High-Throughput Characterization of Listeria monocytogenes Using the OmniLog Phenotypic Microarray.

High-throughput biochemical screening techniques are an important tool in phenotypic analysis of bacteria. New methods, simultaneously measuring many phenotype responses, increase the output of such investigations and allow a more complete overview of the bacterial phenotype, facilitating large-scale correlation to related genotypes. This chapter describes the application of OmniLog phenotype microarray analysis, a high-throughput assay for the phenotypic characterization of bacterial strains across a variety of different traits such as nutrient utilization and antimicrobial sensitivity, to Listeria species.

Whole-Genome Sequencing-Based Characterization of 100 Listeria monocytogenes Isolates Collected from Food Processing Environments over a Four-Year Period.

Listeria monocytogenes is frequently found in foods and processing facilities, where it can persist, creating concerns for the food industry. Its ability to survive under a wide range of environmental conditions enhances the potential for cross-contamination of the final food products, leading to possible outbreaks of listeriosis. In this study, whole-genome sequencing (WGS) was applied as a tool to characterize and track 100 L. monocytogenes isolates collected from three food processing environments. These WGS data from environmental and food isolates were analyzed to (i) assess the genomic diversity of L. monocytogenes, (ii) identify possible source(s) of contamination, cross-contamination routes, and persistence, (iii) detect absence/presence of antimicrobial resistance-encoding genes, (iv) assess virulence genotypes, and (v) explore in vivo pathogenicity of selected L. monocytogenes isolates carrying different virulence genotypes. The predominant L. monocytogenes sublineages (SLs) identified were SL101 (21%), SL9 (17%), SL121 (12%), and SL5 (12%). Benzalkonium chloride (BC) tolerance-encoding genes were found in 62% of these isolates, a value that increased to 73% among putative persistent subgroups. The most prevalent gene was emrC followed by bcrABC, qacH-Tn6188, and qacC. The L. monocytogenes major virulence factor inlA was truncated in 31% of the isolates, and only one environmental isolate (L. monocytogenes CFS086) harbored all major virulence factors, including Listeria pathogenicity island 4 (LIPI-4), which has been shown to confer hypervirulence. A zebrafish embryo infection model showed a low (3%) embryo survival rate for all putatively hypervirulent L. monocytogenes isolates assayed. Higher embryo survival rates were observed following infection with unknown virulence potential (20%) and putatively hypovirulent (53 to 83%) L. monocytogenes isolates showing predicted pathogenic phenotypes inferred from virulence genotypes.IMPORTANCE This study extends current understanding of the genetic diversity among L. monocytogenes from various food products and food processing environments. Application of WGS-based strategies facilitated tracking of this pathogen of importance to human health along the production chain while providing insights into the pathogenic potential for some of the L. monocytogenes isolates recovered. These analyses enabled the grouping of selected isolates into three putative virulence categories according to their genotypes along with informing selection for phenotypic assessment of their pathogenicity using the zebrafish embryo infection model. It has also facilitated the identification of those isolates with genes conferring tolerance to commercially used biocides. Findings from this study highlight the potential for the application of WGS as a proactive tool to support food safety controls as applied to L. monocytogenes.

Susceptibility (re)-testing of a large collection of Listeria monocytogenes from foods in China from 2012 to 2015 and WGS characterization of resistant isolates.

OBJECTIVES: Our aim was to determine the antimicrobial susceptibilities of 2862 Listeria monocytogenes cultured from various foods in China and to use WGS to characterize the antimicrobial resistance and virulence genotypes of those expressing a resistance phenotype. METHODS: The susceptibilities of 2862 L. monocytogenes were determined by broth microdilution. Twenty-eight L. monocytogenes were found to be resistant to one to four antibiotics. All 28 resistant isolates were subsequently sequenced using short-read high accuracy protocols. The corresponding genomes were assembled and further analysis was carried out using appropriate bioinformatics pipelines. RESULTS: All 28 resistant L. monocytogenes were classified into five STs (ST3, ST8, ST9, ST155 and ST515). Both ST9 and ST155 were dominant and their genotypes correlated with their resistance phenotypes. All ST9 isolates were MDR and could be phylogenetically classified into two clusters. One was relatively close to clinical origins and one to food. Downstream analysis of the genetic contexts in which these resistance genotypes were found suggested that these may have been acquired from other bacteria by horizontal transfer or insertion into the chromosome. All isolates harboured Listeria pathogenicity island (LIPI)-1 and LIPI-2, and only two harboured LIPI-3. CONCLUSIONS: This study reported on the antimicrobial susceptibilities of 2862 foodborne L. monocytogenes along with the genomic characterization of 28 resistant isolates, 11 of which expressed an MDR phenotype. These data showed that this bacterium can acquire resistance by horizontal gene transfer in and between species. This study may necessitate a re-evaluation of risk to public health, associated with this bacterial species.

A Comparative Study of the Susceptibility of Listeria Species to Sanitizer Treatments When Grown under Planktonic and Biofilm Conditions.

Listeria species are ubiquitous in nature and can adapt to survive in a variety of niches, including food processing environments. Listeria species that colonize these environments may also have the potential to persist. Food safety strategies designed to manage these niches include regular cleaning and disinfection with proven sanitizers containing biocide-active compounds. Typically, these sanitizers are effective against bacteria growing under planktonic conditions, but their efficacy may be compromised when bacteria are contained in biofilms. The susceptibility of persistent Listeria isolates, i.e., those capable of forming biofilms, to a selection of sanitizers was investigated. A quaternary ammonium compound-based sanitizer was the biocide most effective against planktonic bacteria, with a MIC of 0.0015 to 0.006%. In contrast, ethanol-based sanitizers were the least effective. Although, no triclosan tolerance was observed for planktonic Listeria isolates, triclosan was the only biocide that resulted in a significant biomass reduction. Differences between Listeria species were observed; L. monocytogenes and L. welshimeri biofilms were more tolerant to quaternary ammonium compound-based sanitizers than were L. innocua biofilms. These findings extend our understanding of the application of commonly used sanitizers in the food industry and the efficacy of these sanitizers against Listeria species and their associated biofilms.

Antimicrobial Resistance in Listeria Species.

For nearly a century the use of antibiotics to treat infectious diseases has benefited human and animal health. In recent years there has been an increase in the emergence of antibiotic-resistant bacteria, in part attributed to the overuse of compounds in clinical and farming settings. The genus Listeria currently comprises 17 recognized species found throughout the environment. Listeria monocytogenes is the etiological agent of listeriosis in humans and many vertebrate species, including birds, whereas Listeria ivanovii causes infections mainly in ruminants. L. monocytogenes is the third-most-common cause of death from food poisoning in humans, and infection occurs in at-risk groups, including pregnant women, newborns, the elderly, and immunocompromised individuals.

A 3-year multi-food study of the presence and persistence of Listeria monocytogenes in 54 small food businesses in Ireland.

The problem of assessing the occurrence of the food-borne pathogen Listeria monocytogenes in the food chain, and therefore the risk of exposure of the human population, is often challenging because of the limited scope of some studies. In this study the occurrence of L. monocytogenes in food from four major food groups, dairy products, meats, seafood and vegetables, and associated food processing environments in Ireland was studied over a three-year period. Fifty-four small food businesses participated in the study and sent both food and environmental samples every 2months between 2013 and 2015. L. monocytogenes was isolated using the ISO11290 standard method. Confirmation of L. monocytogenes and identification of serogroups were achieved using a multiplex PCR assay, and for some isolates serotype was determined using commercial antisera. Pulsed- field gel electrophoresis (PFGE) analysis was performed on all isolates allowing the relatedness of isolates from different food businesses to be compared nationwide. In total, 86 distinct pulsotypes were identified. The overall occurrence of L. monocytogenes in food samples was 4.2%, while in environmental samples it was 3.8%. In general, the occurrence of L. monocytogenes in food businesses decreased over the course of the study, presumably reflecting increased awareness and vigilance. The majority of the pulsotypes detected were unique to a particular food group (63/86), while only three pulsotypes were found in all four food groups investigated. The highest occurrence in food was found in the meat category (7.5%) while seafood had the lowest rate of occurrence (1.8%). Seventeen of the pulsotypes detected in the study were persistent, where persistence was defined as repeated isolation from a single facility with a minimum time interval of 6months. Using PFGE, 11 of the pulsotypes identified in this study were indistinguishable from those of 11 clinical isolates obtained from patients in Ireland over the last 4years, highlighting the fact that these pulsotypes are capable of causing disease. Overall, the study shows the diversity of L. monocytogenes strains in the Irish food chain and highlights the ability of many of these strains to persist in food processing environments. The finding that a significant proportion of these pulsotypes are also found in clinical settings highlights the need for continued vigilance by food producers, including frequent sampling and typing of isolates detected.

Pulsed-field gel electrophoresis for Listeria monocytogenes.

Pulsed-Field Gel Electrophoresis (PFGE) is a molecular subtyping method with high discriminatory power, reproducibility, and epidemiological concordance for the subtyping of Listeria monocytogenes and other bacteria. PFGE uses rare-cutting restriction enzymes (macrorestriction) that cut the genomic DNA, usually resulting in 6-25 DNA fragments ranging between 30 and 600 kb. Bacterial cells are immobilized in agarose plugs and subsequently lysed to release genomic DNA, which is then subjected to DNA digestion. AscI and ApaI restriction enzymes are typically used for L. monocytogenes. Electrophoresis using an alternating electric field direction results in a DNA banding pattern, or fingerprint, which is used to classify isolates into different pulsotypes. PFGE is currently the CDC's gold standard method for epidemiological studies in foodborne outbreaks.

Comparison of Listeria monocytogenes isolates across the island of Ireland.

Building a comprehensive knowledge base of the association of Listeria monocytogenes isolates across national food chains, clinical cases, and environments can play a key role in helping control the incidence of listeriosis. Today, many food chains cross national borders and are often shared by neighboring countries. This study characterized L. monocytogenes isolated from food samples in Northern Ireland and investigated whether similarities in the population and associations of L. monocytogenes strains exist in the neighboring countries of Northern Ireland and the Republic of Ireland, which together constitute the island of Ireland. Listeria monocytogenes isolates were characterized using serotyping and pulsed-field gel electrophoresis subtyping. This data was then interrogated against existing data for the Republic of Ireland, to identify any shared trends in the ecology and contamination patterns of L. monocytogenes strains. The results of this study indicated that contaminated food products often shared L. monocytogenes strains with other products. A total of six different strain subtypes were identified among 18 contaminated products. Overall strain diversity in positive samples was low, with no sample yielding more than one L. monocytogenes strain, as determined by pulsed-field gel electrophoresis subtyping. When comparisons against an Irish strain database were performed, many related strain subtypes were also shared by a variety of sources in the Republic of Ireland. This study highlights the potential benefits that a whole-island surveillance approach may present to food safety and public health in both Northern Ireland and the Republic of Ireland.