Snehal Jadhav

Methods used for the detection and subtyping of Listeria monocytogenes

Listeria monocytogenes is an important foodborne pathogen responsible for non-invasive and invasive diseases in the elderly, pregnant women, neonates and immunocompromised populations. This bacterium has many similarities with other non-pathogenic Listeria species which makes its detection from food and environmental samples challenging. Subtyping of L. monocytogenes strains can prove to be crucial in epidemiological investigations, source tracking contamination from food processing plants and determining evolutionary relationships between different strains. In recent years there has been a shift towards the use of molecular subtyping. This has led to the development of new subtyping techniques such as multi-locus variable number tandem repeat analysis (MLVA) and multi-locus sequence based typing (MLST). This review focuses on the available methods for Listeria detection including immuno-based techniques and the more recently developed molecular methods and analytical techniques such as matrix-assisted laser desorption/ionisation time-of-flight based mass spectrometry (MALDI-TOF MS). It also includes a comparison and critical analysis of the available phenotypic and genotypic subtyping techniques that have been investigated for L. monocytogenes.

Detection of Listeria monocytogenes from selective enrichment broth using MALDI-TOF mass spectrometry

UNLABELLED 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.

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

ABSTRACT 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.

Comparing the identification of Clostridium spp. by two Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometry platforms to 16S rRNA PCR sequencing as a reference standard: a detailed analysis of age of culture and sample preparation.

We compared the identification of Clostridium species using mass spectrometry by two different Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) platforms (Bruker MS and Vitek MS) against 16S rRNA sequencing as the reference standard. We then examined the impact of different sample preparations and (on one of those platforms) age of bacterial colonial growth on the performance of the MALDI-TOF MS systems. We identified 10 different species amongst the 52 isolates by 16S rRNA sequencing, with Clostridium perfringens the most prevalent (n=30). Spectrometric analysis using Vitek MS correctly speciated 47/52 (90.4%) isolates and was not affected by the sample preparation used. Performance of the Bruker MS was dependent on sample preparation with correct speciation obtained for 36 of 52 (69.2%) isolates tested using the Direct Transfer [DT] protocol, but all 52 (100%) isolates were correctly speciated using either an Extended Direct Transfer [EDT] or a Full Formic Extraction [EX] protocol. We then examined the effect of bacterial colonial growth age on the performance of Bruker MS and found substantial agreement in speciation using DT (Kappa=0.62, 95% CI: 0.46-0.75), almost perfect agreement for EDT (Kappa=0.94, 95% CI: 0.86-1.00) and exact agreement for EX (Kappa=1.00) between different days.

Omics-based approaches and their use in the assessment of microbial-influenced corrosion of metals

Abstract Microbial-influenced corrosion (MIC) has been known to have economic, environmental, and social implications to offshore oil and gas pipelines, concrete structures, and piped water assets. While corrosion itself is a relatively simple process, the localised manner of corrosion makes in situ assessments difficult. Furthermore, corrosion assessments tend to be measured as part of a forensic investigation. Compounding the issue further is the impact of microbiological/biofilm processes, where corrosion is influenced by the complex processes of different microorganisms performing different electrochemical reactions and secreting proteins and metabolites that can have secondary effects. While traditional microbiological culture-dependent techniques and electrochemical/physical assessments provide some insight into corrosion activity, the identity and role of microbial communities that are related to corrosion and corrosion inhibition in different materials and in different environments are scarce. One avenue to explore MIC and MIC inhibition is through the application of omics-based techniques, where insight into the bacterial population in terms of diversification and their metabolism can be further understood. As such, this paper discusses the recent progresses made in a number of fields that have used omics-based applications to improve the fundamental understanding of biofilms and MIC processes.

Species-Level Discrimination of Psychrotrophic Pathogenic and Spoilage Gram-Negative Raw Milk Isolates Using a Combined MALDI-TOF MS Proteomics–Bioinformatics-based Approach

Identification of psychrotrophic pathogenic and spoilage Gram-negative bacteria using rapid and reliable techniques is important in commercial milk processing, as these bacteria can produce heat-resistant proteases and act as postprocessing contaminants in pasteurized milk. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is a proven technology for identification of bacteria in food, however, may require optimization for identification of pathogenic and spoilage bacteria in milk and dairy products. The current study evaluated the effects of various culture conditions and sample preparation methods on assigning of raw milk isolates to the species level by MALDI-TOF MS. The results indicated that culture media, incubation conditions (temperature and time), and sample preparation significantly affected the identification rates of bacteria to the species level. Nevertheless, the development of spectral libraries of isolates grown on different media using a web tool for hierarchical clustering of peptide mass spectra (SPECLUST) followed by a ribosomal protein based bioinformatics approach significantly enhanced the assigning of bacteria, with at least one unique candidate biomarker peak identified for each species. Phyloproteomic relationships based on spectral profiles were compared to phylogenetic analysis using 16S rRNA gene sequences and demonstrated similar clustering patterns with significant discriminatory power. Thus, with appropriate optimization, MALDI-TOF MS is a valuable tool for species-level discrimination of pathogenic and milk spoilage bacteria.