Cortes et al. Listeria Stress Transcriptomics σ B regulon, particularly lmo2230 and the non-coding RNA Rli47, play an integral role in the response of L. monocytogenes to acid stress. Furthermore, we report the first global transcriptome sequencing analysis of L. monocytogenes plasmid gene expression and identify a putative, plasmid-encoded riboswitch with potential involvement in response to acid exposure.

The food-borne pathogen Listeria monocytogenes is known for its capacity to cope with multiple stress conditions occurring in food and food production environments (FPEs). Plasmids can provide benefits to their host strains, and it is known that various Listeria strains contain plasmids. However, the current understanding of plasmid frequency and function in L. monocytogenes strains remains rather limited. To determine the presence of plasmids among L. monocytogenes strains and their potential contribution to stress survival, a comprehensive dataset was established based on 1,921 published genomes from strains representing 14 L. monocytogenes sequence types (STs). Our results show that an average of 54% of all L. monocytogenes strains in the dataset contained a putative plasmid. The presence of plasmids was highly variable between different STs. While some STs, such as ST1, ST2, and ST4, contained few plasmid-bearing strains (<15% of the strains per ST), other STs, such as ST121, ST5, ST8, ST3, and ST204, possessed a higher proportion of plasmid-bearing strains with plasmids found in >71% of the strains within each ST. Overall, the sizes of plasmids analyzed in this study ranged from 4 to 170 kbp with a median plasmid size of 61 kbp. We also identified two novel groups of putative Listeria plasmids based on the amino acid sequences of the plasmid replication protein, RepA. We show that highly conserved plasmids are shared among Listeria strains which have been isolated from around the world over the last few decades. To investigate the potential roles of plasmids, nine genes related to stress-response were selected for an assessment of their abundance and conservation among L. monocytogenes plasmids. The results demonstrated that these plasmid genes exhibited high sequence conservation but that their presence in plasmids was highly variable. Additionally, we identified a novel transposon, Tn7075, predicted to be involved in mercury-resistance. Here, we provide the largest plasmid survey of L. monocytogenes to date with a comprehensive examination of the distribution of plasmids among L. monocytogenes strains. Our results significantly increase our knowledge about the distribution, composition, and conservation of L. monocytogenes plasmids and suggest that plasmids are likely important for the survival of L. monocytogenes in food and FPEs.

The survival of Listeria (L.) monocytogenes in foods and food production environments (FPE) is dependent on several genes that increase tolerance to stressors; this includes competing with intrinsic bacteria. We aimed to uncover genes that are differentially expressed (DE) in L. monocytogenes sequence type (ST) 121 strain 6179 when co-cultured with cheese rind bacteria. L. monocytogenes was cultivated in broth or on plates with either a Psychrobacter or Brevibacterium isolate from cheese rinds. RNA was extracted from cocultures in broth after two or 12 hours and from plates after 24 and 72 hours. Broth co-cultivations with Brevibacterium or Psychrobacter yielded up to 392 and 601 DE genes, while plate co-cultivations significantly affected the expression of up to 190 and 485 L. monocytogenes genes, respectively. Notably, the transcription of virulence genes encoding the Listeria adhesion protein and Listeriolysin O were induced during plate and broth cocultivations. The expression of several systems under the control of the global stress gene regulator, σ B , increased during co-cultivation. A cobalamin-dependent gene cluster, responsible for the catabolism of ethanolamine and 1,2-propanediol, was upregulated in both broth and plate co-cultures conditions. Finally, a small non-coding (nc)RNA, Rli47, was induced after 72 hours of co-cultivation on plates and accounted for 50-90% of the total reads mapped to L. monocytogenes. A recent study has shown that Rli47 may contribute to L. monocytogenes stress survival by slowing growth during stress conditions through the suppression of branch-chained amino acid biosynthesis. We hypothesize that Rli47 may have an impactful role in the response of L. monocytogenes to co-cultivation by regulating a complex network of metabolic and virulence mechanisms.

The genus Brevibacterium harbors many members important for cheese ripening. We performed real-time quantitative PCR (qPCR) to determine the abundance of Brevibacterium on rinds of Vorarlberger Bergkäse, an Austrian artisanal washed-rind hard cheese, over 160 days of ripening. Our results show that Brevibacterium are abundant on Vorarlberger Bergkäse rinds throughout the ripening time. To elucidate the impact of Brevibacterium on cheese production, we analysed the genomes of three cheese rind isolates, L261, S111, and S22. L261 belongs to Brevibacterium aurantiacum , whereas S111 and S22 represent novel species within the genus Brevibacterium based on 16S rRNA gene similarity and average nucleotide identity. Our comparative genomic analysis showed that important cheese ripening enzymes are conserved among the genus Brevibacterium . Strain S22 harbors a 22 kb circular plasmid which encodes putative iron and hydroxymethylpyrimidine/thiamine transporters. Histamine formation in fermented foods can cause histamine intoxication. We revealed the presence of a putative metabolic pathway for histamine degradation. Growth experiments showed that the three Brevibacterium strains can utilize histamine as the sole carbon source. The capability to utilize histamine, possibly encoded by the putative histamine degradation pathway, highlights the importance of Brevibacterium as key cheese ripening cultures beyond their contribution to cheese flavor production.

The survival of Listeria (L.) monocytogenes in foods and food production environments (FPE) is dependent on several genes that increase tolerance to stressors; this includes competing with intrinsic bacteria. We aimed to uncover genes that are differentially expressed (DE) in L. monocytogenes sequence type (ST) 121 strain 6179 when co-cultured with cheese rind bacteria. L. monocytogenes was cultivated in broth or on plates with either a Psychrobacter or Brevibacterium isolate from cheese rinds. RNA was extracted from co-cultures in broth after two or 12 hours and from plates after 24 and 72 hours. Broth co-cultivations with Brevibacterium or Psychrobacter yielded up to 392 and 601 DE genes, while plate co-cultivations significantly affected the expression of up to 190 and 485 L. monocytogenes genes, respectively. Notably, the transcription of virulence genes encoding the Listeria adhesion protein and Listeriolysin O were induced during plate and broth co-cultivations. The expression of several systems under the control of the global stress gene regulator, σB, increased during co-cultivation. A cobalamin-dependent gene cluster, responsible for the catabolism of ethanolamine and 1,2-propanediol, was upregulated in both broth and plate co-cultures conditions. Finally, a small non-coding (nc)RNA, Rli47, was induced after 72 hours of co-cultivation on plates and accounted for 50-90% of the total reads mapped to L. monocytogenes. A recent study has shown that Rli47 may contribute to L. monocytogenes stress survival by slowing growth during stress conditions through the suppression of branch-chained amino acid biosynthesis. We hypothesize that Rli47 may have an impactful role in the response of L. monocytogenes to co-cultivation by regulating a complex network of metabolic and virulence mechanisms.

Several genes of the eut, pdu, and cob/cbi operons are responsible for the metabolism of ethanolamine (EA) and 1,2-propanediol (PD) and are essential during the pathogenic lifecycles of various enteric pathogens. Studies concerning EA and PD metabolism have primarily focused on bacterial genera from the family Enterobacteriaceae, especially the genus Salmonella. Listeria monocytogenes is a member of the Firmicutes phylum and is the causative agent of the rare but highly fatal foodborne disease listeriosis. The eut, pdu, and cob/cbi operons are organized as a single large locus collectively referred to as the cobalamin-dependent gene cluster (CDGC). The CDGC is well conserved in L. monocytogenes; however, functional characterization of the genes in this cluster and how they may contribute to Listeria virulence and stress tolerance in food production environments is highly limited. Previous work suggests that the degradation pathway of PD is essential for L. monocytogenes establishment in the gastrointestinal tract. In contrast, EA metabolism may be more important during intracellular replication. Other studies indicate that the CDGC is utilized when L. monocytogenes is exposed to food and food production relevant stress conditions. Perhaps most noteworthy, L. monocytogenes exhibits attenuated growth at cold temperatures when a key EA utilization pathway gene was deleted. This review aims to summarize the current knowledge of these pathways in L. monocytogenes and their significance in virulence and stress tolerance, especially considering recent developments.

We previously demonstrated that dairy calves having access to drinking water since birth (W0) achieved greater body weight, fiber digestibility, and feed efficiency than those that first received drinking water at 17 d of age (W17). Since gut microbiota composition could be linked to growth and development of animals, the objective of this study was to examine the effect of offering drinking water to newborn calves on composition of bacteria in the gut using a fecal microbiota analysis. Fresh feces were collected directly from the rectum of calves in W0 (n = 14) and W17 (n = 15) at 2, 6, and 10 wk of age. All of the calves were fed pasteurized waste milk, weaned at 7 wk of age, and offered tap water according to the treatment. The DNA was sequenced using 16S rRNA gene-amplicon sequencing on an Illumina MiSeq system (Illumina Inc., San Diego, CA). The sequences were clustered into operational taxonomic units (OTU) with a 99% similarity threshold. Treatment effects on α-diversity indices and relative abundance of the 10 most abundant genera were analyzed using GLIMMIX procedure of SAS (SAS Institute Inc., Cary, NC). Statistical significance (q-value) of treatment effects on the 50 most abundant OTU was determined with a false discovery rate analysis. At 2 wk of age, W0 had a greater number of observed OTU (5,908 vs. 4,698) and species richness (Chao 1 index) than W17. The number of OTU and richness indices increased from wk 2 to 6, but the increment of W17 was greater than that of W0. The Shannon and inverse-Simpson indices increased linearly with age, but no difference was observed between W0 and W17 at any time point. The Firmicutes to Bacteroidetes ratios were also similar at every time point but decreased markedly when calves were weaned. The relative abun-dance of genera Faecalibacterium and Bacteroides was greater in W0 than W17 at 2 wk of age. The genus Faecalibacterium continued to be more abundant in W0 than W17 at 6 wk of age but had similar abundance 3 wk after weaning (10 wk of age). The abundance of Faecalibacterium at wk 6 was positively correlated with apparent total-tract digestibility of acid detergent fiber at 10 wk of age. Calves receiving water since birth had greater abundance of OTU related to Faecalibacterium prausnitzii, and Bifidobacterium breve at 6 wk of age (q < 0.085). These species are known to improve growth in preweaned calves. The abundance of none of the genera and OTU was different between W0 at W17 at 10 wk of age (q > 0.100). Overall, beginning to offer drinking water at birth has a potential to modulate gut microbiota composition and thereby positively affect performance of young dairy heifer calves (≤10 wk of age).

Listeria monocytogenes is the causative agent of the highly fatal foodborne disease listeriosis and can persist in food production environments. Recent research highlights the involvement of L. monocytogenes plasmids in different stress response mechanisms, which contribute to its survival in food production facilities. Ultraviolet (UV) light in the UVC spectrum (200 to 280 nm) is used in food production to control microbial contamination. Although plasmid-encoded UV resistance mechanisms have been described in other bacteria, no research indicates that L. monocytogenes plasmids contribute to the UV stress response. The plasmids of L. monocytogenes strains 6179, 4KSM, and R479a are genetically distinct and were utilized to study the roles of plasmids in the UV response. Wildtype and plasmid-cured variant cells were grown to logarithmic or late-stationary phase, plated on agar plates, and exposed to UVC for 60 or 90 s, and colony-forming units (CFUs) were determined. CFUs of 6179 and 4KSM, bearing pLM6179 and p4KSM, respectively, were significantly (p-value &lt; 0.05) higher than the plasmid-cured strains in both logarithmic and stationary phases. No difference in survival was observed for the R479a strain. Our data show for the first time that certain L. monocytogenes plasmids contribute to the survival of UVC light stress.