Journal of Medical Microbiology Papers in Press. Published June 25, 2014 as doi:10.1099/jmm.0.071712-0
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Rapid Identification of Acinetobacter baumannii, Acinetobacter nosocomialis, and
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Acinetobacter pittii by a Multiplex PCR Assay
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Running title: Rapid identification of Acb complex
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Contents Category: Diagnostics, typing, and identification
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Te-Li Chen,1,2 Yi-Tzu Lee,1,3 Shu-Chen Kuo,1,2,4 Su-Pen Yang,2,5 Chang-Phone
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Fung,1,2,5 and Shou-Dong Lee6*
10 11
This work was performed at Taipei Veterans General Hospital, No. 201, Section 2,
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Shih-Pai Road, Taipei 11217, Taiwan.
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1
14
University, Taipei, Taiwan
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2
Division of Infectious Diseases, Taipei Veterans General Hospital, Taipei, Taiwan
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3
Department of Emergency, Taipei Veterans General Hospital, Taipei, Taiwan
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4
National Institute of Infectious Diseases and Vaccinology, National Health Research
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Institutes, Miaoli County, Taiwan
Institutes of Clinical Medicine, School of Medicine, National Yang-Ming
1
19
5
20
Taiwan
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6
Emergency and Critical Care Medicine , National Yang-Ming University, Taipei,
Department of Medicine, Cheng Hsin General Hospital, Taipei, Taiwan
22 23
Corresponding author: Shou-Dong Lee
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Cheng Hsin General Hospital, No.45, Cheng Hsin Street, Taipei 11217, Taiwan
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Phone: +886-2-28264400
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Fax: +886-2-28730052
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E-mail: [email protected]
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2
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ABSTRACT
31 32
Acinetobacter baumannii, Acinetobacter nosocomialis, and Acinetobacter pittii are
33
clinically relevant members of the Acinetobacter calcoaceticus–A. baumannii (Acb)
34
complex and emerge as important nosocomial pathogens. These three species are
35
genetically closely related and phenotypically similar; however, they differ in their
36
epidemiology, antibiotic resistance, and pathogenicity. We described a multiplex
37
polymerase chain reaction (PCR)-based assay designed to detect internal fragments of
38
the 16S–23S ribosomal RNA intergenic region, gyrB and recA genes. This assay is
39
capable of differentiating these three species in a reliable manner. In 23 different
40
reference strains and 89 clinical isolates of Acinetobacter spp., this multiplex PCR
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assay accurately identified clinically relevant Acb complex except those ‘Between 1
42
and 3’ or ‘Close to 13TU’. None of the non-Acb complex were misidentified. The
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assay had a sensitivity of 92.4% and a specificity of 98.2% for Acb complex
44
identification in the analysis of 1034 positive blood cultures. Our results showed that
45
a single multiplex PCR assay can reliably differentiate among clinically relevant Acb
46
complex. Thus, this method may be used to better understand the clinical differences
47
between the infections caused by these species.
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3
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Keywords: Acinetobacter baumannii, Acinetobacter nosocomialis, Acinetobacter
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pittii, blood culture, PCR
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4
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INTRODUCTION
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Acinetobacter baumannii, A. nosocomialis and A. pittii have become important
54
nosocomial pathogens worldwide (Dijkshoorn et al., 2007; Peleg et al., 2008). These
55
three clinically relevant Acinetobacter spp., as well as an environmental species, A.
56
calcoaceticus (Nemec et al., 2011), cannot be differentiated reliably by phenotypic
57
tests. Because of their similar phenotypic characteristics, these four species are
58
grouped as A. calcoaceticus–A. baumannii (Acb) complex (Gerner-Smidt et al., 1991).
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It has been demonstrated that A. baumannii is distinct from A. nosocomialis and A.
60
pittii because of its resistance to more classes of antimicrobial agents, its association
61
with a relatively worse clinical outcome, and different responses to appropriate
62
therapy (Lee et al., 2010; Chuang et al., 2011; Kuo et al., 2012; Lee et al., 2012b;
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Wisplinghoff et al., 2012). Methods that can differentiate among these three clinically
64
relevant Acinetobacter spp. are critical for advancing our knowledge of the biology,
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pathogenicity, and therapy of these individual species.
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The use of polymerase chain reaction (PCR) assays offers the potential for rapid
67
detection and species identification of pathogens (Fredricks & Relman, 1998). In this
68
study, we described a multiplex PCR-based assay that can differentiate A. baumannii,
69
A. nosocomialis, and A. pittii from bacterial colonies and positive blood culture
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media.
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METHODS
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Bacterial isolates and identification
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Three sets of bacteria were examined in this study. The first set of bacteria
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included 23 reference strains and 89 clinical isolates of Acinetobacter spp. with
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known genomic species identification (Table 1). The reference strains were purchased
78
from public culture collections or kindly provided by T.-C. Chang (Chang et al.,
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2005) and Alexandr Nemec (Nemec et al., 2009; Nemec et al., 2010). The clinical
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isolates were collected from 10 different medical centers in Taiwan (Chen et al.,
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2010) or generously provided by Alexandr Nemec (Nemec et al., 2009; Nemec et al.,
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2010). The second set of bacteria comprised 100 clinical Acb complex isolates that
83
were obtained from the Taipei Veterans General Hospital (TVGH), without
84
knowledge of the genomic species prior to PCR identification. These two sets of
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Acinetobacter isolates were used to verify the practicality of the multiplex PCR
86
method. Acinetobacter species were identified by sequence analysis of the 16S–23S
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ribosomal RNA intergenic spacer (ITS) region (Chang et al., 2005), amplified
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ribosomal DNA (rDNA) restriction analysis (Dijkshoorn et al., 1998; Nemec et al.,
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2003; Nemec et al., 2011), and rpoB sequence cluster analysis (Nemec et al., 2009).
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Clonality of the clinical isolates of A. baumannii, A. pittii, and A. nosocomialis was
6
91
determined by pulsed-field gel electrophoresis (PFGE) by using the ApaI enzyme as
92
previously described (Huang et al., 2008).
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The third set was comprised of 1034 microorganisms of different genera and
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species based on the result of VITEK 2 system (bioMérieux, Marcy l’Etoile, France)
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(Table 2) that were prospectively collected from the TVGH from July 2010 to June
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2012. This set was used to test whether the multiplex PCR method could directly
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detect and identify Acb complex in blood culture media.
98 99
Development of multiplex PCR assay
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Three pairs of primers that had been previously designed and verified were included
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in the multiplex PCR assay. The primers P-rA1
102
(5 -CCTGAATCTTCTGGTAAAAC-3 ) and P-rA2
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(5 -GTTTCTGGGCTGCCAAACATTAC-3 ), which target a highly conserved
104
425-bp region of the recA gene of Acinetobacter spp., were used as a reaction control
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(Krawczyk et al., 2002). Primers sp4F (5 -CACGCCGTAAGAGTGCATTA-3 ) and
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sp4R (5 -AACGGAGCTTGTCAGGGTTA-3 ) yielded an amplicon of 294 bp from
107
the gyrB gene of A. baumannii and A. nosocomialis (Higgins et al., 2007). Primers
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P-Ab-ITSF (5 -CATTATCACGGTAATTAGTG-3 ) and P-Ab-ITSB
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(5 -AGAGCACTGTGCACTTAAG-3 ) were used to specifically amplify an internal
′
′
′
′
′
′
′
′
′
′
′
′
7
110
208-bp fragment from the ITS region of A. baumannii (Chen et al., 2007). For
111
identification of A. pittii, all the available ITS sequences of different Acinetobacter
112
spp. deposited at GenBank were downloaded and aligned using ClustalW software.
113
After considering the specificity and annealing temperature, primers P-AGS3-F
114
(5 -CTCAAGAGTTTAGATTAAGCAAT-3 ) and P-AGS3-R
115
(5 -GTCCGTGCGATTCTTCATCG-3 ) were selected for the amplification of a
116
150-bp internal fragment from the ITS region of A. pittii.
117
′
′
′
′
GoTaq Flexi DNA polymerase (Promega, Madison, WI) was used for PCR
118
assays performed in the GeneAmp PCR System 2700 (Applied Biosystems, Foster
119
City, CA). The PCR amplification consisted of an initial denaturation step at 94 °C
120
for 5 min, 35 cycles at 94 °C for 1 min, 58 °C for 30 s, and 72 °C for 30 s, followed
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by a final extension at 72 °C for 10 min. For the PCR assays of the first and second
122
sets of Acb complex isolates, the DNA template was prepared by boiling
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(Vaneechoutte et al., 1995). For the bacterial PCR assays from blood culture media,
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the DNA templates were prepared as described in the following section.
125 126
DNA purification from blood culture media
127
When bacterial growth was detected in the culture bottle by the culture system
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(BacT/Alert; Organon-Teknika, Durham, NC), 0.5 ml of the culture medium was 8
129
collected from the bottle and analyzed by Gram staining. The samples that yielded
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Gram-negative coccobacilli or bacilli were subjected to DNA purification followed by
131
multiplex PCR identification. Gram-positive organisms and yeast that were collected
132
in the first 2 weeks were also used to validate the PCR identification method. DNA
133
purification from positive culture media was performed using the benzyl
134
alcohol-guanidine hydrochloride organic extraction method as previously described
135
(Fredricks & Relman, 1998). Briefly, 0.1 ml of the inoculated blood culture media
136
was treated with lysis buffer containing guanidine hydrochloride in Tris buffer and
137
then mixed with 99% benzyl alcohol (Sigma-Aldrich, St. Louis, MO). DNA templates
138
in the aqueous supernatant were precipitated with sodium acetate and isopropanol.
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RESULTS AND DISCUSSION
141 142
Validation of the multiplex PCR assay for identification of A. baumannii, A.
143
nosocomialis, and A. pittii
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Among the 23 reference and 89 clinical Acinetobacter isolates in the first set of
145
bacteria analyzed (Table 1), the method showed 100% sensitivity for the identification
146
of A. baumannii (n = 22), A. nosocomialis (n = 13), and A. pittii (n = 15). However,
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this method cannot identify Acinetobacter spp. belonging to ‘Between 1 and 3’ or
148
‘Close to 13TU’ (generous gifts from Dr. Alexandr Nemec). Isolates belonging to
149
‘Between 1 and 3’ were identified as A. pittii whereas for those belonging to ‘Close to
150
13TU’, some were identified as A. nosocomialis, and the others showed the pattern
151
combining that of A. nosocomialis and A. pittii (data not shown). Since these
152
Acinetobacter spp. are rare and the clinical importance has not been delineated, this
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rapid method is still appropriate to be used for identification of Acb complex.
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Among the 56 non-Acb complex isolates, none were misidentified as any of
155
these three species. For Acinetobacter isolates not belonging to the Acb complex, only
156
a 425 bp-fragment corresponding respectively to the recA gene was found (Fig. 1,
157
lane 1). Two bands, one corresponding to the recA gene and the other to the gyrB
158
gene (294 bp), were found in A. baumannii and A. nosocomialis isolates. A. 10
159
baumannii was differentiated from A. nosocomialis by the presence of another
160
fragment corresponding to its ITS region (208 bp) (Fig. 1, lane 2). Two fragments
161
were observed for the A. pittii isolate and “Between 1 and 3”, one corresponding to
162
the recA gene and the other to the ITS region (150 bp) (Fig. 1, lane 3).
163
The multiplex PCR method was then validated using the second set of 100
164
clinical Acb complex isolates. This method correctly identified 48 isolates as A.
165
baumannii, 15 as A. pittii, and 35 as A. nosocomialis. In addition, two non-Acb
166
complex Acinetobacter spp. (A. lwoffii and A. johnsonii) were found. The
167
identification of all species was confirmed by ITS sequences analysis. PFGE results
168
showed that the clinical Acb complex isolates in the first and second sets had diverse
169
pulsotypes (data not shown).
170
At present, there are PCR-based methods that identify A. baumannii with primers
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that specifically amplify a fragment from blaOXA51-like genes, which are intrinsic to
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this species (Turton et al., 2006), and the ITS region of this species (Chen et al.,
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2007). However, blaOXA51-like genes had spread to other Acinetobacter spp. and may
174
not be a fully reliable target for identification of A. baumannii (Lee et al., 2012a).
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Another PCR method targeting gyrB can differentiate between A. baumannii and A.
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nosocomialis (Higgins et al., 2007), and this gyrB multiplex has been expanded to
177
enable the identification of A. calcoaceticus and A. pittii (Higgins et al., 2010). In this 11
178
study, we developed a multiplex PCR method that is able to differentiate the three
179
clinically relevant Acb complex in a single reaction. Although matrix-assisted laser
180
desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been
181
increasingly used for species identification, only bacterial colonies instead of blood
182
samples could be applied to (Sedo et al., 2013). In addition, The accuracy of
183
MALDI-TOF MS has been reported as “not acceptable for species-level identification
184
of Acinetobacter spp (Sedo et al., 2013).”
185 186
Application of the multiplex PCR method for identification of A. baumannii, A.
187
nosocomialis, and A. pittii from positive blood culture media
188
A total of 1034 positive blood culture samples were prospectively collected over a
189
period of 24 months, consisting of 131 blood cultures showing growth of Acb
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complex strains (70 A. baumannii, 51 A. nosocomialis, and 10 A. pittii) and 903
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positive blood cultures showing growth of organisms other than Acb complex (Table
192
2). The genomic species identifications of the Acb complex isolates were verified by
193
using a DNA template prepared from colonies. The genomic species identification of
194
other Acinetobacter isolates were performed by different molecular methods, as stated
195
in the footnote in Table 2. Among the 1034 positive blood culture samples, the
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multiplex PCR assay had a sensitivity and specificity of 92.4% (121/131) and 98.2% 12
197
(887/903), respectively. Among the 981 blood culture specimens positive for
198
Gram-negative organisms, the multiplex PCR assay had a sensitivity and specificity
199
of 92.4% (121/131) and 98.5% (837/850), respectively. The reactions were completed
200
within 4 h.
201
False-positive and false negative may be a significant drawback to the multiplex
202
method. False-positive results were observed in 16 blood culture samples, showing
203
growth of Aeromonas hydrophila (n = 1), Enterobacter cloacae (n = 5), Klebsiella
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pneumoniae (n = 4), K. oxytoca (n = 1), Proteus mirabilis (n = 1), Pseudomonas
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putida (n = 1), Corynebacterium sp. (n = 1), Enterococcus sp. (n = 1), and
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Staphylococcus aureus (n = 1). One possible explanation is that Acb complex were
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present in these blood culture media but in very small numbers to be detected on agar
208
plates. In any case, the multiplex PCR assay is more sensitive than routine blood
209
culture methods. To prove this assumption, multiple sets of blood cultures or a larger
210
volume of blood may be needed to increase the diagnostic yield. However, the clinical
211
significance of the presence of such a small amount of bacteria in the blood needs to
212
be further investigated. An alternative explanation is that the primers nonspecifically
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bind to the DNA of organisms other than Acb complex. However, this finding is
214
refuted by the negative multiplex PCR results obtained with these non-Acinetobacter
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bacterial colonies as templates. 13
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False-negative results were observed in 10 blood culture samples, which showed
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the growth of A. baumannii (n = 5), A. nosocomialis (n = 3), and A. pittii (n = 2). The
218
results may be attributed to the contamination of Acb complex during the phenotypic
219
identification process or the presence of PCR inhibitors in the samples even after
220
DNA purification with benzyl alcohol extraction.
221 222
In conclusion, we developed a convenient, rapid, and cost-effective method that
223
can identify the three clinically relevant Acb complex. This method provides an
224
opportunity to better understand the biology, pathogenicity, and ecology of individual
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Acinetobacter spp. of the Acb complex.
226 227
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ACKNOWLEDGMENTS
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This work was supported by grants from the Fund of Cheng Hsin General Hospital
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and Yang-Ming University (102F218C06), the Taipei Veterans General Hospital
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(V101E4-003 and V101C-021), the National Science Council
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(NSC98-2314-B-010-010-MY3), and the Yen Tjing Ling Medical Foundation
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(CI-100-35).
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Higgins, P. G., Wisplinghoff, H., Krut, O. & Seifert, H. (2007). A PCR-based method to differentiate between Acinetobacter baumannii and Acinetobacter genomic
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Acinetobacter genomic species 3. Journal of clinical microbiology 48, 4592-4594.
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Huang, L. Y., Chen, T. L., Lu, P. L., Tsai, C. A., Cho, W. L., Chang, F. Y., Fung, C. P. & Siu, L. K. (2008). Dissemination of multidrug-resistant, class 1 integron-carrying Acinetobacter baumannii isolates in Taiwan. Clin Microbiol Infect
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288 289 290 291
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Syst Evol Microbiol 59, 118-124. Nemec, A., Dijkshoorn, L., Cleenwerck, I., De Baere, T., Janssens, D., Van Der Reijden, T. J., Jezek, P. & Vaneechoutte, M. (2003). Acinetobacter parvus sp. nov., a small-colony-forming species isolated from human clinical specimens. Int J Syst
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Evol Microbiol 53, 1563-1567. Nemec, A., Musilek, M., Sedo, O., De Baere, T., Maixnerova, M., van der Reijden, T. J., Zdrahal, Z., Vaneechoutte, M. & Dijkshoorn, L. (2010). Acinetobacter bereziniae sp. nov. and Acinetobacter guillouiae sp. nov., to
17
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accommodate Acinetobacter genomic species 10 and 11, respectively. Int J Syst Evol
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Microbiol 60, 896-903. Nemec, A., Krizova, L., Maixnerova, M., van der Reijden, T. J., Deschaght, P., Passet, V., Vaneechoutte, M., Brisse, S. & Dijkshoorn, L. (2011). Genotypic and phenotypic characterization of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex with the proposal of Acinetobacter pittii sp. nov. (formerly Acinetobacter genomic species 3) and Acinetobacter nosocomialis sp. nov. (formerly
315 316 317 318 319 320
Acinetobacter genomic species 13TU). Research in microbiology 162, 393-404. Peleg, A. Y., Seifert, H. & Paterson, D. L. (2008). Acinetobacter baumannii: emergence of a successful pathogen. Clinical microbiology reviews 21, 538-582. Sedo, O., Nemec, A., Krizova, L., Kacalova, M. & Zdrahal, Z. (2013). Improvement of MALDI-TOF MS profiling for the differentiation of species within the Acinetobacter calcoaceticus-Acinetobacter baumannii complex. Syst Appl
321 322 323 324 325
Microbiol 36, 572-578. Turton, J. F., Woodford, N., Glover, J., Yarde, S., Kaufmann, M. E. & Pitt, T. L. (2006). Identification of Acinetobacter baumannii by detection of the blaOXA-51-like carbapenemase gene intrinsic to this species. Journal of clinical microbiology 44, 2974-2976.
326 327 328
Vaneechoutte, M., Dijkshoorn, L., Tjernberg, I., Elaichouni, A., de Vos, P., Claeys, G. & Verschraegen, G. (1995). Identification of Acinetobacter genomic species by amplified ribosomal DNA restriction analysis. Journal of clinical
329 330 331 332
microbiology 33, 11-15. Wisplinghoff, H., Paulus, T., Lugenheim, M., Stefanik, D., Higgins, P. G., Edmond, M. B., Wenzel, R. P. & Seifert, H. (2012). Nosocomial bloodstream infections due to Acinetobacter baumannii, Acinetobacter pittii and Acinetobacter
333
nosocomialis in the United States. The Journal of infection 64, 282-290.
334 335 336
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337
Table 1. Reference and clinical isolates used in the validation of the multiplex
338
PCR assay for the identification of Acinetobacter baumannii, A. nosocomialis, and
339
A. pittii
340 No. of clinical Genomic species
Reference strains isolates
A. calcoaceticus
ATCC 17987
0
ATCC 19606T and 15151
20
A. pittii
ATCC 17922T
12
A. haemolyticus
ATCC 17906T
1
A. junii
ATCC 17908T
5
Acinetobacter genomic
ATCC 17979
A. baumannii
0 species 6 ATCC 17909T
0
A. lwoffii
0
2
A. bereziniae
0
3
A. guillouiae *
LMG 988T
1
A. radioresistens
ATCC 43998T
3
A. nosocomialis
ATCC 17903
14
A. johnsonii
Acinetobacter genomic
0 2
species 13BJ ⁄ 14TU Acinetobacter genomic
CCUG 26390 4
species 15TU Acinetobacter genomic
CCUG 34436 19
0
species 15BJ Acinetobacter genomic
BCRC 15883 0
species 16 Acinetobacter genomic
CCUG 34437 0
species 17 A. baylyi
ATCC 33305
7
A. beijerinckii *
NIPH 838T
1
A. gyllenbergii *
NIPH 2150T
1
A. parvus
CCUG 48800T
0
A. schindleri
LMG 19576T
1
A. soli *
CCUG 59023T
1
A. ursingii
LMG 19575T
5
A. venetianus
CCUG 45561T
0
A. belonging to 'Close to
0 4
13TU' A. belonging to 'Between
0 2
1 and 3' Total
89
341 342
* Generous gifts from Prof. Nemec A.
343
20
344
Table 2. Clinical isolates used in the validation of the multiplex PCR
345
assay for detection and identification of A. baumannii, A.
346
nosocomialis, and A. pittii from positive blood cultures Multiplex PCR result Microorganism*
No. A. b
Gram-negative bacteria
A. n
A. p
Negative
981
76
50
8
847
Acinetobacter baumannii
70
65
0
0
5
Acinetobacter nosocomialis
51
0
48
0
3
Acinetobacter pittii
10
0
0
8
2
Acinetobacter junii
1
0
0
0
1
Acinetobacter lwoffii
2
0
0
0
2
Other Acinetobacter spp.†
9
0
0
0
9
Achromobacter spp.
4
0
0
0
4
Aeromonas hydrophila
10
1
0
0
9
Aeromonas sorbia
1
0
0
0
1
Bacteroides fragilis group
9
0
0
0
9
Burkholderia cepacia
12
0
0
0
12
Chryseobacterium indologenes
10
0
0
0
10
Chryseobacterium meningosepticum
12
0
0
0
12
Citrobacter diversus
9
0
0
0
9
Citrobacter freundii
6
0
0
0
6
Comamonas testosteroni
1
0
0
0
1
Escherichia coli
372
0
0
0
372
Enterobacter aerogenes
18
0
0
0
18
Enterobacter asburiae
1
0
0
0
1
Enterobacter cloacae
36
4
1
0
31
Fusobacterium spp.
1
0
0
0
1
Klebsiella oxytoca
7
0
1
0
6
Klebsiella ozaenae
1
0
0
0
1
21
Klebsiella pneumoniae
180
4
0
0
176
Kluyvera ascorbata
1
0
0
0
1
Morganella morganii
4
0
0
0
4
Pantoea spp.
1
0
0
0
1
Plesiomonas shigelloides
1
0
0
0
1
Prevotella spp.
2
0
0
0
2
Proteus mirabilis
24
1
0
0
23
Proteus penneri
2
0
0
0
2
Providencia stuartii
1
0
0
0
1
Pseudomonas aeruginosa
40
0
0
0
40
Pseudomonas putida
3
1
0
0
2
Ralstonia mannitolilytica
1
0
0
0
1
Salmonella group B
3
0
0
0
3
Salmonella group D
30
0
0
0
30
Salmonella paratyphi
1
0
0
0
1
Serratia marcescens
13
0
0
0
13
Shewanella algae
1
0
0
0
1
Sphingomonas paucimobilis
2
0
0
0
2
Stenotrophomonas maltophilia
15
0
0
0
15
Vibrio fluvialis
2
0
0
0
2
Vibrio mimicus
1
0
0
0
1
46
2
0
1
43
Bacillus spp.
2
0
0
0
2
Coagulase-negative Staphylococcus spp.
18
0
0
0
18
Corynebacterium spp.
2
1
0
0
1
Enterococcus faecalis
1
0
0
0
1
Enterococcus gallinarum
1
0
0
0
1
Enterococcus spp.
4
1
0
0
3
Lactobacillus spp.
1
0
0
0
1
Rhodococcus equi
1
0
0
0
1
Gram-positive bacteria
22
Staphylococcus aureus
11
0
0
1
10
Staphylococcus hominis
1
0
0
0
1
Streptococcus agalactiae
1
0
0
0
1
Streptococcus group C
1
0
0
0
1
Streptococcus group D
1
0
0
0
1
Viridans streptococcus group
1
0
0
0
1
Yeast
7
0
0
0
7
Total
1034
78
50
9
897
347
A. b, Acinetobacter baumannii; A. n, Acinetobacter nosocomialis; A. p, Acinetobacter pittii.
348
* Acinetobacter spp. identification of the Acb complex was performed by the PCR method; A. junii and
349
A. lwoffii were identified by amplified rDNA restriction analysis and rpoB sequence cluster analysis.
350
† Including Acinetobacter haemolyticus (n=1), Acinetobacter johnsonii (n=2), Acinetobacter bereziniae
351
(n=1), Acinetobacter radioresistens (n=1), Acinetobacter genomic species 13BJ ⁄ 14TU (n=1),
352
Acinetobacter baylyi (n=1), and Acinetobacter ursingii (n=2). All were identified by rpoB sequence
353
cluster analysis.
354
23
355
Figure Legend
356
FIG. 1. Multiplex PCR products resolved by agarose gel electrophoresis. PCR
357
analyses were performed with A. baumannii-specific primers (P-Ab-ITSF and
358
P-Ab-ITSB), internal control primers (P-rA1 and P-rA2) specific for the recA gene of
359
all Acinetobacter spp., A. nosocomialis-specific primers (gyrB-directed primers: sp4F
360
and sp4R), and A. pittii-specific primers (AGS3-R and AGS3-F). Lanes: M, 100-bp
361
DNA ladder; 1, A. calcoaceticus; 2, A. baumannii; 3, A. pittii; 4, A. nosocomialis; 5,
362
negative control.
363
24
1
Rapid Identification of Acinetobacter baumannii, Acinetobacter nosocomialis, and
2
Acinetobacter pittii by a Multiplex PCR Assay
3 4
Running title: Rapid identification of Acb complex
5 6
Contents Category: Diagnostics, typing, and identification
7 8
Te-Li Chen,1,2 Yi-Tzu Lee,1,3 Shu-Chen Kuo,1,2,4 Su-Pen Yang,2,5 Chang-Phone
9
Fung,1,2,5 and Shou-Dong Lee6*
10 11
This work was performed at Taipei Veterans General Hospital, No. 201, Section 2,
12
Shih-Pai Road, Taipei 11217, Taiwan.
13
1
14
University, Taipei, Taiwan
15
2
Division of Infectious Diseases, Taipei Veterans General Hospital, Taipei, Taiwan
16
3
Department of Emergency, Taipei Veterans General Hospital, Taipei, Taiwan
17
4
National Institute of Infectious Diseases and Vaccinology, National Health Research
18
Institutes, Miaoli County, Taiwan
Institutes of Clinical Medicine, School of Medicine, National Yang-Ming
1
19
5
20
Taiwan
21
6
Emergency and Critical Care Medicine , National Yang-Ming University, Taipei,
Department of Medicine, Cheng Hsin General Hospital, Taipei, Taiwan
22 23
Corresponding author: Shou-Dong Lee
24
Cheng Hsin General Hospital, No.45, Cheng Hsin Street, Taipei 11217, Taiwan
25
Phone: +886-2-28264400
26
Fax: +886-2-28730052
27
E-mail: [email protected]
28 29
2
30
ABSTRACT
31 32
Acinetobacter baumannii, Acinetobacter nosocomialis, and Acinetobacter pittii are
33
clinically relevant members of the Acinetobacter calcoaceticus–A. baumannii (Acb)
34
complex and emerge as important nosocomial pathogens. These three species are
35
genetically closely related and phenotypically similar; however, they differ in their
36
epidemiology, antibiotic resistance, and pathogenicity. We described a multiplex
37
polymerase chain reaction (PCR)-based assay designed to detect internal fragments of
38
the 16S–23S ribosomal RNA intergenic region, gyrB and recA genes. This assay is
39
capable of differentiating these three species in a reliable manner. In 23 different
40
reference strains and 89 clinical isolates of Acinetobacter spp., this multiplex PCR
41
assay accurately identified clinically relevant Acb complex except those ‘Between 1
42
and 3’ or ‘Close to 13TU’. None of the non-Acb complex were misidentified. The
43
assay had a sensitivity of 92.4% and a specificity of 98.2% for Acb complex
44
identification in the analysis of 1034 positive blood cultures. Our results showed that
45
a single multiplex PCR assay can reliably differentiate among clinically relevant Acb
46
complex. Thus, this method may be used to better understand the clinical differences
47
between the infections caused by these species.
48
3
49
Keywords: Acinetobacter baumannii, Acinetobacter nosocomialis, Acinetobacter
50
pittii, blood culture, PCR
51
4
52
INTRODUCTION
53
Acinetobacter baumannii, A. nosocomialis and A. pittii have become important
54
nosocomial pathogens worldwide (Dijkshoorn et al., 2007; Peleg et al., 2008). These
55
three clinically relevant Acinetobacter spp., as well as an environmental species, A.
56
calcoaceticus (Nemec et al., 2011), cannot be differentiated reliably by phenotypic
57
tests. Because of their similar phenotypic characteristics, these four species are
58
grouped as A. calcoaceticus–A. baumannii (Acb) complex (Gerner-Smidt et al., 1991).
59
It has been demonstrated that A. baumannii is distinct from A. nosocomialis and A.
60
pittii because of its resistance to more classes of antimicrobial agents, its association
61
with a relatively worse clinical outcome, and different responses to appropriate
62
therapy (Lee et al., 2010; Chuang et al., 2011; Kuo et al., 2012; Lee et al., 2012b;
63
Wisplinghoff et al., 2012). Methods that can differentiate among these three clinically
64
relevant Acinetobacter spp. are critical for advancing our knowledge of the biology,
65
pathogenicity, and therapy of these individual species.
66
The use of polymerase chain reaction (PCR) assays offers the potential for rapid
67
detection and species identification of pathogens (Fredricks & Relman, 1998). In this
68
study, we described a multiplex PCR-based assay that can differentiate A. baumannii,
69
A. nosocomialis, and A. pittii from bacterial colonies and positive blood culture
70
media.
71 72
5
73
METHODS
74
Bacterial isolates and identification
75
Three sets of bacteria were examined in this study. The first set of bacteria
76
included 23 reference strains and 89 clinical isolates of Acinetobacter spp. with
77
known genomic species identification (Table 1). The reference strains were purchased
78
from public culture collections or kindly provided by T.-C. Chang (Chang et al.,
79
2005) and Alexandr Nemec (Nemec et al., 2009; Nemec et al., 2010). The clinical
80
isolates were collected from 10 different medical centers in Taiwan (Chen et al.,
81
2010) or generously provided by Alexandr Nemec (Nemec et al., 2009; Nemec et al.,
82
2010). The second set of bacteria comprised 100 clinical Acb complex isolates that
83
were obtained from the Taipei Veterans General Hospital (TVGH), without
84
knowledge of the genomic species prior to PCR identification. These two sets of
85
Acinetobacter isolates were used to verify the practicality of the multiplex PCR
86
method. Acinetobacter species were identified by sequence analysis of the 16S–23S
87
ribosomal RNA intergenic spacer (ITS) region (Chang et al., 2005), amplified
88
ribosomal DNA (rDNA) restriction analysis (Dijkshoorn et al., 1998; Nemec et al.,
89
2003; Nemec et al., 2011), and rpoB sequence cluster analysis (Nemec et al., 2009).
90
Clonality of the clinical isolates of A. baumannii, A. pittii, and A. nosocomialis was
6
91
determined by pulsed-field gel electrophoresis (PFGE) by using the ApaI enzyme as
92
previously described (Huang et al., 2008).
93
The third set was comprised of 1034 microorganisms of different genera and
94
species based on the result of VITEK 2 system (bioMérieux, Marcy l’Etoile, France)
95
(Table 2) that were prospectively collected from the TVGH from July 2010 to June
96
2012. This set was used to test whether the multiplex PCR method could directly
97
detect and identify Acb complex in blood culture media.
98 99
Development of multiplex PCR assay
100
Three pairs of primers that had been previously designed and verified were included
101
in the multiplex PCR assay. The primers P-rA1
102
(5 -CCTGAATCTTCTGGTAAAAC-3 ) and P-rA2
103
(5 -GTTTCTGGGCTGCCAAACATTAC-3 ), which target a highly conserved
104
425-bp region of the recA gene of Acinetobacter spp., were used as a reaction control
105
(Krawczyk et al., 2002). Primers sp4F (5 -CACGCCGTAAGAGTGCATTA-3 ) and
106
sp4R (5 -AACGGAGCTTGTCAGGGTTA-3 ) yielded an amplicon of 294 bp from
107
the gyrB gene of A. baumannii and A. nosocomialis (Higgins et al., 2007). Primers
108
P-Ab-ITSF (5 -CATTATCACGGTAATTAGTG-3 ) and P-Ab-ITSB
109
(5 -AGAGCACTGTGCACTTAAG-3 ) were used to specifically amplify an internal
′
′
′
′
′
′
′
′
′
′
′
′
7
110
208-bp fragment from the ITS region of A. baumannii (Chen et al., 2007). For
111
identification of A. pittii, all the available ITS sequences of different Acinetobacter
112
spp. deposited at GenBank were downloaded and aligned using ClustalW software.
113
After considering the specificity and annealing temperature, primers P-AGS3-F
114
(5 -CTCAAGAGTTTAGATTAAGCAAT-3 ) and P-AGS3-R
115
(5 -GTCCGTGCGATTCTTCATCG-3 ) were selected for the amplification of a
116
150-bp internal fragment from the ITS region of A. pittii.
117
′
′
′
′
GoTaq Flexi DNA polymerase (Promega, Madison, WI) was used for PCR
118
assays performed in the GeneAmp PCR System 2700 (Applied Biosystems, Foster
119
City, CA). The PCR amplification consisted of an initial denaturation step at 94 °C
120
for 5 min, 35 cycles at 94 °C for 1 min, 58 °C for 30 s, and 72 °C for 30 s, followed
121
by a final extension at 72 °C for 10 min. For the PCR assays of the first and second
122
sets of Acb complex isolates, the DNA template was prepared by boiling
123
(Vaneechoutte et al., 1995). For the bacterial PCR assays from blood culture media,
124
the DNA templates were prepared as described in the following section.
125 126
DNA purification from blood culture media
127
When bacterial growth was detected in the culture bottle by the culture system
128
(BacT/Alert; Organon-Teknika, Durham, NC), 0.5 ml of the culture medium was 8
129
collected from the bottle and analyzed by Gram staining. The samples that yielded
130
Gram-negative coccobacilli or bacilli were subjected to DNA purification followed by
131
multiplex PCR identification. Gram-positive organisms and yeast that were collected
132
in the first 2 weeks were also used to validate the PCR identification method. DNA
133
purification from positive culture media was performed using the benzyl
134
alcohol-guanidine hydrochloride organic extraction method as previously described
135
(Fredricks & Relman, 1998). Briefly, 0.1 ml of the inoculated blood culture media
136
was treated with lysis buffer containing guanidine hydrochloride in Tris buffer and
137
then mixed with 99% benzyl alcohol (Sigma-Aldrich, St. Louis, MO). DNA templates
138
in the aqueous supernatant were precipitated with sodium acetate and isopropanol.
139
9
140
RESULTS AND DISCUSSION
141 142
Validation of the multiplex PCR assay for identification of A. baumannii, A.
143
nosocomialis, and A. pittii
144
Among the 23 reference and 89 clinical Acinetobacter isolates in the first set of
145
bacteria analyzed (Table 1), the method showed 100% sensitivity for the identification
146
of A. baumannii (n = 22), A. nosocomialis (n = 13), and A. pittii (n = 15). However,
147
this method cannot identify Acinetobacter spp. belonging to ‘Between 1 and 3’ or
148
‘Close to 13TU’ (generous gifts from Dr. Alexandr Nemec). Isolates belonging to
149
‘Between 1 and 3’ were identified as A. pittii whereas for those belonging to ‘Close to
150
13TU’, some were identified as A. nosocomialis, and the others showed the pattern
151
combining that of A. nosocomialis and A. pittii (data not shown). Since these
152
Acinetobacter spp. are rare and the clinical importance has not been delineated, this
153
rapid method is still appropriate to be used for identification of Acb complex.
154
Among the 56 non-Acb complex isolates, none were misidentified as any of
155
these three species. For Acinetobacter isolates not belonging to the Acb complex, only
156
a 425 bp-fragment corresponding respectively to the recA gene was found (Fig. 1,
157
lane 1). Two bands, one corresponding to the recA gene and the other to the gyrB
158
gene (294 bp), were found in A. baumannii and A. nosocomialis isolates. A. 10
159
baumannii was differentiated from A. nosocomialis by the presence of another
160
fragment corresponding to its ITS region (208 bp) (Fig. 1, lane 2). Two fragments
161
were observed for the A. pittii isolate and “Between 1 and 3”, one corresponding to
162
the recA gene and the other to the ITS region (150 bp) (Fig. 1, lane 3).
163
The multiplex PCR method was then validated using the second set of 100
164
clinical Acb complex isolates. This method correctly identified 48 isolates as A.
165
baumannii, 15 as A. pittii, and 35 as A. nosocomialis. In addition, two non-Acb
166
complex Acinetobacter spp. (A. lwoffii and A. johnsonii) were found. The
167
identification of all species was confirmed by ITS sequences analysis. PFGE results
168
showed that the clinical Acb complex isolates in the first and second sets had diverse
169
pulsotypes (data not shown).
170
At present, there are PCR-based methods that identify A. baumannii with primers
171
that specifically amplify a fragment from blaOXA51-like genes, which are intrinsic to
172
this species (Turton et al., 2006), and the ITS region of this species (Chen et al.,
173
2007). However, blaOXA51-like genes had spread to other Acinetobacter spp. and may
174
not be a fully reliable target for identification of A. baumannii (Lee et al., 2012a).
175
Another PCR method targeting gyrB can differentiate between A. baumannii and A.
176
nosocomialis (Higgins et al., 2007), and this gyrB multiplex has been expanded to
177
enable the identification of A. calcoaceticus and A. pittii (Higgins et al., 2010). In this 11
178
study, we developed a multiplex PCR method that is able to differentiate the three
179
clinically relevant Acb complex in a single reaction. Although matrix-assisted laser
180
desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been
181
increasingly used for species identification, only bacterial colonies instead of blood
182
samples could be applied to (Sedo et al., 2013). In addition, The accuracy of
183
MALDI-TOF MS has been reported as “not acceptable for species-level identification
184
of Acinetobacter spp (Sedo et al., 2013).”
185 186
Application of the multiplex PCR method for identification of A. baumannii, A.
187
nosocomialis, and A. pittii from positive blood culture media
188
A total of 1034 positive blood culture samples were prospectively collected over a
189
period of 24 months, consisting of 131 blood cultures showing growth of Acb
190
complex strains (70 A. baumannii, 51 A. nosocomialis, and 10 A. pittii) and 903
191
positive blood cultures showing growth of organisms other than Acb complex (Table
192
2). The genomic species identifications of the Acb complex isolates were verified by
193
using a DNA template prepared from colonies. The genomic species identification of
194
other Acinetobacter isolates were performed by different molecular methods, as stated
195
in the footnote in Table 2. Among the 1034 positive blood culture samples, the
196
multiplex PCR assay had a sensitivity and specificity of 92.4% (121/131) and 98.2% 12
197
(887/903), respectively. Among the 981 blood culture specimens positive for
198
Gram-negative organisms, the multiplex PCR assay had a sensitivity and specificity
199
of 92.4% (121/131) and 98.5% (837/850), respectively. The reactions were completed
200
within 4 h.
201
False-positive and false negative may be a significant drawback to the multiplex
202
method. False-positive results were observed in 16 blood culture samples, showing
203
growth of Aeromonas hydrophila (n = 1), Enterobacter cloacae (n = 5), Klebsiella
204
pneumoniae (n = 4), K. oxytoca (n = 1), Proteus mirabilis (n = 1), Pseudomonas
205
putida (n = 1), Corynebacterium sp. (n = 1), Enterococcus sp. (n = 1), and
206
Staphylococcus aureus (n = 1). One possible explanation is that Acb complex were
207
present in these blood culture media but in very small numbers to be detected on agar
208
plates. In any case, the multiplex PCR assay is more sensitive than routine blood
209
culture methods. To prove this assumption, multiple sets of blood cultures or a larger
210
volume of blood may be needed to increase the diagnostic yield. However, the clinical
211
significance of the presence of such a small amount of bacteria in the blood needs to
212
be further investigated. An alternative explanation is that the primers nonspecifically
213
bind to the DNA of organisms other than Acb complex. However, this finding is
214
refuted by the negative multiplex PCR results obtained with these non-Acinetobacter
215
bacterial colonies as templates. 13
216
False-negative results were observed in 10 blood culture samples, which showed
217
the growth of A. baumannii (n = 5), A. nosocomialis (n = 3), and A. pittii (n = 2). The
218
results may be attributed to the contamination of Acb complex during the phenotypic
219
identification process or the presence of PCR inhibitors in the samples even after
220
DNA purification with benzyl alcohol extraction.
221 222
In conclusion, we developed a convenient, rapid, and cost-effective method that
223
can identify the three clinically relevant Acb complex. This method provides an
224
opportunity to better understand the biology, pathogenicity, and ecology of individual
225
Acinetobacter spp. of the Acb complex.
226 227
14
228
ACKNOWLEDGMENTS
229 230
This work was supported by grants from the Fund of Cheng Hsin General Hospital
231
and Yang-Ming University (102F218C06), the Taipei Veterans General Hospital
232
(V101E4-003 and V101C-021), the National Science Council
233
(NSC98-2314-B-010-010-MY3), and the Yen Tjing Ling Medical Foundation
234
(CI-100-35).
15
235
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236 237 238
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Syst Evol Microbiol 59, 118-124. Nemec, A., Dijkshoorn, L., Cleenwerck, I., De Baere, T., Janssens, D., Van Der Reijden, T. J., Jezek, P. & Vaneechoutte, M. (2003). Acinetobacter parvus sp. nov., a small-colony-forming species isolated from human clinical specimens. Int J Syst
304 305 306 307
Evol Microbiol 53, 1563-1567. Nemec, A., Musilek, M., Sedo, O., De Baere, T., Maixnerova, M., van der Reijden, T. J., Zdrahal, Z., Vaneechoutte, M. & Dijkshoorn, L. (2010). Acinetobacter bereziniae sp. nov. and Acinetobacter guillouiae sp. nov., to
17
308
accommodate Acinetobacter genomic species 10 and 11, respectively. Int J Syst Evol
309 310 311 312 313 314
Microbiol 60, 896-903. Nemec, A., Krizova, L., Maixnerova, M., van der Reijden, T. J., Deschaght, P., Passet, V., Vaneechoutte, M., Brisse, S. & Dijkshoorn, L. (2011). Genotypic and phenotypic characterization of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex with the proposal of Acinetobacter pittii sp. nov. (formerly Acinetobacter genomic species 3) and Acinetobacter nosocomialis sp. nov. (formerly
315 316 317 318 319 320
Acinetobacter genomic species 13TU). Research in microbiology 162, 393-404. Peleg, A. Y., Seifert, H. & Paterson, D. L. (2008). Acinetobacter baumannii: emergence of a successful pathogen. Clinical microbiology reviews 21, 538-582. Sedo, O., Nemec, A., Krizova, L., Kacalova, M. & Zdrahal, Z. (2013). Improvement of MALDI-TOF MS profiling for the differentiation of species within the Acinetobacter calcoaceticus-Acinetobacter baumannii complex. Syst Appl
321 322 323 324 325
Microbiol 36, 572-578. Turton, J. F., Woodford, N., Glover, J., Yarde, S., Kaufmann, M. E. & Pitt, T. L. (2006). Identification of Acinetobacter baumannii by detection of the blaOXA-51-like carbapenemase gene intrinsic to this species. Journal of clinical microbiology 44, 2974-2976.
326 327 328
Vaneechoutte, M., Dijkshoorn, L., Tjernberg, I., Elaichouni, A., de Vos, P., Claeys, G. & Verschraegen, G. (1995). Identification of Acinetobacter genomic species by amplified ribosomal DNA restriction analysis. Journal of clinical
329 330 331 332
microbiology 33, 11-15. Wisplinghoff, H., Paulus, T., Lugenheim, M., Stefanik, D., Higgins, P. G., Edmond, M. B., Wenzel, R. P. & Seifert, H. (2012). Nosocomial bloodstream infections due to Acinetobacter baumannii, Acinetobacter pittii and Acinetobacter
333
nosocomialis in the United States. The Journal of infection 64, 282-290.
334 335 336
18
337
Table 1. Reference and clinical isolates used in the validation of the multiplex
338
PCR assay for the identification of Acinetobacter baumannii, A. nosocomialis, and
339
A. pittii
340 No. of clinical Genomic species
Reference strains isolates
A. calcoaceticus
ATCC 17987
0
ATCC 19606T and 15151
20
A. pittii
ATCC 17922T
12
A. haemolyticus
ATCC 17906T
1
A. junii
ATCC 17908T
5
Acinetobacter genomic
ATCC 17979
A. baumannii
0 species 6 ATCC 17909T
0
A. lwoffii
0
2
A. bereziniae
0
3
A. guillouiae *
LMG 988T
1
A. radioresistens
ATCC 43998T
3
A. nosocomialis
ATCC 17903
14
A. johnsonii
Acinetobacter genomic
0 2
species 13BJ ⁄ 14TU Acinetobacter genomic
CCUG 26390 4
species 15TU Acinetobacter genomic
CCUG 34436 19
0
species 15BJ Acinetobacter genomic
BCRC 15883 0
species 16 Acinetobacter genomic
CCUG 34437 0
species 17 A. baylyi
ATCC 33305
7
A. beijerinckii *
NIPH 838T
1
A. gyllenbergii *
NIPH 2150T
1
A. parvus
CCUG 48800T
0
A. schindleri
LMG 19576T
1
A. soli *
CCUG 59023T
1
A. ursingii
LMG 19575T
5
A. venetianus
CCUG 45561T
0
A. belonging to 'Close to
0 4
13TU' A. belonging to 'Between
0 2
1 and 3' Total
89
341 342
* Generous gifts from Prof. Nemec A.
343
20
344
Table 2. Clinical isolates used in the validation of the multiplex PCR
345
assay for detection and identification of A. baumannii, A.
346
nosocomialis, and A. pittii from positive blood cultures Multiplex PCR result Microorganism*
No. A. b
Gram-negative bacteria
A. n
A. p
Negative
981
76
50
8
847
Acinetobacter baumannii
70
65
0
0
5
Acinetobacter nosocomialis
51
0
48
0
3
Acinetobacter pittii
10
0
0
8
2
Acinetobacter junii
1
0
0
0
1
Acinetobacter lwoffii
2
0
0
0
2
Other Acinetobacter spp.†
9
0
0
0
9
Achromobacter spp.
4
0
0
0
4
Aeromonas hydrophila
10
1
0
0
9
Aeromonas sorbia
1
0
0
0
1
Bacteroides fragilis group
9
0
0
0
9
Burkholderia cepacia
12
0
0
0
12
Chryseobacterium indologenes
10
0
0
0
10
Chryseobacterium meningosepticum
12
0
0
0
12
Citrobacter diversus
9
0
0
0
9
Citrobacter freundii
6
0
0
0
6
Comamonas testosteroni
1
0
0
0
1
Escherichia coli
372
0
0
0
372
Enterobacter aerogenes
18
0
0
0
18
Enterobacter asburiae
1
0
0
0
1
Enterobacter cloacae
36
4
1
0
31
Fusobacterium spp.
1
0
0
0
1
Klebsiella oxytoca
7
0
1
0
6
Klebsiella ozaenae
1
0
0
0
1
21
Klebsiella pneumoniae
180
4
0
0
176
Kluyvera ascorbata
1
0
0
0
1
Morganella morganii
4
0
0
0
4
Pantoea spp.
1
0
0
0
1
Plesiomonas shigelloides
1
0
0
0
1
Prevotella spp.
2
0
0
0
2
Proteus mirabilis
24
1
0
0
23
Proteus penneri
2
0
0
0
2
Providencia stuartii
1
0
0
0
1
Pseudomonas aeruginosa
40
0
0
0
40
Pseudomonas putida
3
1
0
0
2
Ralstonia mannitolilytica
1
0
0
0
1
Salmonella group B
3
0
0
0
3
Salmonella group D
30
0
0
0
30
Salmonella paratyphi
1
0
0
0
1
Serratia marcescens
13
0
0
0
13
Shewanella algae
1
0
0
0
1
Sphingomonas paucimobilis
2
0
0
0
2
Stenotrophomonas maltophilia
15
0
0
0
15
Vibrio fluvialis
2
0
0
0
2
Vibrio mimicus
1
0
0
0
1
46
2
0
1
43
Bacillus spp.
2
0
0
0
2
Coagulase-negative Staphylococcus spp.
18
0
0
0
18
Corynebacterium spp.
2
1
0
0
1
Enterococcus faecalis
1
0
0
0
1
Enterococcus gallinarum
1
0
0
0
1
Enterococcus spp.
4
1
0
0
3
Lactobacillus spp.
1
0
0
0
1
Rhodococcus equi
1
0
0
0
1
Gram-positive bacteria
22
Staphylococcus aureus
11
0
0
1
10
Staphylococcus hominis
1
0
0
0
1
Streptococcus agalactiae
1
0
0
0
1
Streptococcus group C
1
0
0
0
1
Streptococcus group D
1
0
0
0
1
Viridans streptococcus group
1
0
0
0
1
Yeast
7
0
0
0
7
Total
1034
78
50
9
897
347
A. b, Acinetobacter baumannii; A. n, Acinetobacter nosocomialis; A. p, Acinetobacter pittii.
348
* Acinetobacter spp. identification of the Acb complex was performed by the PCR method; A. junii and
349
A. lwoffii were identified by amplified rDNA restriction analysis and rpoB sequence cluster analysis.
350
† Including Acinetobacter haemolyticus (n=1), Acinetobacter johnsonii (n=2), Acinetobacter bereziniae
351
(n=1), Acinetobacter radioresistens (n=1), Acinetobacter genomic species 13BJ ⁄ 14TU (n=1),
352
Acinetobacter baylyi (n=1), and Acinetobacter ursingii (n=2). All were identified by rpoB sequence
353
cluster analysis.
354
23
355
Figure Legend
356
FIG. 1. Multiplex PCR products resolved by agarose gel electrophoresis. PCR
357
analyses were performed with A. baumannii-specific primers (P-Ab-ITSF and
358
P-Ab-ITSB), internal control primers (P-rA1 and P-rA2) specific for the recA gene of
359
all Acinetobacter spp., A. nosocomialis-specific primers (gyrB-directed primers: sp4F
360
and sp4R), and A. pittii-specific primers (AGS3-R and AGS3-F). Lanes: M, 100-bp
361
DNA ladder; 1, A. calcoaceticus; 2, A. baumannii; 3, A. pittii; 4, A. nosocomialis; 5,
362
negative control.
363
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