Abstract
Molecular methodologies were used to identify 28 Achromobacter spp. from patients with cystic fibrosis (CF). Multilocus sequence typing (MLST) identified 17 Achromobacter xylosoxidans isolates (all blaOXA-114 positive), nine Achromobacter ruhlandii isolates (all blaOXA-114 positive), one Achromobacter dolens isolate, and one Achromobacter insuavis isolate. All less common species were misidentified as A. xylosoxidans by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Chronic colonization by clonally related A. ruhlandii isolates was demonstrated.
TEXT
Achromobacter is an emerging pathogen in cystic fibrosis (CF) patients, but accurate species identification of isolates is difficult. Most Achromobacter clinical isolates are designated as Achromobacter xylosoxidans; however, the commonly used phenotypic tests are not reliable (1–3). Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis is used in clinical microbiology laboratories as an emerging technology for bacterial species identification (4–8). Genotypic methods have also been developed for Achromobacter species. The amplification of an inner fragment of the blaOXA-114 gene has been proposed for A. xylosoxidans identification (9). Furthermore, the presence of blaOXA-258, blaOXA-364, and blaOXA-243 has been detected in Achromobacter ruhlandii, Achromobacter dolens, and Achromobacter insuavis, respectively (2, 3). Multilocus sequence typing (MLST) schemes that increase the accuracy of the characterization and identification of Achromobacter strains and species have also been developed (10, 11). This study compares the identification of Achromobacter species from CF patients by blaOXA-114 gene sequencing, MLST, and MALDI-TOF MS.
Twenty-eight archived isolates that had been collected between 2003 and 2008 from 16 CF patients attending two reference centers in Rio de Janeiro, Brazil, were included. The isolates were identified as A. xylosoxidans by 16S rRNA gene sequencing (12, 13); blaOXA-114-like gene amplification and sequencing were performed as described by Turton et al. (9). MLST analysis by sequencing of seven housekeeping genes (nusA, rpoB, eno, gltB, lepA, nuoL, and nrdA) was performed as described previously (11). Allelic profiles and sequence types (STs) were analyzed according to the PubMLST databases (http://pubmlst.org/achromobacter). MALDI-TOF MS identification was performed with a Microflex LT instrument (Bruker Daltonics, GmbH, Germany) using FlexControl 3.0 software (Bruker Daltonics). Scores of ≥2.0 indicated species-level identification, scores of 1.7 to 1.9 indicated genus-level identification, and scores of <1.7 indicated no reliable identification (14, 15).
Positive blaOXA-114 amplification was obtained for 26 isolates. In 17 isolates, amplicons showed 99 to 100% identity with the blaOXA-114 reference sequence. In 9 isolates, identified as A. ruhlandii, sequences displayed 99% identity with blaOXA-258. Two isolates (isolates 4530 and 7393) were negative for blaOXA. As observed by Papalia et al. (2), in our study not only A. xylosoxidans but also A. ruhlandii yielded positive amplification for the A. xylosoxidans species-specific marker blaOXA-114. Amplification of the inner fragment without sequence analysis, as initially described by Turton et al. (9), thus may result in the misidentification of some Achromobacter species; by this criterion, we would have identified 26 isolates instead of 17 as A. xylosoxidans. MALDI-TOF MS identified 89% of the isolates (25/28 isolates) as A. xylosoxidans, while 7% (2/28 isolates) were identified at the probable genus level and one (with a score of 1.666) could not be identified (Table 1). MLST analysis identified 4 different species and 14 different STs among 28 isolates; 17 (61%) were A. xylosoxidans (ST2, ST13, ST198, ST200, ST201, ST202, ST205, ST206, and ST207), 9 (32%) A. ruhlandii (ST35, ST36, and ST204), one A. dolens (ST199), and one A. insuavis (ST203). Ten new STs and two new allelic arrangements were identified and added to the PubMLST database (http://pubmlst.org/perl/bigsdb/bigsdb.pl?db=pubmlst_achromobacter_seqdef&page=query&scheme_id=1) (Table 2).
TABLE 1.
Comparison of 16S rRNA gene sequencing, blaOXA-114 detection, and MLST analysis in characterization of Achromobacter species
| Isolate no. | Patient no. | Isolation date (day/mo/yr) | Origin | ST | 16S rRNA identification | MLST identification | OXA variant identification | MALDI-TOF MS |
|
|---|---|---|---|---|---|---|---|---|---|
| Best match | Score | ||||||||
| 6161 | 1 | 4/5/2007 | Center I | 207 | A. xylosoxidans | A. xylosoxidans | OXA-114 | A. xylosoxidans | 2.058 |
| 4747 | 2 | 9/5/2005 | Center I | 200 | A. xylosoxidans | A. xylosoxidans | OXA-114 | A. xylosoxidans | 2.052 |
| 4400 | 3 | 28/10/2004 | Center I | 198 | A. xylosoxidans | A. xylosoxidans | OXA-114 | A. xylosoxidans | 2.119 |
| 6531 | 5 | 24/8/2007 | Center II | 35 | A. xylosoxidans | A. ruhlandii | OXA-258 | A. xylosoxidans | 2.178 |
| 6956 | 5 | NDa | Center II | 35 | A. xylosoxidans | A. ruhlandii | OXA-258 | A. xylosoxidans | 2.109 |
| 7292 | 5 | 25/3/2008 | Center II | 35 | A. xylosoxidans | A. ruhlandii | OXA-258 | A. xylosoxidans | 2.016 |
| 8173 | 5 | 9/9/2008 | Center II | 35 | A. xylosoxidans | A. ruhlandii | OXA-258 | A. xylosoxidans | 2.267 |
| 4481 | 6 | 2/2/2004 | Center I | 13 | A. xylosoxidans | A. xylosoxidans | OXA-114 | A. xylosoxidans | 2.23 |
| 8054 | 7 | 20/10/2008 | Center I | 205 | A. xylosoxidans | A. xylosoxidans | OXA-114 | A. xylosoxidans | 2.01 |
| 6081 | 8 | 21/3/2007 | Center I | 202 | A. xylosoxidans | A. xylosoxidans | OXA-114 | A. xylosoxidans | 2.099 |
| 4530 | 9 | 27/1/2005 | Center I | 199 | A. xylosoxidans | A. dolens | −b | A. xylosoxidans | 2.074 |
| 6042 | 9 | 15/2/2007 | Center I | 201 | A. xylosoxidans | A xylosoxidans | OXA-114 | A. xylosoxidans | 2.315 |
| 7437 | 9 | 5/6/2008 | Center I | 201 | A. xylosoxidans | A. xylosoxidans | OXA-114 | A. xylosoxidans | 2.222 |
| 6694 | 10 | 11/10/2007 | Center I | 36 | A. xylosoxidans | A. ruhlandii | OXA-258 | A. xylosoxidans | 2.222 |
| 7022 | 10 | 17/1/2008 | Center I | 36 | A. xylosoxidans | A. ruhlandii | OXA-258 | A. xylosoxidans | 2.100 |
| 3446 | 11 | 18/6/2003 | Center I | 206 | A. xylosoxidans | A. xylosoxidans | OXA-114 | A. xylosoxidans | 2.00 |
| 4984 | 12 | 19/8/2005 | Center I | 200 | A. xylosoxidans | A. xylosoxidans | OXA-114 | A. xylosoxidans | 2.325 |
| 6016 | 12 | 15/2/2007 | Center I | 201 | A. xylosoxidans | A. xylosoxidans | OXA-114 | A. xylosoxidans | 2.124 |
| 8240 | 12 | 27/11/2008 | Center I | 201 | A. xylosoxidans | A. xylosoxidans | OXA-114 | A. xylosoxidans | 2.311 |
| 6241 | 13 | 21/5/2007 | Center I | 35 | A. xylosoxidans | A. ruhlandii | OXA-258 | A. xylosoxidans | 2.109 |
| 7291 | 14 | 31/3/2008 | Center II | 204 | A. xylosoxidans | A. ruhlandii | OXA-258 | A. xylosoxidans | 2.376 |
| 7863 | 14 | 22/8/2008 | Center II | 204 | A. xylosoxidans | A. ruhlandii | OXA-258 | NRc | 1.666 |
| 4168 | 15 | 17/6/2004 | Center I | 2 | A. xylosoxidans | A. xylosoxidans | OXA-114 | Achromobacter spp. | 1.84 |
| 4928 | 15 | 14/7/2005 | Center I | 200 | A. xylosoxidans | A. xylosoxidans | OXA-114 | A. xylosoxidans | 2.264 |
| 5038 | 17 | 22/9/2005 | Center I | 13 | A. xylosoxidans | A. xylosoxidans | OXA-114 | Achromobacter spp. | 1.907 |
| 5131 | 18 | 20/10/2005 | Center I | 201 | A. xylosoxidans | A. xylosoxidans | OXA-114 | A. xylosoxidans | 2.057 |
| 5146 | 18 | 20/10/2005 | Center I | 13 | A. xylosoxidans | A. xylosoxidans | OXA-114 | A. xylosoxidans | 2.054 |
| 7393 | 18 | 12/5/2008 | Center I | 203 | A. xylosoxidans | A. insuavis | −b | A. xylosoxidans | 2.018 |
ND, date not disclosed.
−, negative amplification.
NR, not reliable.
TABLE 2.
New alleles and STs of Achromobacter species described in this study
| ST | Allele |
No. of isolates | Isolate no(s). | Identification | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| nusA | rpoB | eno | gltB | lepA | nuoL | nrdA | nrdA_765 | ||||
| 198 | 6 | 89a | 2 | 65 | 59 | 4 | 2 | 36 | 1 | 4400 | A. xylosoxidans |
| 199 | 22 | 90a | 12 | 7 | 49 | 16 | 9 | 92 | 1 | 4530 | A. dolens |
| 200 | 9 | 91a | 2 | 65 | 87 | 2 | 2 | 36 | 4 | 4747, 4728, 4984, 5131 | A. xylosoxidans |
| 201 | 91a | 26 | 2 | 2 | 62 | 8 | 2 | 36 | 4 | 6016, 6042, 7437, 8240 | A. xylosoxidans |
| 202 | 91a | 92a | 2 | 2 | 59 | 8 | 2 | 111 | 1 | 6081 | A. xylosoxidans |
| 203 | 92a | 51 | 20 | 68a | 57 | 23 | 19 | 148 | 1 | 7393 | A. insuavis |
| 204 | 17 | 28 | 24 | 15 | 46 | 76a | 11 | 160a | 2 | 7291, 7863 | A. ruhlandii |
| 205 | 1 | 26 | 83a | 4 | 59 | 8 | 2 | 36 | 1 | 8054 | A. xylosoxidans |
| 206 | 14 | 21b | 2 | 4 | 59 | 2 | 2 | 111b | 1 | 3446 | A. xylosoxidans |
| 207 | 6b | 26 | 2 | 4 | 59 | 8 | 2 | 36 | 1 | 6161 | A. xylosoxidans |
Alleles described in the study.
Allelic variant determinant of new allelic profile.
Bacterial misidentification is especially problematic and presents a challenge to effective infection control in CF (11). With 16S rRNA gene sequencing as the sole criterion, 11 (39%) of our isolates would be wrongly identified as A. xylosoxidans. Furthermore, comparative analyses of 16S rRNA gene sequences are of limited value, as all known Achromobacter species exhibit less than 1% sequence dissimilarity with respect to each other (16). The MALDI-TOF MS results were 39% discordant with MLST identification. All A. ruhlandii (n = 9), A. dolens (n = 1), and A. insuavis (n = 1) isolates were incorrectly identified as A. xylosoxidans (Table 1), probably due to limitations of the database used, such as the lack of identification spectra for the reference strains (6). In our samples, we found ST2 and ST13, STs that were detected in CF patients in the United States between 2009 and 2010. We also detected ST36, an ST that was previously isolated in the United States (2004) and Russia (2013), in a non-CF patient and a CF patient, respectively (http://pubmlst.org/perl/bigsdb/bigsdb.pl?db=pubmlst_achromobacter_seqdef&page=query&scheme_id=1). Furthermore, we also observed ST35, which was first described in 1998 in a non-CF patient in the United States (http://pubmlst.org/achromobacter). This ST, identified as A. ruhlandii, was shared by two patients (patients 5 and 13) who attended different reference centers. This is the first report of this ST in CF patients. Perhaps this ST originated from a common source of contamination; however, further studies are needed to demonstrate the mode and source of ST acquisition. A group of Danish patients attending two different CF centers (in Copenhagen and Aarhus) were reported to share the same Achromobacter clone for several years (17, 18). This Danish epidemic strain (DES) was an exceptionally resistant clone capable of causing cross-infection even after brief indirect contact between infected and noninfected CF patients (19). Although it was not associated with any known ST, DES was recently identified as A. ruhlandii by MLST (10). We identified novel STs and, because the Achromobacter MLST database is a recently developed database that still lacks a large number of submitted data, our work contributes to update it with 10 new alleles and STs.
Our study demonstrated a concordance between blaOXA-114 gene sequencing and MLST analysis for A. xylosoxidans and A. ruhlandii identification. However, the misidentification of A. dolens and A. insuavis highlights the use of MLST as a robust identification scheme for novel and less common species. Interestingly, MLST analysis also showed the successive recovery of isolates from some patients. For three patients, A. ruhlandii isolates with identical characteristics were identified over 3-month to 1-year periods (patient 5, ST35; patient 10, ST36; patient 14, ST204). Two patients showed successive isolation of different species (patient 9, A. xylosoxidans [ST201] and A. dolens [ST199]; patient 18, A. xylosoxidans [ST201/ST13, isolated in the same culture] and A. insuavis [ST203]). For the remaining two patients, successive identification of A. xylosoxidans of different STs was made (patient 12, ST200/ST201; patient 15, ST2/ST200). These STs differ by five alleles in five genes, suggesting that no intrapatient adaptation occurred. Chronic colonization by clonally related A. xylosoxidans isolates has been described (13, 20), but the isolation of different species or different STs within a species is original. Moreover, for the first time we demonstrate chronic colonization by clonally related A. ruhlandii isolates in Brazilian CF patients.
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