymerase and 4 l.tg template DNA in a total reaction volume of 6 ~tl. Samples were submitted to 60 cycles of 45 s at 95°C, 2 min and 30 sec at 55°C and 1 rain at 75°C in a "GeneAmp PCR System 2400" (PE Applied Biosystems, USA). Reaction products were resolved on denaturing 4 % polyacrylamide gels and stained with silver nitrate following the instructions of the manufacturers (Promega). DNA was sequenced in both directions. Sequence analysis was carried out using the Inteliigenetics Suite software package (lntelligenetics Inc., USA).

Dot-blot analysis Amounts of 1 I.tg of DNA extracted from bacteria of genera different from Acinetobacter and 500 ng of DNA extracted from A c i n e t o b a c t e r strains were spotted on positively charged nylon membrane (ZetaProbe, Bio-Rad, USA) and fixed by UV irradiation. O l i g o n u c l e o t i d e probes were synthesized by "Primm" (Milano, Italy) and HPLC-purified. They were marked by polyA tailing using the DIG (digoxigenin) oligonucleotide 3'-end-labelling kit (Boehringer GmbH, Mannheim, Germany). Hybridization, with a probe concentration of 5 pmol/ml, was prolonged for 150 min at 45°C for Aci0 and Aci2, and at 42°C for Aci3. Hybridization buffer was as follows: NaCI 750 mM, sodium citrate 75 mM, pH 7.0, blocking reagent (Boehringer GmbH, Mannheim, Germany) 1% (w/v), N-lauroylsarcosine 0.1% (w/v), SDS 0.02% (w/v). Detection of hybrids was obtained by reaction with alkaline phosphatase-conjugated anti-DIG Fab fragments followed by addition of the colourimetric substrates of alkaline phos-

560

C. L A G A T O L L A E T AL.

phatase, X-phosphate and NitroBlue tetrazolium chloride, in the conditions suggested by the manufacturers (Boehringer Mannheim GmbH, 1993).

RESULTS

Amplification and cloning of the 16S-23S rRNA intergenic spacer regions Amplification of the spacer regions gave rise to the production of a single DNA fragment for each genomic s ~ i e s , as revealed by agarose gel electrophoresis (not shown). Both the presence of a single band and the amplicon lengths were consistent with previous results obtained with an external pair of primers (Dolzani et al., 1995). Amplicon lengths were evaluated to be 720 for Acinetobacter genomic species 2 and 13, 730 bp for genornic

species 3 and 740 bp for genomic species 1. Their insertion in the polylinker region of pUCI9 and subsequent transformation of recombinant plasmids into E. coil DH5ct generated several tens of clones for each construct. One of these clones was sequenced for each genomic species.

Sequence of the 16S-23S intergenic spacer regions Nucleotide sequences of the 16S-23S rDNA spacer regions have been given the following GenBank accession numbers: U60278 for genomic species 1, U60279 for genomic species 2, U60280 for genomic species 3 and U60281 for genomic species 13. A synthetic picture of the 16S-23S spacer organization in Acinetobacter is given in figure 1.

Aci0: ~TCC~Ca.~C~QT'rO:TCT:C~T~ Aci2

rRNA.I.6.S~-~IRNAIIe~

--

Aci3

23S

-,i rRNA L .

.

.

.

.

.

.

.

.

: : : 25 bp Aci2:Gs13

370 AGcaTGAcCTGACGAAGgCgtgTTtAtT

GS2

370 AGtgTGAtCTGACGAAGsCa©aTTaAQT

GS3

370 AGCATGACCTGACGAAGGCgTGTTTATT

GS1

371 AGCATGACCTGACGAAGGCaTGTTTATT

II Ill lilllllll i

Aci3:Gs13 GS2

il i I

II ill lllillill i II I i llilllilillilllilll lillill]

562 TaCTCTAAACTGAA

ATGTT

I llilllllilll TgCTCTAAACTGAA llll

ATGTT

558

lIIil

GS3

565 AtagtagcGATGAAcgaatcGcAcg

GS1

569 AactgtaaGAgattgatcgaGaAat

i

II

II

Fig, 1, Organization of the 16S-23S rRNA intergenic spacer region in Acinetobacter belonging to the Acb complex and sequences of the probes tested in this work. The nucleotide sequence of Aci0 was identical in the four genomic species of the complex. The nucleotide sequences of Aci2 and Aci3 are shown in bold, aligned with the corresponding regions of the other genomic species, to show their specificity for genomic species 2 (A. baumannii) and genomic species 3, respectively. Nucleotide numbering is the same as the sequences deposited in GenBank.

DNA PROBES FOR IDENTIFYING ACINETOBACTER

Alignment of the four sequences showed a nearly perfect homology from nucleotide 1 to 317. Analysis of this region disclosed the presence of two tRNA genes within each spacer: tRNAne from base 70 to base 147 and tRNAAI a from base 204 to base 279. DNA regions between the 3' end of the 16S rRNA gene and the 5' end of the tRNAne gene and between the two tRNA genes were found to be highly conserved in these four strains. Searching these sequences against public databases revealed that both sequences are unique to Acinetobacter. In contrast, the region downstream from the tRNAAIa gene, approximately from nucleotide 318 to the end of the spacers, was much less well conserved. This region contained some short sequences, which were found to be particular to the different genomic species and therefore of potential use for species differentiation. To assess this possibility, we synthesized two probes, called Aci2 and Aci3, which were designed to be specific, respectively, for genomic species 2 and 3. A third oligonucleotide, Aci0, reproducing a part of the sequence situated between the 3' end of the 16S rRNA gene and the 5' end of the tRNAne gene, was also constructed, to be used as a genus-specific probe.

Genus-specific probe Aci0 is complementary to a 24-bp sequence in the conserved region upstream of the tRNA ne gene (fig. 1). To verify whether this sequence could really be considered specific for the genus Acinetobacter, as apparent from sequence analysis, Aci0 was tested for hybridization with the DNA of 53 Acinetobacter strains (table I) and of 78 isolates of bacteria belonging to other genera (table II). Results showed that Aci0 could hybridize with the DNA of all the Acinetobacter strains tested, which included both reference strains of 13 different genomic species and clinical isolates. On the contrary, Aci0 did not hybridize with any DNA extracted from strains listed in table II, indicating that this sequence was typical of the genus Acinetobacter. Representative results are shown in figure 2. The sensitivity of this probe varied with the genomic species, but was generally between 10 and 50 ng (data not shown).

561

Genomic species-specific probes The sequences of probes Aci2 and Aci3 were copied from the less conserved region downstream from tRNAAI a and were designed to be genomic species-specific (fig. 1). Hybridization tests of these probes with DNA of the type or reference strains of the genomic species 1, 2, 3 and 13TU are shown in figure 3a. No cross-reaction was noted between the different genomic species. The two probes were subsequently tested on the DNA of all the Acinetobacter strains listed in table I. Aci2 gave a positive hybridization signal only with the 14 strains of genomic species 2, thus demonstrating its specificity. Correspondingly, Aci3 hybridized only with DNA of genomic species 3 strains, with three exceptions, which were represented by one strain of the "Close to 13TU" and two strains of the "Between 1 and 3" DNA groups. Thus, these three strains were unexpectedly recognized by Aci3. To our k n o w l e d g e , only two strains are hitherto allocated to each of the genomic species "Close to 13TU" and "Between 1 and 3" (Gerner-Smidt and Tjernberg, 1993; Garcia-Arata et al., 1997). Both strains of the genomic species "Between 1 and 3" were recognized by the Aci3 probe. In a previous study (Dolzani et al., 1995), it was shown that restriction analysis performed by two different enzymes on amplified 16S-23S rDNA spacers of strains belonging to genomic species 3 and "Between 1 and 3" gave identical patterns. These results suggest that the spacers of the two genomic species have substantially similar nucleotide sequences. In contrast, only one of the two strains belonging to the genomic species "Close to 13TU" is recognized by the Aci3 probe. The two strains also performed differently in other situations, such as restriction analysis of the 16S-23S rDNA spacers (Dolzani et al., 1995) or of the entire rRNA transcription units (Garc]a-Arata et al., 1997). In this context, therefore, genomic species "Close to 13TU" appears to be nonhomogeneous. The sensitivity of the probes was also tested. Digoxigenin-labelled probes could detect 20 ng of

C. I.~GATOLLA E T AL.

562

T a b l e I. Hybridization o f Acinembacter D N A with genus- and species-specific probes. No. o f strains tested

A. calcoaceticus A. baumannii

A. genomic species 3

A. haemolyticus A. junii A.

genomic species 6

A. johnsonii A. lwoffii A. genomic species 9 A. genomic species 10 A. genomic species 11

A. radioresistens A. genomic species 13TU "Close to 13TU" "Between

1 and 3"

A T C C 23055 T(a) A T C C 17902 (a) A T C C 14987 (a) A T C C 19606 T(a) A T C C 17961 (a) A T C C 15149Ca) A T C C 17978 ca) Clinical isolates (b) A T C C 19004(a) A T C C 17922~a) A D A M 53 1984 (a) A D A M 202 OKI 55732 (a~ Clinical isolates (b) A T C C 17906 Tea) A T C C 17977 Ca) S E I P 23.77 Ca) A T C C 17908 Ta S E I P 883 (a) ATCC 17979Ca) A T C C 17909 Tea) A T C C 15309 T(a) Clinical isolates (b) A T C C 9957 Ca) A T C C 17924 ta) A T C C 11171 (a) C I P 1 0 3 7 8 8 TM A T C C 17903 (a) Clinical isolatestb) 10090 td) 5804 ~d) 10169
Hybridization with Aci0 Aci2 Aci3

1 1 1 1 1 1 1 i0 1 1

+ + + + + + + + + +

+ + + + + -

+ +

1

+

-

+

1 10 1 1 1 1

+ + + + + +

-

+ + --

1

+

-

-

1 1 1 5 1 1 1 1 1 5

+ + + + + + + + + +

-

i

+

-

1 1 1

+ +

-

+ +

+

-

+

-

Ca~Bouvet and Grimont, 1986, ~b)This work, cc~Strain from the Collection des bact6ries de I'lnstitut Pasteur, corresponding to FO-1T (Nishimura et al., 1987). ednA. calcoaceticus-baumannii complex (Gerner-Smidt et a1.,1991: Gerner-Smidt and Tjernberg, 1993).

c h r o m o s o m a l D N A in the case o f A c i 2 a n d 100 ng o f c h r o m o s o m a l D N A in the case o f A c i 3 (fig. 3b).

Acinetobacter g e n o m i c s p e c i e s 1 3 T U h a v e b e e n sequenced and analysed. After alignment of the four sequences, a common organization of the spacers became

DISCUSSION

In t h i s w o r k , t h e 1 6 S - 2 3 S r D N A i n t e r g e n i c s p a c e r r e g i o n s o f f o u r t y p e a n d r e f e r e n c e strains o f the Acb c o m p l e x , i . e . A , calcoaceticus, A. baumanni, Acinetobacter g e n o m i c s p e c i e s 3 a n d

evident. Two coding regions,

the tRNAne and the tRNAAla genes, were identified within the spacers. Perhaps more interesti n g , t h e s p a c e r s c o u l d b e d i v i d e d into t w o p a r t s , t h e first o f w h i c h , c o r r e s p o n d i n g r o u g h l y to t h e f r a g m e n t s i t u a t e d o n t h e 16S side, w a s a p p a r ently h i g h l y c o n s e r v e d a m o n g the four strains,

DNA PROBES FOR IDENTIFYING ACINETOBACTER

Table II. Gram-negative bacteria tested tbr hybridization with Aci0.

EL coli Proteus mirabilis Serratia marcescens S. blodev Klebsiella pneumoniae Citrobacter freundii Enterobacter cloacae Pantoea agglomerans Salmonella enteritidis S. ~,phimurium S. derby S. heidelberg Pseudomonas aeruginosa Burkholderia cepacia B. pickettii Comamonas acidovorans Stenotrophomonas maltophilia Alcaligenes xylosoxidans Ochrobactrum anthrophi Hafnia alvei

1

No. of strains tested

A

10

B

2 7 1 6 1 ! I 12 !2 I 1 13 5 2 1 5 3 1 1

All strains were clinical isolates identified by conventional phenotypic tests.

while the other part showed a higher variability among the four genomic species. The biological s i g n i f i c a n c e of this d i f f e r e n c e is p r e s e n t l y unknown. However, the first half of the spacer contains the tRNA genes quite close to each other, and it is known (Srivastava and Schlessinger, 1990) that the regions at the 3' and 5' ends of the tRNA and rRNA genes are generally well conserved because they are involved in RNA processing. Three oligonucleotide probes were synthesized and tested on a preliminary set of reference strains and clinical isolates. Two of them, Aci2 and Aci3, were designed, respectively, for genomic species 2 and 3. Both appeared to be specific for the genomic species they were designed for, with the exception of detection by Aci3 of three strains belonging to the "Close to 13TU" and "Between 1 and 3" DNA groups. To our knowledge, these genomic

C

D

2

563

3

4

5

6

~::

@

Fig. 2. Dot blot hybridization of DNA from Acinetobacter spp. (0.5 lag) and of DNA from bacteria of other genera (1 lag) with Aci0. Lines A-B: type strains of the different genomic species of Acinetobacter. From the left to the right: line A = genomic species 1 to genomic species 6; line B = genomic species 7 to genomic species 11, and genomic species 13TU. Lines C-D: clinical isolates. From the left to the right: line C = Serratia marcescens, Serratia blodev, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter cloacae and Acinetobacter genomic species 3; line D =Acinetobatter genomic species 2, Salmonella enteritidis, Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia and Alcaligenes xylosoxidans.

groups occur infrequently. Thus, this fact should not represent a problem. The sensitivity of the two probes differed considerably, with Aci2 being able to detect about five times less DNA than Aci3. This could be due to differences in the chemico-physical properties of the two oligonucleotides. In fact, despite an identical theoretical Tm, they have different optimal annealing temperatures. Moreover, this difference could be related to the copy number of the target sequence. In fact, at least 5-6 copies of rRNA transcriptional units are present in Acinetobacter (Dolzani et al., 1995). Restriction analysis of the spacer regions of genomic species 2 indicated that the nucleotide sequence is probably very well conserved in the different cepies, since a single restriction pattern was observed with all the endonucleases and in all the tested strains. The same kind of analysis for genomic species 3 demonstrated the existence of three different

564

C. L A G A T O L L A E T AL.

gs gs gs gs

a

1

2

3

13

A B

0

b

0 I~

A B

0

0 ,e-

0

ql'

N

A

iiii :!i~!i~i+i :+~~;

Fig. 3. Specificity (a) and sensitivity (b) of Aci2 and Aci3. a) Hybridization of DNA from four type strains of the Acb complex with Aci2 (line A) and Aci3 (line B). b) Hybridization of different amounts of DNA from the type strains of A. baumannii (line A) and Acinetobacter genomic species 3 (line B) with Aci2 and Aci3, respectively.

restriction patterns for Ndell and the coexistence of different spacer sequences within the same bacterial chromosome (Doizani et al., 1995). Since Aci3 is based on the nucleotide sequence of a single spacer region, it is possible that it recognizes only some of the spacer copies present in the chromosome of each isolate. The third oligonucleotide probe, Aci0, was designed to recognize bacteria of the genus Acinetobacter. Dot blot analysis showed that this probe was able to recognize all tested strains belonging to this genus, while it did not hybridize with DNA from other Gram-negative bacteria, whether fermentative or non-fermentative. Usually, in clinical practice, the identification of Acinetobacter at the genus level is achieved by conventional methods, so the need for a probe is questionable, even if it could be sometimes employed in confirmative assays for doubtful cases. It is worthwhile to note that the only unam-

biguous method |br genus identification currently available relies on the ability of extracted DNA to restore the wild-type phenotype to a mutant Acinetobacter strain in a transformation assay (Juni, 1972). Hybridization with the genus-specific probe is also useful as a control test for those specimens which do not hybridize with the genomic species-specific probes, as a positive signal indicates good sample quality. Finally, a possible use of the Aci0 probe in environmental studies could also be postulated, as acinetobacters are ubiquitous organisms that can be isolated from soil, water and sewage. An rRNA-targeted oligonucleotide probe specific for the genus A c i n e t o b a c t e r has recently been employed to investigate, by in situ hybridization, the role of Acinetobacter spp. in an activated sludge (Wagner et ai., 1994). In conclusion, the three probes tested in this

work appear to be very promising. This is especially true for Aci2, which is the most sensitive. Moreover, it enables discrimination between the most frequently isolated pathogen, i.e.A, baumannii (Bouvet and Grimont 1987; BergogneBErrzin and Towner, 1996) and the other components of the A c b complex, to which it is phenotypically closely related (Gerner-Smidt et al., 1991). Diagnostic use of these probes would need the extension of the tests to a greater number of strains and validation. However, if the results obtained in this work are confirmed, the availability of specific oligonucleotide probes could be the first step toward innovative methods for the identification of Acinetobacter strains at the genomic species level. Moreover, possible use of these three specific oligonucleotide probes in new technologies, like DNA chips (Caviani Pease et al., 1994), could be hypothesized.

Acknowledgments We are grateful to Dr. P. Gerner-Smidt (Statens Serum Institute, Copenhagen, Denmark) for providing some of the strains used in this work and to the Unit6 des Agents Antibact6riens (lnstitut Pasteur, Paris) for supplying the A. radioresistens strain. This work was supported by grants from Italian MURST.

DNA P R O B E S FOR I D E N T I F Y I N G ACINETOBACTER

Caract~risation de sondes o|igonucl~otidiques p o u r I'idenfificafion de A c i n e t o b a c t e r spp., A. b a u m a n n i i el Acinetobacter de |'esp~ce g~notypique 3 Les r6gions interg6niques 16S-23S de 4 esp6ces g6notypiques de Acinetobacter provenant du complexe A c b (A. c a i c o a c e t i c u s - A , b a u m a n n i i ) , c'est-?~-dire l'esp6ce g6notypique ! (A. calcoaceticus), l'espbce gfnotypique 2 (A. baumannii), l'esp~ce g6notypique 3 et l'esp6ce g6notypique 13 de Tjernberg et Ursing (TU) ont 6t6 c l o n f e s et les s 6 q u e n c e s ont 6t6 d6termin6es. L'analyse de la s6quence a permis de dfcouvrir dans chaque r6gion interg6nique la pr6sence, en s i m p l e copie, de gbnes c o d a n t I ' A R N t - l l e et I'ARNt-Ala. Les comparaisons de s6quence ont permis la raise en 6vidence d'un fragment hautement conserv6 de 192 paires de bases, entre l'extr6mit6 3' du g6ne codant I'ARNr 16S et l'extr6mit6 5' du g~ne c o d a n t I ' A R N t - A I a . De plus, 2 p e t i t e s r f g i o n s spfcifiques des esp~ces g6notypiques 2 et 3 ont 6t6 identifi6es. Des oligonucl6otides sp6cifiques de ces sfquences ont 6t6 choisis et testfs pour leur aptitude h hybrider avec de I'ADN chromosomique extrait de A c i n e t o b a c t e r p r o v e n a n t de d i f f 6 r e n t e s esp~ces g6notypiques et avec de I'ADN chromosomique provenant d ' a u t r e s genres bact6riens. Un de ces oligonucl6otides s'est montr6 utilisable comme sonde sensible et sp6cifique de A. baunumnii. Une sonde moins sensible a 6galement 6t6 d6velopp6e pour l'espbce g6notypique 3 de Acinetobacter. Mots-cl~s : Sonde oligonucl6otidique, Acinetobacter, Acinetobacter baumannii ; Identification.

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