Dionyssios Nicholas Sgouras

Helicobacter species affect humans and animals, mainly causing gastrointestinal but also extra-gastrointestinal pathologies. Besides Helicobacter pylori, which is the main human pathogen, Non-Helicobacter pylori Helicobacters (NHPH) are also associated with human diseases, thus raising concern about their zoonotic potential. Veterinarians are considered a risk group for NHPH infections and act as first-line communicators to animal owners about their prophylaxis. Therefore, we aimed to assess the knowledge and perception of veterinarians working in Greece about Helicobacter pylori and NHPH by asking them to participate anonymously in an online 34-question survey. The questionnaire consisted of three sections regarding environmental exposure to Helicobacter spp.; know-how about clinical signs in various species, including personal human experience; and willingness to get updated information about NHPH. Of the 111 respondents, 41.4% had not heard of H. suis (NHPH), and 35.0% were unaware of the species that could be affected. Almost 60.0% of companion animal veterinarians rarely suspect and 20.0% never suspect Helicobacter spp. infections in the case of gastritis. Nevertheless, 41.0% of respondents considered Helicobacter as zoonotic, and 87.0% wanted to receive information via professional channels and brochures. Despite the limited number of respondents and the exploratory nature of our study, as with similar data from Portugal, we emphasize the need to train veterinarians to have a more targeted focus on the zoonotic potential of Helicobacter within a One Health approach.

Background and aim: We conducted an equivalence trial of quadruple non-bismuth “concomitant” and “hybrid” regimens for H. pylori eradication in a high clarithromycin resistance area. Methods: There were 321 treatment-naïve H. pylori-positive individuals in this multicenter clinical trial randomized to either the hybrid (esomeprazole 40 mg/bid, amoxicillin 1 g/bid for 7 days, then 7 days esomeprazole 40 mg/bid, amoxicillin 1 g/bid, clarithromycin 500 mg/bid, and metronidazole 500 mg/bid) or the concomitant regimen (all medications given concurrently bid for 10 days). Eradication was tested using histology and/or a 13C-urea breath test. Results: The concomitant regimen had 161 patients (90F/71M, mean 54.5 years, 26.7% smokers, 30.4% ulcer) and the hybrid regimen had 160 (80F/80M, mean 52.8 years, 35.6% smokers, 31.2% ulcer). The regimens were equivalent, by intention to treat 85% and 81.8%, (p = 0.5), and per protocol analysis 91.8% and 87.8%, (p = 0.3), respectively. The eradication rate by resistance, between concomitant and hybrid regimens, was in susceptible strains (97% and 97%, p = 0.6), clarithromycin single-resistant strains (86% and 90%, p = 0.9), metronidazole single-resistant strains (96% and 81%, p = 0.1), and dual-resistant strains (70% and 53%, p = 0.5). The side effects were comparable, except for diarrhea being more frequent in the concomitant regimen. Conclusions: A 14-day hybrid regimen is equivalent to a 10-day concomitant regimen currently used in high clarithromycin and metronidazole resistance areas. Both regimens are well tolerated and safe.

Background and Aims: Helicobacter pylori (H. pylori)‐induced gastric pathology involves remodeling of extracellular matrix mediated by aberrant activity of matrix metalloproteinases (MMPs). We have previously shown that in vitro H. pylori infection leads to MMP‐3 and MMP‐9 overexpression, associated with phosphorylation of bacterial oncoprotein CagA. We extended these findings in an in vivo model of H. pylori infection and further assessed the involvement of MAPK pathways in MMP expression.Materials and Methods: C57BL/6 mice were infected with H. pylori strains HPARE, HPARE ΔCagA, and SS1, for 6 and 9 months. Transcriptional expression of Mmp‐3 and Mmp‐9 was evaluated via qPCR while respective protein levels in the gastric mucosa were determined immunohistochemically. Epithelial cell lines AGS and GES‐1 were infected with H. pylori strain P12 in the presence of chemical inhibitors of JNK, ERK1/2, and p38 pathways, for 24 h. mRNA and protein expression of MMP‐3 and MMP‐9 were determined via qPCR and Western blot, respectively.Results: We observed transcriptional activation of Mmp‐3 and Mmp‐9 as well as aberrant MMP‐3 and MMP‐9 protein expression in murine gastric tissue following H. pylori infection. CagA expression was associated with MMP upregulation, particularly during the early time points of infection. We found that inhibition of ERK1/2 resulted in reduced mRNA and protein expression of MMP‐3 and MMP‐9 during H. pylori infection, in both cell lines. Expressed protein levels of both MMPs were also found reduced in the presence of JNK pathway inhibitors in both cell lines. However, p38 inhibition resulted in a more complex effect, probably attributed to the accumulation of phospho‐p38 and increased phospho‐ERK1/2 activity due to crosstalk between MAPK pathways.Conclusions: H. pylori colonization leads to the upregulation of MMP‐3 and MMP‐9 in vivo, which primarily involves ERK1/2 and JNK pathways. Therefore, their inhibition may potentially offer a protective effect against gastric carcinogenesis and metastasis.

Helicobacter pylori lives in the human stomach and has a population structure resembling that of its host. However, H. pylori from Europe and the Middle East trace substantially more ancestry from modern African populations than the humans that carry them. Here, we use a collection of Afro-Eurasian H. pylori genomes to show that this African ancestry is due to at least three distinct admixture events. H. pylori from East Asia, which have undergone little admixture, have accumulated many more non-synonymous mutations than African strains. European and Middle Eastern bacteria have elevated African ancestry at the sites of these mutations, implying selection to remove them during admixture. Simulations show that population fitness can be restored after bottlenecks by migration and subsequent admixture of small numbers of bacteria from non-bottlenecked populations. We conclude that recent spread of African DNA has been driven by deleterious mutations accumulated during the original out-of-Africa bottleneck.

Targeted virome enrichment and sequencing (VirCapSeq-VERT) utilizes a pool of oligos (baits) to enrich all known—up to 2015—vertebrate-infecting viruses, increasing their detection sensitivity. The hybridisation of the baits to the target sequences can be partial, thus enabling the detection and genomic reconstruction of novel pathogens with <40% genetic diversity compared to the strains used for the baits’ design. In this study, we deploy this method in multiplexed mixes of viral extracts, and we assess its performance in the unbiased detection of DNA and RNA viruses after cDNA synthesis. We further assess its efficiency in depleting various background genomic material. Finally, as a proof-of-concept, we explore the potential usage of the method for the characterization of unknown, emerging human viruses, such as SARS-CoV-2, which may not be included in the baits’ panel. We mixed positive samples of equimolar DNA/RNA viral extracts from SARS-CoV-2, coronavirus OC43, cytomegalovirus, influenza A virus H3N2, parvovirus B19, respiratory syncytial virus, adenovirus C and coxsackievirus A16. Targeted virome enrichment was performed on a dsDNA mix, followed by sequencing on the NextSeq500 (Illumina) and the portable MinION sequencer, to evaluate its usability as a point-of-care (PoC) application. Genome mapping assembly was performed using viral reference sequences. The untargeted libraries contained less than 1% of total reads mapped on most viral genomes, while RNA viruses remained undetected. In the targeted libraries, the percentage of viral-mapped reads were substantially increased, allowing full genome assembly in most cases. Targeted virome sequencing can enrich a broad range of viruses, potentially enabling the discovery of emerging viruses.

Background: Helicobacter pylori (Hp) colonizes the human stomach and can induce gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma. Clinical observations suggest a role for the Hp virulence factor cytotoxin-associated gene A (CagA) in pathogenesis. The pathogenic activity of CagA is partly regulated by tyrosine phosphorylation of C-terminal Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs in host cells. However, CagA differs considerably in EPIYA motifs, whose functions have been well characterized in epithelial cells. Since CagA is fragmented in immune cells, different CagA variants may exhibit undetected functions in B cells. Methods: B cells were infected with Hp isolates and isogenic mutants expressing different CagA EPIYA variants. CagA translocation and tyrosine phosphorylation were investigated by Western blotting. Apoptosis was analyzed by flow cytometry and metabolic activity was detected by an MTT assay. Results: Isogenic CagA EPIYA variants are equally well translocated into B cells, followed by tyrosine phosphorylation and cleavage. B cell apoptosis was induced in a CagA-independent manner. However, variants containing at least one EPIYA-C motif affected metabolic activity independently of phosphorylation or multiplication of EPIYA-C motifs. Conclusions: The diverse structure of CagA regulates B cell physiology, whereas B cell survival is independent of CagA.

Background: The spatiotemporal profiling of molecular transmission clusters (MTCs) using viral genomic data can effectively identify transmission networks in order to inform public health actions targeting SARS-CoV-2 spread. Methods: We used whole genome SARS-CoV-2 sequences derived from ten European regions belonging to eight countries to perform phylogenetic and phylodynamic analysis. We developed dedicated bioinformatics pipelines to identify regional MTCs and to assess demographic factors potentially associated with their formation. Results: The total number and the scale of MTCs varied from small household clusters identified in all regions, to a super-spreading event found in Uusimaa-FI. Specific age groups were more likely to belong to MTCs in different regions. The clustered sequences referring to the age groups 50–100 years old (y.o.) were increased in all regions two weeks after the establishment of the lockdown, while those referring to the age group 0–19 y.o. decreased only in those regions where schools’ closure was combined with a lockdown. Conclusions: The spatiotemporal profiling of the SARS-CoV-2 MTCs can be a useful tool to monitor the effectiveness of the interventions and to reveal cryptic transmissions that have not been identified through contact tracing.

Helicobacter pylori infection induces a plethora of DNA damages. Gastric epithelial cells, in order to maintain genomic integrity, require an integrous DNA damage repair (DDR) machinery, which, however, is reported to be modulated by the infection. CagA is a major H. pylori virulence factor, associated with increased risk for gastric carcinogenesis. Its pathogenic activity is partly regulated by phosphorylation on EPIYA motifs. Our aim was to identify effects of H. pylori infection and CagA on DDR, investigating the transcriptome of AGS cells, infected with wild-type, ΔCagA and EPIYA-phosphorylation-defective strains. Upon RNA-Seq-based transcriptomic analysis, we observed that a notable number of DDR genes were found deregulated during the infection, potentially resulting to base excision repair and mismatch repair compromise and an intricate deregulation of nucleotide excision repair, homologous recombination and non-homologous end-joining. Transcriptome observations were further investigated on the protein expression level, utilizing infections of AGS and GES-1 cells. We observed that CagA contributed to the downregulation of Nth Like DNA Glycosylase 1 (NTHL1), MutY DNA Glycosylase (MUTYH), Flap Structure-Specific Endonuclease 1 (FEN1), RAD51 Recombinase, DNA Polymerase Delta Catalytic Subunit (POLD1), and DNA Ligase 1 (LIG1) and, contrary to transcriptome results, Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APE1) upregulation. Our study accentuates the role of CagA as a significant contributor of H. pylori infection-mediated DDR modulation, potentially disrupting the balance between DNA damage and repair, thus favoring genomic instability and carcinogenesis.

The characteristics of fermented milk are affected by the type of milk used and the microorganisms involved in the fermentation process. Goat milk has been widely suggested as a possible alternative to cow milk in allergic subjects, because of the high genetic variability in alpha-s1 casein (CSN1S1) content, which is associated with different technological and nutritional properties of milk. The aim of the study was to evaluate the suitability of goat milk with low and high CSN1S1 to produce fermented milk. In addition, the performance as starter of selected Lactobacillus paracasei FS109 strain compared to no-selected L. paracasei strains was investigated. Initially, the selected L. paracasei FS109 strain was tested for adhesion ability to HT-29 and Caco-2 cells and immunomodulation effect. Then, the strain was used to produce fermented milk from goat milk with a low and high casein CSN1S1 genotype. The results indicated that greater acidifying activity was obtained for L. paracasei FS109 after 24 h of fermentation than the other two strains tested independently by the CSN1S1 genotype. L. paracasei FS109 grew well during fermentation, reaching a higher value (>8.5 log CFU/mL). Interestingly, the same strain maintained a high viable population (about 9 log CFU/mL) during the 30-day cold storage of the product. The present study shows for the first time the suitability of the goat milk with low CSN1S1 genotypes to produce fermented milk and highlight the importance of strain selection in determination of technological and beneficial traits. Combining goat milk with low CSN1S1 and selected strains could be a strategy of improving traditional and functional fermented milk market.