1. Email: mandy.wootton@nphs.wales.nhs.uk Table 1: Resistance profile
C1-097b
49th Interscience Conference on Antimicrobial Agents and Chemotherapy
September 12-15, 2009
San Francisco
A Novel GES carbapenemase mutation (Thr232Ala) in Acinetobacter baumannii from the UK M. WOOTTON1, V. E. DANIEL1, L. E. DAVIES1, M. A. TOLEMAN2, T. R. WALSH2, E. KUBIAK3 & R. A. HOWE1 1NPHS Microbiology, University Hospital Wales, Cardiff, UK. 2 Infection Immunity & Biochemistry, University of Cardiff. 3 Royal Gwent Hospital, Newport, UK
Introduction
Table 1: Resistance profile
Fig. 2: Genetic locus of first UK blaGES genes and GES-11
Acinetobacter baumannii (AB) is frequently associated with nosocomial infections. Multiple resistance, either intrinsic or acquired, leads to limited therapeutic options. GES is an Ambler class A ESBL enzyme with carbapenemase activity. It has been reported in Klebsiella pneumoniae & AB in France, Japan, Brazil, Korea, & Greece. Here we report both a novel GES sequence distinct from other published GES & also the first GES reported in the UK from AB (S ).
intI1
dfrA7
qacEΔ1/sul
aac6’-lb
blaGES
intI1
dfrA7
qacEΔ1/sul
aac6’-lb
blaGES
aGI= Glycopeptide intermediate
GES sequence differs from GES-11 by A694G mutation (Fig. 1) leading to an amino acid change from Thr237Ala (Fig. 3). This sequence has now been designated GES-12.
Methods
Picture 1: Phenotypic and genotypic characterisation
AB was isolated from sputum and CVC tip of a 50yr old female returned from a short stay in Egypt. Susceptibilities of imipenem (IMI), meropenem (MER), ertapenem (ERT), ceftazidime (CAZ), tigecycline (TIG), Timentin (TIM), Aztreonam (AZT) & colistin (COL) were established using Etest and BSAC breakpoints. Phenotypic assays were performed for carbapenemase (Hodge-H), oxacillinase & ampC β-lactamases. PCR assays were performed for TEM, SHV, CTX-M, GES, IMP, VIM & OXA. blaGES and class 1 integron variable region were analysed by PCR and sequencing. A qualitative MER hydrolysis assay (HA) was performed on crude cell lysates of S , ATCC and SPM-1 using ΔA decrease at 299nm.
Hodge test
GES PCR
IMP PCR
VIM PCR
Results
Fig.3: From Smith et al The substitution Thr237Ala will affect carbapenemase activity as Thr237 sits above Ser70 and plays a key role in interacting with the β-lactam carboxyl moiety
AB was resistant to all agents tested except COL (MIC 1mg/L); MICs of IMI, MER, ERT, & CAZ were >32mg/L (Table 1). Phenotypic tests for MBLs & carbapenemase were positive (Picture 1). The IMI MIC was reduced from 32mg/L to 3mg/L when combined with EDTA suggesting a metallo-ß-lactamase, however false positives are well documented with AB containing oxacillinases.
PCR assays for VIM and IMP were negative, however a positive result was seen for GES. The integron variable region consisted of the following gene array: blaGES, aac6-Ib, dfrA7 (Fig. 2). HA indicated the presence of carbapenemase activity.
Conclusions
This is the first report of GES enzyme in the UK.
The novel substitution Thr237Ala probably carbapenemase activity
As the source of the AB is from Egypt, global surveillance networks are mandatory.
Fig. 1: Sequence alignment of S with published GES sequences
Reference. Acta Crystallogr D Biol Crystallogr : Structure of GES-1 at atomic resolution: insights into the evolution of carbapenamase activity in the class A extended-spectrum -lactamases. Smith CA, Caccamo M, Kantardjieff KA, Vakulenko S.