Epidemiological surveillance and characterisation of TEM-, SHV-, and CTX-M-type ESBLs in Russian nosocomial strains of Enterobacteriaceae using real-time PCR and melting-curve analysis techniques

Abstract number: 1732_130

Stepanova M., Nikulin A., Sukhorukova M., Edelstein M.

Objectives: To determine the prevalence of major molecular class A extended-spectrum b-lactamases (ESBLs) of TEM, SHV and CTX-M families among nosocomial Enterobacteriaceae from Russian ICUs.

Methods: A total of 1373 consecutive nosocomial isolates of Enterobacteriaceae collected as part of the national surveillance study of antimicrobial resistance in ICUs of 32 Russian hospitals in 2002–2004 were screened for ESBL production using the MIC and double-disk synergy tests with cefotaxime, ceftazidime, cefepime and clavulanic acid. All strains with ESBL phenotype were tested for the presence of TEM, SHV and CTX-M ESBL-encoding genes using three real-time PCR assays. Two of these assays employed postamplification melting curve analysis with differentially labeled fluorogenic probes to detect all the known mutations associated with ESBL activity in blaTEM and blaSHV genes, respectively. The third assay targeting blaCTX-M genes utilised melting curve analysis with SYBR Green I to distinguish the members of CTX-M-1, -2, -8, and -9 clusters according to melting temperatures of PCR products generated with nested set of primers. Strains producing penicillinases SHV-1, TEM-1, SHV ESBLs with aa substitutions 146(V), 149(S), 156(D), 179(A,G,N), 238(A,S), 240(K), TEM ESBLs with aa substitutions 104(K), 164(C,H,S), 237(T), 238(S), 240(K) and CTX-M enzymes (-2, -3, -5, -8, -9, -14, -15) representing all four clusters were used as controls. Selected b-lactamase genes from isolates with unusual real-time PCR melting profiles were cloned and sequenced.

Results: ESBL phenotype was detected in 718 (52.3%) strains, members of 10 genera, most commonly in Klebsiella pneumoniae (81.4%), Escherichia coli (49.5%), Citrobacter spp. (59.3%) and Proteus mirabilis (38.9%). The distribution of various ESBL types and their combinations is summarised in the Table. In addition to the detection of known ESBL types, real-time PCR and sequencing allowed the identification of new SHV variants, each containing one of the following substitutions: 156D, 157E, 240K (alone), but lacking ESBL activity.

b-Lactamase typesNumberPercent in all ESBL producers
TEM (His164)10.1
SHV-2-like (Ser238)182.5
SHV-5-like (Ser238; Lys240)7810.9
CTX-M-1-cluster + SHV-2-like446.1
CTX-M-1-cluster + SHV-5-like9813.6
CTX-M-2-cluster + SHV-2-like30.4
CTX-M-2-cluster + SHV-5-like10.1
CTX-M-9-cluster + SHV-5-like81.1
Other ESBLs71.0
Total TEMs/TEM ESBLs373/151.9/0.1
Total SHVs/SHV ESBLs425/25059.3/34.8
Total CTX-M ESBLs61485.5

Conclusions: This study demonstrates the usefulness and efficiency of real-time PCR techniques for epidemiological typing of molecular class A ESBLs. It reveals the extremely high incidence of ESBLs, the dominance of CTX-M-1-cluster enzymes, frequent co-occurrence of CTX-M and SHV ESBLs, and surprisingly low incidence of TEM ESBLs among nosocomial Enterobacteriaceae isolated in Russian ICUs.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Location: ICC, Munich, Germany
Presentation type:
Back to top