Molecular characterization of extended-spectrum cephalosporinase-producing Salmonella enterica serovar Choleraesuis isolates from patients in Thailand and Denmark

Abstract

The objective of this study was to characterize extended-spectrum cephalosporinase (ESC)-producing isolates of Salmonella enterica serovar Choleraesuis recovered from patients in Thailand and Denmark. Twenty-four blood culture isolates from 22 patients were included in the study, of which 23 isolates were recovered from 21 Thai patients during 2003, 2007, or 2008 and one isolate was recovered from a Danish traveler to Thailand. ESC production was confirmed in 13 out of the 24 isolates by MIC testing. Microarray and plasmid profiling (replicon typing and restriction fragment length polymorphism [RFLP]) were used to characterize the genetic mechanisms of antimicrobial resistance in the 13 ESC-producing isolates. Pulsed-field gel electrophoresis (PFGE) and MIC testing were used to compare the clonality between the 13 ESC-producing isolates and the 11 non-ESC-producing isolates. Based on susceptibility patterns, the ESC-producing isolates were more closely related than non-ESC-producing isolates. Microarray, PCR, plasmid profiling, and replicon typing revealed that the 13 ESC-producing isolates harbored either bla(CMY-2) containing incA/C or bla(CTX-M-14) containing incFIIA, incFrepB, and an unknown replicon located on plasmids ranging in size from 75 to 200 kb. The RFLP and replicon typing clustered the isolates into four distinct groups. PFGE revealed 16 unique patterns and five clusters; each cluster contained two or three of the 24 isolates. The isolate from the Danish patient was indistinguishable from two Thai clinical isolates by PFGE. This study revealed the emergence of the bla(CTX-M-14) gene among several clones of Salmonella serovar Choleraesuis. Numerous plasmids were identified containing up to two different ESC genes and four distinct replicons. A "travel-associated" spread was confirmed. Overall, a high degree of clonal diversity between isolates resistant and susceptible to cephalosporins was observed. The findings represent a serious threat to public health for the Thai people and tourists.

FIG. 1.

Dendrographic analysis of PFGE (XbaI) data for Salmonella Choleraesuis isolates from Thailand and Denmark. Black and gray spots in the dendrogram represent the isolates as being either resistant or intermediate, respectively. Antimicrobials are abbreviated as follows: ampicillin, AMP; amoxicillin-clavulanic acid, AMC; apramycin, APR; cefalothin, CEP; cefazolin, CFZ; cefepime, FEP; cefoxitin, FOX; cefpodoxime, CPD; cefotaxime, CTX; ceftazidime, CAZ; ceftiofur, XNL; ceftriaxone, CRO; chloramphenicol, CHL; ciprofloxacin, CIP; colistin, COL; florfenicol, FFN; gentamicin, GEN; imipenem, IPM; meropenem, MEM; nalidixic acid, NAL; neomycin, NEO; spectinomycin, SPT; streptomycin, STR; sulfamethoxazole, SMX; tetracycline, TET; trimethoprim, TMP. Data with the same numbers of asterisks are from the same patient. Dates of isolations are shown as month-day-year. Patient ages are in years. M, male; F, female.

FIG. 2.

Dendrographic analysis of plasmid replicons from Salmonella Choleraesuis transformants originating from Thai and Danish patients and subjected to RFLP. Plasmid sizes are in kb. Data with the same numbers of asterisks are from the same patient.