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Rapid and accurate identification of carriers of resistant microorganisms is an important aspect of efficient infection control in hospitals. Traditional identification methods of antibiotic-resistant bacteria usually take at least 3 to 4 days after sampling. A duplex real-time PCR assay was developed for rapid detection of ampicillin-resistant Enterococcus faecium (ARE). Primers and probes that are used in this assay specifically detected the D-Ala-D-Ala ligase gene of E. faecium and the modified penicillin-binding protein 5 gene (pbp5) carrying the Glu-to-Val substitution at position 629 (Val-629) in a set of 129 tested E. faecium strains with known pbp5 sequence. Presence of the Val-629 in the strain set from 11 different countries was highly correlated with ampicillin resistance. In a screening of hospitalized patients, the real-time PCR assay yielded a sensitivity and a specificity for the detection of ARE colonization of 95% and 100%, respectively. The results were obtained 4 h after samples were harvested from overnight broth of rectal swab samples, identifying both species and the resistance marker mutation in pbp5. This novel assay reliably identifies ARE 2 to 3 days more quickly than traditional culture methods, thereby increasing laboratory throughput, making it useful for rectal screening of ARE. The assay demonstrates the advantages of real-time PCR for detection of nosocomial pathogens.

The genetic relationship of 81 ampicillin-resistant and 21 ampicillin-susceptible Enterococcus faecium isolates from clinical infections and rectal screening in hospitalized patients in Norway was studied by pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP). PFGE showed 55 different banding patterns, and 65 of the isolates could be grouped into one large group. With AFLP, 46 patterns were discerned, and 74 isolates clustered in one group. In general, the isolates had a higher degree of similarity than with PFGE. The purK gene, which is one of the targets of the E. faecium multilocus sequence typing scheme, was sequenced. Eleven different purK alleles could be discerned, with the majority of isolates (n = 80) harboring allele 1. With only two exceptions, all strains carrying purK-1 clustered in the same PFGE and AFLP groups, indicating a good correlation between PFGE type, AFLP type, and purK allele. Genetic polymorphism of a 571-bp PCR fragment of the C-terminal domain of the penicillin-binding protein 5 gene (pbp5) was determined, and sequence differences were associated with the level of ampicillin resistance. This study indicates that the majority of ampicillin-resistant E. faecium strains in Norway belong to a distinct genetic lineage of closely related genotypes. Rectal and clinical isolates were generally indistinguishable, and differences in clonal distribution and allele polymorphism were found mainly between ampicillin-resistant and -susceptible isolates.