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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
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Molecular Epidemiology of Imipenem Resistant Acinetobacter baumannii
carrying the ISAba1–blaOXA-51-like genes
Bidur Prasad Chaulagain a,1, Sook-Jin Janga,b
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
• A non-fermentative, aerobic, Gram-negative coccobacillus. (Coelho 2006, Jubelle, 2006;
Kraniotaki 2006, Aygun, 2002, Nemec, 2000)
• Associated with post-surgical, ventilator-associated pneumonia, burn wounds, ICU pts, device-associated infections.
Acinetobacter baumannii
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
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.
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
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.
Materials and Methods
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).
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.
Table 1. Number and sites of environmental culture in ICU
Besides clinical samples from case control patients environmental samplings were also done
Sampling site N Sampling site N
Both hand of Health care worker 124 EKG 6
Ventilator 13 Chart 3
Tracheostomy site 6 Recording sheet 4
Fluid in bottle containing suction tip 6 Table 11
Suction 2 Computer 3
O2 supplier 2 Dresser shelf 1
IV fluid line 1 Dressing car 9
Bed 10 Canister lid 2
Washing stands 2 Humidifier 2
waste water disposal sink 4 Door handle 2
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.
J Clin Microbiol. 2003; 41: 4623–4629
IPM-imipenem SMA=sodium mercaptoacetic acid DDST=Double disk synergy test MBL=metallo-β-lactamases
Carbapenemase assay
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
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
PCR mapping
ISAba1 blaOXA-51-like
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
• 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.
Infection control intervention program
Results
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
0
2
4
6
8
10
12
14
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
2003 2004 2005
SICU
MICU
MICU+SICU
Table 2. Results of univariate analysis for the
occurrence of imipenem-resistant A. baumannii
case control
(n=77) (n=77)
Demographics
male gender 58 (75.3) 57 (74.0) 1
age(years)* 56.0 (15.8) 55.6 (16.7) 0.866
Comorbidity
cardiac disease 4 ( 5.2) 20 (26.0) 0.001
diabetes 13 (16.9) 9 (11.7) 0.49
malignancy 5 (6.5) 2 (2.6) 0.442
CVA 17 (22.1) 0 (0.0) 0
pulmonary disease 17 (22.1) 5 (6.5) 0.01
hepatic disease 5 (6.5) 7 (9.1) 0.765
renal disease 2 (2.6) 0 (0.0) 0.497
Variable p-value
Table 3. Results of univariate analysis for the occurrence of
imipenem-resistant A. baumannii
Variable case control p-value
(n=77) (n=77)
Related to hospitalization
Time at risk(days) 24.2(32.4) 6.2(4.5) 0
surgery 48(62.3) 35(45.5) 0.052
bladder catheter 77(100.0) 58(75.3) 0
IV catheter 46(60.5) 34(44.2) 0.052
mechanical ventilation 52(67.5) 8(10.4) 0
intubation 67(87.0) 32(41.6) 0
Antibiotics
imipenem 10(13.0) 5(6.5) 0.277
piperacillin 0(0.0) 3(3.9) 0.245
ampicillin-sulbactam 1(1.3) 0(0.0) 1
vancomycin 8(10.4) 13(10.4) 0.348
cephalosporins
1st generation 0(0.0) 15(19.5) 0
2nd generation 35(45.5) 35(45.5) 1
3rd generation 18(23.4) 36(46.8) 0.004
aminoglycosides 46(59.7) 50(65.8) 0.505
quinolone 4(5.2) 5(6.5) 1
cabapenem 6(7.8) 1(1.3) 0.116
duration of anti use(days) 10.8(10.0) 15.3(12.9) 0.008
Table 4. Results of multivariate analysis for the
occurrence of imipenem-resistant A. baumannii
Variable Crude OR (95% CI) Adjusted OR (95% CI)
Comorbidity
Cardiac disease(yes/no) 0.16 (0.05- 0.48) 0.32 (0.06- 1.58)
Pulmonary disease(yes/no) 4.08 (1.42-11.71) 6.03 (0.88-41.08)
Related to hospitalization
Time at risk(days) 1.31 (1.19- 1.45) 1.29 (1.14- 1.46)
Surgery(yes/no) 1.99 (1.04- 3.78) 2.13 (0.65- 6.93)
IV catheter(yes/no) 1.94 (1.02- 3.69) 0.42 (0.12- 1.45)
Mechanical ventilation(yes/no) 17.94 (7.49-42.98) 6.41 (1.71-24.06)
Intubation(yes/no) 9.42 (4.21-21.06) 2.55 (0.69- 9.44)
Antibiotics
3rd generation(yes/no) 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.
Table 5. Number and sites of positive culture for IRAB from
environmental culture
Sampling site
No.
test
ed
No.
posit
ive
Sampling site
No.
test
ed
No.
posit
ive
Both hand of Health care worker 124 5 EKG 6 1
Ventilator 13 7 Chart 3 0
Tracheostomy site 6 4 Recording sheet 4 0
Fluid in bottle containing suction tip 6 2 Table 11 0
Suction 2 0 Computer 3 0
O2 supplier 2 0 Dresser shelf 1 1
IV fluid line 1 1 Dressing car 9 2
Bed 10 7 Canister lid 2 0
Washing stands 2 0 Humidifier 2 0
Table 6. Result of surveillance culture
Date
No.
test
ed
No.
positive
for IRAB
Positive
rate for
IRAB
No.
test
ed
No.
positive
for IRAB
Positive
rate for
IRAB10/7 36 0 0.0 22 2 9.1
10/14 32 2 6.3 34 0 0.0
10/21 34 0 0.0 30 0 0.0
10/27 35 2 5.7 38 1 2.6
11/4 28 2 7.1 36 0 0.0
11/10 32 3 9.4 32 0 0.0
11/18 24 0 0.0 32 1 3.1
11/24 24 0 0.0 26 1 3.8
12/2 34 1 2.9 30 0 0.0
12/15 69 0 0.0 129 0 0.0
Total 348 10 2.9 409 5 1.2
MICU SICU
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.
Isolate PFGE IPM MEM ISAba1–OXA-
51 OXA-51 OXA-23 OXA-24 OXA-58 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 A 32 64 P P N N N N N N N N N N N N N N 119 A - - - P N N N N N N N N N N N N N N 121 A 32 64 P P N N N N N N N N N N N N N N 124 A - - - P N N N N N N N N N N N N N N 125 A - - P P N N N N N N N N N N N N N N 128 A 16 64 P P N N N N N N N N N N N N N N 129 A 64 64 P P N N N N N N N N N N N N N N 130 A 32 64 P P N N N N N N N N N N N N N N 133 A 16 - P P N N N N N N N N N N N N N N 134 A 16 64 P P N N N N N N N N N N N N N N 135 A 32 64 P P N N N N N N N N N N N N N N 137 A 16 16 P P N N N N N N N N N N N N N N 140 A 32 64 P P N N N N N N N N N N N N N N 141 A 16 - P P N N N N N N N N N N N N N N 143 A 32 64 P P N N N N N N N N N N N N N N 145 A 32 - P P N N N N N N N N N N N N N N 111 A1 16 64 P P 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 A2 32 64 P P N N N N N N N N N N N N N N 144 A2 - - P P N N N N N N N N N N N N N N 117 A3 - - P P N N N N N N N N N N N N N N 113 B 16 1 N P N N N P N N N N N N N N N N 115 B 16 - N P N N N P N N N N N N N N N N 101 C 32 64 P P N N N N N N N N N N N N N N 106 C - - P P N N N N N N N N N N N N N N 136 D - - - P N N N N N N N N N N N N N N
E-IRAB
302 A - - P P N N N N N N N N N N N N N N 304 A 32 64 P P N N N N N N N N N N N N N N 306 A 32 64 P P N N N N N N N N N N N N N N 309 A 32 64 P P N N N N N N N N N N N N N N 312 A 32 64 P P N N N N N N N N N N N N N N 314 A 32 64 P P N N N N N N N N N N N N N N 316 J 32 64 P P N N N N N N N N N N N N N N
ISAB
213 F 1 32 N P N N N N N N N N N N N N N N 214 I 1 2 N P N N N N N N N N N N N N N N 220 K 2 2 N P N N N N N N N N N N N N N N 224 M 2 2 N P N N N N N N N N N N N N N N 227 G 4 2 N P N N N N N N N N N N N N N N 230 H 4 2 N P 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
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
Table 8. Carbapenemase assay strain Modified
Hodge Test
(for carbapenemase)
IPM-Zn DDST
(imipenem specific)
ISAba1-Oxa-51
like gene PCR
blaOXA-51-
like gene PCR
PFGE type
C-IRAB 25/25 2/25 (8 %) 23/25 (92 %)
25/25 A, A1, A2
(23/28)
E-IRAB 7/7 0 7/7 7/7 A (6/7)
C-ISAB 9/9 0 0 9/9 different
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.
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 e-IRAB strains ranged from 16 to >128 mg/L.
• The meropenem MICs of 15 c-IRAB and six e-IRAB ranged from 1 to 128 mg/L.
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.
• 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
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.
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
probably
Nature Reviews Microbiology 8:251-259 , 2010)
Mechanisms of antibiotic resistance in a Gram-negative bacterium
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
Thank you
Acknowledgements
The present study was supported by grants from the
Clinical Medicine Research Institute at Chosun
University Hospital (2006).