Download Colonization of a Medical Center in Southern Taiwan by Epidemic

Jpn. J. Infect. Dis., 62, 155-157, 2009
Short Communication
Colonization of a Medical Center in Southern Taiwan by Epidemic Strains
of Carbapenem- and Multidrug-Resistant Acinetobacter baumannii and
the Genetic Organization of Their Integrons
Yao-Shen Chen1,2, Hsi-Hsun Lin3, Chia-Hsuang Wu4, Yu-Shan Hsiao4,
Ning-Shan Hsu4, and Ya-Lei Chen4*
1
Section of Infectious Disease, Kaoshiung Veterans General Hospital, Kaoshiung; 2National Yang-Ming
Medical University, Taipei; 3Department of Infectious Disease, E-DA Hospital/I-Shou University, Kaoshiung;
and 4Department of Biotechnology, National Kaoshiung Normal University, Kaoshiung, Taiwan
(Received August 1, 2008. Accepted January 19, 2009)
SUMMARY: A total of 46 carbapenem- and multidrug-resistant (CR- and MDR-)Acinetobacter baumannii
bacteremic isolates from a Taiwanese medical center were investigated over the period 2000 to 2006 using
randomly amplified polymorphic DNA (RAPD) profiling and by analysing the genetic organization of their
integrons. The results of RAPD patterns revealed that before 2003 each CR- and MDR-A. baumannii bacteremic
isolate was independent, but after 2003 the isolates appeared to belong in four epidemic strains and persisted in
the hospital. All the CR- and MDR-A. baumannii strains harbored class I integron (intI1) genes. PCR amplification and nucleotide sequencing showed that the cassette genes of intI1 were found to form four different antibioticresistant gene alignments in those strains. The blaIMP-1 gene in the cassette genes of intI1 was identified in a
clone, which raised great concern that clonal spread of this strain or of an integron-mediated horizontal gene
may have occurred.
The medical records of 40 of these patients were available
for review, and 87.5% (35/40) of cases were mechanical
ventilation users. All the CR-A. baumannii bacteremic isolates
were also MDR strains as defined. Those strains were confirmed by an automatic system (BD Phoenix 100 Automated
Microbiology System; Becton, Dickinson and Company,
Franklin Lakes, N.J., USA) as well as by the nucleotide sequences of their 16S-23S rRNA gene spacer region (7). An
antimicrobial susceptibility test was performed against a range
of commercially available antibiotics, and the identification
of this resistance profile followed the guidelines provided by
the Clinical and Laboratory Standards Institute (CLSI) (8).
MDR strains were defined as resistant to at least four of the
five following groups: (i) gentamicin or amikacin; (ii) cefepime
or ceftazidime; (iii) piperacillin, piperacillin--tazobactam or
ticarcillin--clavulanic acid; (iv) ciprofloxacin or levofloxacin;
(v) meropenem or imipenem.
Each strain was grown in 3 ml of Luria-Bertani (LB) broth
medium at 37°C. After 16 h, the genomic DNA of the bacteria was isolated using a genomic DNA extraction kit (Promega,
Madison, Wis., USA). The primer designs for randomly
amplified polymorphic DNA (RAPD) (5´-CCTCATGACC3´) and differential integron (intIf: 5´-CAGTGGACATAAGC
CTGTTC-3´ and intIr: CCCGAGGCATAGACTGTA; intIIf:
5´-TTGCGAGTATCCATAACCTG and intIIr: TTACCTGCA
CTGGATTAAGC; 5´-CS: 5´-GGCATCCAAGCAGCAAG3´ and 3´-CS 5´-AAGCAGACTTGACCTGA-3´) and PCR
conditions were followed as standard protocols (9). The reproducible PCR-RAPD amplicon profiles were visualized by
1.5% agarose electrophoresis by UV illumination. The RAPD
profiles were captured and analyzed using the Quantity One
software, version 4.4.0 (BioRad, Hercules, Calif., USA). If
the isolates had the same RAPD profiles, the second random
primer (5´-TGCGCGCGGG-3´) was used to confirm their
identical genetic backgrounds.
Carbapenem-resistant (CR-) or multidrug-resistant (MDR-)
Acinetobacter baumannii has emerged as a major nosocomial
pathogen worldwide. The prevalence of nosocomial infection in hospital respiratory care wards due to CR- or MDRA. baumannii was 32.1 or 8.9%, respectively, of all Gramnegative bacterial infection in Taiwan (1). Such infections
are beset by the problem that these bacteria offer very few
therapeutic options and sometimes can develop into an epidemic strain, which produces repeated outbreaks over a long
time period in a hospital (2). Developing MDR strains have
been suggested to acquire their antibiotic-resistant genes
via class I integrons (IntI1) that carry within them a single
or multiple gene cassette(s); most often among MDR-A.
baumannii isolates, these are found in the internal variable
regions between the integron’s two conserved regions (the
5´-CS and the 3´-CS) (3). Aminoglycoside resistance genes,
such as aac (acetyltransferase), aph (phosphotransferase), and
aad (adenylyltransferase), which contribute to the enzymatic
inactivation of the aminoglycoside antibiotics, are the genes
usually found among the cassette genes (4). In special instances, A. baumannii can give rise to the spread of β-lactam
antibiotic drug resistance in hospital if the bacteria harbor
a bla gene variant among the cassette genes (5,6). In this
report, we identified colonization by epidemic strains of CRand MDR-A. baumannii in a 1,400-bed teaching hospital,
Kaoshiung Veterans General Hospital (KVGH), and investigated the prevalence of antibiotic-resistant genes within the
IntI1 cassettes from those isolates.
A total of 46 CR-A. baumannii strains were isolated from
individual bacteremic patients over the period 2000 to 2006.
*Corresponding author: Mailing address: Department of Biotechnology, National Kaoshiung Normal University, Kaoshiung,
Taiwan. Tel: +886-7-6051366, Fax: +886-7-6051353, E-mail:
[email protected]
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The CR- and MDR-A. baumannii bacteremic strains isolated at KVGH included as few as four isolates per year
before 2003, but this dramatically increased to over 10 after
2003. A total of 46 individual isolates of CR- and MDR-A.
baumannii were obtained over the period from 2000 to 2006
and were analyzed for genetic differentiability (Fig. 1). The
results indicated that the RAPD patterns of each CR- and
MDR-A. baumannii isolate before 2003 were independent,
but after 2003, the isolates appeared to be grouped into four
identical patterns (A-D). An epidemic strain (pattern A) initially appeared in 2003, reached a peak in 2004, but then
decreased in 2005. The epidemic strain (pattern B) was found
in 2004 and continuously appeared in the hospital. Two emerging epidemic strains (patterns C and D) only occurred in 2006
(Table 1). These results indicate that the origin of the CRand MDR-A. baumannii strains in KVGH changed over time.
Integron-specific PCR amplification revealed that all the
CR- and MDR-A. baumannii strains harbored class 1
integrons and that no class 2 integrons were present in the
chromosomes of these strains. The cassette genes found within
those strains were shown to form four types by PCR amplification and nucleotide sequencing (Table 1). All types harbored aadA1a, and Types I and IV harbored aacA4. This
result was associated with the known resistance of these A.
baumannii isolates to aminoglycoside antibiotics. The open
reading frames coding for as-yet-undetermined products were
presented in all types. An open reading frame orfC in Type III
was identical to the sequence of an unknown functional protein which was found in environmental isolates of Acinetobacter
spp. (3). It is noted that one strain harbored an intI1 encoding
the blaIMP-1 gene.
Our group and others (10) have reported that MDR strains
harboring blaIMP-1 existed where this gene forms part of the
intI1 cassette genes found in A. baumannii. This finding raised
great concern because integron-associated imipenem-resistant
A. baumannii has been repeatedly found in Taiwanese hospitals, and it suggested that antibiotic resistance might be disseminated throughout populations by the epidemic spread of
this clone or through the exchange of genetic material. However, due to natural transformation, loss or altered outer-mem-
Fig. 1. Dendrogram and RAPD patterns of A. baumannii isolates. The PCR-RAPD amplicon profiles were amplified with
random primer (5´-CCTCATGACC-3´). The RAPD profiles were captured and analyzed using the software of Quantity
One, version 4.4.0 (BioRad). The typical RAPD patterns (A-D) of related strains and the profiles of independent strains
(strain number) were presented in this figure.
Table 1. Summary for RAPD typing and integron organization analysis of Acinetobacter baumannii isolates
Isolation
Year No.
2000
2001
2002
2003
2004
3
3
0
2
13
2005
2006
10
15
pattern A
Epidemic strain (Strain no.integron type)
pattern B
pattern C
pattern D
Independent strain
(Strain no.integron type)
1II 2I 3IV
4III5III7III
9III*10III**
11 13III14III15III
16III17III19III20III22III
27III29III 30III 31III
III
23IV
12III18III21IV
25IV26IV32IV
24IV28IV33IV
III
46IV
III
III
III
34 35 36 39
43III44III45III47III
48III49III
37III41III
40III42III
*Roman letters indicated the genetic organization of integron type following as
Type I IntI1
arrA3
accA4
ORF
aadAla qacEΔ1 Type II
IntI1
blaIMP-1
ORF aadAla qacEΔ1
dfrA1 ORFC ORF aadAla qacEΔ1 Type IV IntI1
accA4 catB8 ORF aadAla qacEΔ1
**The underline indicated that the clinical isolates were obtained from mechanical ventilator users.
Type III IntI1
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brane protein and endogenous metallo-β-lactamases variants,
CR strains were probably generated (11-13). Cross-transmission of colonized MDR-A. baumannii between patients who
are hospitalized in intensive-care units has been well documented (14). In this study, we reported an epidemic strain
that caused a hospital-wide outbreak over the period 2003 to
2005. The results of molecular typing revealed that the origin of epidemic strains after 2005 may have changed in this
hospital. The restriction of antibiotics use, the intensification
and modification of the cleaning procedures for contaminated
equipment and the cohorting of patients infected or colonized
with MDR-A. baumannii need to be seriously considered as
control measures to confine the spread of these bacteria.
ACKNOWLEDGMENTS
This project was supported by the grants (DOH95-DC-1023; DOH96DC-1203).
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