1. C-158
Genetic and Biochemical Characterisation of OXA-405, a New Extended Spectrum Class D -Lactamase from Serratia marcescens
L. Dortet1,2, S. Oueslati1, K. Jeannot2,3, D. Tandé4, F. Deschamps5, T. Naas1,2 and P. Nordmann1,2,6
1INSERM U914, 2Antibiotic Resistance Reference Center, 3Besançon hospital, 4Brest hospital, 5Reims hospital, France, 6Med Microbiol Unit, Dept Medicine, University Fribourg, Switzerland
Background
Results
Antibiograms of the two S. marcescens isolates
Negative Carba NP test
Positive Carba NP test
AMX, Amoxicillin; AMC, Amoxicillin + clavulanate; TIC, Ticarcillin; TCC, Ticarcillin + clavulanate; PIP, Piperacillin; PTZ, Piperacillin + tazobactam; TEM, Temocillin; FOX, Cefoxitin; CTX, Cefotaxime; CAZ, Ceftazidime; FEP, Cefepime; ATM, aztréonam; IMP, Impenem; MEM Meropenem; ETP, Ertapenem; AKM, Amikacin, TMN, Tobramycin; GMI, Gentamicin; NET Netilmicin; TET, Tetracycline; TGC Tigecycline; CHL, Chloramphenicol; FTN, Nitrofurane; CST, Colistin; RAM, Rifampicin; FSF, Fosfomycin; OFX, Ofloxacin; LVX, Levofloxacin; CIP, Ciprofloxacin
Protein alignment of OXA-163, OXA-405, OXA-247 and OXA-48
The aim of this study was to investigate the mechanisms responsible for extended-spectrum generation cephalosporins and carbapenem resistance from two Serratia marcescens isolates recovered from the same patient.
S. marcescens OXA-405
PTZ
PIP
TIC
AMX
ETP
TCC
CAZ
TEM
FOX
IMP
AMC
CTX
MOX
MEM
ATM
FEP
TET
TMN
AKM
GMI
NET
CHL
SXT
FTN
CST
TGC
RAM
SUL
FSF
OFX
LVX
NOR
Methods
Susceptibility testing
Antibiotic susceptibilities were determined by the disk diffusion method and Etest. Results were interpreted according to CLSI guidelines updated in 2014.
Strain comparison
Genotypic comparison of the two S. marcescens was done by rep-PCR technology using Diversilab (bioMérieux)
Carbapenemase detection
Carbapenemase production was assessed using the Carba NP test, MALDI-TOF mass spectrometry and UV-spectrophotometry.
Plasmid content and conjugation assays
Plasmid DNA obtained from the clinical isolates were extracted by using Kieser method. Whole plasmid preparations were electroporated into E. coli TOP10. E. coli NCTC50192, harboring plasmids of 154,66,48 and 7 kb was usued as size marker. Direct ransfert of plasmids in E. coli J53 were attempted using matting out assays at 37°C.
Plasmid characterization
Plasmid DNA was obtained using a Maxiprep plasmid purification kit (Qiagen). Plasmid incompatibility groups were determined by PCR-based replicon typing scheme.
Genetic environment
The genetic surrounding structures of -lactamase encoding genes were characterized by PCR mapping and DNA sequencing.
Cloning experiments
In order to express the different blaOXA-48-like genes in an isogenic background, cloning of the blaOXA-48, blaOXA-405, and blaOXA-163 was performed using a PCR-BluntII-TOPO cloning kit (Invitrogens, followed by selection of clones on plates containing 100mg/L of ticarcillin and 30 mg/L of kanamycin. The PCR fragments comprising the entire blaOXA-48-like genes were obtained with primers preOXA48A (5’-TATATTGCATTAAGCAAGGG-3’) and preOXA48B (5’-CACACAAATACGCGCTAACC3’)
Kinetic studies
Biochemical characterization was performed using UV spectrophotometry on the supernatant of a whole cell crude extract obtained from an overnight culture of the different clones.
Amino-acids involved in the active site of the carbapenemase OXA-48
Antibiotic susceptibility of clinical isolates of
S. marcescens and blaOXA-48-like clones in E. coli
Specific enzymatic activities of OXA-48, OXA-405 and OXA-163
S. marcescens OXA-48
PTZ
PIP
TIC
AMX
ETP
TCC
CAZ
TEM
FOX
IMP
AMC
CTX
MOX
MEM
ATM
FEP
TET
TMN
AKM
GMI
NET
CHL
SXT
FTN
CST
TGC
RAM
SUL
FSF
OFX
LVX
NOR
β-Lactams
MIC (mg/L)
S. marcescens
E.coli TOP10
OXA-48
OXA-405
pTOPO-OXA-48
pTOPO-OXA-405
pTOPO-OXA-163
no plasmid
Amoxicillin
>256 2
Amoxicillin+ Ac clav
192 96
Piperacillin
128
1.5
Piperacillin + Tazo 12 24 32 1
Temocillin 8 4
Ticarcillin
Cefepime
0,25 3
0,032
0.5
0.023
Cefotaxime
1,5 6
0.19
0.06
Ceftazidime
0.25 16
0.12
Imipenem
0,5
Meropenem
0,19
0.094
0,023
0.01
Ertapenem
>32
0,75
0.032
Doripenem
0,125
0.064
Aztreonam
0.047
β-Lactams
Specific Activity
(mU/mg of protein)
OXA-48
OXA-405
OXA-163
Amoxicillin
981
485
795
Amoxicillin+ Ac clav
364
643
Piperacillin
450
436
214
Piperacillin + Tazo
168
149 39
Temocillin
10,6 5
4,8
Ticarcillin
647 63 80
Cefepime
4,6 27 30
Cefotaxime 60
117
167
Cefoxitine
1,9
1,2
1,1
Ceftazidime
1,8
9,2 53
Cephalotine
75,4
139,5
130
Imipenem 57
2,6 2
Meropenem
2,8
1,6
Ertapenem
2,2
1,4
Doripenem
1,3
Aztreonam
4,7
13,8 18
Genetic environment of blaOXA-48 and blaOXA-405
Conclusions
IncL/M pOXA-48
This is the very first example of successive identification in a same patient of an OXA-48 enzyme with carbapenemase activity (without extended-spectrum activity) followed by identification of a novel OXA-48 variant, OXA-405, losing its carbapenemase activity but gaining a hydrolysis activity against extended-spectrum cephalosporins
blaOXA-48
Genomic comparison of the two S. marcescens isolates using rep-PCR
IncL/M pOXA-405
S. marcescens OXA-405
S. marcescens OXA-48
Unrelated S. marcescens
blaOXA-405