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

• 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).