Phillip Brown

Listeria monocytogenes is a Gram-positive bacterial foodborne pathogen responsible for the severe illness listeriosis. Of the 14 L. monocytogenes serotypes, serotype 4b is a major contributor to human listeriosis and encompasses all four leading hypervirulent clonal complexes (CCs), including the ancient, ubiquitous CC2. CC2 is globally dominated by sublineage (SL) 2, responsible for most human CC2-associated cases. Here we describe two other CC2 SLs, SLs 782 and 1039. These SLs are newly recognized, having been reported only since 2002, and to date are encountered exclusively in North America. Phylogenetic analysis revealed that they are strikingly divergent from each other as well as from SL2. SL782 and SL1039 have been implicated in human listeriosis and have also been repeatedly isolated from surface water and wildlife in North America, with several of these environmental strains exhibiting high genomic similarity (≤7 core genome allelic mismatches) to strains from human listeriosis. They share an unusual resistance profile towards a panel of Listeria wide-host-range-phages and exhibit several distinct lineage-specific traits. Specifically, SL782 universally lacks a gene otherwise unique to and conserved in serotype 4b and harbors the Listeria pathogenicity island LIPI-4, while SL1039 harbors LIPI-3 and is almost always resistant to tetracycline, harboring the novel Tn 916 -like transposon Tn 916 . 1039 . These and other traits may have driven clonal emergence of SL782 and SL1039, potentially via adaptations in natural ecosystems. IMPORTANCE: Listeria monocytogenes causes the severe foodborne illness listeriosis and the hypervirulent clonal complex (CC) 2 is a major contributor to human disease. Most CC2 strains belong to the long-recognized, ubiquitous sublineage (SL) 2. Surprisingly, the two other leading SLs within CC2, SL782 and 1039, have been isolated exclusively from North America, and only subsequently to 2002. They differ remarkably from each other and other CC2 strains and exhibit unusual genomic and phenotypic traits. They have been repeatedly isolated from watersheds and wildlife, with such environmental isolates exhibiting high genomic similarity to those from human listeriosis. The findings are important in identifying novel, unexpected features in the composition and evolution of a major, hypervirulent L. monocytogenes CC. They will also serve as platforms for further studies to further elucidate the reservoirs, emergence, persistence, and dissemination of novel Listeria sublineages and their roles in the continuum between natural ecosystems and human disease.

The process of Sanger sequencing can be a challenging and unintuitive concept for students to master. In order to improve student learning, we developed a hands-on Sanger sequencing activity using 3D-printed models to incorporate tactile learning. These 3D models and the accompanying activity demonstrate the differences between gene amplification polymerase chain reaction (PCR) and Sanger sequencing, including the purpose and function of dNTPs and ddNTPs, both in terms of building and terminating the chain and in how the DNA sequence is read. After completing the activity, students self-reported high levels of both learning and enjoyment from the activity. Students were also asked to discuss what misconceptions they had prior to this activity that were addressed and provide suggestions for improving this activity. A majority of the misconceptions are related to the function and differences between dNTPs and ddNTPs, with others related to the function of primers, the high-quality region of sequencing, and the purpose of DNA fragment sizes. Overall, student responses indicate that this activity was enjoyable, improved student learning, and addressed specific misconceptions regarding Sanger sequencing. The use of online dice rolling software or additional computational analysis was a common suggestion from students to improve this activity further in future semesters.

In September 2018, Hurricane Florence resulted in major flooding in North Carolina, USA. Efforts to isolate Listeria monocytogenes and other Listeria spp. from Hurricane Florence floodwaters repeatedly yielded non-haemolytic Listeria-like isolates that could not be readily assigned to known Listeria taxa. Whole-genome sequence analyses against the 28 currently known Listeria species confirmed that the isolates constitute two new taxa within the genus Listeria. Taxon I, with one isolate, showed the highest similarity to Listeria goaensis, with an average nucleotide identity blast of 85.3±4.4% and an in silico DNA–DNA hybridization (isDDH) of 32.4% (range: 30–35%), differing from the latter by its ability to reduce nitrate, ferment d-ribose and sucrose, and by its inability to produce catalase or ferment d-trehalose and d-lactose. Taxon II, represented by 11 isolates, showed the highest similarity to Listeria rocourtiae, with an average nucleotide identity blast of 92.64±3.8% and an isDDH of 49.9% (range: 47.3–52.5%), differing from the latter by its ability to ferment l-arabinose and its inability to ferment l-rhamnose, d-galactose, d-lactose and d-melibiose. The names Listeria tempestatis sp. nov. and Listeria rocourtiae subsp. hofi subsp. nov. are proposed for taxon I and II, respectively, with type strains CLIP 2022/01175^T (F6L-1A=CIP 112444^T = DSM 117029^T) and CLIP 2022/01000^T (F66L-1A=CIP 112443^T = DSM 117030^T), respectively. Both taxa lack known Listeria pathogenic islands, suggesting a lack of pathogenicity for humans.

Listeria monocytogenes presents a significant concern for the food industry due to its ability to persist in the food processing environment. One of the factors contributing to its persistence is decreased sensitivity to disinfectants. Our objective was to assess the diversity of L. monocytogenes sensitivity to food industry disinfectants by testing the response of 1,671 L . monocytogenes isolates to quaternary ammonium compounds (QACs) and 414 isolates to peracetic acid (PAA) using broth microdilution and growth curve analysis assays, respectively, and to categorize the isolates into sensitive and tolerant. A high phenotype-genotype concordance (95%) regarding tolerance to QACs was obtained by screening the genomes for the presence of QAC tolerance-associated genes bcrABC , emrE , emrC, and qacH . Based on this high concordance, we assessed the QAC genes’ dissemination among publicly available L. monocytogenes genomes ( n = 39,196). Overall, QAC genes were found in 23% and 28% of the L. monocytogenes collection in this study and in the global data set, respectively. bcrABC and qacH were the most prevalent genes, with bcrABC being the most detected QAC gene in the USA, while qacH dominated in Europe. No significant differences ( P > 0.05) in the PAA tolerance were detected among isolates belonging to different lineages, serogroups, clonal complexes, or isolation sources, highlighting limited variation in the L. monocytogenes sensitivity to this disinfectant. The present work represents the largest testing of L. monocytogenes sensitivity to important food industry disinfectants at the phenotypic and genomic level, revealing diversity in the tolerance to QACs while all isolates showed similar sensitivity to PAA. IMPORTANCE: Contamination of Listeria monocytogenes within food processing environments is of great concern to the food industry due to challenges in eradicating the isolates once they become established and persistent in the environment. Genetic markers associated with increased tolerance to certain disinfectants have been identified, which alongside other biotic and abiotic factors can favor the persistence of L. monocytogenes in the food production environment. By employing a comprehensive large-scale phenotypic testing and genomic analysis, this study significantly enhances the understanding of the L. monocytogenes tolerance to quaternary ammonium compounds (QACs) and the genetic determinants associated with the increased tolerance. We provide a global overview of the QAC genes prevalence among public L. monocytogenes sequences and their distribution among clonal complexes, isolation sources, and geographical locations. Additionally, our comprehensive screening of the peracetic acid (PAA) sensitivity shows that this disinfectant can be used in the food industry as the lack of variation in sensitivity indicates reliable effect and no apparent possibility for the emergence of tolerance.

Listeria monocytogenes is a Gram-positive facultative intracellular bacterium that is ubiquitous in nature and the causative agent of listeriosis. The outbreak-derived serotype 4b strain L. monocytogenes strain WS1, sequence type (ST) 558, sublineage (SL) 558, was previously found to have unusual pathogenicity, with ability to cause fetal damage in the first trimester of pregnancy. Search of the WS1 genome for novel and unique genomic features identified a putative lantibiotic island on the chromosome of WS1 and all tested strains of SL558 and two other putative emerging serotype 4b clones, clonal complex 554 (SL554 and SL555) and ST782 (SL782), but absent from all other major clones of L. monocytogenes. The island was deleted from four strains, including two each of ST558 and ST554. The deletions did not impact virulence in a Galleria mellonella model but consistently resulted in reduced hemolytic activity. In addition, we noted strain-dependent impacts on biofilm formation. Additional studies will be necessary to further elucidate the roles of this genomic island in the adaptive physiology and virulence of L. monocytogenes.

Premature stop codons in the internalin virulence determinant inlA are common in serotype 1/2a Listeria monocytogenes from food/food processing environments but rare among human clinical isolates. Here, we report the genome sequences of serotype 1/2a (STs 121 and 3258) human listeriosis isolates from Sweden harboring such mutations in inlA .

Listeria monocytogenes is notorious for persistence in food facilities. Phages can significantly impact the ecology of Listeria , but there is a dearth of genome sequence data for Listeria phages from food processing ecosystems. We report the genome sequences of two Listeria phages from turkey processing facilities in the USA.

Listeria monocytogenes is a facultative intracellular foodborne pathogen and the causative agent of the severe disease listeriosis. It is found ubiquitously in the environment and exhibits innate resistance to certain antimicrobials, but acquired antimicrobial resistance remains relatively uncommon. Given the potentially dire health outcomes associated with listeriosis, acquisition of antimicrobial resistance (AMR) by this pathogen is of considerable public health concern. AMR in L. monocytogenes has been surveyed frequently in strains of clinical and food origin, but much less commonly in wildlife. We analyzed 158 strains of L. monocytogenes and 27 of non-pathogenic Listeria spp. isolated from wild black bears ( Ursus americanus ) for resistance to a panel of antimicrobials. AMR was uncommon and noted mostly for tetracycline. Tetracycline resistance was more common in Listeria innocua than in L. monocytogenes . All tetracycline-resistant L. monocytogenes strains belonged to sequence type ST1039 and harbored the Tn 916 -like tet (M) transposon Tn 916.1039 in a conserved chromosomal location. In contrast, three different tetracycline resistance elements, i.e., the tet (M) elements Tn 5801.UAM and Tn 5801.551 and the tet (S) element Tn 6000.205 , were identified among tetracycline-resistant strains of L. innocua . The greater prevalence and diversity of tetracycline resistance elements among bear-derived non-pathogenic Listeria strains suggest the potential of the latter to serve as reservoirs for retention and diversification of AMR determinants in this wildlife host and warrant their further monitoring and study. IMPORTANCE: Listeria monocytogenes causes severe foodborne illness and is the only human pathogen in the genus Listeria . Previous surveys of AMR in Listeria focused on clinical sources and food or food processing environments, with AMR in strains from wildlife and other natural ecosystems remaining under-explored. We analyzed 185 sequenced strains from wild black bears ( Ursus americanus ) from the United States, including 158 and 27 L . monocytogenes and L. innocua , respectively. Tetracycline resistance was the most prevalent resistance trait. In L . monocytogenes, it was encountered exclusively in serotype 4b strains with the novel Tn 916 -like element Tn 916.1039 . In contrast, three distinct, novel tetracycline resistance elements (Tn 5801.UAM , Tn5801.551 , and Tn 6000.205 ) were identified in L. innocua . Interestingly, Tn5801.551 was identical to elements in L. monocytogenes from a major foodborne outbreak in the United States in 2011. The findings suggest the importance of wildlife and non-pathogenic Listeria species as reservoir for resistance elements in Listeria .

Listeria monocytogenes can persistently contaminate food processing environments and tolerate sanitizers. Most sequenced strains are from clinical and environmental sources in the contemporary era, with relatively few prior to extensive food processing and sanitizer use. We report the genome sequences of a diverse panel of 83 strains from 1926 to 1964.