Molecular Characterization of Achromobacter Isolates from Cystic Fibrosis and Non-Cystic Fibrosis Patients in Madrid, Spain

Abstract

Multilocus sequence typing and nrdA sequence analysis identified 6 different species or genogroups and 13 sequence types (STs) among 15 Achromobacter isolates from cystic fibrosis (CF) patients and 7 species or genogroups and 11 STs among 11 isolates from non-CF patients. Achromobacter xylosoxidans was the most frequently isolated species among CF patients.

TEXT

Achromobacter spp. are nonfermenting, Gram-negative rods that are widely distributed in nature, some of which are associated with opportunistic infection of humans (1). Because phenotypic methods for identification are inadequate, multilocus sequence typing (MLST) (2) and nrdA 765 DNA sequencing (3) have recently been developed to elucidate the taxonomy and epidemiology of Achromobacter isolates. In addition to seven previously named Achromobacter species, 14 new taxa have now been described, some of which are associated with infections in cystic fibrosis (CF) patients (2–5). The recent changes in the knowledge of the biodiversity of Achromobacter infections and the novel taxonomy mean that there are few published data on the clinical impact of different Achromobacter species. The aims of this study were to determine the prevalence and clinical importance of Achromobacter infection in CF patients in Spain and to understand the distribution of this genus among CF and non-CF patients.

We undertook a retrospective study of all CF patients who were colonized or infected with Achromobacter and who were treated at the CF reference center of the Hospital Universitario 12 de Octubre (Madrid) between 2002 and 2012. Patients were considered chronically colonized if at least three positive cultures were obtained in 1 year and sporadically colonized if there were less than three positive cultures. Clinical records were reviewed to collect demographic, clinical, and microbiology data. Sputum samples were inoculated onto different culture media (Columbia 5% blood agar, MacConkey agar, mannitol-salt agar, chocolate agar, and Burkholderia cepacia selective agar [BCSA] medium). Phenotypic identification was performed using the Wider system (Francisco Soria-Melguizo, Spain) and API 20 NE (bioMérieux, Marcy l′Etoile, France). Antimicrobial susceptibility was determined by broth microdilution and the ε-test method and interpreted according to CLSI guidelines (6). Sequence analysis of 16S rRNA was used to confirm the phenotypic identification of Achromobacter spp., and MLST (2) and nrdA 765 DNA sequencing (3) were performed on all isolates. We tested for the presence of the bla_OXA-114-like gene by PCR for the identification of A. xylosoxidans (7). Taxonomic status was obtained by comparison of results to those in the GenBank (http://www.ncbi.nlm.nih.gov/BLAST) and MLST (http://pubmlst.org/achromobacter/) databases. Achromobacter isolates were also genotyped by pulsed-field gel electrophoresis (PFGE) (8). Univariate analysis was performed using the t test for continuous variables and the χ² or Fisher's exact test for categorical variables.

Over the study period, 5.6% (14/250) of CF patients were colonized with Achromobacter spp., 9 (3.6%) with chronic colonization and 5 (2.0%) with sporadic colonization, compared with 1.1% to 29.3% in other studies (8–15). Clinical characteristics of chronically and sporadically colonized patients are shown in Table 1. Although other reports have shown A. xylosoxidans colonization to be associated with older age, more pronounced lung damage, and lower lung function values (9, 16), we were unable to establish a relationship between chronic Achromobacter colonization and lung function, probably because of the small sample size and limited duration of follow-up (1 year). Another study that evaluated patients 3 years after the first isolate suggested that A. xylosoxidans infection might lead to a decline in lung function in a subgroup of chronically infected CF patients (17).

Table 1.

Clinical characteristics of 14 CF patients colonized with Achromobacter species

Characteristic(s) (n = 14)	Colonization result for characteristic
	Chronic (n = 9)	Sporadic (n = 5)
Mean ± SD age (yr)	24.3 ± 14.5	10.9 ± 3
No. female (%)	6/9 (66.6)	2/5 (40)
Body mass index (%)	20.3 ± 4.7	16.8 ± 2.3
No. (%) with CF transmembrane regulator mutation type:
F508del homozygote	4/9 (44.4)	2/5 (40)
F508del heterozygote	3/9 (33.3)	2/5 (40)
Others	2/9 (22.2)	1/5 (20)
No. (%) with:
Pancreatic insufficiency	7/9 (77.7)	5/5 (100)
Diabetes mellitus	2/9 (22.2)	2/5 (40)
Liver disease	2/9 (22.2)	0/5 (0)
Mean ± SD pulmonary function (%)a
FEV1	66.1 ± 15.9	61.8 ± 17.5
FVC	75.3 ± 15.6	69 ± 16.9
No. (%) of patients hospitalized 1 yr before	4/9 (44.4)	1/5 (20)
No. (%) with antimicrobial exposure tob:
≥3 groups	4/9 (44.4)	3/5 (60)
1 or 2 groups	5/9 (55.5)	2/5 (40)
Tobramycin (inhaled)	3/9 (33.3)	2/5 (40)
Colistin (inhaled)	2/9 (22.2)	1/5 (20)
No. (%) coinfected with other microorganism(s)
Staphylococcus aureus	5/9 (55.5)	2/5 (40)
Pseudomonas aeruginosa	4/9 (44.4)	1/5 (20)
Burkholderia cepacia complex	1/9 (11.1)	0/5 (0)
Enterobacteriaceae	0/9 (0)	1/5 (20)
Other nonfermentative Gram-negative bacilli	1/9 (11.1)	2/5 (40)
Haemophilus influenzae	2/9 (22.2)	1/5 (20)

^aFEV1, forced expiratory volume in 1 s; FVC, forced vital capacity.

^bAntimicrobial exposure in the previous year of acquisition and 1 year after the first isolate. The groups of antimicrobial agents were β-lactams, macrolides, fluoroquinolones, aminoglycosides, trimethoprim-sulfamethoxazole, and others.

Molecular characterization based on MLST and nrdA 765 sequencing identified 6 different species or genogroups and 13 different sequence types (STs) among 15 Achromobacter isolates from 14 patients: 10 (71.4%) A. xylosoxidans (ST3, ST20, ST27, ST153, ST154, ST156, ST158, ST160), and one (7.1%) each of Achromobacter genogroup 2a (ST144), Achromobacter genogroup 2b (ST150), Achromobacter genogroup 7 (ST152), Achromobacter genogroup 14 (ST162), and Achromobacter genogroup 17 (ST149) (Table 2) (18). Similar diversity of species was also observed in a recent study that included 341 isolates from 86 CF centers in the United States. This study identified 14 different species or genogroups, the most prevalent being A. xylosoxidans (42%), Achromobacter ruhlandii (23.5%), and Achromobacter genogroup 14 (17%) (3). In our study, A. xylosoxidans was more frequently isolated from patients with chronic than sporadic colonization (88.9% versus 20%; P = 0.023).

Table 2.

Characteristics and distribution of Achromobacter species or genogroups among 26 isolates obtained from 25 CF and non-CF patients

Source	Patient ID no.	Species	Date of first isolation	Colonization typea	Sample type	PFGE pattern	Sequence type	blaOXA-114-like gene	Resistance patternb
CF	1	A. xylosoxidans	2003	CC	Sputum	K	ST154	+	FEP, IPM, CIP, CO
	2	A. xylosoxidans	2004	CC	Sputum	P	ST27	+	FEP, CAZ, IPM, MEM, CIP, CO
	3	A. xylosoxidans	2005	CC	Sputum	P	ST27	+	FEP, CAZ, IPM, MEM, CIP, SXT
	4	A. xylosoxidans	2005	CC	Sputum	Q	ST158	+	FEP, CIP
	5	A. xylosoxidans	2005	SC	Sputum	T	ST160	+	FEP, CIP
	6	A. xylosoxidans	2006	SC	Sputum	E	ST20	+
	7	A. xylosoxidans	2007	CC	Sputum	J	ST153	+	FEP, CAZ, IPM, MEM, CIP
	8	A. xylosoxidans	2007	CC	Sputum	N	ST156	+	FEP, CAZ, CIP, CO
	9	A. xylosoxidans	2012	CC	Sputum	C	ST3	+
	10	A. xylosoxidans	2012	CC	Sputum	J	ST153	+	FEP, CAZ, CO
	11	Achromobacter genogroup 2a	2007	SC	Sputum	S	ST144	+
	12	Achromobacter genogroup 2b	2009	SC	Sputum	F	ST150	-	CO
	13	Achromobacter genogroup 7	2012	CC	Sputum	I	ST152	-
	14	Achromobacter genogroup 14	2011	SC	Sputum	V	ST162	+
	13	Achromobacter genogroup 17	2007	CC	Sputum	B	ST149	+	FEP, CAZ, IPM
Non-CF	15	A. xylosoxidans	2003		Blood culture	P	ST27	+
	16	A. xylosoxidans	2010		Ear discharge	A	ST19	+	FEP, CAZ, CO
	17	A. xylosoxidans	2010		Blood culture	H	ST16	+
	18	A. xylosoxidans	2010		Nasal aspirate	O	ST157	+	FEP, CO
	19	A. xylosoxidans	2012		Biopsy	R	ST159	+	FEP, CIP
	20	Achromobacter genogroup 2a	2005		Blood culture	U	ST161	+
	21	Achromobacter genogroup 7	2002		Blood culture	I	ST152	−
	22	A. spiritinus (genogroup 10)	2006		Sputum	G	ST151	−	FEP, CO
	23	A. pulmonis (genogroup 11)	2006		Aqueous Humor	L	ST155	−	FEP
	24	Achromobacter genogroup 12	2010		Blood culture	D	ST71	+	CIP
	25	A. insolitus	2011		Blood culture	M	ST93	−

^aCC, chronic colonization; SC, sporadic colonization.

^bA. xylosoxidans exhibits innate resistance to many antibiotics, including cephalosporins (except ceftazidime), aztreonam, and aminoglycosides (18). FEP, cefepime; CAZ, ceftazidime; IPM, imipenem; MEM, meropenem; CIP, ciprofloxacin; SXT, trimethoprim-sulfamethoxazole; CO, colistin; GEN, gentamicin; TOB, tobramycin; AMK, amikacin.

Because of the genetic diversity of Achromobacter spp. identified, we investigated whether this was also evident among isolates from non-CF patients. Eleven Achromobacter isolates from 11 patients were available for analysis, among which we found 7 different species or genogroups and 11 different sequence types (STs): 5 (45.4%) A. xylosoxidans (ST16, ST19, ST27, ST157, ST159) and 1 (9.1%) each of Achromobacter genogroup 2a (ST161), Achromobacter genogroup 7 (ST152), Achromobacter spiritinus (genogroup 10; ST151), Achromobacter pulmonis (genogroup 11; ST155), Achromobacter genogroup 12 (ST71), and Achromobacter insolitus (ST93) (Table 2).

PFGE analysis showed good correlation between the results of MLST analysis and nrdA 765 sequencing. Each ST corresponded to a different PFGE pattern (Table 2). ST27 was identified in two CF patients and one non-CF patient, while ST152 was identified in one CF patient and one non-CF patient. In all cases in which a common ST was found, the PFGE patterns were also indistinguishable, although no epidemiologic link was established between the patients. It is possible that the common strains identified among CF patients could be the result of person-to-person spread, as has been reported elsewhere (13, 14, 17, 19).

In order to evaluate whether chronic colonization involves the same or different strains over time, we used PFGE to analyze 40 isolates from 9 chronically colonized patients with infections spanning 1 to 9 years. Eight patients harbored the same strain over time. One who was initially colonized with Achromobacter genogroup 17 (PFGE pattern B) was found to carry a different strain of Achromobacter genogroup 7 (PFGE pattern I) 5 years after the initial isolate. Although both long-term colonization with a single strain and acquisition of different strains over time have been described (14, 20), our results suggest that chronic colonization is more often due to persistence than reinfection (1).

The intrinsic β-lactamase bla_OXA-114-like gene is reported to provide a means to identify A. xylosoxidans (7, 21). Nevertheless, all of our isolates of A. xylosoxidans and the recently described Achromobacter species or genogroups 2a, 12, 14, and 17 were also shown to carry this gene (Table 2). Sequencing of the PCR products from 3 representative A. xylosoxidans and 5 non-A. xylosoxidans isolates confirmed our results, suggesting that detection of the β-lactamase bla_OXA-114-like gene is not specific for A. xylosoxidans.

In summary, we demonstrated colonization and/or infection in both CF and non-CF patients by a wide diversity of Achromobacter species. We found A. xylosoxidans to be the most common species involved in chronic colonization of CF patients, although further studies are necessary to understand the epidemiology, virulence, and clinical impact of other Achromobacter species.