Molecular Epidemiology of Imipenem Resistant Acinetobacter baumannii carrying the ISAba1–blaOXA-51-like genes A story of a molecular epidemiological analysis to know : # Imipenem resistance in A. baumannii # the cause of outbreak in hospital setting and, # an intervention to control the outbreak

1. Molecular Epidemiology of Imipenem Resistant
Acinetobacter baumannii
carrying the ISAba1–blaOXA-51-like genes
Bidur Prasad Chaulagain a,1, Sook-Jin Janga,b
bpchaula@gmail.com
aResearch
Center for Resistant Cells, Chosun University Medical
School, Gwang-Ju, Korea
bDepartment of Laboratory Medicine, Chosun University Medical
School, Gwang-Ju, Korea
1Biosystem Engineering Laboratory, Department of Biological
Engineering, Inha University, Incheon, 402-751, Korea

2. Acinetobacter baumannii
• A non-fermentative, aerobic, Gramnegative coccobacillus. (Coelho 2006, Jubelle, 2006;
Kraniotaki 2006, Aygun, 2002, Nemec, 2000)
• Associated with post-surgical,
ventilator-associated pneumonia,
burn wounds, ICU pts, deviceassociated infections.

3. our A. baumannii strains
• have phenotype for IMIPENEM resistance
with other Multi Drug resistance
• Show carbapenemase activity as detected by
Modified Hodge test
• But carbapenemase negative for Imipenem
specific IPM- EDTA-double disk synergy test
• No carbapenemase genes in the chromosome
or in integron when assayed with beta
lactamase PCR

4. Outbreak of IRAB with ISAba1–blaOXA-51-like gene
• Imipenem and meropenem are among
the drugs of choice used to treat
nosocomial infections caused by MRAB.
• Emergence of carbapenem-hydrolyzing
β-lactamases of molecular Ambler class B
(metalloenzymes) and D enzymes
(oxacillinases) challenged their efficacy.

5. Thus here is a story of a molecular epidemiological
analysis to know :
 Imipenem resistance in A. baumannii
 the cause of outbreak in hospital
setting and,
an intervention to control the outbreak

6. To investigate the outbreak,
a case-control study,
environmental culture,
genotyping of A. baumannii by PFGE
•
and compared with integron -PCR typing
assay for resistance determinants for
carbapenemase
were performed.

7. Materials and Methods

8. Definition of Case and Control
Case : patients from whom ACB (IRAB) had been
isolated in clinical cultures and were admitted in
MICU) and SICU of Chosun University Hospital from
Jan. 2004 to Dec. 2004.
Control : patients from whom IRAB had not been
isolated and were admitted during the same time
period and place as the case group.
Case and control patients were matched by
gender and age (±5 years).

9. Statistical analysis
•
Statistical analyses: SPSS version 12.0 (SPSS, Chicago, IL).
•
Univariate analyses


•
Qualitative variables : chi-square test or Fisher's exact test.
Quantitative variables : Student's t test.
Multiple logistic regression analysis,

•
Odds ratios (ORs) and 95% confidence intervals (CIs)
p-value of < 0.05 was considered statistically
significant.

10. Table 1. Number and sites of environmental culture in ICU
Besides clinical samples from case control patients environmental samplings
were also done
Sampling site
Both hand of Health care worker
Ventilator
N
Sampling site
N
124
EKG
6
13
Chart
3
4
Tracheostomy site
6
Recording sheet
Fluid in bottle containing suction tip
6
Table
Suction
2
Computer
3
O2 supplier
2
Dresser shelf
1
IV fluid line
1
Dressing car
9
10
Canister lid
2
Washing stands
2
Humidifier
2
waste water disposal sink
4
Door handle
2
Bed
11

11. Strain identification and antimicrobial
susceptibility test
•
Strain identification :
•
•
•
Vitek ID-GNB card
API 20 NE identification kit (BioMerieux)
Antimicrobial susceptibility test :
•
Vitek AST-No. 22 kit or AST-N017 kit.

12. Carbapenemase assay
IPM-imipenem
SMA=sodium mercaptoacetic acid
DDST=Double disk synergy test
MBL=metallo-β-lactamases
J Clin Microbiol. 2003; 41: 4623–4629

13. carbapenemases PCR assay
OXA-type Class-D
carbapenemases
genes
• blaOXA-23-like,
• blaOXA-24-like ,
• blaOXA-58-like ,
and
blaOXA-51-like
genes
Ambler class B and A
carbapenemases genes
• blaIMP-1 , blaIMP-2 ,
blaIMP-4 ,
• blaIMP-11 , blaIMP-18 ,
blaIMP-19 ,
• blaVIM-1, blaVIM-2,
blaGIM-1 ,
• blaSPM-1 ,
• blaGES

14. PCR mapping
ISAba1
blaOXA-51-like
To determine whether the ISAba1 is present
in upstream of the blaOXA-51-like gene or not,
forward primer - ISAba1 insertion DNA sequence
reverse primers -OXA-51-like gene DNA sequence

15. Molecular typing by PFGE
• Plug Preparation- agarose gel embedded genomic DNA
• Proteolysis- proteinase-K lysis method, plug then
washing with 1x TE buffer for 24 h.
• Restriction Digestion- ApaI, 25 OC , 16 h.
• Gel electrophoresis:
-CHEF DR-III Genepath
- Pulse time: 5.3 s to 49.9 s at 120O angle
- 20 h at 6V/cm at 14 OC
-0.5X TBE Buffer
-Staining with Eth-Br
- UV light visulization
Takes at least 3 days to complete the process

16. Infection control intervention program
•
•
•
Extensive decontamination
Continued ICU personnel educational programs
Rigorous open surveillance for adequate
compliance with barrier precautions
•
•
•
Cleaning protocols
House-keeping procedures
Adequate compliance with the control program was
supervised by a team who attended to the ICUs daily
after the implementation of the infection control
program.

17. Results

18. 14
12
SICU
10
MICU
MICU+SICU
8
6
4
2
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
0
2003
2004
2005
Figure 5. Epidemic curve showing the number of SICU plus
MICU patients colonizing or infecting with
imipenem resistant A. baumannii at 1-month intervals
from January 2003 to December 2005

19. Table 2. Results of univariate analysis for the
occurrence of imipenem-resistant A. baumannii
Variable
Demographics
male gender
age(years)*
Comorbidity
cardiac disease
diabetes
malignancy
CVA
pulmonary disease
hepatic disease
renal disease
case
(n=77)
control
(n=77)
p-value
58 (75.3)
57 (74.0)
56.0 (15.8) 55.6 (16.7)
1
0.866
4 ( 5.2)
13 (16.9)
5 (6.5)
17 (22.1)
17 (22.1)
5 (6.5)
2 (2.6)
20 (26.0)
9 (11.7)
2 (2.6)
0 (0.0)
5 (6.5)
7 (9.1)
0 (0.0)
0.001
0.49
0.442
0
0.01
0.765
0.497

20. Table 3. Results of univariate analysis for the occurrence of
imipenem-resistant A. baumannii
Variable
Related to hospitalization
Time at risk(days)
surgery
bladder catheter
IV catheter
mechanical ventilation
intubation
Antibiotics
imipenem
piperacillin
ampicillin-sulbactam
vancomycin
cephalosporins
1st generation
2nd generation
3rd generation
aminoglycosides
quinolone
cabapenem
duration of anti use(days)
case
(n=77)
control p-value
(n=77)
24.2(32.4)
48(62.3)
77(100.0)
46(60.5)
52(67.5)
67(87.0)
6.2(4.5)
35(45.5)
58(75.3)
34(44.2)
8(10.4)
32(41.6)
0
0.052
0
0.052
0
0
10(13.0)
0(0.0)
1(1.3)
8(10.4)
5(6.5)
3(3.9)
0(0.0)
13(10.4)
0.277
0.245
1
0.348
0(0.0)
35(45.5)
18(23.4)
46(59.7)
4(5.2)
6(7.8)
10.8(10.0)
15(19.5)
35(45.5)
36(46.8)
50(65.8)
5(6.5)
1(1.3)
15.3(12.9)
0
1
0.004
0.505
1
0.116
0.008

21. Table 4. Results of multivariate analysis for the
occurrence of imipenem-resistant A. baumannii
Variable
Comorbidity
Cardiac disease(yes/no)
Pulmonary disease(yes/no)
Related to hospitalization
Time at risk(days)
Surgery(yes/no)
IV catheter(yes/no)
Mechanical ventilation(yes/no)
Intubation(yes/no)
Antibiotics
3rd generation(yes/no)
Crude OR (95% CI)
Adjusted OR (95% CI)
0.16 (0.05- 0.48)
4.08 (1.42-11.71)
0.32 (0.06- 1.58)
6.03 (0.88-41.08)
1.31 (1.19- 1.45)
1.99 (1.04- 3.78)
1.94 (1.02- 3.69)
17.94 (7.49-42.98)
9.42 (4.21-21.06)
1.29 (1.14- 1.46)
2.13 (0.65- 6.93)
0.42 (0.12- 1.45)
6.41 (1.71-24.06)
2.55 (0.69- 9.44)
0.35 (0.17- 0.69)
0.34 (0.11- 1.07)
* Adjusted ORs were adjusted for comorbid condition such as cardiac
disease and pulmonary disease, time at risk, surgery, IV catheter,
intubation, and antibiotics usage in a logistic regression model.

22. Table 5. Number and sites of positive culture for IRAB from
environmental culture
Sampling site
Both hand of Health care worker
Ventilator
Tracheostomy site
Fluid in bottle containing suction tip
Suction
O2 supplier
IV fluid line
Bed
Washing stands
No. No.
test posit
ed ive
124
13
6
6
2
2
1
10
2
5
7
4
2
0
0
1
7
0
Sampling site
EKG
Chart
Recording sheet
Table
Computer
Dresser shelf
Dressing car
Canister lid
Humidifier
No. No.
test posit
ed ive
6
3
4
11
3
1
9
2
2
1
0
0
0
0
1
2
0
0

23. Table 6. Result of surveillance culture
Date
10/7
10/14
10/21
10/27
11/4
11/10
11/18
11/24
12/2
12/15
Total
No.
test
ed
36
32
34
35
28
32
24
24
34
69
348
MICU
No.
Positive
positive rate for
for IRAB IRAB
0
0.0
2
6.3
0
0.0
2
5.7
2
7.1
3
9.4
0
0.0
0
0.0
1
2.9
0
0.0
10
2.9
No.
test
ed
22
34
30
38
36
32
32
26
30
129
409
SICU
No.
Positive
positive
rate for
for IRAB
IRAB
2
9.1
0
0.0
0
0.0
1
2.6
0
0.0
0
0.0
1
3.1
1
3.8
0
0.0
0
0.0
5
1.2

24. Figure 8. Representative PFGE patterns of ApaI (gggccc/c) digested genomic
DNA from A. baumannii strains. Lanes: 1 and 14, molecular size marker;
lanes 2 to 6, clinical imipenem resistant A. baumannii (IRAB) outbreak
isolates; lanes 7 to 9, environmental IRAB isolates; lanes 10 to 13, clinical
imipenem susceptible A. baumannii non-outbreak isolates.

25. Table 7 The results of PFGE analysis, MIC determination for imipenem and meropenem, and PCR assay for carbapenemase
in 44 strains tested including 28 c-IRAB, 9 ISAB and 7 e-IRAB strains
Isolate
PFGE
IPM
MEM
ISAba1–OXAOXA-51 OXA-23 OXA-24 OXA-58
51
IMP-1
IMP-2
IMP-4
IMP-11 IMP-18 IMP-19
VIM-1
VIM-2
GIM-1
SPM-1
GES-1
C-IRAB
108
A
16
64
P
P
N
N
N
N
N
N
N
N
N
N
N
N
N
N
110
A
>256
128
P
P
N
N
N
N
N
N
N
N
N
N
N
N
N
N
203
119
121
124
125
128
129
130
133
134
135
137
140
141
143
145
111
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A1
32
32
16
64
32
16
16
32
16
32
16
32
32
16
64
64
64
64
64
64
64
16
64
64
64
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
112
A1
32
128
P
P
N
N
N
N
N
N
N
N
N
N
N
N
N
N
139
144
117
113
115
101
106
136
A2
A2
A3
B
B
C
C
D
32
16
16
32
-
64
1
64
-
P
P
P
N
N
P
P
-
P
P
P
P
P
P
P
P
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
P
P
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
A
A
A
A
A
A
J
32
32
32
32
32
32
64
64
64
64
64
64
P
P
P
P
P
P
P
P
P
P
P
P
P
P
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
213
214
220
224
227
230
F
I
K
M
G
H
1
1
2
2
4
4
32
2
2
2
2
2
N
N
N
N
N
N
P
P
P
P
P
P
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
114
E
<=0.5
1
N
P
N
N
N
N
N
N
N
N
N
N
P
N
N
N
118
E
<=0.5
1
N
P
N
N
N
N
N
N
N
N
N
N
N
N
N
N
226
L
-
-
-
P
N
N
N
N
N
N
N
N
N
N
N
N
N
N
E-IRAB
302
304
306
309
312
314
316
ISAB

26. Table 8. Carbapenemase assay
strain
Modified
Hodge
Test
(for
carbapenemase)
IPM-Zn
DDST
ISAba1Oxa-51
(imipenem like gene
specific)
PCR
blaOXA-51like gene
PCR
PFGE
type
A, A1,
A2
(23/28)
A (6/7)
different
C-IRAB
25/25
2/25 (8 %)
23/25
(92 %)
25/25
E-IRAB
C-ISAB
7/7
9/9
0
0
7/7
0
7/7
9/9
PCR with ISAba1-OXA-51-like primer exhibited 99% identity with
sequences of insertion sequence ISAba1 transposase gene and
beta-lactamase OXA-66/OXA-51-like carbapenemase gene of
several A. baumannii strains.

27. Antimicrobial assay
• The rates of resistance of 35 IRAB to almost all
the antibiotics tested > 90%. (e.g., all the beta-lactams including
imipenem, gentamicin, netilmicin, tobramycin, isepamicin, ciprofloxacin, pefloxacin and
trimethoprim/sulfamethoxazole)
• The imipenem MICs of 21 c-IRAB and six eIRAB strains ranged from 16 to >128 mg/L.
• The meropenem MICs of 15 c-IRAB and six eIRAB ranged from 1 to 128 mg/L.

28. Summary
• 4.0% of health care worker’s (HCW) hands
culture IRAB positive
• 23.6% of ICU environmental culture E-IRAB
positive, especially from ventilator-associated
surfaces.
• the risk factors for IRAB acquisition were
mechanical ventilation practice and time at
risk.

29. • All isolates for MHT were positive showing
carbapenemase hinting for blaOXA-51-like gene
oxacillinase activity
•
but All isolates except 2 were negative to
IPM Zn –DDST showing absence of imipenem
specific carbapenemase
• All IRAB show positive for ISAba1-OXA-51-like
primer
But all ISAB showed negative reaction

30. Only A. baumannii with ISAba1 adjacent to
blaOXA-51-like were carbapenem resistant among
isolates with blaOXA-51-like as sole
carbapenemase gene. FEMS Microbiol Lett 2006;258:72-77
Probably,
ISAba1, working as promoter for blaOXA-51-like, for
the overproduction of carbapenemase
resulting carbapenem resistance.

31. Mechanisms of antibiotic resistance in a Gram-negative bacterium
probably
ISAba1
Oxa-51- like
Insertion of ISAba1 upstream
of Oxa-51-like intrinsic gene in
the chromosme may have
facilitated the over production
of carbapenemase
Nature Reviews Microbiology 8:251-259 , 2010)

32. conclusion
• After the infection control strategy, steady
reduction of attack rate to the basal level.
• First report of carbapenemase activity in
IRAB outbreak with insertion sequence
ISAba1 upstream of the blaOXA-51-like gene in
Acinetobacter baumannii

33. Acknowledgements
The present study was supported by grants from the
Clinical Medicine Research Institute at Chosun
University Hospital (2006).
Thank you