A Taxonomically Unique Acinetobacter Strain with Proteolytic and Hemolytic Activities Recovered from a Patient with a Soft Tissue Injury

Abstract

A taxonomically unique bacterial strain, Acinetobacter sp. A47, has been recovered from several soft tissue samples from a patient undergoing reconstructive surgery owing to a
traumatic amputation. The results of 16S rRNA, rpoB, and gyrB gene comparative sequence analyses showed that A47 does not belong to any of the hitherto-known taxa and may represent an
as-yet-unknown Acinetobacter species. The recognition of this novel organism contributes to our knowledge of the taxonomic complexity underlying infections
caused by Acinetobacter.

Full text

A Taxonomically Unique Acinetobacter Strain with Proteolytic and
Hemolytic Activities Recovered from a Patient with a Soft Tissue
Injury
Marisa Almuzara,
b,c
German Matías Traglia,
a
Lenka Krizova,
d
Claudia Barberis,
b
Sabrina Montaña,
a
Romina Bakai,
c
Alicia Tuduri,
c
Carlos Vay,
b
Alexandr Nemec,
d
María Soledad Ramírez
a,e
Instituto de Microbiología y Parasitología Médica (IMPaM, UBA-CONICET),
a
Laboratorio de Bacteriología Clínica, Departamento de Bioquímica Clínica, Hospital de Clínicas
José de San Martín, Facultad de Farmacia y Bioquímica,
b
and Laboratorio de Bacteriología, Hospital Interzonal de Agudos Eva Perón, San Martín,
c
Buenos Aires, Argentina;
Laboratory of Bacterial Genetics, National Institute of Public Health, Šrobárova, Prague, Czech Republic
d
; Center for Applied Biotechnology Studies, Department of
Biological Science, California State University, Fullerton, Fullerton, California, USA
e
A taxonomically unique bacterial strain, Acinetobacter sp. A47, has been recovered from several soft tissue samples from a pa-
tient undergoing reconstructive surgery owing to a traumatic amputation. The results of 16S rRNA, rpoB, and gyrB gene com-
parative sequence analyses showed that A47 does not belong to any of the hitherto-known taxa and may represent an as-yet-un-
known Acinetobacter species. The recognition of this novel organism contributes to our knowledge of the taxonomic complexity
underlying infections caused by Acinetobacter.
CASE REPORT
A59-year-old female patient was admitted to an emergency
room owing to severe left forearm trauma and traumatic am-
putation of her left foot secondary to a road accident. On the day
of admission, she underwent a surgical toilette of the forearm and
infrapatellar leg amputation. At that moment, samples of skin,
soft tissue, and bone were sent for culture with negative results. Six
days after the surgical debridement, wound dehiscence with pu-
rulent discharge of the amputation stump with exposed bone was
observed. A new surgical debridement was performed, and a
wound secretion sample and two soft tissue samples were sent for
bacteriological examination. In the Gram stain, abundant Gram-
negative (GN) bacilli together with Gram-positive cocci arranged
in chains were seen. After 24 h of incubation at 35°C, colonies
formed by both a Gram-negative organism and Enterococcus
faecalis were observed on sheep blood agar. The colonies of the
Gram-negative bacterium were about 2 mm in diameter, with an
irregular spreading edge surrounded by wide zones of beta-hemo-
lysis. Both organisms were considered the possible causative agent
of the infection because of their association with clinical signs in
the infected tissue (purulent secretion and wound dehiscence). As
these microorganisms were isolated 6 days after the patient’s ad-
mission to hospital, the infection was considered hospital ac-
quired.
The Gram-negative strain (designated A47) was first investi-
gated by conventional biochemical tests. Positive results were ob-
tained for D-glucose oxidation and gelatin hydrolysis whereas neg-
ative results were recorded for oxidase, swimming motility,
dissimilative nitrate reduction, urease, or acetamide hydrolysis.
Identification by the use of an automated Vitek 2 compact system
(bioMérieux) using a GN Colorimetric Identification Card
yielded a biocode of 0001012100500300 which corresponds to
Acinetobacter baumannii with a probability of 94%. Species iden-
tification was also carried out by using matrix-assisted laser de-
sorption ionization–time of flight mass spectrometry (MALDI-
TOF MS) (Bruker Daltonik) and the current Bruker Daltonics
database (MBT-BDAL-5627 MSP library). Using this approach,
the organisms was identified only at the genus level (maximum
score of ⬍2.0) although the first identification hit was Acinetobac-
ter parvus with a score of 1.93. However, the appearance of its
colonies (see above) was not congruent with the description of A.
parvus, which is a small-colony-forming, nonhemolytic species
(1). Furthermore, we determined the 16S rRNA gene sequence
(1,332 bp) of A47 (NCBI accession no. KM386667). A BLAST-
based search using NCBI databases for this sequence revealed its
highest similarity (95%) to be with that of A. parvus X32
(KC477206). Altogether, these results indicated that A47 be-
longed to the genus Acinetobacter but its identity at the species
level remained obscure.
Antibiotic susceptibility testing was performed using a Vitek 2
system and an AST-079 panel (Gram-negative-susceptibility
[GNS] card). MIC results were interpreted using the CLSI break-
points for Acinetobacter except for ampicillin, cephalothin, and
cefoxitin, for which the breakpoints for Enterobacteriaceae were
used (2). Strain A47 was susceptible to ampicillin-sulbactam, pip-
eracillin-tazobactam, ceftazidime, cefepime, imipenem, mero-
penem, amikacin, gentamicin, ciprofloxacin, colistin, and trim-
ethoprim-sulfamethoxazole, whereas it appeared resistant to
Received 10 September 2014 Returned for modification 2 October 2014
Accepted 29 October 2014
Accepted manuscript posted online 12 November 2014
Citation Almuzara M, Traglia GM, Krizova L, Barberis C, Montaña S, Bakai R,
Tuduri A, Vay C, Nemec A, Ramírez MS. 2015. A taxonomically unique
Acinetobacter strain with proteolytic and hemolytic activities recovered from a
patient with a soft tissue injury. J Clin Microbiol 53:349 –351.
doi:10.1128/JCM.02625-14.
Editor: G. A. Land
Address correspondence to Alexandr Nemec, anemec@szu.cz, or María Soledad
Ramírez, msramirez@fullerton.edu.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/JCM.02625-14.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JCM.02625-14
CASE REPORT
January 2015 Volume 53 Number 1 jcm.asm.org 349Journal of Clinical Microbiology

ampicillin, cefalotin, cefoxitin, and cefotaxime. Based on these
results, treatment with imipenem at 500 mg/6 h of intravenous
(i.v.) administration and with ampicillin at 2 g/6 h (owing to the
presence of ampicillin-susceptible Enterococcus faecalis) for 30
days was conducted. After completing treatment and because of
the good clinical course, the patient was discharged to her home in
stable condition.
Given the conflicting results of the species identification based
on routine and commercial diagnostic systems, we decided to as-
sess precisely the taxonomic position of strain A47 using a set of
taxonomic methods relevant for the genus Acinetobacter. First, we
performed the comparative sequence analysis of the rpoB gene,
which is which is the single most frequently used gene taxonomic
marker for the genus (3–7). The similarity calculations and cluster
analysis were performed for a variable 861-bp region that included
nucleotide positions 2915 to 3775 of the rpoB coding region of A.
baumannii CIP 70.34
T
(NCBI accession no. APRG00000000)as
described previously (3,6). The rpoB-based dendrogram for A47
and strains representing all known validly or provisionally named
species of the genus is shown in Fig. S1A in the supplemental
material. The intraspecies similarity values between A47 and the
other members of the genus were in the range of 77.1% (Acineto-
bacter qingfengensis) to 90.9% (genomic species 17), which indi-
cates the uniqueness of A47 at the species level. To verify the tax-
onomic distinctness of A47 inferred from rpoB sequences, we
carried out comparative analysis of the partial sequences of the
gyrB gene using the primers described by Yamamoto et al. (8). The
similarity calculations and cluster analysis were performed for an
804-bp region corresponding to positions 421 to 1224 of the gyrB
gene of A. baumannii CIP 70.34
T
(see Fig. S1B). The identity val-
ues between A47 and the representatives of known Acinetobacter
spp. ranged from 74.9% (Acinetobacter bohemicus) to 86.6%
(Acinetobacter gyllenbergii), which further supports the taxonomic
uniqueness of A47.
To define the phenotype of A47, we performed the in-house
nutritional and physiological tests targeted to the Acinetobacter
genus (3,5). A47 lysed both sheep erythrocytes and gelatin, pro-
duced acid from D-glucose, and grew at 41°C but not at 44°C. It
utilized acetate, trans-aconitate, 4-aminobutyrate, adipate, L-argi-
nine, azelate, ␤-alanine, citrate, glutarate, L-histidine, 4-hydroxy-
benzoate, DL-lactate, L-leucine, L-ornithine, phenylacetate, phe-
nylalanine, putrescine, D-ribose, and tricarballylate as sole sources
of carbon but did not grow on L-arabinose, L-aspartate, benzoate,
butanediol, histamine, levulinate, citraconate, L-tartrate, gentisate, D-
glucose, D-gluconate, trigonelline, or tryptamine. The combination
of these properties is unique among those of nearly 800 phenotypi-
cally well-characterized strains which are deposited in the Acineto-
bacter collection of the Laboratory of Bacterial Genetics and which
represent all validly named species and a number of novel tenta-
tive species (A. Nemec and L. Krizova, unpublished data).
The genotypic results described above clearly indicate that A47
is distinct from the hitherto known Acinetobacter taxa at the spe-
cies level. Based on both the rpoB and gyrB sequences (see Fig. S1
in the supplemental material), this strain appears to be most
closely related to the Acinetobacter spp. which typically include
hemolytic and/or proteolytic strains, such as A. gyllenbergii,Acin-
etobacter venetianus, and genomic species described by Bouvet and
Jeanjean et al. (9,10). A47 clustered with these proteolytic/hemolytic
species also using MALDI-TOF MS whole-cell profiling based on our
in-house database (5) which encompasses the representatives of all
validly named species and a number of provisionally termed genomic
species of the genus Acinetobacter (Krizova and Nemec, unpub-
lished). The hemolytic-proteolytic phenotype and MALDI-TOF
MS protein spectrum of A47 are thus consistent with its taxo-
nomic position based on genotypic markers.
In conclusion, the present report provides another example
that illustrates the difficulties of the identification of Acinetobacter
isolates in routine diagnostic laboratories. Such laboratories
mostly depend on commercial systems which generally suffer
from a small number of diagnostically relevant characteristics
and/or the insufficient quality of reference data (11). This report
also demonstrates the usefulness of Acinetobacter-targeted taxo-
nomic methods such as rpoB- and gyrB-based comparative anal-
ysis, which may help in the elucidation of the taxonomic position
of unusual strains and to uncover novel, clinically relevant species.
Nucleotide sequence accession numbers. The 16S rRNA,
gyrB, and rpoB genes of Acinetobacter sp. A47 were deposited in
GenBank under accession numbers KM386667,KM386668, and
KM386669, respectively.
ACKNOWLEDGMENTS
M.S.R. is a career investigator of CONICET, Argentina. G.M.T. has doc-
toral fellowships from CONICET. This study was supported by grant PIP
11420100100152 to M.S.R. and grant PICT 2012-00120 to M.S.R. and by
grants from the “Secretaría de Ciencia y Técnica de la Universidad de
Buenos Aires” (UBACyT) to C.V. A.N. and L.K. were supported by grant
13-26693S from the Czech Science Foundation.
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