Sequence types of Portuguese carbapenem-resistant Acinetobacter baumannii isolates collected over 10 years

A. baumannii, MLST, IMP-5 β-lactamase, European clone II

Sir,

Acinetobacter baumannii is recognized as one of the most problematic nosocomial pathogens, whose infections are often associated with epidemic spread and outbreaks of multidrug-resistant strains. The identification of resistant clones with epidemic potential is crucial to understanding the epidemiology of this microorganism, and to enable the review and implementation of adequate infection control guidelines. In Portugal, a sublineage of A. baumannii European clone II was identified by PFGE and amplified fragment length polymorphism in different Portuguese hospitals. The sublineage was responsible for the spread of the multidrug-resistant phenotype associated with this strain, including resistance to imipenem.¹ Despite the good discrimination obtained by these typing methods, they are laborious and fingerprinting results are not always transferable between laboratories, as protocols and thresholds may differ. The development of multilocus sequence typing (MLST) using housekeeping genes for the characterization of A. baumannii isolates allows the comparison of results between laboratories, contributing to a global epidemiological understanding of the relationship between A. baumannii phenotypes and genotypes.^2,3

The main objective of this study was, therefore, to identify the sequence types (STs) of carbapenem-resistant isolates of A. baumannii collected in Portugal between 1998 and 2009. Our carbapenem-resistant A. baumannii collection (540 isolates) showed three antimicrobial susceptibility patterns: A, resistant to all β-lactams, including carbapenems, but susceptible to quinolones and aminoglycosides (collected from 1998 to 1999); B, resistant to all antibiotics, except tobramycin and/or amikacin for some isolates and colistin (collected from 1999 to 2009); and C, resistant to β-lactam antibiotics and susceptible to gentamicin, netilmicin, tobramycin, amikacin and colistin (collected from 2004 to 2009). Fifteen isolates were selected from these groups according to the antimicrobial susceptibility profile, the date of isolation, the geographical location of the hospital and the genotype profile (Table 1). Antimicrobial susceptibility was confirmed using the disc diffusion method for β-lactams, quinolones and aminoglycosides. MICs of ceftazidime and imipenem were determined by Etest. MLST was performed using two schemes,^2,3 and sequences for each gene were compared with those available at Acinetobacter MLST websites http://pubmlst.org/abaumannii/ and http://www.pasteur.fr/recherche/genopole/PF8/mlst/Abaumannii.html.^2,3

Table 1.

Data for the carbapenem-resistant A. baumannii representative isolates collected from 1998 to 2009 in several Portuguese hospitals and results obtained by MLST

					Susceptibility profile													Institute Pasteur MLST scheme3								Bartual et al.2 MLST scheme
Month/year	Ward	Sample	Hospital	Isolate	AMX	TIC	CAZ	FOX	ATM	IPM	GEN	NET	TOB	AMK	CIP	CST	PFGEa	cpn60	fusA	gltA	pyrG	recA	rplB	rpoB	ST	gltA	gyrB	gdhB	recA	cpn60	gpi	rpoD	ST
122HUC	3/98	medical unit	urine	HUC	R	R	R	R	R	R	S	S	S	S	S	S	A	1	1	2	2	3	4	4	32	1	3	6	1	4	3	6	120
65HUC	9/98	neurotraumatology	urine	HUC	R	R	R	R	R	R	S	S	S	S	S	S	A	1	1	2	2	3	4	4	32	1	3	6	1	4	3	6	120
140HUC	1/99	ICU	blood	HUC	R	R	R	R	R	R	S	S	S	S	S	S	A	1	1	2	2	3	4	4	32	1	3	6	1	4	3	6	120
142HUC	1/99	burn unit	wound	HUC	R	R	R	R	R	R	R	R	S	I	R	S	B4	2	2	2	2	2	2	2	2	1	12	3	2	2	3	3	98
11HSM	2/99	orthopaedic unit	blood	HSM	R	R	R	R	R	R	R	R	R	R	R	S	B1	2	2	2	2	2	2	2	2	1	12	3	2	2	3	3	98
159HCR	11/99	medical unit	urine	HCR	R	R	R	R	R	R	R	R	R	R	R	S	B1	2	2	2	2	2	2	2	2	1	12	3	2	2	3	3	98
4HSB	8/00	ICU	pulmonary secretion	HSB	R	R	R	R	R	R	R	R	R	R	R	S	B1	2	2	2	2	2	2	2	2	1	12	3	2	2	3	3	98
189HUC	10/00	ICU	pulmonary secretion	HUC	R	R	R	R	R	R	R	R	S	I	R	S	B1	2	2	2	2	2	2	2	2	1	12	3	2	2	3	3	98
40HSFX	2/02	ICU	pus	HSFX	R	R	R	R	R	R	R	R	R	R	R	S	B2	2	2	2	2	2	2	2	2	1	12	3	2	2	3	3	98
130HSM	6/02	ICU	pulmonary secretion	HSM	R	R	R	R	R	R	R	R	R	R	R	S	B3	2	2	2	2	2	2	2	2	1	12	3	2	2	3	3	98
2396HP	2/04	medical unit	pulmonary secretion	HP	R	R	R	R	R	R	R	R	S	I	R	S	B1	2	2	2	2	2	2	2	2	1	12	3	2	2	3	3	98
A-2Ev	1/09	ICU	pulmonary secretion	HES	R	R	R	R	R	R	R	R	S	I	R	S	B1	2	2	2	2	2	2	2	2	1	12	3	2	2	3	3	98
2HSAC	4/04	ICU	pulmonary secretion	HSAC	R	R	R	R	R	R	S	S	S	S	S	S	B5	2	2	2	2	2	2	2	2	1	12	3	2	2	3	3	98
437HUC	12/07	ICU	blood	HUC	R	R	R	R	R	R	S	S	S	S	S	S	B6	2	2	2	2	2	2	2	2	1	12	3	2	2	3	3	98
N-1Ev	2/09	ICU	urine	HES	R	R	R	R	R	R	S	S	S	S	S	S	B5	2	2	2	2	2	2	2	2	1	12	3	2	2	3	3	98

ICU, intensive care unit; HUC, Hospitals of the University of Coimbra; HSFX, Hospital S. Francisco Xavier, Lisboa; HSM, University Hospital of Santa Maria, Lisboa; HSB, Hospital S. Bernardo, Setúbal; HCR, Hospital das Caldas da Rainha; HP, Hospital de Pombal; HSAC, Hospital S. António dos Capuchos, Lisboa; HES, Hospital Espírito Santo, Évora; AMX, amoxicillin; TIC, ticarcillin; CAZ, ceftazidime; FOX, cefoxitin; ATM, aztreonam; IPM, imipenem; GEN, gentamicin; NET, netilmicin; TOB, tobramycin; AMK, amikacin; CIP, ciprofloxacin; CST, colistin; R, resistant; I, intermediate; S, susceptible.

^aDNA fingerprinting of profiles B2 to B6 differ from B1 by one to three bands.

The three isolates of antimicrobial susceptibility group A, producing IMP-5 metallo-β-lactamase,⁴ were recovered from only one hospital and were not detected in further years. Nine isolates were selected from group B and three from group C according to the date of isolation, hospital, ward and type of sample. Isolates of groups B and C, producing OXA-40 enzyme, were recovered from seven hospitals located in the centre of Portugal and Lisbon region (HUC, HSM, HCR, HSB, HSFX, HP and HSAC) and, recently (2008–09), from a hospital located in the south of Portugal (HES). All isolates showed MICs of imipenem and ceftazidime of >32 and >256 mg/L, respectively.

According to the http://pubmlst.org/abaumannii/ database,² isolates of groups B and C (the sublineage of European clone II)¹ are ST98 (gltA-1, gyrB-12, gdhB-3, recA-2, cpn60-2, gpi-3 and rpoD-3), described for the first time in Portugal. ST98 shared six alleles with ST4, ST6, ST27, ST76, ST118 and ST130 genotypes, isolates recovered between 1982 and 2009 in Italy, Spain, Germany, Japan and Australia. The IMP-5 producer revealed a new ST, deposited in this database as ST120 (gltA-1, gyrB-3, gdhB-6, recA-1, cpn60-4, gpi-3 and rpoD-6). ST120 was not clonally related to ST98, but was clonally related to ST40, a Danish isolate from 1990, sharing five alleles.

According to the Institute Pasteur database,³ the isolates of groups B and C showed the same allelic profile (cpn60-2, fusA-2, gltA-2, pyrG-2, recA-2, rpIB-2 and rpoB-2). This allelic profile corresponds to European clone II, recommended to be designated by ST2 or CC2 (where CC stands for clonal complex) for uniform nomenclature, and is widely disseminated in the world.³ The IMP-5 producer was shown to be ST32 (cpn60-1, fusA-1, gltA-2, pyrG-2, recA-3, rpIB-4 and rpoB-4), corresponding to CC32 isolated in Denmark in 1990.³ The closest matches were ST28 and ST53 collected in Sweden in 1980–81, with only one different allele (recA and rpoB, respectively).³

Recently, it has been suggested that the gyrB and gpi loci, genes used in the Bartual et al.² scheme, may be affected by horizontal gene transfer, and are thus not good candidates for phylogeny studies.⁵ In this work, the two databases showed identical results.

The successful spread of ST2 (CC2)/ST98 may be related to the ability to develop multidrug resistance. Differences in resistance profiles may be due to local antibiotic selective pressures. Furthermore, the high dissemination of CC2 might be related to virulence factors. Recently, the presence of putative alien islands (pA_ICU20 and pA_ICU32) that may contribute to the virulence of this clone was shown.⁶ In contrast, the spread of the ST32 (CC32)/ST120 clone is limited. We describe for the first time that the IMP-5 producer strain belongs to ST32, according to the Institute Pasteur database, and apparently does not have epidemic potential.

Overall, not all of the carbapenem-resistant A. baumannii strains have the same ability to spread. However, the surveillance of carbapenem-resistant A. baumannii strains and better knowledge of well-identified clones with defined characteristics are crucial to enable the tracking of potential epidemic strains and to improve infection control measures, thus preventing further dissemination.

Funding

This work was supported financially by iMed.UL, University of Lisbon and by the Center of Pharmaceutical Sciences from Fundação para a Ciência e a Tecnologia, Lisbon, Portugal. N. M. is supported by grant SFRH/BPD/45815/2008 from Fundação para a Ciência e a Tecnologia, Lisbon, Portugal.

Transparency declarations

None to declare.

Acknowledgements

Part of this work was presented at the Twentieth European Congress of Clinical Microbiology and Infectious Diseases, Vienna, Austria, 2010 (Poster 1299).