Rapid identification and source-tracking of Listeria monocytogenes using MALDI-TOF mass spectrometry.

Listeria monocytogenes is an important foodborne pathogen responsible for the sometimes fatal disease listeriosis. Public health concerns and stringent regulations associated with the presence of this pathogen in food and food processing environments underline the need for rapid and reliable detection and subtyping techniques. In the current study, the application of matrix assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) as a single identification and source-tracking tool for a collection of L. monocytogenes isolates, obtained predominantly from dairy sources within Australia, was explored. The isolates were cultured on different growth media and analysed using MALDI-TOF MS at two incubation times (24 and 48 h). Whilst reliable genus-level identification was achieved from most media, identification at the species level was found to be dependent on culture conditions. Successful speciation was highest for isolates cultured on the chromogenic Agar Listeria Ottaviani Agosti agar (ALOA, 91% of isolates) and non-selective horse blood agar (HBA, 89%) for 24h. Chemometric statistical analysis of the MALDI-TOF MS data enabled source-tracking of L. monocytogenes isolates obtained from four different dairy sources. Strain-level discrimination was also observed to be influenced by culture conditions. In addition, t-test/analysis of variance (ANOVA) was used to identify potential biomarker peaks that differentiated the isolates according to their source of isolation. Source-tracking using MALDI-TOF MS was compared and correlated with the gold standard pulsed-field gel electrophoresis (PFGE) technique. The discriminatory index and the congruence between both techniques were compared using the Simpsons Diversity Index and adjusted Rand and Wallace coefficients. Overall, MALDI-TOF MS based source-tracking (using data obtained by culturing the isolates on HBA) and PFGE demonstrated good congruence with a Wallace coefficient of 0.71 and comparable discriminatory indices of 0.89 and 0.86, respectively. MALDI-TOF MS thus represents a rapid and cost-effective source-tracking technique for L. monocytogenes.

Phenotypic detection of carbapenemase-producing Enterobacteriaceae by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry and the Carba NP test.

We compared the diagnostic accuracy of the Carba NP test with that of a straightforward matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method for detecting carbapenemase-producing Enterobacteriaceae (CPE). Using PCR as the reference method, both tests demonstrated a sensitivity of 87% and a specificity of 100%. MALDI-TOF MS offers a potential alternative for the rapid detection of CPE in the clinical laboratory setting.

Detection of Listeria monocytogenes from selective enrichment broth using MALDI-TOF Mass Spectrometry.

Conventional methods used for primary detection of Listeria monocytogenes from foods and subsequent confirmation of presumptive positive samples involve prolonged incubation and biochemical testing which generally require four to five days to obtain a result. In the current study, a simple and rapid proteomics-based MALDI-TOF MS approach was developed to detect L. monocytogenes directly from selective enrichment broths. Milk samples spiked with single species and multiple species cultures were incubated in a selective enrichment broth for 24h, followed by an additional 6h secondary enrichment. As few as 1 colony-forming unit (cfu) of L. monocytogenes per mL of initial selective broth culture could be detected within 30h. On applying the same approach to solid foods previously implicated in listeriosis, namely chicken pâté, cantaloupe and Camembert cheese, detection was achieved within the same time interval at inoculation levels of 10cfu/mL. Unlike the routine application of MALDI-TOF MS for identification of bacteria from solid media, this study proposes a cost-effective and time-saving detection scheme for direct identification of L. monocytogenes from broth cultures.This article is part of a Special Issue entitled: Trends in Microbial Proteomics. BIOLOGICAL SIGNIFICANCE: Globally, foodborne diseases are major causes of illness and fatalities in humans. Hence, there is a continual need for reliable and rapid means for pathogen detection from food samples. Recent applications of MALDI-TOF MS for diagnostic microbiology focused on detection of microbes from clinical specimens. However, the current study has emphasized its use as a tool for detecting the major foodborne pathogen, Listeria monocytogenes, directly from selective enrichment broths. This proof-of-concept study proposes a detection scheme that is more rapid and simple compared to conventional methods of Listeria detection. Very low levels of the pathogen could be identified from different food samples post-enrichment in selective enrichment broths. Use of this scheme will facilitate rapid and cost-effective testing for this important foodborne pathogen.

Hepatocytes: the powerhouse of biotransformation.

Liver is the most important organ involved in biotransformation of xenobiotics. Within the main organisational unit, the hepatocyte, is an assembly of enzymes commonly classified as phase I and phase II enzymes. The phase I enzymes principally cytochrome P450 catalyse both oxidative and reductive reactions of a bewildering number of xenobiotics. Many of the products of phase I enzymes become substrates for the phase II enzymes, which catalyse conjugation reactions making use of endogenous cofactors. As xenobiotic metabolising enzymes are responsible for the toxicity of many chemicals and drugs, testing the role of the biotransformation enzymes and the transporters within the hepatocyte is critical. New methodologies may be able to provide information to allow for better in vitro to in vivo extrapolation of data.