The foodborne pathogen Listeria monocytogenes is able to survive a variety of stress conditions leading to the colonization of different niches like the food processing environment. This study focuses on the hypervariable genetic hot spot lmo0443 to lmo0449 haboring three inserts: the stress survival islet 1 (SSI-1), the single-gene insert LMOf2365_0481 , and two homologous genes of the nonpathogenic species Listeria innocua : lin0464 , coding for a putative transcriptional regulator, and lin0465 , encoding an intracellular PfpI protease. Our prevalence study revealed a different distribution of the inserts between human and food-associated isolates. The lin0464-lin0465 insert was predominantly found in food-associated strains of sequence type 121 (ST121). Functional characterization of this insert showed that the putative PfpI protease Lin0465 is involved in alkaline and oxidative stress responses but not in acidic, gastric, heat, cold, osmotic, and antibiotic stresses. In parallel, deletion of lin0464 decreased survival under alkaline and oxidative stresses. The expression of both genes increased significantly under oxidative stress conditions independently of the alternative sigma factor σ ^B . Furthermore, we showed that the expression of the protease gene lin0465 is regulated by the transcription factor lin0464 under stress conditions, suggesting that lin0464 and lin0465 form a functional unit. In conclusion, we identified a novel stress survival islet 2 (SSI-2), predominantly present in L. monocytogenes ST121 strains, beneficial for survival under alkaline and oxidative stresses, potentially supporting adaptation and persistence of L. monocytogenes in food processing environments. IMPORTANCE Listeria monocytogenes strains of ST121 are known to persist for months and even years in food processing environments, thereby increasing the risk of food contamination and listeriosis. However, the molecular mechanism underlying this remarkable niche-specific adaptation is still unknown. Here, we demonstrate that the genomic islet SSI-2, predominantly present in L. monocytogenes ST121 strains, is beneficial for survival under alkaline and oxidative stress conditions, which are routinely encountered in food processing environments. Our findings suggest that SSI-2 is part of a diverse set of molecular determinants contributing to niche-specific adaptation and persistence of L. monocytogenes ST121 strains in food processing environments.

We showed that a combination of a short primary enrichment combined with MDA and Nanopore sequencing can accelerate the traditional process of cultivation and identification of L. monocytogenes . The use of Illumina MiSeq sequencing additionally allowed us to predict the presence of cooccurring L. monocytogenes strains.

The pathogenicity of the human foodborne pathogen Listeria (L.) monocytogenes relies on virulence factors such as internalins. In 2009/2010 two L. monocytogenes strains were responsible for a serious listeriosis outbreak in Austria, Germany and the Czech Republic. One of these clones, QOC1, which caused 14 cases including five fatalities, encodes the novel internalins inlP1, inlPq and inlP4 and the novel internalin-like protein inlP3 in the genomic region of hypervariable genetic hotspot 9 in addition to the standard set of virulence genes. The in silico prevalence study revealed that these genes rarely occur in L. monocytogenes and mainly in minor clonal complexes. To obtain first insights of the role of these genes in the pathogenicity of L. monocytogenes, we studied the gene expression under conditions mimicking the ingestion in the host. Expression of inlP1, inlP3, inlPq and inlP4 was increased under gastric stress and in intracellular bacteria grown in intestinal epithelial cells. Furthermore, colonization of the liver and the spleen was slightly, but significantly reduced 72 hours post infection in an oral mouse infection model when inlP1 or inlP4 was deleted. Moreover, the impact of InlP1 and InlP3 in virulence was shown in vitro in human intestinal epithelial cells. In this study we conclusively demonstrate a potential contribution of uncommon novel internalins and an internalin-like protein to the pathogenicity of L. monocytogenes.

BACKGROUND aGVHD, a common complication of allogeneic stem cell transplant (alloSCT), is driven by proinflammatory cytokines and chemokines that activate the immune system, resulting in end-organ damage. Steroids are first-line treatment but up to 50% of pts are steroid refractory (SR), resulting in high mortality and morbidity. RUX, a JAK1/JAK2 inhibitor, inhibits cytokine-dependent activation of the JAK-STAT pathway and cell proliferation and differentiation, which prevents worsening of aGVHD and allows recovery. JAK pathway inhibition by RUX may also lead to modulation of proinflammatory cytokines and prognostic GVHD biomarkers. The phase 3 randomized REACH2 trial (NCT02913261) in SR aGVHD demonstrated superiority of RUX vs BAT, with a significantly higher overall response rate (ORR; complete [CR] + partial response [PR]) at D28 (62% vs 39%; P < .001) and higher durable ORR at D56 (40% vs 22%; P < .001) (Zeiser R, et al. N Engl J Med. 2020). In this exploratory analysis of REACH2, we assessed whether baseline (BL) levels of proinflammatory cytokines and GVHD markers were prognostic for response and how this changed over time related to treatment. METHODS Pts (N = 309; ≥ 12 y old with grade II-IV SR aGVHD after alloSCT) were randomized 1:1 to RUX (starting at 10 mg bid) or investigator-selected BAT and stratified by aGVHD grade; 295 pts had valid biomarker levels. Serum samples were collected at BL, D14, and D28 and biomarkers, including inflammatory cytokines, soluble receptors of cytokines, chemokines, and tissue-specific markers for gastrointestinal (GI), liver, and skin GVHD, were assessed (Table). Biomarker levels at BL were stratified by response (CR, PR, none [NR]) at D28 for each treatment arm; those that differed by response were analyzed by logistic regression (LR) to assess the association (CR+PR vs NR), adjusting for treatment. The analysis was repeated, adjusting for key covariates that had significant impact on the biomarker-response relationship. Change in biomarkers over time (BL to D28) was assessed via geometric mean values at each visit, along with fold change from BL for each treatment arm. RESULTS Of 22 GVHD biomarkers assessed, 17 (77%) were evaluable (Table). Higher median BL levels of proinflammatory cytokines (IL-6, IL-8, TNF-α), soluble cytokine receptors (IL2RA, TNFRSF1A, ST2) and tissue-specific GVHD markers (REG3A, HGF) were generally observed in NR vs CR pts (Table) and were further analyzed. Higher BL levels of these markers were significantly associated with a lower probability of OR (P[OR]) in the LR analysis (Figure). An interaction term was investigated to assess whether there was a differing biomarker effect within treatment groups; however, this was insignificant and not retained. The addition of skin involvement to the LR for all biomarkers had an impact on P(OR), with increased P(OR) for pts with skin involvement at BL. Liver involvement at BL was added to the LR for HGF, IL6, REG3A, TNFα, and TNFRSF1A. The LR models showed a significant decrease in P(OR) for pts with liver involvement at BL. GI involvement did not have a significant impact for any biomarker. Geometric mean levels of TNFα, TNFRSF1A, IL2RA, REG3A, and ST2 decreased from BL to D14 in CR pts in the RUX arm but increased with BAT. NR pts had increases in these biomarkers regardless of treatment; however, longer follow-up is needed. Treatment had an underlying impact on P(OR) but no further impact on the effect of biomarkers on P(OR). Maximum change from BL was observed at D14; a slight return to BL levels was observed at D28 in CR pts. Surprisingly, IL6 levels increased with RUX regardless of response. IL6, like TNFα, is directly modulated by the JAK-STAT pathway and was therefore expected to decrease with RUX. CONCLUSIONS This is the first biomarker study in a phase 3 trial in GVHD. BL levels of IL6, IL8, TNFα, IL2RA, TNFRSF1A, ST2, REG3A, and HGF tended to be higher in NR vs CR pts; higher levels were associated with lower P(OR) when adjusted for treatment arm. Skin and liver involvement also had an impact on response. Skin involvement was associated with higher P(OR), regardless of biomarker BL levels, possibly because no skin involvement implied other organ involvement and tended to be associated with a higher overall severity grade at BL. CR pts in the RUX group had lower BL levels of TNFα and showed trends toward decreases from BL to D14 in the soluble cytokine receptors ST2, TNFRSF1A, and IL2RA and tissue-specific marker REG3A.