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Aldert Bart

Birgitta Duim

Christina M. V

Evert De Jonge

J. Clin Microbiol

Lodewijk Spanjaard

Menno D. De Jong

Nashwan Al Naiemi

Paul H. M. Savelkoul

PubMed

Naiemi, N.A.

Duim, B.

Savelkoul, P.H.M.

Spanjaard, L.

de Jonge, E.

Bart, A.

Vandenbroucke-Grauls, C.M.J.E.

de Jong, M.D.

NARCIS 

Widespread transfer of resistance genes between bacterial species in an intensive care unit: implications for hospital epidemiology

N. A. Naiemi

B. Duim

P. H. M. Savelkoul

L. Spanjaard

E. de Jonge

A. Bart

C. M. Vandenbroucke-Grauls

M. D. de Jong

Crossref

Widespread Transfer of Resistance Genes between Bacterial Species in an Intensive Care Unit: Implications for Hospital Epidemiology

Jonge, E. de

Jong, M.D. de

DSpace at VU

JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2005, p. 4862–4864 Vol. 43, No. 9
0095-1137/05/$08.000 doi:10.1128/JCM.43.9.4862–4864.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Widespread Transfer of Resistance Genes between Bacterial Species in
an Intensive Care Unit: Implications for Hospital Epidemiology
Nashwan Al Naiemi,1 Birgitta Duim,1* Paul H. M. Savelkoul,3 Lodewijk Spanjaard,1 Evert de Jonge,2
Aldert Bart,1 Christina M. Vandenbroucke-Grauls,1,3 and Menno D. de Jong1†
Academic Medical Center, Departments of Medical Microbiology1 and Intensive Care,2 Amsterdam, The Netherlands,
and VU University Medical Center, Medical Microbiology and Infection Control, Amsterdam, The Netherlands3
Received 15 June 2005/Accepted 23 June 2005
A transferable plasmid encoding SHV-12 extended-spectrum -lactamase, TEM-116, and aminoglycoside
resistance was responsible for two sequential clonal outbreaks of Enterobacter cloacae and Acinetobacter bau-
mannii bacteria. A similar plasmid was present among isolates of four different bacterial species. Recognition
of plasmid transfer is crucial for control of outbreaks of multidrug-resistant nosocomial pathogens.
Antibiotic resistance genes are often carried in mobile ge-
netic elements (1, 3, 6, 8, 16). The transfer of resistance genes
between different bacterial species may go unnoticed by tradi-
tional infection control and epidemiological methods, thereby
undermining hospital infection control policies (4, 5, 11). We
describe the occurrence of two sequential outbreaks of multi-
drug resistant (MDR) Enterobacter cloacae and Acinetobacter
baumannii bacteria at our Intensive Care Department (ICU)
and provide evidence that both clones and other Enterobacte-
riaceae at the ICU harbored similar plasmids.
Between June and November 2000, aminoglycoside-resis-
tant and extended-spectrum -lactamase (ESBL)-producing E.
cloacae isolates were isolated from 10 patients. In November
2000, this was followed by the isolation of A. baumannii strains
with similar resistance patterns from three patients. Aminogly-
coside resistance was determined by the disk diffusion test. For
FIG. 1. Dendrogram showing the relatedness of AFLP patterns. AFLP patterns of MDR E. cloacae isolates (A) and MDR A. baumannii
isolates (B) are shown. The numbers denote the patient numbers as given in Table 1.
* Corresponding author. Mailing address: Academic Medical Cen-
ter, Dept. of Medical Microbiology, L1-244, Meibergdreef 9, 1105 AZ
Amsterdam, The Netherlands. Phone: 31 20 5665714. Fax: 31 20
5669745. E-mail: b.duim@amc.uva.nl.
† Present address: Oxford University Clinical Research Unit, Hos-
pital for Tropical Diseases, Ho Chi Minh City, Vietnam.
4862
ESBL production the double-disk synergy test was used, with
disks containing cefotaxime, ceftazidime, cefpodoxime, and
cefepime at distances of 30 and 20 mm (center to center) from
a disk with amoxicillin and clavulanate (7, 9). AFLP analysis
(15) confirmed the clonality of the E. cloacae strains and the
clonality of the A. baumannii strains (Fig. 1).
The outbreak of E. cloacae bacteria had been noticed in
August 2000 and was managed by standard infection control
measures. During the period from April until November 2000,
an increase of aminoglycoside-resistant gram-negative isolates
in surveillance cultures from patients admitted to the ICU was
observed. This ICU served as a control unit in an ongoing
study to evaluate the effectiveness of selective decontamination
of the digestive tract (2). Twenty-nine strains of eight different
bacterial species were obtained from 26 patients admitted to
the ICU. Nineteen isolates exhibited phenotypic evidence of
ESBL production and aminoglycoside resistance, similar to the
outbreak strains (Table 1).
PCR and sequence analysis of ESBL-encoding genes was
performed. An 827-bp gene fragment of the blaSHV gene was
amplified with primers SHV-F1 (5-CTT TAC TCG CCT TTA
TCG-3) and SHV-R1 (5-TCC CGC AGA TAA ATC ACC
A-3) and sequenced with amplification primers and primers
blaSHV-F2 (5-ACT GCC TTT TTG CGC CAG AT-3) and
blaSHV-R2 (5-CAG TTC CGT TTC CCA GCG GT-3). An
862-bp gene fragment of blaTEM was amplified with primers
TEM-F1 (5-ATG AGT ATT CAA CAT TTC CG-3) (14) and
TEM-R1 (5-GAC AGT TAC CAA TGC TTA ATC A-3)
(10) and sequenced with amplification primers and primers
blaTEM-F2 (5-TAA CCA TGA GTG ATA ACA CT-3) and
TABLE 1. Characteristics of isolated strains
Isolateb Species Mo ofisolation
Patient
(identification no.)
Site or
source of
isolation
Presence ofa:
ESBL
phenotype
Beta-lactamase gene encoding indicated
enzyme
Similar plasmid
with five PCR
reactionsSHV-2 SHV-12 TEM-1 TEM-116
1 E. cloacae April 13 Rectum      
2 E. cloacae May 12 Rectum      
3 E. cloacae* June 1 Sputum      
4 E. cloacae June 11 Throat      
5 Enterobacter agglomerans June 15 Sputum      
6 Citrobacter freundii June 11 Sputum      
7 E. coli June 22 Sputum      
8 E. coli June 23 Sputum      
9 A. baumannii June 21 Throat      
10 E. cloacae* July 2 Sputum      
11 E. coli July 24 Sputum      
12 Pseudomonas aeruginosa July 34 Sputum      
13 Klebsiella oxytoca July 36 Throat      
14 E. cloacae* August 3 Sputum      
15 E. cloacae* August 8 Rectum      
16 E. cloacae* August 5 Throat      
17 E. cloacae* August 7 Sputum      
18 E. cloacae August 14 Throat      
19 E. coli August 3 Rectum      
20 E. coli August 25 Throat      
21 E. cloacae* September 9 Sputum      
22 E. cloacae* September 4 Sputum      
23 E. cloacae* September 6 Sputum      
24 E. coli September 26 Throat      
25 E. coli September 32 Sputum      
26 E. cloacae* October 10 Sputum      
27 E. coli October 33 Rectum      
28 A. baumannii* November 16 Sputum      
29 A. baumannii* November 17 Throat      
30 A. baumannii* November 18 Throat      
31 P. aeruginosa June 11 Sputum      
32 E. coli October 30 Rectum      
33 C. freundii July 6 Sputum      
34 A. baumannii August 20 Sputum      
35 P. aeruginosa August 35 Sputum      
36 E. coli September 27 Rectum      
37 E. coli September 28 Rectum      
38 P. aeruginosa September 14 Rectum      
39 E. coli October 31 Rectum      
40 Aeromonas spp. October 37 Sputum      
41 A. baumannii August 19 Sputum      
42 E. coli October 29 Rectum      
a , present; , absent.
b All strains are gentamicin resistant. *, clonal outbreak strains.
VOL. 43, 2005 NOTES 4863
blaTEM-R2 (5-CCG ATC GTT GTC AGA AGT AA-3). To
prevent carryover contamination, the Uracil system (Roche)
was applied. Both outbreak strains (E. cloacae and A. bauman-
nii) proved to harbor the SHV-12 ESBL gene (12) and the
TEM-116 beta-lactamase gene (http://www.lahey.org/studies/
webt.htm). These two genes were also present in 18 of the 29
nonoutbreak gram-negative bacteria (Table 1).
Plasmids were isolated with the QIAgen Plasmid Midi kit
(QIAGEN, Westburg B.V., The Netherlands) and were ana-
lyzed by 0.7% agarose Tris-borate-EDTA gel electrophoresis.
Most isolates contained multiple plasmids, but a plasmid of
approximately 65 kb in size was observed in the outbreak
strains and in 18 nonoutbreak gram-negative bacteria. Trans-
formation of plasmids from both outbreak strains and two
other MDR gram-negative bacteria to Escherichia coli K-12
strains conveyed resistance to aminoglycosides and cephalo-
sporins. All transformants shared a 65-kb plasmid, indicating
that this plasmid contained genes for aminoglycoside resis-
tance and for ESBL production. The 65-kb plasmid of a re-
combinant strain was digested with EcoRV and fragments
were subcloned in pUC19. Selection for ceftazidime resistance
yielded a clone with an insert of approximately 20 kb. The
nucleotide sequences of both ends of the insert were deter-
mined with M13 sequencing primers specific for the cloning
vector. One end of the insert contained the 5 end of the
aac(3)-II gene, which confers aminoglycoside resistance, fol-
lowed by the 3 end of the blaTEM-116 gene. The opposite end
of the insert contained two putative open reading frames,
ORF20 and ORF21, which are present in the known antibiotic
resistance plasmids pEL60 and pCTX-M13 (GenBank acces-
sion numbers AY422214 and AF550415, respectively). Based
on these sequences, five new primers specific for the insert
were developed (ABNN01 [5-GAT ATC TCC TCT AAA
CTG CAA A], ABNN02 [5-GTT TAC TCA TAT ATA CTT
TAG ATT], ABNN03 [5-CGT AAC GCG GCA ACG ACC
GTC T], ABNN04 [5-CTG ATA GCC GGG CGA AAT CA],
and ABNN05 [5-ATG ATT AAG ATG GTT AGC GTC
GTT]) and used for PCR on isolates. By using combinations of
these and blaTEM-F2 primers in independent PCRs, we deter-
mined that similar plasmids with identical organizations of
blaTEM and aaC(3)-II and of ORF20 and ORF21, respectively,
were present in the outbreak strains and in five other gram-
negative isolates of four different species (Table 1). The data
suggest that widespread ongoing dissemination of a similar
plasmid among strains in the ICU ward occurred and supports
previous published reports on (interspecies) plasmid transfer
(13, 17).
The infection control measures taken after the E. cloacae
outbreak, including extra attention for hand hygiene, wearing
gloves and gowns during patient care activities, and isolation of
patients who had MDR E. cloacae infections in private rooms,
seem to have failed to prevent the clonal outbreak of MDR A.
baumannii bacteria. Other MDR gram-negative bacilli may
have acted as a reservoir for the resistance plasmid. Since the
infection control measures were directed only at preventing
the spread of the resistant E. cloacae strain, cross-infection
with other strains was still possible. The subsequent outbreak
of MDR A. baumannii bacteria led to stricter infection control
measures: (i) control measures which were extended to all
patients in the ICU, (ii) closure of the ICU to new admissions,
and (iii) a dedicated nursing team for the patients colonized
with the resistant strain. These measures were successful in
halting the A. baumannii outbreak. A significant decrease in
the prevalence of all MDR gram-negative bacilli was noted at
the same time.
In conclusion, our study shows not only that plasmid out-
breaks may go unrecognized but also that stringent measures
directed against all MDR gram-negative bacilli can be effective
in controlling such outbreaks.
We thank Josje de Bruijn, Monique Feller, Mark van Passel, and
Wim van Est for their technical support.
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4864 NOTES J. CLIN. MICROBIOL.


A transferable plasmid encoding SHV-12 extended-spectrum beta-lactamase, TEM-116, and aminoglycoside resistance was responsible for two sequential clonal outbreaks of Enterobacter cloacae and Acinetobacter baumannii bacteria. A similar plasmid was present among isolates of four different bacterial species. Recognition of plasmid transfer is crucial for control of outbreaks of multidrug-resistant nosocomial pathogen

Al Naiemi, Nashwan

Duim, Birgitta

Savelkoul, Paul H. M.

Spanjaard, Lodewijk

de Jonge, Evert

Bart, Aldert

Vandenbroucke-Grauls, Christina M.

de Jong, Menno D.

Widespread transfer of resistance genes between bacterial species in an intensive care unit: Implications for hospital epidemiology

http://citeseerx.ist.psu.edu/viewdoc/summary?doi=10.1.1.335.6323

Widespread transfer of resistance genes between bacterial species in an intensive care unit: implications for hospital epidemiology

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