ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2006, p. 3222–3224
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0066-4804/06/$08.00⫹0 doi:10.1128/AAC.00284-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Emergence of PER-2 and VEB-1a in Acinetobacter baumannii Strains in the Americas
Antimicrobial treatment of Acinetobacter infections may be
limited because of the emergence of extended-spectrum ␤-lac-
tamase (ESBL)- and carbapenemase-producing multiresistant
strains (7, 13). Non-TEM-, non-SHV-derived ESBLs (such as
PER and VEB) have been documented in Acinetobacter iso-
lates from Europe and Asia (7) but not yet from the Americas.
Recently, an increasing trend has been documented in
carbapenem and extended-spectrum cephalosporin resistance
in Acinetobacter isolates in Buenos Aires. Indeed, data from
the Whonet-Argentina Network showed that, in 2004, more
than 80% of Acinetobacter isolates were resistant to extended-
spectrum cephalosporins. In addition, imipenem resistance in-
creased from 5% to 54%, in the period from 2000 to 2004 (M.
Galas, unpublished results). National ESBL surveillance is per-
formed by Whonet-Argentina Network participants using a
modification of the CLSI antibiogram (8), consisting of a syn-
ergy test with ceftazidime or cefepime and amoxicillin-clavu-
lanic acid (distance between disks, 20 mm, center to center).
FAV-1 and M5179 were the first putative ESBL-producing
Acinetobacter baumannii strains isolated at two hospitals in
Buenos Aires in 2000 and 2003, respectively. FAV-1 was a
colonizer from the urine of a patient hospitalized for lung
transplantation. M5179 was from the peritoneal fluid of a
patient with hemolytic-uremic syndrome. Strains were con-
firmed to be A. baumannii by using the API 20NE system
(bioMérieux, Marcy l’Etoile, France) combined with their
ability to grow at 44°C.
MICs (micrograms per milliliter) for FAV-1 and M5179
determined by agar dilution (10) were, respectively, as follows:
ticarcillin, 1,024 and 1,024; piperacillin, 512 and 256; piperacil-
lin-tazobactam, 512 and 0.5; ampicillin-sulbactam, 8 and 8;
cefotaxime, 64 and 64; cefotaxime-clavulanate, 64 and 8; cef-
tazidime, 64 and 512; ceftazidime-clavulanate, 16 and 2;
cefepime, 64 and 64; cefepime-clavulanate, 16 and 4; aztreo-
nam, 512 and 512; imipenem, 8 and 0.5; meropenem, 8 and 0.5;
amikacin, 128 and 32; gentamicin, 2 and 32; trimethoprim-
sulfamethoxazole, 128 and 8; ciprofloxacin, 32 and 0.12; ri-
fampin, 2 and ⬎32; minocycline, 0.5 and ⬍0.25. EDTA (0.4
mM) did not affect carbapenem MICs.
Attempts to transfer the ceftazidime (FAV-1 and M5179) or
imipenem (FAV-1) resistance marker by conjugation or elec-
troporation to Escherichia coli ER1793 (6) were unsuccessful.
Isoelectric focusing analysis (6) revealed ESBL bands at pIs
of 5.4 (FAV-1) and 7.4 (M5179) and narrow-spectrum ␤-lac-
tamase bands at pIs of 6.9, 9.4 (FAV-1), and 5.4 (M5179). A
band at a pI of 9.4 was inhibited by oxacillin (1 mM) (probable
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AmpC-like enzyme). A band at a pI of 6.9 showed weak ac-
tivity against imipenem and was not inhibited by oxacillin,
clavulanate (1 mM), or EDTA (30 mM).
In the PCR screening for ESBL genes (Table 1), amplifica-
tions of blaPER (FAV-1) or blaTEM and blaVEB (M5179) were
positive. DNA sequencing identified the genes as blaPER-2,
blaTEM-1, and blaVEB-1a (11) (the latter unequivocally identi-
fied by full-length amplification and sequencing). PCR map-
ping with class 1 integron-blaVEB primer combinations (5⬘-CS–
VEB-R, IntI1-F–VEB-R, VEB-F–3⬘-CS, and VEB-F–sulI-R)
under either conventional or long amplification (Elongase
Amplification System, Invitrogen, California) conditions yielded
negative results. A PCR assay with primers 5⬘-CS and 3⬘-CS
resulted in a unique amplicon of 1,395 bp that carried arr-2 (first
report in Argentina) and aacA4 cassettes (4). PCRs with primers
targeting intI2 and intI3 or with primer combinations 3⬘-CS–
VEB-R, 3⬘-CS–VEB-Rc, sulI-R–VEB-R, and sulI-R–VEB-Rc, to
target antisense insertion of blaVEB-1a (1), also failed to generate
amplicons. These data suggested an unusual localization of the
blaVEB-1a gene in M5179.
The PCR characterization of the carbapenemase-encod-
ing genes in FAV-1 (Table 1) yielded positive amplifications
for blaOXA subgroup 3 and blaOXA-58. The identities of
blaOXA-51 and blaOXA-58 were determined by amplification
and sequencing of complete genes and flanking regions as
described previously (2) (GenBank accession numbers
DQ385606 and DQ385607, respectively). The region up-
stream of blaOXA-51 showed 79% identity with a fragment of
the Acinetobacter sp. strain ADP1 genome (positions
2,028,863 to 2,029,156 in the sequence with GenBank acces-
sion number CR543861), supporting its previously proposed
chromosomal location (3). The genetic environment of
blaOXA-58 was the same as that in an A. baumannii isolate
from France, i.e., bracketed by two ISAba3 elements in
opposite orientations (10).
This is the first report of VEB- and PER-producing Acineto-
bacter strains in the Americas and also the first documentation
of a VEB-type enzyme on this continent. Since 2003, a nation-
wide surveillance effort to address the degree of dissemination
of ESBLs among Acinetobacter strains in Argentina has re-
vealed the occurrence of 21 ESBL-producing isolates, PER-2
(n ⫽ 11) and VEB (n ⫽ 10), in four provinces. Therefore, the
emergence of both VEB and PER-2 in Acinetobacter strains in
Argentina constitutes a public health concern.
Fernando Pasterán and Melina Rapoport contributed equally to
this work.
 TABLE 1. Primers used in this study
a
Purpose and target Primer name Use(s) Oligonucleotide sequence Reference

␤-Lactamase characterization
ESBLs
blaTEM OT-1 Bothe TTGGGTGCACGAGTGGGTTA 6
OT-2 TAATTGTTGCCGGGAAGCTA

blaSHV OS-1 Screening TCGGGCCGCGTAGGCATGAT 6

OS-2 AGCAGGGCGACAATCCCGCG

blaPER PER-F Both GTAGTATCAGCCCAATCCCC 6

PER-R CCAATAAAGGCCGTCCATCA

blaCTX-M-2 CTX-M-2 plus Screening CGGAATTCATGATGACTCAGAGCATTCG 6

CTX-M-2 minus GCTCTAGATTATTGCATCAGAAACCGTG

blaGES GES-F Screening GAAAAAGCAGCTCAGATCG 9

GES-R CAACAACCCAATCTTTAGGA

blaVEB VEB-F Screening GGAACAACTTTGACGATTGA This work

VEB-R CCCTGTTTTATGAGCAACAA
VEB-Fc Sequencing GATAGGAGTACAGACATATG
VEB-Rc TTTATTCAAATAGTAATTCCACG

blaOXA-10 OG1-F Screening TCAACAAATCGCCAGAGAAG This work

OG1-R TCCCACACCAGAAAAACCAG

Carbapenemases
Ambler class Db

blaOXA subgroup 1 O23-F Screening TCGGATTGGAGAACCAGAAAA This work

O23/27-R GTATAGATGCCGGCATTTCTGA

blaOXA subgroup 2 O25/26-F Screening GATGAAGCTCAAACACAGGG This work

O26-R ATGATTCCAAGATTTTCTAGCG

blaOXA subgroup 3 OG6-F Both CTCGTGCTTCGACCGAGTAT This work

OG6-R GCTGAACAACCCATCCAGTT
OXA51-R 278–299 Sequencing TCTGTGGTGGTTGCCTTATGGT
OXA51-R 237–260 GCATTAAGCATTTTGAAGGTCGAA
OXA51-F 532–553 GAGGCACAGTTTGCTTACAAGC
OXA51-F 586–607 GTCCAAGATGAAGTGCAATCCA

blaOXA-58 OXA58-F Both AAGTATTGGGGCTTGTGCTG This work

OXA58-R TACGACGTGCCAATTCTTGA
OXA58-R 153–176 Sequencing GTGACAAACACAGCATCAGCTGAG
OXA58-R 119–139 GCGCTTGAACATTCTGATCGA
OXA58-F 318–337 GTGGGATGGAAAGCCACGTT
OXA58-F 369–391 GGGCGAAGCCATGCAAGCATCTA

blaOXA flanking sequences FPO (outer) Sequencing CAGTTCAAGCTTGTCCAGGAATTCN7CCGGA This work

FPI (inner) Sequencing CAGTTCAAGCTTGTCCAGGAATTC

Ambler class B

blaVIM VIM-F Screening AGTGGTGAGTATCCGACAG 9

VIM-R ATGAAAGTGCGTGGAGAC

blaIMP IMP-UF Screening GGYGTTTWTGTTCATACWTCKTTYGA This work

IMP-UR GGYARCCAAACCACTASGTTATCT

blaSPM-1 SPM-F Screening AGACCGCGATTTCTATTCTT This work

SPM-R AGTTCCTTCGGCTTTATCAT

Integron characterization
5⬘ CSc 5⬘-CS Both GGCATCCAAGCAGCAAG 6
3⬘ CSd 3⬘-CS AAGCAGACTTGACCTGA

intI1 IntI1-F Screening ATCATCGTCGTAGAGACGTCGG 12

IntI1-R GTCAAGGTTCTGGACCAGTTGC

intI2 IntI2-F Screening GCAAATGAAGTGCAACGC 12

IntI2-R ACACGCTTGCTAACGATG

intI3 IntI3-F Screening GCAGGGTGTGGACGAATACG 12

IntI3-R ACAGACCGAGAAGGCTTATG

sulI sulI-R Screening TGAAGGTTCGACAGCAC 5

a
F, forward; R, reverse. Numbers to the right of OXA-51 and OXA-58 (R or F) primers indicate the corresponding binding regions (positions on blaOXA-51 and
blaOXA-58 encoding genes, respectively).
b
Subgroup 1, OXA-23, OXA-27, and OXA-49; subgroup 2, OXA-24 to OXA-26, OXA-40, and OXA-72; subgroup 3, OXA-51, OXA-64 to OXA-66, OXA-68 to
OXA-71, and OXA-75 to OXA-78; subgroup 4, OXA-58.
c
5⬘ conserved segment of class 1 integrons.
d
3⬘ conserved segment of class 1 integrons.
e
Both screening and sequencing.

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3224 LETTERS TO THE EDITOR
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Fernando Pasterán
Melina Rapoport
Alejandro Petroni
Diego Faccone
Alejandra Corso
Marcelo Galas*
Servicio Antimicrobianos
Departamento Bacteriologı́a
Instituto Nacional de Enfermedades Infecciosas-ANLIS
“Carlos G. Malbrán”
Ciudad Autónoma de Buenos Aires, Argentina
Miryam Vázquez
Adriana Procopio
Sección Microbiologı́a
Hospital de Niños “Ricardo Gutiérrez”
Gobierno de Buenos Aires
Ciudad Autónoma de Buenos Aires, Argentina
Marta Tokumoto
Viviana Cagnoni
Instituto de Cardiologı́a y Cirugı́a Cardiovascular
“Fundación Favaloro”
Ciudad Autónoma de Buenos Aires, Argentina
*Phone and fax: 54 11 4 303 2812
E-mail: mgalas@anlis.gov.ar