Triton Hodge Test: Improved Protocol for Modified Hodge Test for Enhanced Detection of NDM and Other Carbapenemase Producers

Abstract

Accurate detection of carbapenemase-producing Gram-negative bacilli is of utmost importance for the control of nosocomial spread and the initiation of appropriate antimicrobial therapy. The modified Hodge test (MHT), a carbapenem inactivation assay, has shown poor sensitivity in detecting the worldwide spread of New Delhi metallo-␤-lactamase (NDM). Recent studies demonstrated that NDM is a lipoprotein anchored to the outer membrane in Gram-negative bacteria, unlike all other known carbapenemases. Here we report that membrane anchoring of ␤-lactamases precludes detection of carbapenemase activity by the MHT. We also show that this limitation can be overcome by the addition of Triton X-100 during the test, which allows detection of NDM. We propose an improved version of the assay, called the Triton Hodge test (THT), which allows detection of membrane-bound carbapenemases with the addition of this nonionic surfactant. This test was challenged with a panel of 185 clinical isolates (145 carrying known carbapenemase-encoding genes and 40 carbapenemase nonproducers). The THT displayed test sensitivity of >90% against NDM-producing clinical isolates, while improving performance against other carbapenemases. Ertapenem provided the highest sensitivity (97 to 100%, depending on the type of carbapenemase), followed by meropenem (92.5 to 100%). Test specificity was not affected by the addition of Triton (87.5% and 92.5% with ertapenem and meropenem, respectively). This simple inexpensive test confers a large improvement to the sensitivity of the MHT for the detection of NDM and other carbapenemases. Detection of carbapenemase producers in clinical laboratories is of major importance to define appropriate empirical antimicrobial therapy and to implement infection control measures. Acquired carbapenemases belong to three of the four known classes of ␤-lactamases, namely, Ambler class A (KPC, SME, NMC-A, IMI-1, and some allelic variants of GES), Ambler class B or metallo-␤-lactamases (MBLs) (e.g., VIM, IMP, NDM, and SPM), and Ambler class D or oxacillinases (OXAs) (e.g., OXA-48 and OXA-181) (1).The modified Hodge test (MHT) is a phenotypic screening test to identify carbapenemase producers, being recommended by the Clinical and Laboratory Standards Institute (CLSI) for Enterobacteriaceae with elevated carbapenem MICs or reduced disk diffusion inhibition zones (2). This test is based on the inactivation of a carbapenem by carbapenemase-producing strains, which enables a susceptible indicator strain to extend growth toward a disk containing this antibiotic, along the streak of inoculum of the tested strain. The MHT has shown excellent sensitivity in the detection of class A and class D carbapenemase producers (3-6). Unfortunately, the MHT performs poorly in the detection of NDM-producing isolates, with sensitivity below 50% (3-7). Because NDMs are Zn(II)-dependent enzymes, it has been suggested that the deficits of this cation in commercial media could be responsible for these false-negative results (4). Indeed, Zn(II) availabili...

Phenotypic and molecular identification of carbapenemase-producing Enterobacteriaceae - challenges in diagnosis and treatment

“…Also, our results revealed 2 strains of P. stuartii, 1 strain of S. marcescens and 1 isolate of K. pneumoniae harbouring gene bla NDM , which were negative for the MHT with both meropenem and ertapenem disks. Similar reports recognized the MHT unsatisfying performance in the detection of NDM-carriers, with sensitivity below 50% (2,8,14,21,22). Recent studies demonstrated that NDM is a lipoprotein anchored to the outer membrane in Gram-negative bacteria, unlike all other known carbapenemases (21).…”

The rapid spread of carbapenem-resistant Enterobacteriaceae

“…However, the MHT has poor sensitivity for class B carbapenemases (sensitivity <50 %) which is marginally improved by addition of ZnSO 4 (sensitivity 87%) [Girlich 2012]. As reported in Argentina in 2016, addition of 0.2% Triton X-100 [vol/vol] to Mueller Hinton Agar plates improved sensitivity (97–100%) for detecting class B carbapenemase expressing CPE [Pasteran 2016]. The proposed mechanism relies on the anionic detergent disturbing the hydrophobic interactions between the lipoprotein carbapenemases and the outer membrane [Gonzalez 2016, Pasteran 2016].…”

A simple protocol to characterize bacterial cell‐envelope lipoproteins in a native‐like environment

“…The genes coding for these enzymes were cloned with their peptide leaders in a plasmid designed for the controlled expression of MBLs . This system allows the induction of MBL expression with IPTG, in such a way that the enzymes are expressed at levels similar to those found in clinical strains with their native promoter, avoiding artifacts due to overexpression …”

“…20 This system allows the induction of MBL expression with IPTG, in such a way that the enzymes are expressed at levels similar to those found in clinical strains with their native promoter, avoiding artifacts due to overexpression. 20,22 F I G U R E 1 Localization of NDM-1, NDM-1 C26A and NDM-8 in the periplasm of Escherichia coli. The New Delhi Metallo-β-lactamase (NDM-1) has a lipobox sequence in its N-terminal, which is recognized by the lipidation machinery of Gram-negative bacteria.…”