Identification of Enterococcus species using an automated microarray-based nucleic acid test

Image of Enterococcus sp. Photo courtesy of Janice Haney Carr.

Cara N. Wilder, Ph.D.

Enterococcus faecalis is a Gram-positive commensal bacterium known to inhabit the gastrointestinal tract of humans and animals. In immunologically compromised individuals, E. faecalis is a leading cause of urinary tract infections and nosocomial bacteraemia. This latter condition is of particular concern as it can lead to septic shock or the hematogenous spread of bacteria to other parts of the body, resulting in high rates of mortality and organ failure.

Unfortunately, the treatment of E. faecalis infection is not straightforward as a number of strains exhibit intrinsic and acquired resistance to a variety of antibiotics, including aminoglycosides, cephalosporins, and penicillins. Presently, these drug-resistant strains are commonly treated with vancomycin; however, in some instances the extended use of this antimicrobial drug has resulted in the emergence of vancomycin-resistant strains. To make matters worse, current treatment options for vancomycin-resistant strains is limited. Therefore, the rapid identification of vancomycin-resistant strains is imperative to the successful treatment of infection.

In recent years, advances in diagnostics have yielded a number of sensitive assays for the detection of E. faecalis, including fluorescent in situ hybridization (FISH), matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS), and the microarray-based Verigene Gram-Positive Blood Culture test (BC-GP) (Nanosphere). This latter method was recently validated through an examination of 12 Gram-positive targets including E. faecalis, as well as associated drug-resistance determinants. In this study, Buchan et al. used the Verigene BC-GP test to analyze 1,252 positive blood culture broths collected from five clinical centers throughout the United States. The resultant data indicated that the BC-GP test can sensitively and specifically identify E. faecalis and other leading causes of Gram-positive bacteremia directly from positive blood cultures. Further, this assay was capable of detecting the genetic markers vanA and vanB, which confer resistance to vancomycin. Overall, the Verigene BC-GP assay may prE. faecalis, ultimately enhancing public health.
ovide diagnosticians with a more sensitive means of detecting vancomycin-resistant

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Rapid and sensitive detection of blaKPC in Klebsiella pneumoniae

Cara N. Wilder, Ph.D.

Klebsiella pneumoniae is a Gram-negative bacterium found within the mouth, skin, and intestines. Although this organism is a common inhabitant of the human microflora, it can behave as an opportunistic pathogen in immunologically compromised individuals, causing a variety of clinical diseases including pneumonia, thrombophlebitis, and septicemia. Current therapies for K. pneumoniae infection include treatment with β-lactam antibiotics such as third-generation cephalosporins, carbapenems, and penicillins.

Over the last decade, a number of K. pneumoniae strains exhibiting multidrug resistance have emerged in numerous healthcare settings, resulting in prolonged hospitalization and increased mortality. These strains were found to produce Klebsiella pneumoniae carbapenemase (KPC), which is a β-lactamase enzyme that confers resistance to all β-lactam antibiotics. More troubling was the discovery that KPC is encoded by the Tn₃-type transposon-localized gene, bla_KPC. Mobilization of this genetic element has permitted the diffusion of bla_KPC between various plasmids, allowing for inter-species dispersion via horizontal transfer. To date, plasmids harboring bla_KPC have been isolated among Enterobacteriaceae genera commonly found in the human microbiome, including Klebsiella pneumoniae, Enterobacter spp., and Escherichia coli. Regrettably, these strains are often not detectable through routine susceptibility screening and are easily disseminated, further contributing to escalating morbidity and mortality rates.

In order to control the spread of KPC strains, a rapid and sensitive diagnostic tool is necessary. In a recent report, Mosca et al. evaluated the performance of NucliSens EasyQKPC (bioMérieux, France), which is a new molecular assay designed to rapidly detect the presence of the bla_KPC gene via real-time nucleic acid sequenced-based amplification (NASBA™). In this study, 38 non-duplicate, carbapenem-resistant K. pneumoniae isolates were first analyzed using the modified Hodge test (MHT) and the carbapenemase inhibitor test, which are traditional culture-based methods often recommended for the detection of carbapenem-resistant strains. While all strains exhibited the production of KPC when examined by the carbapenemase inhibitor test, only 84% of the strains evaluated by MHT exhibited carbapenemase-production. In contrast, NucliSens EasyQKPC was able to detect the bla_KPC gene in each of the isolates evaluated. The accuracy of this assay was confirmed when each of the isolates tested positive for the presence of the gene following PCR analysis. Overall, NucliSens EasyQKPC provides a sensitive and reliable assay for the identification of the bla_KPC gene, allowing for the rapid detection of KPC strains in clinical samples.

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