Development of a real-time SYBRGreen PCR assay for rapid detection of acquired AmpC in Enterobacteriaceae.
BACKGROUND
Acquired AmpC enzymes, classified as miscellaneous extended-spectrum beta-lactamase (ESBL(M)) enzymes according to a recently proposed beta-lactamase classification, are increasing according to several publications. Simple and rapid methods for detection of ESBL(M) are needed for appropriate infection control. A gel-based multiplex PCR method for acquired bla(AmpC) detection and subtype classification has been available for several years. Here, we describe a modification of the protocol to suit real-time PCR platforms and to include novel genotypes.


METHODS
Clinical isolates with clavulanic acid non-reversible non-susceptibility to extended-spectrum cephalosporins were subjected to combination disk testing with cefoxitin +/- cloxacillin at Malmö University Hospital. Phenotypical AmpC production was defined as cloxacillin reversible cefoxitin resistance. In this study 51 phenotypical AmpC-producing isolates, were subjected to the acquired bla(AmpC) real-time PCR assay. The acquired blaAmpC positive isolates were further characterized by DNA sequencing of the acquired AmpC encoding gene, Pulsed-Field Gel Electrophoresis (PFGE) and PCR-based replicon typing.


CONCLUSIONS
The real-time PCR assay was able to detect and sub-classify all acquired bla(AmpC) genes described to date. The assay can be performed in less than 3h, including pre-PCR preparations. Analysis of the isolate collection resulted in 18 of 51 phenotypical AmpC-producing isolates being positive in the acquired bla(AmpC) real-time multiplex PCR assay; 17 of subtype CIT and one DHA. Sequence analysis identified 16 isolates as blaCMY-2, one as blaCMY-16 and one as blaDHA-1. Detected plasmid replicon types were I1 and B/O. Two of the E. coli isolates were identical according to PFGE and the others were unrelated.
Brolund
Alma
A
Swedish Institute for Infectious Disease Control, Stockholm, Sweden. alma.brolund@smi.se
Wisell
Karin Tegmark
KT
Edquist
Petra J
PJ
Elfström
Lisbeth
L
Walder
Mats
M
Giske
Christian G
CG
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics*; D052160:Benzothiazoles; D003959:Diamines; D004755:Enterobacteriaceae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification*; D004756:Enterobacteriaceae Infections / Q000382:microbiology*; D006801:Humans; D008826:Microbial Sensitivity Tests; D009930:Organic Chemicals / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012680:Sensitivity and Specificity; D001618:beta-Lactamases / Q000235:genetics*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; C048795:AmpC beta-lactamases; D001618:beta-Lactamases