Download Rapid identification of Acinetobacter baumannii, Acinetobacter

Journal of Medical Microbiology (2014), 63, 1154–1159
DOI 10.1099/jmm.0.071712-0
Rapid identification of Acinetobacter baumannii,
Acinetobacter nosocomialis and Acinetobacter
pittii with a multiplex PCR assay
Te-Li Chen,1,2 Yi-Tzu Lee,1,3 Shu-Chen Kuo,1,2,4 Su-Pen Yang,2,5
Chang-Phone Fung1,2,5 and Shou-Dong Lee6
Correspondence
1
Shou-Dong Lee
2
[email protected]
Institutes of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan
Division of Infectious Diseases, Taipei Veterans General Hospital, Taipei, Taiwan
3
Department of Emergency, Taipei Veterans General Hospital, Taipei, Taiwan
4
National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes,
Miaoli County, Taiwan
5
Emergency and Critical Care Medicine, National Yang-Ming University, Taipei, Taiwan
6
Department of Medicine, Cheng Hsin General Hospital, Taipei, Taiwan
Received 9 December 2014
Accepted 24 June 2014
Acinetobacter baumannii, Acinetobacter nosocomialis and Acinetobacter pittii are clinically
relevant members of the Acinetobacter calcoaceticus–A. baumannii (Acb) complex and important
nosocomial pathogens. These three species are genetically closely related and phenotypically
similar; however, they differ in their epidemiology, antibiotic resistance and pathogenicity. In this
study, we investigated the use of a multiplex PCR-based assay designed to detect internal
fragments of the 16S–23S rRNA intergenic region and the gyrB and recA genes. The assay was
capable of differentiating A. baumannii, A. nosocomialis and A. pittii in a reliable manner. In 23
different reference strains and 89 clinical isolates of Acinetobacter species, the assay accurately
identified clinically relevant Acb complex species except those ‘between 1 and 3’ or ‘close to
13TU’. None of the non-Acb complex species was misidentified. In an analysis of 1034 positive
blood cultures, the assay had a sensitivity of 92.4 % and specificity of 98.2 % for Acb complex
identification. Our results show that a single multiplex PCR assay can reliably differentiate
clinically relevant Acb complex species. Thus, this method may be used to better understand the
clinical differences between infections caused by these species.
INTRODUCTION
Acinetobacter baumannii, Acinetobacter nosocomialis and
Acinetobacter pittii have become important nosocomial
pathogens worldwide (Dijkshoorn et al., 2007; Peleg et al.,
2008). These three clinically relevant Acinetobacter species,
as well as an environmental species, Acinetobacter calcoaceticus (Nemec et al., 2011), cannot be reliably differentiated
with phenotypic tests. Because of their similar phenotypic
characteristics, these four species are grouped as A.
calcoaceticus–A. baumannii (Acb) complex (Gerner-Smidt
et al., 1991). A. baumannii is distinct from A. nosocomialis
and A. pittii, being resistant to more classes of antimicrobial
agents, associated with a relatively worse clinical outcome
and responding differently to appropriate therapy (Lee et al.,
2010, 2012b; Chuang et al., 2011; Wisplinghoff et al., 2012;
Abbreviations: Acb, Acinetobacter calcoaceticus–Acinetobacter baumannii; ITS, intergenic spacer; MALDI-TOF MS, matrix-assisted laser
desorption ionization–time of flight mass spectrometry.
1154
Kuo et al., 2013). Methods that can differentiate these
three clinically relevant Acinetobacter species are critical for
advancing our knowledge of the biology, pathogenicity and
therapy of these individual species.
The use of PCR assays offers the potential for rapid
detection and species identification of pathogens (Fredricks
& Relman, 1998). In this study, we describe a multiplex
PCR-based assay that can differentiate A. baumannii, A.
nosocomialis and A. pittii in bacterial colonies and positive
blood culture media.
METHODS
Bacterial isolates and identification. Three sets of bacteria were
examined in this study. The first set of bacteria included 23 reference
strains and 89 clinical isolates of Acinetobacter species with known
genomic species identification (Table 1). The reference strains were
purchased from public culture collections or kindly provided by
T.-C. Chang (Chang et al., 2005) and Alexandr Nemec (Nemec et al.,
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Rapid identification of the Acb complex
Table 1. Reference and clinical isolates used to validate the multiplex PCR assay for the identification of A. baumannii, A.
nosocomialis and A. pittii
Genomic species
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Total
Reference strains
calcoaceticus
baumannii
pittii
haemolyticus
junii
genomic species 6
johnsonii
lwoffii
bereziniae
guillouiae*
radioresistens
nosocomialis
genomic species 13BJ/14TU
genomic species 15TU
genomic species 15BJ
genomic species 16
genomic species 17
baylyi
beijerinckii*
gyllenbergii*
parvus
schindleri
soli*
ursingii
venetianus
belonging to ‘close to 13TU’
belonging to ‘between 1 and 3’
ATCC 17987
ATCC 19606T and 15151
ATCC 17922T
ATCC 17906T
ATCC 17908T
ATCC 17979
ATCC 17909T
0
0
LMG 988T
ATCC 43998T
ATCC 17903
0
CCUG 26390
CCUG 34436
BCRC 15883
CCUG 34437
ATCC 33305
NIPH 838T
NIPH 2150T
CCUG 48800T
LMG 19576T
CCUG 59023T
LMG 19575T
CCUG 45561T
0
0
No. clinical isolates
0
20
12
1
5
0
0
2
3
1
3
14
2
4
0
0
0
7
1
1
0
1
1
5
0
4
2
89
*Generous gifts from Professor A. Nemec.
2009, 2010). The clinical isolates were collected from 10 different
medical centres in Taiwan (Chen et al., 2010) or generously provided
by Alexandr Nemec (Nemec et al., 2009, 2010). The second set of
bacteria comprised 100 clinical Acb complex isolates that were
obtained from the Taipei Veterans General Hospital, without
knowledge of the genomic species prior to PCR identification. These
two sets of Acinetobacter isolates were used to verify the practicality of
the multiplex PCR method. Acinetobacter species were identified by
sequence analysis of the 16S–23S rRNA intergenic spacer (ITS) region
(Chang et al., 2005), amplified rDNA restriction analysis (Dijkshoorn
et al., 1998; Nemec et al., 2003, 2011) and rpoB sequence cluster
analysis (Nemec et al., 2009). Clonality of the clinical isolates of A.
baumannii, A. pittii and A. nosocomialis was determined by PFGE by
using the ApaI enzyme as described previously (Huang et al., 2008).
The third set comprised 1034 micro-organisms of different genera
and species (Table 2), based on results of the VITEK 2 system
(bioMérieux), that were prospectively collected from the Taipei
Veterans General Hospital from July 2010 to June 2012. This set was
used to test whether the multiplex PCR method could directly detect
and identify Acb complex in blood culture media.
Development of the multiplex PCR assay. Three pairs of primers
that had been previously designed and verified were included in the
multiplex PCR assay. The primers P-rA1 (59-CCTGAATCTTCTGGTAAAAC-39) and P-rA2 (59-GTTTCTGGGCTGCCAAACATTAC-39),
http://jmm.sgmjournals.org
which target a highly conserved 425 bp region of the recA gene of
Acinetobacter species, were used as a reaction control (Krawczyk et al.,
2002). Primers sp4F (59-CACGCCGTAAGAGTGCATTA-39) and
sp4R (59-AACGGAGCTTGTCAGGGTTA-39) yielded an amplicon of
294 bp from the gyrB genes of A. baumannii and A. nosocomialis
(Higgins et al., 2007). Primers P-Ab-ITSF (59-CATTATCACGGTAATTAGTG-39) and P-Ab-ITSB (59-AGAGCACTGTGCACTTAAG39) were used to specifically amplify an internal 208 bp fragment from
the ITS region of A. baumannii (Chen et al., 2007). For identification of
A. pittii, all of the available ITS sequences of different Acinetobacter
species deposited in GenBank were downloaded and aligned using
CLUSTAL W software. After considering specificity and annealing
temperature, primers P-AGS3-F (59-CTCAAGAGTTTAGATTAAGCAAT-39) and P-AGS3-R (59-GTCCGTGCGATTCTTCATCG-39)
were selected to amplify a 150 bp internal fragment from the ITS region
of A. pittii.
GoTaq Flexi DNA polymerase (Promega) was used for PCR assays
performed in the GeneAmp PCR System 2700 (Applied Biosystems).
PCR amplification consisted of an initial denaturation step at 94 uC
for 5 min; 35 cycles at 94 uC for 1 min, 58 uC for 30 s and 72 uC for
30 s; and a final extension at 72 uC for 10 min. For the PCR assays of
the first and second sets of Acb complex isolates, the DNA template
was prepared by boiling (Vaneechoutte et al., 1995). For the bacterial
PCR assays from blood culture media, the DNA templates were
prepared as described in the following section.
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T.-L. Chen and others
Table 2. Clinical isolates used in the validation of the multiplex PCR assay for detection and identification of A. baumannii, A.
nosocomialis and A. pittii from positive blood cultures
Micro-organism*
n
Multiplex PCR result
A. b
Gram-negative bacteria
A. baumannii
A. nosocomialis
A. pittii
A. junii
A. lwoffii
Other Acinetobacter spp.D
Achromobacter spp.
Aeromonas hydrophila
Aeromonas sobria
Bacteroides fragilis group
Burkholderia cepacia
Chryseobacterium indologenes
Chryseobacterium meningosepticum
Citrobacter diversus
Citrobacter freundii
Comamonas testosteroni
Escherichia coli
Enterobacter aerogenes
Enterobacter asburiae
Enterobacter cloacae
Fusobacterium spp.
Klebsiella oxytoca
Klebsiella ozaenae
Klebsiella pneumoniae
Kluyvera ascorbata
Morganella morganii
Pantoea spp.
Plesiomonas shigelloides
Prevotella spp.
Proteus mirabilis
Proteus penneri
Providencia stuartii
Pseudomonas aeruginosa
Pseudomonas putida
Ralstonia mannitolilytica
Salmonella group B
Salmonella group D
Salmonella paratyphi
Serratia marcescens
Shewanella algae
Sphingomonas paucimobilis
Stenotrophomonas maltophilia
Vibrio fluvialis
Vibrio mimicus
Gram-positive bacteria
Bacillus spp.
Coagulase-negative Staphylococcus spp.
Corynebacterium spp.
Enterococcus faecalis
Enterococcus gallinarum
Enterococcus spp.
Lactobacillus spp.
1156
981
70
51
10
1
2
9
4
10
1
9
12
10
12
9
6
1
372
18
1
36
1
7
1
180
1
4
1
1
2
24
2
1
40
3
1
3
30
1
13
1
2
15
2
1
46
2
18
2
1
1
4
1
76
65
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
4
0
0
0
4
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
2
0
0
1
0
0
1
0
A. n
A. p
Negative
50
0
48
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
8
0
0
8
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
847
5
3
2
1
2
9
4
9
1
9
12
10
12
9
6
1
372
18
1
31
1
6
1
176
1
4
1
1
2
23
2
1
40
2
1
3
30
1
13
1
2
15
2
1
43
2
18
1
1
1
3
1
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Journal of Medical Microbiology 63
Rapid identification of the Acb complex
Table 2. cont.
Micro-organism*
n
Multiplex PCR result
A. b
Rhodococcus equi
Staphylococcus aureus
Staphylococcus hominis
Streptococcus agalactiae
Streptococcus group C
Streptococcus group D
Viridans group streptococci
Yeast
Total
1
11
1
1
1
1
1
7
1034
A. n
A. p
Negative
0
0
0
0
0
0
0
0
50
0
1
0
0
0
0
0
0
9
1
10
1
1
1
1
1
7
897
0
0
0
0
0
0
0
0
78
A. b, A. baumannii; A. n, A. nosocomialis; A. p, A. pittii.
*Acinetobacter species identification of the Acb complex was performed using the PCR method; A. junii and A. lwoffii were identified using
amplified rDNA restriction analysis and rpoB sequence cluster analysis.
DIncluding A. haemolyticus (n51), A. johnsonii (n52), A. bereziniae (n51), A. radioresistens (n51), Acinetobacter genomic species 13BJ/14TU
(n51), A. baylyi (n51) and A. ursingii (n52). All were identified using rpoB sequence cluster analysis.
DNA purification from blood culture media. When bacterial
growth was detected in the culture bottle by the culture system (BacT/
Alert; Organon Teknika), 0.5 ml culture medium was collected from
the bottle and analysed by Gram staining. Samples that yielded Gramnegative coccobacilli or bacilli were subjected to DNA purification
followed by multiplex PCR identification. Gram-positive organisms
and yeast collected in the first 2 weeks were used to validate the PCR
identification method. DNA purification from positive culture media
was performed using the benzyl alcohol–guanidine hydrochloride
organic extraction method, as described previously (Fredricks &
Relman, 1998). Briefly, 0.1 ml inoculated blood culture media was
treated with lysis buffer containing guanidine hydrochloride in Tris
buffer and then mixed with 99 % benzyl alcohol (Sigma-Aldrich).
DNA templates in the aqueous supernatant were precipitated with
sodium acetate and 2-propanol.
A. pittii. For Acinetobacter isolates not belonging to the
Acb complex, only a 425-bp fragment corresponding to
the recA gene was found (Fig. 1, lane 1). Two bands, one
corresponding to the recA gene and the other to the
gyrB gene (294 bp), were found in A. baumannii and A.
M
1
2
3
4
5
M
RESULTS AND DISCUSSION
Validation of the multiplex PCR assay in identifying
A. baumannii, A. nosocomialis and A. pittii
Among the 23 reference and 89 clinical Acinetobacter
isolates in the first set of bacteria analysed (Table 1), the
PCR method showed 100 % sensitivity for the identification of A. baumannii (n522), A. nosocomialis (n513) and
A. pittii (n515). However, this method could not identify
Acinetobacter species belonging to ‘between 1 and 3’ or
‘close to 13TU’ (generous gifts from Alexandr Nemec).
Isolates belonging to ‘between 1 and 3’ were identified as
A. pittii, whereas those belonging to ‘close to 13TU’ were
identified as A. nosocomialis or showed patterns combining
A. nosocomialis and A. pittii (data not shown). Since these
Acinetobacter species are rare and their clinical importance
has not been delineated, this rapid method is still
appropriate for identifying Acb complex.
Among the 56 non-Acb complex isolates, none was
misidentified as any of A. baumannii, A. nosocomialis or
http://jmm.sgmjournals.org
500 bp
Fig. 1. Multiplex PCR products resolved by agarose gel
electrophoresis. PCR analyses were performed using A. baumannii-specific primers (P-Ab-ITSF and P-Ab-ITSB), internal control
primers (P-rA1 and P-rA2) specific for the recA gene of all
Acinetobacter species, A. nosocomialis-specific primers (gyrBdirected primers: sp4F and sp4R) and A. pittii-specific primers
(AGS3-R and AGS3-F). Lanes: M, 100 bp DNA ladder; 1, A.
calcoaceticus; 2, A. baumannii; 3, A. pittii; 4, A. nosocomialis; 5,
negative control.
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T.-L. Chen and others
nosocomialis isolates. A. baumannii was differentiated
from A. nosocomialis by the presence of another fragment
corresponding to its ITS region (208 bp) (Fig. 1, lane 2).
Two fragments were observed for the A. pittii isolate and
‘between 1 and 3’, one corresponding to the recA gene and
the other to the ITS region (150 bp) (Fig. 1, lane 3).
The multiplex PCR method was then validated using
the second set of 100 clinical Acb complex isolates. This
method correctly identified 48 isolates as A. baumannii, 15
as A. pittii and 35 as A. nosocomialis. In addition, two nonAcb complex Acinetobacter species (Acinetobacter lwoffii
and Acinetobacter johnsonii) were found. The identification
of all species was confirmed by ITS sequence analysis.
PFGE results showed that the clinical Acb complex isolates
in the first and second sets had diverse pulsotypes (data not
shown).
At present, PCR-based methods to identify A. baumannii
use primers that specifically amplify a fragment from
blaOXA51-like genes, which are intrinsic to this species
(Turton et al., 2006), and the ITS region of this species
(Chen et al., 2007). However, blaOXA51-like genes have
spread to other Acinetobacter species and may not be fully
reliable targets for identifying A. baumannii (Lee et al.,
2012a). Another PCR method targeting gyrB can differentiate between A. baumannii and A. nosocomialis (Higgins
et al., 2007), and this gyrB multiplex has been expanded to
enable the identification of A. calcoaceticus and A. pittii
(Higgins et al., 2010). In the present study, we developed a
multiplex PCR method that is able to differentiate the three
clinically relevant Acb complex species in a single reaction.
Although matrix-assisted laser desorption ionization–time
of flight mass spectrometry (MALDI-TOF MS) is increasingly used for species identification, it can only be applied
to bacterial colonies and not to blood samples (Šedo et al.,
2013). In addition, the accuracy of MALDI-TOF MS has
been described as ‘not acceptable for species-level identification of Acinetobacter spp.’ (Šedo et al., 2013).
Application of the multiplex PCR method to
identify A. baumannii, A. nosocomialis and A.
pittii from positive blood culture media
A total of 1034 positive blood culture samples were prospectively collected over a period of 24 months, consisting
of 131 blood cultures showing growth of Acb complex
isolates (70 A. baumannii, 51 A. nosocomialis and 10 A.
pittii) and 903 positive blood cultures showing growth
of organisms other than Acb complex (Table 2). Genomic
species identifications of the Acb complex isolates were
verified using a DNA template prepared from colonies.
Genomic species identification of other Acinetobacter
isolates was performed using different molecular methods,
as shown in the footnotes to Table 2. Among the 1034
positive blood culture samples, the multiplex PCR assay had
a sensitivity of 92.4 % (121/131) and specificity of 98.2 %
(887/903). Among the 981 blood culture specimens positive
for Gram-negative organisms, the multiplex PCR assay had
1158
a sensitivity of 92.4 % (121/131) and specificity of 98.5 %
(837/850). All reactions were completed within 4 h.
False-positive and -negative results may be a significant
drawback to the multiplex method. False-positive results
were observed in 16 blood culture samples, showing growth
of Aeromonas hydrophila (n51), Enterobacter cloacae
(n55), Klebsiella pneumoniae (n54), Klebsiella oxytoca
(n51), Proteus mirabilis (n51), Pseudomonas putida (n51),
Corynebacterium species (n51), Enterococcus species (n51)
and Staphylococcus aureus (n51). One possible explanation
is that Acb complex species were present in these blood
culture media but in too few numbers to be detected on agar
plates with routine blood culture methods, while the moresensitive multiplex PCR assay was able to identify them. To
prove this hypothesis, multiple sets of blood cultures or
a larger volume of blood may be needed to increase the
diagnostic yield. However, the clinical significance of the
presence of such a small amount of bacteria in blood needs
to be further investigated. An alternative explanation is that
the primers non-specifically bind to the DNA of organisms
other than Acb complex. However, this theory is refuted by
the negative multiplex PCR results obtained with these nonAcinetobacter bacterial colonies as templates.
False-negative results were observed in 10 blood culture
samples, which showed the growth of A. baumannii (n55),
A. nosocomialis (n53) and A. pittii (n52). These results
may be attributed to contamination with Acb complex
species during the phenotypic identification process or the
presence of PCR inhibitors in the samples, even after DNA
purification with benzyl alcohol extraction.
In conclusion, we have developed a convenient, rapid and
cost-effective method that can identify the three clinically
relevant Acb complex species. This method provides an
opportunity to better understand the biology, pathogenicity and ecology of individual Acinetobacter species of the
Acb complex.
ACKNOWLEDGEMENTS
This work was supported by grants from the Fund of Cheng
Hsin General Hospital and Yang-Ming University (102F218C06), the
Taipei Veterans General Hospital (V101E4-003 and V101C-021),
the National Science Council (NSC98-2314-B-010-010-MY3) and the
Yen Tjing Ling Medical Foundation (CI-100-35). This work was
performed at Taipei Veterans General Hospital, No. 201, Section 2,
Shih-Pai Road, Taipei 11217, Taiwan.
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