Helicobacter pylori (H. pylori) is of major importance. The invasion, survival, colonization, and stimulation of further inflammation within the gastric mucosa are possible due to several evasive mechanisms induced by the virulence factors that are expressed by the bacterium. The knowledge concerning the mechanisms of H. pylori pathogenicity is crucial to ameliorate eradication strategies preventing the possible induction of carcinogenesis. This review highlights the current state of knowledge and the most recent findings regarding H. pylori virulence factors and their relationship with gastric premalignant lesions and further carcinogenesis.Gastric cancer constitutes one of the most prevalent malignancies in both sexes; it is currently the fourth major cause of cancer-related deaths worldwide. The pathogenesis of gastric cancer is associated with the interaction between genetic and environmental factors, among which infection by  Helicobacter pylori (H. pylori) is a Gram-negative, helix-shaped, microaerophilic, flagellated bacterium that is capable of converting its shape from spiral to coccoid, which is believed to be involved in the enhancement of bacterial survival in the host gastric microenvironment. The spiral form of H. pylori enables the successful motility of the bacterium, whereas the coccoid form provides the ability to colonize the mucus layer of the gastric epithelium, enhancing further invasiveness of the bacterium. Furthermore, H. pylori is capable of forming biofilms to decrease its susceptibility to several antibiotics, leading to antibiotic resistance mutations and further difficulties with bacterial eradication.H. pylori has been classified as a class I carcinogen associated with the onset of gastric cancer (GC) by the World Health Organization (WHO) as well as by the International Agency for Research on Cancer [H. pylori colonizes the gastric mucosa of nearly half of the world’s population; however, in the majority of cases, the infected individuals remain asymptomatic. The prevalence of the infections induced by H. pylori is estimated at 85–95% in developing countries and approximately 30–50% in developed countries [H. pylori include the bacterial and environmental factors that enable its survival in the gastric microenvironment where the acidity is at pH even lower than 3.0 [H. pylori is associated with several mechanisms, among which the alterations of the host signaling pathways, indirect inflammatory responses induced within the gastric mucosa, and direct epigenetic outcomes on gastric epithelial cells, are of major importance [Since 1994, n Cancer . H. pylothan 3.0 . The patportance .H. pylori infection occurs most prevalently during childhood, whereas the appearance of gastrointestinal diseases is observed in adulthood [H. pylori significantly contributes to the induction of GC, which is the second most prevalent and the fourth leading cause of death worldwide, leading to more than 740,000 deaths per year [H. pylori is believed to be the major causation of this malignancy in approximately 92% of patients [H. pylori infection enhances the progression of the epithelial–mesenchymal transition (EMT) that induces the oncogenic alterations within the gastric mucosa, constituting one of the hallmarks of gastric carcinogenesis [H. pylori is responsible for the induction and progression of other gastrointestinal impairments and diseases, including gastritis, peptic ulcer disease, dyspepsia, or mucosa-associated lymphoid tissue lymphoma (MALT); contrarily, H. pylori is a protective factor against inflammatory bowel disease (IBD) and gastroesophageal reflux disease (GERD) [The acquisition of dulthood . Infectiper year . Among ppatients ,11. H. pogenesis ,13. Excee (GERD) ,16,17,18H. pylori-related diseases is associated with numerous virulence factors; a particular genotype of the H. pylori strain plays a crucial role. Furthermore, what is of the highest importance is an interplay between the host, gastric microenvironment, as well as bacterial virulence factors. H. pylori virulence factors are not only involved in the induction of inflammatory responses, but they also control and regulate those responses, maintaining chronic inflammation. H. pylori virulence factors enable the colonization and survival of the bacterium within the gastric mucosa, leading to further immune escape and ultimately, the induction of premalignant alterations. H. pylori exhibits an expanded complex of mechanisms that alters host cellular responses and signaling pathways  [Urease is one of the most essential external . Externaic mucus . Except nd AmiF) .H. pylori strains [H. pylori, urease protects the bacterium from gastric acidity due to the increased release of ammonia, promotes bacterial nutrition via the release of the host metabolites, and also generates the proton motive force during the hydrolysis of urea [H. pylori species, urease activity is controlled by both the environmental pH and the availability of the cofactor nickel; bacterial cultures that are supplemented with nickel tend to exhibit significantly increased urease activity [H. pylori in the spiral form presents higher urease activity compared with the bacterium present in the coccoid form [H. pylori, might be impaired at very low pH [flbA gene, the same gene that is involved in flagellar biosynthesis [H. pylori-related diseases [Urease is a critical factor that facilitates bacterial colonization within the gastric mucosa; urease-negative mutants fail in colonizing the gastric mucosa at physiological pH levels as sufficiently as urease-positive  strains . Except  of urea . Further of urea . Urease  of urea . Urease  of urea . In H. pactivity . H. pylooid form . Moreovey low pH . Greatery low pH . Urease ynthesis . The inhdiseases .H. pylori flagellum constitutes a crucial factor enabling bacterial motility and chemotaxis properties. Physiologically, H. pylori possesses a bundle of 2–6 sheathed unipolar flagella; each of the flagella is approximately 3 μm long and the surrounding sheaths enable protection from the gastric acidic microenvironment [H. pylori movement is associated with the exact number of flagella and might differ among species [H. pylori mutants do not produce flagella at all, whereas FlaB-negative mutants might produce flagella; however, they are not fully functional [csrA H. pylori mutants showed decreased motility and adherence properties compared to the wild-type H. pylori presenting the relevance of csrA in the flagellar formation [The ironment ,32. The  species . Two flanctional . Kao et ormation . The motormation ,37.H. pylori pathogenicity specifically at the very first stages of bacterial invasion. It was demonstrated that less motile strains of H. pylori present difficulties with further colonization within the gastric mucosa; furthermore, bacterial motility correlates with bacterial infectivity as well [H. pylori mutants are not capable of colonizing the gastric mucosa, providing an insight that bacterial motility is fundamental for further bacterial pathogenicity [H. pylori-induced inflammation and immune invasion; furthermore, flagella take part in biofilm formation [H. pylori strains that possess highly motile properties induce an increased release of IL-8 [Flagellum, as a factor that provides bacterial motility, enables further colonization in the gastric mucosa; therefore, it constitutes one of the most crucial virulence factors associated with  as well . It was genicity . Flagellgenicity ,41. Exceormation ,43. Seveormation ,45. It w of IL-8 ,47. It w of IL-8 .H. pylori strains can be generally categorized into two subtypes—cytotoxin-associated gene A (CagA)-positive and CagA-negative H. pylori strains. CagA along with a type 4 secretion system (T4SS) are encoded by the cag pathogenicity island (cagPAI) that plays a key role in carcinogenesis. CagA is injected into the cell via the pilus formed by T4SS and induces cellular alterations that impair cell motility, cellular proliferation, and apoptosis, as well as alters the arrangement of the whole cytoskeleton. The adherence of H. pylori to the gastric epithelial cells induces the expression of cagA and the process seems to be controlled by the Fur protein among others [H. pylori.g others . The preH. pylori strains are associated with greater severity and worse clinical outcome of patients. Kyrillos et al. (2015) showed that cagA expression might be associated with the bacteriophage genes (phage orthologous genes) affecting H. pylori virulence [H. pylori strains stimulate the elevation of IL-8 and IL-12 levels in the sera of infected individuals [H. pylori infection are highly associated with the number and the variations within C-terminal Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs; for instance, H. pylori strains with the EPIYA-D motif are prone to release higher amounts of IL-8 compared to other variations [Generally, infections by CagA-positive irulence . CagA-poividuals ,52. The riations . An animriations . Lai et riations .H. pylori, indicating the cooperation between H. pylori motility and CagA presence [P53 mutations are more prevalent in tumors induced by CagA-positive strains than the negative ones [CagA gene presents several variants and there is a correlation between the particular cagA genotype and patients’ ethnicity [cagA genotypes present different severity of enhanced IL-8 and cytotoxin production, inflammatory responses, or apoptosis in gastric epithelial cells. CagA represses host heat shock proteins’ expression by downregulating the levels of host HSPH1 (HSP105), HSPA1A (HSP72), and HSPD1 (HSP60) [CagA-positive strains are more motile compared to the CagA-negative strains, which indicates that CagA is also associated with bacterial motility . Furtherpresence . CagA prpresence . P53 mutive ones . CagA gethnicity ,65,66,67thnicity . Moreovethnicity ,74,75,76 (HSP60) .Infection by the CagA-positive strains promotes the induction of gastritis (active or atrophic), duodenal ulcers, and gastric carcinogenesis; ,78,79. TH. pylori in the host cells [vacA gene is present in all of the H. pylori strains. Receptor-like protein tyrosine phosphatases α and β (RPTP—RPTPα and RPTPβ), low-density lipoprotein receptor-related protein-1 (LRP-1), or sphingomyelin are major types of receptors to which VacA binds [vacA genotypes are distinguished including s1, s2, m1, m2, s1m1, s1m2, s2m2, and s2m1. The VacA s1 genotype is one of the most abundant in H. pylori-infected patients and there is an association between this genotype and peptic ulcer disease [H. pylori-infected patients with chronic gastritis, whereas in H. pylori-induced gastric cancer, usually s1 and m1 VacA genotypes are most prevalent [H. pylori infection.Vacuolating cytotoxin A (VacA) is a cytotoxin involved in the formation of pores where mechanisms of action and final pathogenicity slightly differ depending on the time of exposure to the host cells. In cases of acute exposure, VacA promotes the autophagy pathways in cells, whereas, during chronic exposure, VacA promotes the appearance of impaired autophagosomes as well as induces the formation of intracellular vacuoles that enable the survival of st cells . VacA cocA binds ,84. So f disease . VacA s1revalent ,87. It wH. pylori infection [Except for autophagy and disruption of the endosomal and lysosomal functions, VacA is involved in many other processes that impair gastric epithelial cells such as alterations in mitochondrial functioning, as well as apoptosis and necrosis . Furthernfection . Gastricnfection .H. pylori infection might significantly stimulate cell death via the enhancement of VacA activity [H. pylori pathogenicity [Abdullah et al. (2019) demonstrated a synergy between VacA and CagA, showing that CagA might accumulate within the VacA-induced impaired autophagosomes . This syactivity . Similargenicity .vacA genotypes are further associated with different morbidity incidents of such gastrointestinal disorders as GC or peptic ulcer disease, as well as the clinical outcome of patients [vacA genotypes might also be associated with the severity of H. pylori-induced inflammation [vacA genotypes might induce the enhanced expression of the transforming growth factor-beta 1 (TGF-β1) mRNA levels [cagA, vacA expression might be stimulated in a Fur-dependent manner [vacA polymorphism might correlate with the histological type of GC; this characteristic is not applicable in the case of cagA [vacA s1 strains are far easier to eradicate compared to vacA s2 strains [H. pylori survival in the gastric environment even after eradication therapy [Various patients ,101,102.patients . Differeammation . FurtherA levels . Similart manner . Figura  of cagA . It was  strains . However therapy .2O2) into water (H2O) and molecular oxygen (O2) and is one of the most abundant proteins in both—plant and animal cells. Three unrelated types of catalase can be distinguished and those include the “classical” catalase, catalase-peroxidase, and manganese-containing catalase. Catalase is implicated in numerous pathological processes such as inflammation, apoptosis prevention, as well as induction of mutagenesis resulting in a vast number of tumors. Catalase presents its activity in the cytoplasm and periplasm; it can also be surface-expressed [Catalase is an enzyme that converts hydrogen peroxide  that regulates the complement system [H. pylori survival within the macrophage phagosomes [H. pylori colonization [H. pylori catalase prevents the bacterium from toxic long-chain fatty acids and their metabolites as well as provides H. pylori protection from phagocytosis; catalase is also involved in the modulation of immune responses [H. pylori catalase activity is controlled by Fur protein and iron concentrations; it was demonstrated that strains of H. pylori that were Fur-deficient and grown on a medium with low iron concentrations showed significantly reduced catalase activity [H. pylori catalase suppresses further bacterial colonization and inflammatory responses induced in gastric mucosa; furthermore, humoral immune responses are induced by such a vaccination [Catalase levels are estimated at 4–5% of c mucosa ,110. H.  species . It was  species . Furthert system . The autt system . Catalasagosomes . An animnization . H. pyloesponses ,118,119.activity . It was cination .H. pylori enzymes that prevents the bacterium from reactive oxygen species (ROS), enabling the maintenance of the proper homeostasis. Contrarily to catalase, SOD is located on the cell surface only. SOD facilitates the dismutation of superoxide to oxygen, preventing excessive amounts of toxic superoxide free radicals. SOD, which is a metalloenzyme, can be categorized into three isoforms depending on its properties and chemical composition—copper-zinc SOD (Cu/Zn-SOD), manganese SOD (Mn-SOD), and extracellular matrix SOD (EC-SOD). H. pylori SOD requires iron (Fe) for proper and effective functioning, acting as a SOD cofactor. Any alterations in the cellular Fe concentrations decrease SOD activity, enhancing cellular susceptibility to oxidative damage.Superoxide dismutase (SOD) is one of the H. pylori-infected individuals with moderate to severe gastric mucosa inflammation present greater expression of Mn-SOD than individuals with the intact gastric mucosa. Mn-SOD expression was observed to be more enhanced primarily within the pit cells of the antrum and not the corpus [H. pylori strains results in significantly greater SOD, catalase, and glutathione peroxidase activities compared to exposure to the CagA-negative strains [H. pylori SOD inhibits the production of the pro-inflammatory cytokines (by downregulating the nuclear factor kappa B (NF-κB) activation pathways), as well as macrophage inflammatory protein 2 (MIP-2) (IL-8 homolog) via activation of the macrophages [H. pylori mutants present a greater susceptibility to oxidative damage as well as fail in colonizing the gastric mucosa, presenting the relevance of SOD in the proper bacterial colonization process. Except for colonization, SOD expression is crucial for bacterial growth and survival primarily in the host microenvironment with a high intensity of oxidative stress [e corpus . It was  strains . SOD is  strains . Negovan strains . H. pylorophages . Noguchirophages . SOD-defe stress . Furthere stress . H. pylori O-specific chain of the lipopolysaccharide (LPS) and the majority of H. pylori strains express at least one type of Le antigen. Le antigens are expressed on the gastric epithelial cells; thus, H. pylori molecular mimicry in the form of Le antigens expression on the bacterial cell surface provides one of the most effective mechanisms enabling H. pylori colonization within the gastric mucosa, evoking the host immune responses. The expression of Le antigens on the bacterial cell surface prevents the bacterium from the host defense mechanisms promoting bacterial survival. Le antigens expression within the gastric epithelial cells might differ depending on the clinicopathological features of the infected patients and different Le types might be expressed depending on whether a patient will develop chronic gastritis or GC specifically [H. pylori infection [Lewis (Le) antigens are fucosylated glycolipids that constitute one of the components of the ifically . Except nfection ,132.a and Leb) and type 2 (Lex and Ley). It is estimated that approximately 80–90% of H. pylori strains express Lex or Ley [H. pylori strain [b expressed on the gastric epithelial cells constitutes the major site of the H. pylori attachment; however, there are incidents of bacterial colonization that are independent of particular Le antigen expression [H. pylori Le antigens expression depends on the time of the bacterial growth being mostly expressed in a growth phase-dependent manner. Different patterns of Le antigens’ expression were observed in adults and children—for instance, Leb expression correlates with the bacterial distribution within the antrum and corpus of the adult patients, whereas in children, it might increase the risk of ulceration [H. pylori-infected individuals; asymptomatic individuals tend to present significantly lowered expression of Lex antigen and the complete absence of type 1 Lewis antigens, compared to the infected symptomatic individuals [Gastric epithelial cells and LPS might express various Le antigens at the same time, among which two major Le antigen types can be distinguished—type 1  phenotypes might be more susceptible to H. pylori infection [H. pylori as well as those with an H. pylori-induced GC and according to some research, this response is strictly associated with the bacterial Le phenotype [x IgM antibodies in the gastric lumen enhances the adhesive properties of H. pylori strains, primarily those which express high amounts of the Lex antigen [H. pylori invasion, have the ability to absorb anti-Le antibodies from the sera of infected individuals, enabling further H. pylori growth and colonization [Martin et al. (2006) demonstrated a relationship between the ABO group and Le antigens expression, indicating that individuals with 0 Le (a-b+) and Anfection . Anti-Lehenotype . Further antigen . Howevernization ,144.2+-metalloenzymes belonging to the ureohydrolase family that catalyze the hydrolysis of L-arginine into L-ornithine and urea during the urea cycle [Arginases are Mnea cycle . There aH. pylori arginase is encoded by the RocF gene; arginase is crucial for bacterial colonization since it provides acid resistance in the gastric microenvironment. H. pylori arginase shows a similarity to arginase II, however, with the presence of additional acidic residues near the N-terminus. Contrarily to other arginases, H. pylori arginase exhibits higher catalytic activity with Co2+ or Ni2+ rather than Mn2+ as a cofactor and shows greater activity at the acidic pH instead of the alkaline; the optimal activity of arginase is shown at pH 6.1 [H. pylori are more prone to be killed by the macrophages in a NO-dependent manner; arginase also inhibits the proliferation of the T cells mainly by the reduction in CD3ζ expression [H. pylori infection, the inhibition of arginase increased NO and iNOS production in the macrophages [H. pylori arginase expression alters the immune responses by affecting M1 macrophage activation, diminishing Th1/Th17 T-cell differentiation, as well as controlling the IL-8 transcription in gastric epithelial cells [H. pylori-infected tissues induces the apoptosis of the macrophages [H. pylori survival in the acidic microenvironment; however, it is not crucial for bacterial colonization. t pH 6.1 ,147. Thepression . Furtherpression . The majpression . It was rophages . H. pyloal cells ,153. Morrophages . ArginasH. pylori is involved in the degradation of various lipids, enabling effective bacterial permeability, especially during stress conditions. Furthermore, H. pylori synthesizes phospholipase A2 (PLA2), phospholipase A1 (PLA1), as well as phospholipase C (PLC) and phospholipase D (PLD) that degrade phosphatidylcholine and phosphatidylethanolamine [H. pylori phospholipases, gastric epithelial cells become progressively impaired and lose their physiological functions. Except for mucosal damage, phospholipases promote the chronic inflammation that might further induce the formation of peptic ulcers. This enables further bacterial colonization and survival within the gastric microenvironment. It was demonstrated that H. pylori strains that secrete greater amounts of phospholipases significantly increase the risk of chronic gastritis and subsequent carcinogenesis [H. pylori PLD activates the ERK1/2 signaling pathway in gastric epithelial cells, inducing further cellular impairments [Outer membrane phospholipase A (OMPLA) of nolamine . Due to ogenesis . The secairments . H. pylori outer membrane that constitutes one of the most essential virulence factors, enabling the induction and progression of a chronic bacterial infection. LPS is composed of three major parts, including the core oligosaccharide, the lipid-A region, and the O-antigen; cholesterol is considered to play a crucial role in the maintenance of the LPS structure [H. pylori; it was observed that iron-deficient strains expressed significantly lower amounts of LPS [Lipopolysaccharide (LPS) is the component of the tructure ,159. It tructure ,163,164.s of LPS . One of s of LPS . Furthers of LPS ,168.H. pylori strains primarily by glucan-heptane linker presence or absence, depending on the geographical occurrence of the H. pylori strains [H. pylori from the compounds that could be potentially toxic for the bacterium. Yokota et al. (2012) proposed the division of LPS into two antigenic phenotypes—weakly antigenic epitope-carrying LPS and highly antigenic epitope-carrying LPS that is most prevalently expressed in GC patients with chronic and severe inflammation [LPS was reported to differ among particular  strains . Being eammation .H. pylori LPS is recognized by toll-like receptor 4 (TLR4)/MD-2 where toxic activity is slightly slower and weaker compared to other Gram-negative bacteria species. Some research demonstrated that several H. pylori strains might act as TLR4 antagonists [H. pylori infection [phox also via the stimulation of TLR4 in gastric pit cells [agonists . Apart fagonists ,173. Thenfection . LPS typit cells . Moreoveit cells .H. pylori persistence that significantly contributes to the induction of the host inflammatory responses, resulting in chronic inflammation, peptic ulcers, gastritis, and further gastric carcinogenesis. Furthermore, LPS presents a capacity to activate neutrophils, enhancing the onset of the oxidative stress reactions [IL-1β gene polymorphism is associated with the risk and severity of H. pylori-induced GC [MDM2 gene, H. pylori infection, and further gastric carcinogenesis, and the whole process might be intensified by LPS [LPS enables chronic eactions . Gastriceactions . LPS caneactions . Inflammeactions ,181. LPSeactions . LPS medduced GC ,184. Furduced GC ,186,187.d by LPS . LPS regd by LPS . LPS dysd by LPS . LPS alsd by LPS . Anotherd by LPS . LPS is d by LPS .H. pylori adherence to the gastric epithelium and delivery of toxins (such as CagA or VacA) or other virulence factors into the host cells, promoting either direct or indirect damage of host tissues via inflammatory or immune responses. It was proposed that BabA expression in gastric tissue samples might constitute a biomarker of H. pylori infection [babA gene is strongly associated with the presence of cagA in particular H. pylori strains; it also depends on the particular geographic area where specific strains are more or less prevalent [babA, cagA, and vacA might contribute to the onset and progression of paraneoplastic gastric lesions as well as other gastrointestinal disorders. BabA, CagA, and VacA present a synergy, worsening the severity and clinical outcome of the gastritis patients [H. pylori strains is significantly associated with the onset, progression, as well as severity, and clinical outcome of several gastrointestinal diseases such as peptic ulcer disease or GC [Blood group antigen-binding adhesin (BabA) is a virulence factor that enables nfection . The exprevalent ,196. Furpatients . Apart fpatients . Expressse or GC . BabA exb) blood group antigens expressed in the gastric epithelium facilitating TSS4 activity, releasing excessive amounts of the pro-inflammatory factors stimulating carcinogenesis [H. pylori binding to MUC5AC mucin in the gastric epithelium is also BabA-dependent [H. pylori strains obtained from symptomatic patients . BabA is capable of binding to its receptors both in the mucosa of the oral cavity and gastric mucosa. It was demonstrated that there is a relationship between BabA expression and the severity of gastritis in the gastric antrum [H. pylori strains with low BabA expression and decreased capability of binding to the Leb can be detached from the gastric mucus and stimulate ulceration within the duodenum, increasing the risk of peptic ulcer disease [H. pylori strains are capable of inducing histological alterations within the gastric mucosa and stimulate the infiltration of inflammatory mediators; there are no differences between BabA-positive and -negative strains in terms of colonization properties [babA and cagA genes, as well as bacterial coccoid form, significantly affect the severity of gastritis [H. pylori strains compared to CagA-negative strains [H. pylori adherence to the spasmolytic polypeptide-expressing metaplasia (SPEM) glands, promoting metaplastic alterations that contribute to the onset of carcinogenesis [BabA adheres to H-type 1 and ABO/Lewis b  [H. pylori infection and H. pylori-induced GC [saba gene was demonstrated to be one of the triggers that induce an iron deficiency in H. pylori-infected patients [saba. Genetic diversity of the saba gene depends on particular geographical origin; moreover, a particular genotype might influence the clinical outcome of H. pylori-infected patients [Sialic acid-binding adhesin (SabA) is an adhesin belonging to the outer membrane protein (OMP) family that facilitates nization ,213. Thenization . Except nization . Furtherin (NAP) . Similarduced GC . The enhpatients . Furtherpatients .hopH gene, belongs to the OMPs family and as an H. pylori virulence factor, it is strongly associated with the bacterial adherence, colonization, induction, and progression of gastrointestinal disorders, and might affect the clinical outcome of the infected patients [H. pylori strains are more prone to induce much more severe inflammation in gastric mucosa compared to OipA-negative strains. Binding of the OipA to the gastric epithelial cells activates the apoptotic cascade in the host cells primarily via the Bcl-2 family pathway and increased levels of Bax and intracellular cleaved-caspase 3 [H. pylori [H. pylori strains that co-express the oipa gene with caga, and vaca [H. pylori strains usually express high amounts of Oipa [H. pylori-induced damage [Outer inflammatory protein A (OipA), encoded by the patients . OipA-poaspase 3 . Damage . pylori ,223. The. pylori . OipA is. pylori ,229,230.. pylori . OipA su. pylori . The synand vaca . OipA is of Oipa ,235. Oipd damage . H. pylori virulence factor where pathogenicity is associated with the induction of duodenal ulcers as well as the risk of gastritis; nevertheless, DupA expression is negatively correlated with the risk of GC [dupa strains might contribute to GC via previously induced gastritis [H. pylori strains do not always increase the risk of duodenal ulcers [dupa gene is considered to be involved in the formation of T4SS [H. pylori strains is estimated at 45% and significantly differs depending on the geographic area and the investigated ethnic groups [dupa cluster enables a greater infiltration of the inflammatory cells and is generally associated with greater virulence compared to strains with an incomplete dupa cluster [H. pylori virulence factors, DupA inhibits the proliferation and growth of GC cells, preventing further carcinogenesis primarily via the overactivation of the mitochondria-mediated apoptotic pathway as well as high tolerance of DupA-positive strains to the acidic gastric microenvironment [H. pylori-related gastrointestinal pathologies and provide better clinical outcomes of patients. Furthermore, DupA stimulates gastric acid secretion, which was demonstrated by the presence of higher serum gastrin levels in patients infected by DupA-positive strains [H. pylori eradication therapies [H. pylori-related gastrointestinal diseases; so far, the dupa gene without premature stop codons or the 112 bp region of H. pylori DupA specifically could act as potential candidates [Duodenal ulcer promoting gene A (DupA) is the sk of GC ,238. Morsk of GC . Althougastritis . Interesl ulcers . There i of T4SS . The prec groups ,244. A c cluster . DupA st cluster ,247. Fur cluster . Except  cluster ,250. Conironment . Infecti strains . DupA isherapies . It is andidates ,257,258.H. pylori adherence to the gastric epithelial cells and further stimulation of bacterial colonization [H. pylori mutants show significantly decreased capability to bind to the gastric epithelial cells compared to strains that express high levels of AlpA/B [H. pylori strains independently of geographic localization. AlpA/B can also regulate the release of several cytokines and pro-inflammatory factors including IL-6 and IL-8 and might be involved in the activation of the ERK, c-Fos, and CREB pathways; some of the AlpA/B genotypes might affect JNK and NF-κB pathways as well [H. pylori adherence, the lack of AlpA/B expression results in the enhanced inflammatory responses and more severe inflammation [Adherence-associated lipoprotein A and B (AlpA/AlpB) are proteins involved in nization ,260,261.f AlpA/B . AlpA/B f AlpA/B . AlpA/B  as well . It was  as well . Even thammation . H. pylori binding to the gastric epithelial cells via the recognition of the LacdiNAc  determinant [H. pylori-mediated pathogenicity.LacdiNAc-specific adhesin (LabA) is an adhesin that facilitates erminant ,267. LabHelicobacter pylori outer membrane protein Q (HopQ) is a virulence factor relevant in bacterial survival in the gastric acidic microenvironment as well as the adherence to gastric epithelial cells, colonization, and further progression of gastrointestinal ailments. Two major types of HopQ can be distinguished—type 1 and 2—where expression might influence GC onset and progression in H. pylori-infected individuals. Generally, HopQ type 1 is present in the majority of H. pylori strains, which also possess cagPAI [H. pylori-induced gastrointestinal pathologies; type 1 HopQ is relatively more often found in CagA and s1-VacA-positive strains, increasing the risk of peptic ulcer disease [H. pylori thioloxidoreductase [H. pylori survival in neutrophils [H. pylori-related gastrointestinal pathologies. s cagPAI . The gen disease ,270. Fur disease . The Hop disease . HoQ exp disease . The int disease ,279,280.eductase . HopQ–CEtrophils . Furthertrophils . HopQ-detrophils . HopQ–CEHelicobacter pylori outer membrane protein Z (HopZ) is another member of the OMPs family where pathogenicity is not clearly described yet compared to other OMPs. HopZ is divided into two variants—HopZ1 and HopZ2 [H. pylori colonization in gastric mucosa; however, the exact site of HopZ binding is still unknown. HopZ pathogenicity is considered to be the most essential during the early stages of H. pylori infection; HopZ disturbs gastric acid secretion and is involved in the transient hypochlorhydria [nd HopZ2 . HopZ faorhydria .H. pylori that is still poorly described. IceA levels significantly increase during H. pylori binding with the gastric epithelial cells. There are two major allelic variants of IceA—IceA1 and IceA2. Since H. pylori possess only one iceA locus, IceA1 or IceA can be expressed and the presence of both indicates infection by several different H. pylori strains. Some researchers demonstrated that there is no relationship between IceA expression and the presence of other virulence factors such as CagA or VacA; however, these data are quite contradictory among authors, thus more research is needed in this field [H. pylori binding with the gastric epithelial cells and might be associated with the development of peptic ulcer disease [H. pylori strains, IceA2 presence is associated with greater granulocytic and lymphocytic infiltration, as well as atrophic gastritis [H. pylori.Induced by contact with epithelium gene A (IceA) is a quite recently discovered virulence factor of is field ,288,289.is field . The exp disease ,290,291. disease ,293. Con disease . IceA1-p disease ,296. Eveastritis . Both alastritis . IceA isH. pylori that is involved in the formation of cholesteryl α-glucoside (αCGL) by adding α-glucosyl to the cholesterol of the host cellular membranes. H. pylori does not synthesize cholesterol on its own but extracts it from the plasma membranes of the host cells; αCgT is involved in the subsequent glycosylation of the cholesterol. αCgT is produced by H. pylori in an inactive form which becomes activated after binding to the host cell membrane [αCgT gene is associated with significant dysregulations of the cholesterol uptake by the bacterium [H. pylori from the phagocytosis and immune responses induced by bacterial infection [+ T-cells, as well as the IL-4 and IFN-γ pathways [H. pylori-infected patients are positively correlated with the atrophy score of the gastric tissues [H. pylori-induced inflammation, especially during the early stages of infection. Furthermore, αCgT also seems to be crucial for proper bacterial growth [H. pylori strains deficient in αCgT result in inhibited growth or even enhanced bacterial lethality [H. pylori strains that lack CGs show greater permeability of the cellular walls, decreasing bacterial pathogenic potential [H. pylori infection. Cholesteryl α-glucosyltransferase (αCgT) is a quite poorly characterized enzyme of membrane . The absacterium . Glucosynfection . Furtherpathways . Increaspathways . αCgT ispathways . The lev tissues . Thus, il growth . As an eethality . αCgT-poethality ,309. H. otential . At the H. pylori that stimulates the conversion of glutamine and glutathione into glutamate and ammonia as well as glutamate and cyteinylglycine, respectively. The hydrolysis of the glutamine facilitates the vacuolation process by VacA [2O2), inhibition of cellular proliferation, as well as progression of apoptosis and necrosis of the gastric epithelial cells via the excessive secretion of IL-8 (in an NFκB-dependent manner), IL-10, cyclooxygenase-2 (COX-2), inducible iNOS, and epidermal growth factor-related peptides at the same time, resulting in dysregulated cell cycle processes [c and further overactivation of the caspase pathway (caspase-3 and -9) [2+ levels through the activation of the phospholipase C-inositol 1,4,5-trisphosphate (PLC–IP3) pathway, eventually enhancing apoptotic processes [H. pylori GGT might also contribute to the progression of cholangiocarcinoma [γ-glutamyl-transpeptidase (GGT) is one of the virulent enzymes of  by VacA . GGT prorocesses ,314,315. and -9) . Park etrocesses . Major drocesses ,319. Apoarcinoma .+ cells infiltration into the gastric microenvironment, and preventing differentiation of the dendritic cells, triggering alterations in their phenotype into the tolerogenic one [H. pylori mutants fail in colonizing the gastric epithelium efficiently [H. pylori strains also show an ability to reduce cell viability and promote the loss of survivin [GGT stimulates bacterial colonization and persistence in the gastric microenvironment via regulating immune tolerance by inhibiting CD4-positive T-cell proliferation, facilitating CD8enic one ,323,324.enic one . GGT-negiciently ,327. Furiciently . GGT acticiently . Impaireiciently . GGT-possurvivin . Apart fsurvivin .H. pylori virulence factor that stimulates neutrophil adherence to the gastric epithelial cells, promoting the production of ROS as well as myeloperoxidase primarily during the stationary phases of infection. NAP activates the neutrophils and mast cells as well as promotes monocytes migration. Neutrophils are primarily stimulated due to the overactivation of the ERK and p38-MAPK pathways [H. pylori infection. NAP shows structural similarity to ferritin and belongs to the DNA-protecting protein under severe conditions (Dps) family. It presents ferroxidase activity however, without directly binding to it; due to the presence of the ferroxidase center, NAP incorporates Fe2+ ions that are further oxidized to Fe3+ and the whole process results in hydroxyl radicals that protect H. pylori DNA from damage [Neutrophil-activating protein (NAP) is an pathways . NAP prem damage . Except m damage .NAP stimulates the infiltration of monocytes and polymorphonuclear granulocytes as well as the surface expression of β2-integrins; furthermore, it stimulates the human blood mononuclear cells to secrete tissue factor (TF) and plasminogen activator inhibitor-2 (PAI-2), leading to the imbalance between coagulation and fibrinolysis . MoreoveH. pylori growth during infection [H. pylori survival by its contribution to DNA protection [The release of the above-mentioned factors significantly increases the secretion of gastrin and pepsinogen and subsequent damage to the gastric mucosa. These alterations might contribute to the progression of chronic gastritis via the disrupted processes of fibrin removal and further impaired healing processes. Furthermore, NAP stimulates the mast cells to secrete β-hexosaminidase and IL-6, facilitating a pro-inflammatory cascade . Its invnfection ,346. NAPotection . H. pylori serine protease that targets E-cadherin on the gastric epithelial cells, leading to the cleavage of the E-cadherin extracellular domain and subsequent impairments in epithelial barrier functions [H. pylori OMVs. Several studies reported that inhibition of H. pylori HtrA results in the blockage of H. pylori passage through the gastric epithelium as well as the reduced number of alterations within the gastric epithelial cells [H. pylori migration properties even up to 2.2-fold when compared to HtrA-negative species [H. pylori transmigration through the gastric epithelium, HtrA plays a role in CagA injection into the host cells by interacting with the α5β1 receptor [htrA gene is highly prevalent in numerous H. pylori strains regardless of the geographic areas. What is intriguing is that HtrA inhibition might be lethal for H. pylori strains, indicating that it constitutes a pivotal virulence factor of H. pylori; zinc and copper ions were presented to be effective inhibitors of the HtrA activity [H. pylori strains that are deprived of HtrA are much more sensitive to particular conditions, including elevated temperature, osmotic shock, or treatment with puromycin [H. pylori from numerous stress conditions, enabling effective bacterial colonization within the gastric mucosa. HtrA is suspected to be one of the potential factors that might induce EMT and further tumorigenesis; however, so far, there is not sufficient data to provide an ultimate confirmation of this phenomenon. High temperature requirement A (HtrA) is an unctions ,349,350.unctions . HtrA isal cells ,353. Htr species . The trareceptor . HtrA exactivity ,357. Moractivity . Furtheruromycin ,360. IndH. pylori—HspB (Hsp60), HspA (Hsp10), and Hsp70—and their expression is regulated by two major repressors—HspR and HrcA. HspB affects the Nrf2/Keap1 pathway, inhibiting Nrf2 translocation, subsequent increased release of COX-2, IL-8, and metalloproteinases (MMP3 and MMP7), and enhanced inflammatory responses [H. pylori infection. HspA of H. pylori shares approximately 50% similarity to human homologues of Hsp60 [H. pylori adherence and further colonization within the gastric epithelium. The C-terminal domain of HspA was reported to play a role in nickel sequestration and detoxification and since nickel homeostasis is crucial for H. pylori colonization, this virulence factor might act as a potential molecular target for H. pylori eradication [H. pylori motility is highly associated with the expression of Hsps inhibitors—HspR and HrcA [H. pylori into the gastric epithelial cells, leading to persistent bacterial infection, inflammation, and subsequent carcinogenesis via the enhancement of tumor cell migration and angiogenesis. Hsp60-mediated angiogenesis is primarily promoted by the CXCR2/PLCb2/Ca2+ signaling pathways in gastric epithelial cells [H. pylori eradication on the gastric MALT lymphoma [H. pylori, bacterial colonization in the gastric mucosa significantly decreases the expression of host Hsps such as Hsp70 or Hsp90, and at the same time increases Hsp32 and Hsp27 levels, which additionally increases the susceptibility to further damage [Heat shock proteins (Hsps) constitute a family of proteins that act as molecular chaperones, being responsible for the maintenance of proper structural and functional properties of the cellular proteins. Except for the above-mentioned chaperoning role, Hsps also regulate apoptosis, autophagy, immunity, inflammation, carcinogenesis, as well as the protection against oxidative stress. There are three major Hsps expressed by esponses . HspA isof Hsp60 . HspA biof Hsp60 . HspA60-of Hsp60 ,366,367.of Hsp60 ,369. Furdication . H. pyloand HrcA . It was and HrcA . Hsp60 dal cells . Hsp60 mlymphoma ,375,376.lymphoma . Apart fr damage ,380,381.H. pylori and its impact on the gastric microenvironment in the current state of knowledge. Colonization of different H. pylori strains in the same individual at the same time indicates that H. pylori is capable of inducing various genetic alterations and the expression of numerous virulence factors during its colonization period. Specific virulence factors are associated with the severity of symptoms and the clinical outcome of the infected patients. Several bacterial antigens such as BabA, SabA, OipA, AlpA, DupA, GGT, NAP, catalase, or Hsp60 are considered to be potential candidates for vaccines that might elicit both humoral and cellular immune responses during the infection. H. pylori elicits numerous adaptive mechanisms that enable effective bacterial adherence, colonization, and cellular alterations that provide the induction of further premalignant changes in the gastric microenvironment. Even though H. pylori virulence factors might act synergistically, some of them are crucial in bacterial colonization and thus chronic infection, whereas others might only act as additional triggers that only stimulate further responses. Therefore, more research regarding the H. pylori structure and virulence as well as their association with gastrointestinal diseases is needed. This review provides an insight into the virulence of


E. coli was the most frequent isolate , followed by the Klebsiella genus . The third most frequent isolate was S. aureus . In total, multi-drug resistance (MDR) was present in 23.8% and was most prevalent in A. baumanii (65.5%), P. mirabilis (42.7%), and K. pneumoniae (32.6%). MRSA was isolated in 19.6%, while ESBL-producing Enterobacterales in 17.7%, and these were associated with remarkably higher resistance to other antibacterials as well. Difficult-to-treat resistance (DTR) was detected in 0.5%. The frequent isolation of some ESKAPE bacteria and the detected considerable acquired resistance among ED patients raise concern. The revealed data identified problematic pathogens and will guide us to set up the optimal empiric antibiotic protocol for clinicians.Antibiotic treatments initiated on Emergency Departments (ED) are empirical. Therefore, knowledge of local susceptibility patterns is important. Despite this, data on expected pathogens and their resistance profile are scarce from EDs internationally. The study aim was to assess the epidemiology and resistance patterns of bacterial isolates from a tertiary-care ED over 5 years, focusing on ESKAPE bacteria . After removal of duplicates,  Antimicrobial resistance (AMR) is a serious threat to public health worldwide. Comprehensive knowledge on the burden and associated outcomes of multi-drug-resistant (MDR) infections is still lacking. Nevertheless, several studies revealed the association between infections caused by antibiotic-resistant bacteria and negative patient outcomes, such as longer hospital stays, higher morbidity and mortality ,2,3,4. IEnterococcus faecium, S: Staphylococcus aureus, K: Klebsiella pneumoniae, A: Acinetobacter baumannii, P: Pseudomonas aeruginosa, E: Enterobacter spp [Stenotrophomonas maltophilia, as that has emerged as global opportunistic human Gram-negative pathogen [Based on overall mortality and economic impact, the so-called “ESKAPE” group of pathogens deserve the utmost attention, both from clinical and from research and development aspect ,10. Ricecter spp . These pcter spp ,13. Rececter spp . Hence, cter spp ,16 and tpathogen . One of the main drivers behind the development of AMR is the misuse and overuse of antibacterial agents ,19. As tTherefore, the aim of the present study was to assess the epidemiology and resistance patterns of bacterial isolates from the ED that may help to identify problematic pathogens and to set up an optimal empiric antibiotic guide for clinicians.n = 6887 individual bacterial isolates were recovered from clinical specimens, out of which n = 4974 (72.22%) were ESKAPE pathogens. Overall, as shown in Escherichia coli was the most frequently isolated bacteria , followed by the Klebsiella genus , most frequently K. pneumoniae. The third most frequent isolate was S. aureus , followed by Proteus spp. . Stenotrophomonas maltophilia, A. baumannii, and the Providencia, Salmonella and Serratia genus played limited role, with below 1% of share within the ESKAPE group.During the study period, n = 121) mainly from critically ill patients. We observed the dominance of Escherichia isolates in blood and urine samples. In blood cultures, S. aureus was the second most frequent isolate, whereas in urine the Klebsiella genus. In wound/abscess specimens S. aureus were isolated most often, followed by the Proteus spp.The most frequent clinical specimens were blood cultures, urinary catheter or midstream urine specimens, and deep wound or abscess samples . RespiraA. bauminii) and 84.85%%  among different isolates. Usual drug resistance (UDR) was developed in 44.69%% of isolates, and ranged between 15.15%%  and 87.10%% (A. bauminii). UDR was prevalent among the most frequently isolated bacteria . Multi-drug resistance (MDR) was acquired in almost every fourth isolate and showed considerable range between different ESKAPE isolates . DTR was present in 23 isolates  while we detected only one pandrug-resistant P. aeruginosa isolate.The overall resistance level of the ESKAPE isolates is summarized in isolates . MDR wasE. faecalis species can be effectively treated with first line agent such as ampicillin, as all isolates proved to be susceptible. In contrast, in case of E. faecium isolates, the clinical success can be anticipated in case of tigecycline and linezolid, which are both reserve antibacterial agents based on in-vitro data. Vancomycin resistance among E. faecium was detected in every third case, while MDR can be encountered in nearly every second isolate (45.5%). Methicilin-resistant S. aureus (MRSA) were isolated in 19.6% (95/561). Over the years, no clear increasing or decreasing trend for MRSA has been observed (data not shown). Methicillin-resistant strains (MRSA) were also considerably less susceptible to many other antibacterial agents  to all beta-lactams except carbapenems (indicative of ESBL production) and presence of MDR in every third isolate (32.6%). A. baumannii isolates showed high resistance level to all tested antibacterials, except the reserve antibiotic colistin to which all tested organisms (n = 15) were fully susceptible. P. aeruginosa strains were highly susceptible to ceftazidime (90.6%), cefepime (91.7%), and aminoglycosides (over 90%) and clinical success can be anticipated by also piperacilin-tazobactam empirical treatment, as overall susceptibility rate was 87.2%. Out of the four P. aueruginosa isolates that were tested for colistin, one isolate, considered extensively drug-resistant (XDR) was resistant to colistin. Different cterials . K. pneuE. coli, the most frequently isolated Gram-negative bacteria were susceptible to aminoglycosides in ~90% , while P. vulgaris strains were highly susceptible (over 90%) to many beta-lactam agents  , which cE. coli was the most frequently isolated organism in urine and blood samples, while S. aureus and the Klebsiella genus was the second most frequent organism isolated from blood culture and urine samples, respectively. A study from a US emergency department, that similarly to our study, focused on all clinical specimens, also revealed the dominance of Gram-negative bacteria and especially E. coli [S. aureus and this pathogen spectra has been found in another ED study focusing on blood stream infections (BSI) [E. coli and Klebsiella spp. were the most frequent isolate in other ED studies focusing on urinary tract infections (UTIs) [In this study, the most common clinical specimen was catheter-specimen urine, followed by blood culture samples. ESKAPE organisms were responsible for 72.22% (4974/6887) of all positive bacterial clinical isolates, and we detected the predominance of Gram-negative bacteria. The most common isolates were from the Enterobacterales family, and within the group  E. coli . A surve E. coli  found thns (BSI) . E. colis (UTIs) ,39.Strenotrophomonas maltophilia and A. baumannii isolates from the ED patients; this phenomenon is concerning, as these bacteria are predominantly considered as hospital-acquired pathogens, often associated with infections in immunocompromised individuals. The isolation of Strenotrophomonas maltophilia and Acinetobacter spp. was reported only from one epidemiological study from EDs [Acinetobacter spp. was not involved in any community-acquired infections but was prevalent in hospital acquired BISs [We also isolated from EDs . In an Ired BISs . A. baumanii: 41.94%, Klebsiella spp.: 0.15%, and no DTR in E. coli). Regarding the MDR category, 23.8% of the bacterial isolates were classified as MDR, and highest rates were observed for A. baumannii, P. mirabilis, K. pneumoniae and E. coli, respectively (Existing resistance classifications (MDR-XDR-PDR) have been used worldwide and they have been praised in their utility for microbiological and epidemiological purposes . Neverthectively . No published ED studies has reported the prevalence of UDR, DTR or even MDR, so we limited our comparison to some hospital-based studies. A. baumanni (18.3% and 17.2%) and lowest in E. coli similar to our study (0.04% and 0.07%). In a Korean multicenter study [E. coli was found, and DTR was highest in A. baumannii (79.6%) [E. coli, K. pneumoniae, P. aeruginosa, and Acinetobacter species, so any comparisons should be very cautiously made. Gianella et al. [P. aeruginosa was 34.8% in community-acquired infections (vs. 13.3% in this study), while no Acinetobacter spp. was isolated from community-acquired infections. The MDR rate of the Enterobacterales group was nearly 100% in our study, while it was 40.8 in ESBL positive E. coli and 78.8% in ESBL positive K. pneumoniae.Two multicenter studies on bloodstream infection from US hospitals found DTR in 1% and 1.3% of isolates respectively ,43, and,er study  on Gram- (79.6%) . Howevera et al.  assesseda et al. . RegardiK. pneumoniae were ESBL producers . while E. coli isolates were ESBL producers in 14%. In a recent German ED study [E. coli rate of 5.9%, while in a Taiwan ED study revealed ESBL positivity in 1.7% of E. coli and in 0.9% of K. pneumoniae isolated from blood. Ceftriaxone resistance was reported at 10% in urinary E. coli isolates [E. coli in invasive isolates was 15.1%, and this rate was 31.7% for K. pneumoniae. All these confirm that ESBL-producing Gram-negative pathogens are prevalent in our ED and that this organism has been expanded to the community setting. The study of Liu et al. confirmed that ESBP-producing isolates were more prevalent in nursing home residents admitted to the ED [Since the millennium, several reports showed a substantial increase of Enterobacterales-producing ESBLs and carbapenemases worldwide ,47,48,49ED study  ceftriaxED study  reportedisolates  from a Uo the ED . As nursE. coli and K. pneumoniae isolates from hospital or community onset BSIs [E. coli and K. pneumoniae are associated with increased mortality and length of hospital stay or worse clinical outcome compared to non-ESBL-producing isolates [K. pneumoniae isolate resistant to meropenem, while all other Enterobacterales species were fully susceptible to carbapenems except one P. mirabilis isolate that were resistant to imipenem and ertapenem. However, if the spread of ESBLs will not be contained in our setting, and the use of carbapenems will not be controlled, which may favor the selection of carbapenemase-producing Enterobacterales in the future. Similar carbapenem susceptibility rates among Enterobacterales were reported from other EDs [An Italian study also confirmed the presence of ‘problematic’ pathogens in the outpatient setting: they have found no significant difference in the rate of ESBL-producer phenotypes in set BSIs . The preisolates ,51. Regather EDs ,35,37.E. coli isolates was 89% in the ED study of Jorgensen et al. [E. coli resistance rate in this study and similar to the EU mean of aminoglycoside resistance in E. coli (11.1%) However, for K. pneumoniae, we recorded lower (<80%) gentamicin and amikacin resistance rates, which data correspond well to the data of the ESAC-Net report [K. pneumoniae and P. mirabilis. Ciprofloxacin resistance was 22.9% in the Enterobacterales group in the German ED [E. coli resistance ranged between 23% and 25.2% in American EDs [Gentamicin susceptibility of n et al. , and then et al.  reportedt report . Considet report . The fluerman ED , and E. ican EDs ,36. Simiican EDs . The higican EDs . The higican EDs . All theA. baumannii and P. aeruginosa are both opportunistic pathogens associated primarily with healthcare-associated infections. Both species are intrinsically resistant to many antimicrobial agents. In this study, only colistin, a “reserve antibiotic”, showed to be effective in-vitro for all tested A. baumannii isolates. Only 55.2% of the A. baumannii isolates were susceptible to meropenem. This value is identical to the reported value for Hungary and considerable higher than the European average (31.9% in 2018) in the latest EARS-NET surveillance system report [m report , which, P. aeruginosa isolates in this study, effective antibacterial therapy may be anticipated in most cases using anti-pseudomonal beta-lactam agents. Interestingly, meropenem susceptibility of P. aeruginosa was slightly lower than susceptibility to third generation cephalosporins. The presence of carbapenem-resistant but cephalosporin-susceptible (Car-R/Ceph-S) P. aeruginosa strains has been already reported from our university [Considering iversity . P. aeruginosa recovered from blood stream infections [P. aeruginosa isolates. Gentamicin susceptibility was higher (96%) in P. aeruginosa in the ED study of Draper et al. [However, ciprofloxacin resistance rate exceeded 20% , and this was slightly above the EU mean revealed by the European Surveillance . A recenfections . In our r et al. , while nr et al. .S. aureus isolates, 16.9% were identified as MRSA with no identifiable temporal trends. This is identical to the EU mean (16.4%) reported in 2018 and slightly lower than the Hungarian average (23.1%) reported by the EARS-Net. In comparison, Kao et al. from Taiwan [Among m Taiwan  reportedm Taiwan  reportedm Taiwan  found siEnterococcus spp., no vancomycin-resistant E. faecalis isolates were detected, while vancomycin susceptible E. faecium was only 66.7% (resistance rate 33.3%). This rate is much higher that the EU mean resistance of E. faecium to vancomycin (17.3%) and similar to the value reported for Hungary (39.5%) [E. faecium and vancomycin-resistance was detected in only 1 case, corresponding to 5.3% [E. faecium is suspected, one should consider the possibility of glycopeptide-resistance. Regarding  (39.5%) . In an I to 5.3% . From a  to 5.3% . All theThe present study provides the landscape of ESKAPE pathogens causing a variety of infections in an ED over a 5-year surveillance period to serve as a basis for antimicrobial stewardship interventions. Such an epidemiological snapshot is scarce in the literature for emergency departments, and has not been reported from Hungary. Also, this is one of the first studies that use the novel resistance indicators  in other than an ICU setting and other than blood stream infections.We have to acknowledge the limitations of this research. This is a single-center study based on a retrospective analysis of laboratory data without linkage to clinical outcome or to patient characteristics. However, the heterogeneous patient population of this ED, the large catchment area and high patient-turnover may allow some generalization to other EDs. Prior hospitalization is a well-known risk factor for infection/colonization with drug-resistant bacteria. As some of the patients may have been hospitalized before ED presentation or live in long-term care facility, means that our data do not reflect the true prevalence  the true prevalence of bacteria and AMR in the community. Also, it has been reported that culture sending may be biased by high risk cases and treatment failures . On the The present retrospective study was carried out during the time period between 1st of June 2014 and 31st of July 2019 at the central Emergency Department (ED) of the Albert Szent-Györgyi Clinical Center, a tertiary-care teaching hospital (with approx. 1800 beds), in Southern Hungary. The level I ED of the University of Szeged has opened on the 1st of May, 2014. The ED has annual census of nearly 40,000 adult patient visits for mainly traumatology, internal medicine, and neurological cases. Due to geographical distances, emergency cases of gynecology, urology, ear-nose-throat, and ophthalmology are provided at the dedicated clinics. During the study, all positive microbiological specimens and data collected at the central ED were retrieved. Data collection pertaining to isolates and records of “ESKAPE” bacteria were performed electronically, in the records of the MedBakter laboratory information system (LIS) by the authors. Non-ESKAPE bacteria and contaminants were excluded from the study. In addition, bacteria of the normal skin flora were considered pathogenic bacteria only if the same bacteria, has been recovered from 2 or more blood cultures with the same antibiotic-susceptibility patterns.n = 16) taken for screening purposes specimens positive for fungal isolates only (n = 305) and faeces samples (n = 785) were also excluded. During data collection and analysis, only the first isolate per patient was included in the study; however, isolates with different antibiotic-susceptibility patterns from the same patient or isolation of similar bacteria >7 days apart were considered as different individual isolates. If the same bacteria with identical antibiotic-susceptibility patterns have been isolated from both the urine culture and the blood culture of the same patient on the same day, the urine specimen was removed from the analysis. In addition, anorectal swabs, throat swabs, and vulva specimens , the plates were passed on for further processing. Aerobic and anaerobic blood culture bottles were incubated in the BacT/ALERT 3D  detection system; blood culture bottles were incubated for 5 days . Positive blood cultures, respiratory tract samples, cerebrospinal fluid samples, and other invasive sample types were routinely processed on blood agar, chocolate agar, eosine-methylene blue agar and Sabouraud agar ; bacterial culture media were incubated at 37 °C for 24–48 h, aerobically, while Sabouraud agar was incubated at 37 °C for 24 h and for additional 6 days at room temperature. Positive blood cultures, cerebrospinal fluid samples and other invasive sample types were also processed in Brain Heart Infusion broth  and on plated on Schaedler agar  containing horse blood 5% v/v, haemin, and Vitamin K1, for the isolation of anaerobic organisms . The cultivation of anaerobic bacteria was carried out in line with principles in anaerobic bacteriology, at 37 °C in an anaerobic chamber  for at least 48 h.Processing of clinical samples submitted by the ED to the Institute of Clinical Microbiology was carried out according to routine cultivation and identification guidelines of clinical bacteriology, briefly described below . All samBacterial identification was carried out using the VITEK 2 Compact ID/AST , based on the manufacturer’s recommendations and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). For MALDI-TOF analysis, bacterial cells were transferred to a stainless-steel target with a sterile toothpick, after cultivation of the respective isolates on appropriate culture media, described previously. An on-target extraction was performed by adding 1 µL of 70% formic acid prior to the matrix. After drying at ambient temperature, the cells were covered with 1 µL matrix (α-cyano-4-hydroxy cinnamic acid in 50% acetonitrile/2.5% trifluoro-acetic acid). Mass spectrometry measurements were performed by the Microflex MALDI Biotyper  in positive linear mode across the m/z range of 2 to 20 kDa; for each spectrum, 240 laser shots at 60 Hz in groups of 40 shots per sampling area were collected. The MALDI Biotyper RTC 3.1 software  and the MALDI Biotyper Library 3.1 were used for spectrum analysis . LogaritS. aureus ATCC 29213, E. faecalis ATCC 29212, P. mirabilis ATCC 35659, E. coli ATCC 25922, P. aeruginosa ATCC 27853, A. baumannii ATCC 19606 and S. maltophilia ATCC 13637 were used as quality control strains.Antimicrobial susceptibility testing for the relevant bacterial isolates were carried out based on the methodological recommendations and standards of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) valid at the time of the assays. The Kirby–Bauer disk diffusion method  and gradient tests  were performed on Mueller–Hinton agar (MHA). Colistin susceptibility was performed using the broth microdilution method in a cation-adjusted Mueller-Hinton broth. Susceptibilities to carbapenems and colistin were evaluated using commercial panels , using cation-adjusted Mueller–Hinton broth. In addition, for the verification of discrepant results, the VITEK 2 Compact ID/AST  was also utilized. The interpretation of susceptibility results was performed based on EUCAST breakpoints valid at the time of the interpretation. Intermediate results were grouped with and reported as resistant. S. aureus (MRSA) strains were detected using mannitol salt agar (MSA) and cefoxitin (FOX) disks (<22 mm zone diameter) and PBP2’ Latex Agglutination Test Kit  [Methicillin-resistant Hungary) . If exteHungary) , detectiSusceptibility testing included all relevant antibiotics for the respective bacteria, to allow for their classification into resistance categories based on the criteria detailed in Based on the analysis of the susceptibility testing results, respective isolates were classified into various resistance categories. An isolate was considered as wild-type (Wt) or pan-susceptible, if they were susceptible to all tested antibiotics, excluding those where intrinsic non-susceptibility is present. Isolates were classified in the usual drug resistance (UDR) category, if resistance was observed to at least one agent outside of the group of antibiotics affected by intrinsic non-susceptibility . Multi-dDescriptive statistical analysis (including means or medians with ranges and percentages to characterize data) was performed using Microsoft Excel 2013 . R  program was used for data analysis and visualization.This study is part of the Antimicrobial Stewardship Programme of the University and has been approved by the Regional and Institutional Ethical Board. The frequent isolation of ESKAPE bacteria and the detected considerable acquired resistance among ED patients raise concern and indicate that these organisms are present/has been disseminated in the community as well. Understanding the local epidemiology of bacterial pathogens may provide benefits in establishing local empirical treatment protocols.


Solanum japonense Nakai. (Solanaceae) is a traditional Chinese medicine and animal fodder in Asian continent. Here, the complete chloroplast genome sequence of S. japonense was determined by the Illumina Hiseq technology. The complete chloroplast genome of S. japonense was 155,415 bp and the GC content was 37.81%. The typical circular quadripartite structure was composed with two inverted repeat (IR) regions with 25,588 bp, a large single-copy (LSC) region  and a small single-copy (SSC) region . The chloroplast genome of S. japonense contained 132 unigenes, which contained 86 protein-coding genes, 37 tRNA and 8 rRNA genes and one pseudo-gene. Moreover, 183 SSRs were identified and 65% (119)of them located at LSC region. A Maximum-Likelihood (ML) phylogenetic analysis based on chloroplast genomes indicated that S. japonense was closely related to S.dulcamara, S.nigrum. Our results would provide a valuable resource for resource utilization and the phylogenetic studies of species of Dulcamara sect. in Solanaceae. Solanum japonense Nakai. (Yehaiqie in Chinese) is a widely perennial herb, belongs to Solanaceae family, which distributes in wastelands, slopes or forests ranged 300 m to 2900 m in China (Flora in China Solanum dulcamara both belongs to Sect. Dulcamara (Moench), while the former has deltate, broadly lanceolate or ovate-lanceolate leaves, often with 2 basal lobes .India Li . SolanacWe collected fresh leaves from a single individual of this species from Minhe County , Qinghai, China, and dried leaves with silica gel. Voucher speciements were deposited in the herbarium of Northwest Institute of Plateau Biology, CAS . Total DNA was extracted from the fresh leaves with the DNeasy Plant MiniKit  according to the manufacturer’s instructions. DNA quality was assessed based on spectrophotometry and electrophoresis in 1% (w/v) agarose gel, and then the good integrity and purity DNA was used for library construction and sequencing. The average insertion of the library was 320 bp and whole genome sequencing with 150 bp pair-end reads using the Illumina Hiseq platform  at Genepioneer Biotechnologies Inc., Nanjing, China.S. dulcamara  regions with 25,588 bp in length, which separated by a large single-copy (LSC) region  and a small single-copy (SSC) region .The chloroplast genome of S. japonense contained 132 unigenes, which was composed of 86 protein-coding genes, 37 tRNA and 8 rRNA genes and one psedo-gene. Moreover, 183 SSRs were identified and 65% (119) of them existed in LSC region. Compared the four cp gnome of Solanaceae, most genes were conserved and the variation displayed in the intergeneric regions.The complete chloroplast genome of S. japonense and other 39 species of Solanaceae, the whole chloroplast genome of them were aligned with MAFFT software (Katoh and Standley Scrophularia dentata was set as the outgroup species. Maximum -Likelihood (ML) phylogenetic analysis was conducted adopting HKY85 model by PhyML3.0 (Guindon et al. S. japonense was closer clustered with S. dulcamara while S.nigrum was out of them clearly (S. japonense would facilitate population, genetic identification and cp genetic engineering research of this traditional Chinese herb in the future.To identified the phylogenetic relationship of  clearly . Our cp


Porphyromonas gingivalis, Treponema denticola, and Fusobacterium nucleatum) promote oral cancer via specific host-bacterial interactions, and that bacteriocin/nisin therapy may modulate these responses. All three periodontal pathogens enhanced oral squamous cell carcinoma (OSCC) cell migration, invasion, tumorsphere formation, and tumorigenesis in vivo, without significantly affecting cell proliferation or apoptosis. In contrast, oral commensal bacteria did not affect OSCC cell migration. Pathogen-enhanced OSCC cell migration was mediated via integrin alpha V and FAK activation, since stably blocking alpha V or FAK expression abrogated these effects. Nisin inhibited these pathogen-mediated processes. Further, Treponema denticola induced TLR2 and 4 and MyD88 expression. Stable suppression of MyD88 significantly inhibited Treponema denticola-induced FAK activation and abrogated pathogen-induced migration. Together, these data demonstrate that periodontal pathogens contribute to a highly aggressive cancer phenotype via crosstalk between TLR/MyD88 and integrin/FAK signaling. Nisin can modulate these pathogen-mediated effects, and thus has therapeutic potential as an antimicrobial and anti-tumorigenic agent.Epidemiological studies reveal significant associations between periodontitis and oral cancer. However, knowledge about the contribution of periodontal pathogens to oral cancer and potential regulatory mechanisms involved is limited. Previously, we showed that nisin, a bacteriocin and commonly used food preservative, reduced oral cancer tumorigenesis and extended the life expectancy in tumor-bearing mice. In addition, nisin has antimicrobial effects on key periodontal pathogens. Thus, the purpose of this study was to test the hypothesis that key periodontal pathogens , a subset of HNSCC, is the most common malignant oral neoplasm. Risk factors, including smoking, alcohol consumption and human papilloma virus (HPV) infection alone have not been sufficient in explaining the incidence and aggressive behaviors of OSCC. Thus, other factors, such as oral periodontal pathogens may play an important role in OSCC tumor development, progression and metastasis, yet this has not been well explored. Here, we test whether periodontal pathogens promote oral cancer tumorigenesis, and whether bacteriocin/nisin treatment may modulate these responses. We found that periodontal pathogens promote cancer aggressivity via crosstalk between integrin/FAK and TLR/MyD88 signaling pathways that is reversible by bacteriocin/nisin treatment. Our study offers direct evidence that a bacteriocin, nisin has a broad therapeutic potential as an anti-cancer agent, and as an inhibitor of pathogen-mediated carcinogenesis. Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers worldwide and it is a major health burden –3. Each Treponema denticola, Porphyromonas gingivalis and Tannerella forsythia appear in late stages of oral biofilm development and comprise the bacterial “red complex” that is considered pathogenic in the etiology of periodontal disease [(Bulleidia and Olsenella) and stable periodontitis (Campylobacter and Eubacterium). In addition, at the species level, Bulleidia extructa, Eubacterium infirmum, Fusobacterium periodonticum, Filifactor alocis, Gemella morbillorum, Streptococcus constellatus and Streptococcus intermedius have been found relevant to progressing periodontitis [Periodontitis, a chronic inflammatory disease mediated by a bacterial dysbiosis and unfavorable host-bacterial interactions, is characterized by destruction of periodontal tissues.  disease . Other p disease , 10. Rec disease , 12. Recdontitis . These ddontitis . More imT. denticola, often predominate in periodontal disease, though they are typically below detectable levels in healthy gingival plaque [T. denticola increase with the severity of periodontitis, underscoring its major role in the disease [T. denticola virulence stems, in part, from its protease complex dentilisin. Dentilisin contributes to T. denticola adherence to and cytotoxic effects on epithelial cells and fibroblasts, penetration of epithelial tissue, and it may play a role in T. denticola evasion of complement-mediated bactericidal activity [P. gingivalis is frequently detected in patients with periodontitis and it plays a major role in the pathophysiology of the disease. P. gingivalis colonizes the subgingival crevice, where it mediates its invasive and replicative potential within various cell types including oral cancer cells [F. nucleatum is another important periodontal pathogen, since it is one of the most abundant oral bacteria present in biofilms from periodontal disease [F. nucleatum has strong co-aggregation properties with most oral biofilm colonizers , and it can help other bacteria cross the host epithelial and endothelial barrier [F. nucleatum’s unique properties could play a key role in modulating microbial diversity and microbe-host interactions in the tumor biofilm environment.Oral spirochetes, including l plaque , 15. The disease , 17. T. activity –21. P. ger cells , 23. F.  disease . F. nucl barrier –27. F. nFusobacterium spp. may be one of the etiological factors associated with colorectal carcinoma (CRC) [Fusobacterium species and depletion in species from the phyla Bacteroidetes and Firmicutes relative to the healthy colon tissues [F. nucleatum in their colorectal cancer and oral cavity [Streptococcus species and an increase in Fusobacterium species in the transition from health to carcinogenesis. Similar to the gut, the human oral cavity is a reservoir for hundreds of microbial species, which together coexist as a biofilm consortium [The innate human microbiota has an intimate relationship with the human host in health and disease . Multiplma (CRC) , 34–36.  tissues , 37. Furl cavity . Investil cavity , 40. Schl cavity . Recentll cavity . Importansortium . Changesnsortium . Epideminsortium , 45, 46.nsortium , 47–50. Lactococcus and Streptococcus species [Staphylococcus aureus infections in atopic dermatitis, respiratory tract infections, and experimental gingivitis in dogs [in vivo, and long-term treatment with nisin ZP extended the lifespan of tumor-bearing mice [A few reports have suggested that antimicrobial peptides or bacteriocins have cytotoxic effects against cancer cells –55. Nisi species . Many su species , 58. Nis in dogs –65. Rece in dogs , 67. Fur in dogs . Given t in dogs . We furting mice . In addiing mice . Thus, bT. denticola, P. gingivalis and F. nucleatum, enhance OSCC cell migration, invasion, stemness, and tumor aggressivity via crosstalk between integrin/FAK and TLR/MyDD88 signaling pathways, which can be abrogated by bacteriocin/nisin treatment. This study offers the first direct evidence that a bacteriocin, nisin, inhibits periodontal pathogen-enhanced oral carcinogenesis.The goal of the present study was to test the hypothesis that key periodontal pathogens promote oral cancer tumorigenesis, and that the bacteriocin nisin can mitigate these effects. This study demonstrates that key periodontal pathogens, T. denticola, P. gingivalis, and F. nucleatum, on OSCC cell migration and invasion were evaluated using a scratch migration assay and matrigel invasion assay. All three periodontal pathogens significantly increased OSCC cell migration in two different OSCC cell lines  induced apoptotic effects on OSCC cells  did not mediate any significant changes in cell migration  appeared to be toxic to OSCC cells. We further found that treatment with nisin ZP significantly blocked pathogen-enhanced tumorsphere formation  to focal contacts and downstream signaling. In addition, integrin alpha V and FAK signaling help mediate cell migration. Therefore, we investigated the potential role of FAK in pathogen-induced cell migration. igration . Furtherhallenge .T. denticola-lipooligosaccharide (LOS), P. gingivalis-lipopolysaccharide (LPS) and F. nucleatum-LPS. LOS and LPS derived from T. denticola, P. gingivalis and F. nucleatum promoted significant OSCC cell migration (V. Parvula) did not show any significant changes in cell migration  are pattern recognition receptors (PRRs), which play a pivotal role in the development of periodontal disease and cancer pathogenesis , 75. Thehenotype .H. pylori in gastric cancer, C. trachomatis in cervical cancer, S. typhi in gall bladder cancer, both B. fragilis and F. nucleatum in colon cancer, and P. gingivalis in pancreatic cancer [P. gingivalis, T. forsythia and T. denticola [F. nucleatum is one of the most abundant oral bacteria present in periodontal disease biofilms. F. nucleatum has strong intermicrobial binding properties with most oral biofilm colonizers, and it can help other bacteria cross host-epithelial and -endothelial barriers [P.gingivalis and F. nucleatum have been implicated in the pathogenesis of several chronic diseases and cancer. Although relationships between these bacteria and the etiology and progression of colon and pancreatic cancers has been reported, direct mechanistic evidence for involvement of these oral pathogens in oral cancer is limited [P. gingivalis, F. nucleatum, and T. denticola in OSCC carcinogenesis.The human host and the human microbiota have co-evolved to maintain an intricate balanced relationship . This rec cancer , 78–83. c cancer , 84–87. c cancer , 89. Thebarriers –27. P.gi limited , 90–94. T. denticola, P. gingivalis and F. nucleatum promote oral carcinogenesis. We demonstrated that T. denticola, P. gingivalis and F. nucleatum enhanced OSCC cell migration, invasion, stemness and oral tumorigenesis, without significantly altering cell proliferation or inducing apoptosis, except P. gingivalis induced a low level of apoptosis/necrosis at a high MOI (100). These differences in response (apoptosis vs proliferation) to P. gingivalis at this high MOI likely reflect differences in the timing of these cellular processes and/or differences in the detection sensitivity of the assays. P. gingivalis can increase cell proliferation by accelerating the cell cycle via modulation of apoptosis [P. gingivalis can also induce G1 cell cycle arrest and increase autophagy without affecting apoptosis [P. gingivalis employs a selective form of autophagy mediated via an endoplasmic reticulum network in order to replicate and survive within human primary epithelial cells [P. gingivalis in infected cells may have contributed to the apoptosis. We also found that P. gingivalis at a high MOI (100) is partially toxic to HSC-3 but not to SCC-14A cells, in terms of tumorsphere formation. This likely reflects normal biological and cellular variability. In this regard, bacterial pathogens express several molecules that promote their virulence or adhesion to host cells, and these actions rely on interactions with host cell surface receptors or proteins. Differential expression of host cell receptors or proteins by HSC-3 and SCC-14A may have resulted in different cellular responses to the important P. gingivalis virulence factors, including LPS, fimbriae, gingipains, and outer membrane vesicles. Although P. gingivalis and F. nucleatum can promote migration and invasion of OSCC cells by triggering an epithelial to mesenchymal transition and acquisition of stemness properties, specific bacterial molecules or their structural features have not been specifically linked with migration effects [P. gingivalis LPS is the best studied and it has shown heterogeneity in its acylation patterns, which result in opposite effects on TLR2/4 activation [P. gingivalis strains express at least two different varieties of LPS mostly differing in lipid A composition (designated O-LPS and A-LPS). In addition, growth conditions influence the relative balance between LPS types in single strains [F. nucleatum strains show considerable diversity in LPS O-antigen composition and structure [T. denticola LOS has a diacylglycerol lipid anchor and a hexose-hexosamine-hexose core region, but lacks heptose, 3-deoxy-d-manno-2-octulosonic acid, and β-hydroxy fatty acids, which are core components in LPS [P. gingivalis and F. nucleatum LPS and T. denticola LOS that may help promote cancer cell migration. T. denticola LOS generally has decreased effects compared to the classic E. coli LPS, and not much is known about its host cell effects [P. gingivalis can also promote invasion in OSCC and esophageal cancer [P. gingivalis promotes progression in chemically induced OSCC tumorigenesis and resistance to paclitaxel [T. denticola dentilisin proteinase (Td-CTLP) contributes to oral tonsillar and esophageal squamous cell carcinoma, gastric, pancreatic and colon carcinoma [T. denticola, P. gingivalis and F. nucleatum in the tumor microenvironment modify the biological behavior of OSCC cells and confer an aggressive phenotype in oral carcinogenesis.Oral pathogenic bacteria contribute to oral carcinogenesis via a number of potential mechanisms. However, given the paucity of knowledge in this area, we investigated the mechanisms by which poptosis . P. gingpoptosis . Furtheral cells . Thus, a effects , 97, 98.tivation . Differehigh MOI . These dtructure . T. dents in LPS , 103. Th effects . P. gingl cancer , 104. A clitaxel –107. Niearcinoma . Althougin vivo [in vitro and HNSCC tumor growth in vivo [T. denticola, P. gingivalis or F. nucleatum triggered increased expression of integrin alpha V, and stable suppression of alpha V expression significantly interfered with pathogen-enhanced migration and stemness of OSCC cells in vitro. These findings confirmed that oral pathogenic bacteria mediate a more aggressive OSCC cellular phenotype by modulating integrin alpha V expression. However, the involvement of additional integrins or other cell surface receptors cannot be excluded.Integrins are a large family of heterodimeric transmembrane receptors that mediate cell-extracellular matrix (ECM) interactions. Integrins serve as anchoring molecules by mediating the adhesion of the cellular cytoskeleton to the ECM . Cancer in vivo , 119. Fu in vivo , 121. Ah in vivo . In an eT. denticola enhances OSCC cell migration and tumorsphere formation via integrin/FAK-dependent signaling.Growth factor-mediated FAK phosphorylation promotes cellular adhesion, migration, and survival; all key factors in carcinogenesis. Furthermore, increased expression of phosphorylated FAK (phosphorylation at Tyr 576) enabled identification of HNSCC tumors with a more aggressive phenotype . In addiT. denticola-induced migration of OSCC cells is regulated via TLR/MyD88 triggered activation of integrin/FAK signaling. Taken together, T. denticola promotes OSCC carcinogenesis properties, such as migration, via crosstalk between TLR/MyD88 and integrin/FAK signaling pathways, and thereby contributes to a more aggressive oral cancer phenotype.Toll-like receptors have been implicated in a variety of cancers, including colorectal cancer, breast cancer, gastric cancer, nasopharyngeal carcinoma and oral squamous cell carcinoma., 126–130Staphylococcus aureus and Streptococcus mutans isolates, which inhibited the growth of T. denticola and P. gingivalis by producing a bacteriocin-like inhibitory substance and lactic acid [P. gingivalis strains was inhibited by S. sanguinis, S. cristatus, S. salivarius, S. mitis, Actinomyces naeslundii, and Haemophilus parainfluenzae , 138. Also, clinical isolates from healthy people containing growth-inhibiting strains including, Streptococcus, Actinomyces, and Bifidobacterium strains, inhibited P. gingivalis . Nisin is a polycyclic antimicrobial peptide (bacteriocin) produced by the commensal bacterium Lactococcus lactis [in vivo. Furthermore, we discovered that nisin decreases integrin alpha V expression and FAK phosphorylation and the concomitant negative cancer cell phenotype triggered by T.denticola. Nisin induces lipid II aggregation, thereby causing increased membrane permeability of the targeted cell. Specifically, it exerts its inhibitory activity by inhibiting bacterial cell wall biosynthesis and mediating stable pore formation in the target membrane [T. denticola)-mediated oral carcinogenesis by downregulating integrin alpha V expression and FAK phosphorylation. This suggests that integrin alpha V/FAK signaling could be a novel target and nisin a novel agent in the prevention of periodontal-pathogen (T. denticola)-enhanced oral tumorigenesis.The role of bacteriocins has not been explored in the context of modulating pathogen-mediated carcinogenesis nor in modulation of host cell receptors, such as integrins. The aggregate of microorganisms that reside in the saliva and oral mucosa and the interbacterial interactions and diversity among the microorganism are indicative of oral health . Within tic acid . The adhs lactis . Recentls lactis . In the membrane , 140. Oumembrane . The mecmembrane . As hypoT. denticola)-rich environment profoundly influences the tumor microenvironment by enhancing TLR/MyD88 and integrin/FAK crosstalk and signaling pathways. These signaling events trigger enhanced cancer cell migration and tumorsphere formation; properties that all contribute to a more aggressive OSCC phenotype. Overall, the present study provides novel evidence and insights into the molecular mechanisms of periodontal pathogen (T. denticola)-driven oral tumorigenesis and implicates the contribution of Toll-like receptors in the regulation of extracellular matrix and integrins as part of this process. Nisin alters these pathogen-mediated effects and mechanisms in OSCC, and since nisin also inhibits oral pathogenic bacteria and oral carcinogenesis directly, nisin has a broad therapeutic potential as an antimicrobial and as an anti-cancer agent. Altogether our findings shed new light on molecular mechanisms of pathogen-mediated carcinogenesis and pave the way to better understand the anticancer potential of the bacteriocin, nisin in oral carcinogenesis.In summary, our findings demonstrate that a periodontal pathogen  and HSC-3 (tongue) were provided by Dr. Thomas Carey  and by Dr. Randall Kramer , respectively , 142. OST. denticola (ATCC 35405), P. gingivalis (ATCC 33277) and F. nucleatum (ATCC 25586) were purchased from ATCC. T. denticola, P. gingivalis and F. nucleatum were grown as described previously at 37°C under oxygen deprived anaerobic conditions in New Oral Spirochete Medium or in Brain-heart Infusion (BHI) broth supplemented with hemin (5 μg ml-1) and vitamin K (1 μg ml-1) [S. gordonii (ATCC 10558), S. salivarius (ATCC 13419) and V. parvula (ATCC 10790) were grown overnight at 37°C under anaerobic conditions in BHI broth then harvested for subsequent assays. Gram staining followed by microscopic evaluation and colony morphology on Mitis Salivarius plates were used to confirm purity of cultures. T.denticola lipoologosaccharide (Td-LOS) was a gift from Daniel Grenier, Universite Laval, Quebec, Canada, P.gingivalis lipopolysaccharide (Pg-LPS) and F.nucleatum lipopolysaccharide (Fn-LPS) were provided by Richard P. Darveau, University of Washington, Seattle and V. parvula lipopolysaccharide (Vp-LPS) was provided by Christopher Fenno, University of Michigan, Ann Arbor, MI.Anaerobic bacteria μg ml-1) , 144. PuHigh content nisin ZP  was purchased from Handary . Nisin ZP was reconstituted in water (5 mg/ml) and used for all experiments. Bepridil was obtained from Sigma-Aldrich .8 cells/ml for T. denticola and 1×108 cells/ml for P. gingivalis and F. nucleatum. Oral cancer cells (UM-SCC-14A and HSC-3) were challenged with control medium or media containing different MOIs of T. denticola, P. gingivalis or F. nucleatum for 2 h. After the two-hour challenge, cells were washed with PBS three times then treated with nisin for 24 h.Bacteria in broth cultures were collected by centrifugation then re-suspended in serum- and antibiotic-free DMEM to an optical density of 0.1 at 600 nm, such that the cellular density was approximately 2.4×10T. denticola, P. gingivalis or F. nucleatum for 2 h then washed three times with PBS. A uniform wound or scratch was then placed across the “cell lawn” using plastic pipette tips and cells were then cultured for 24 h in the presence of nisin. Standard photographs were taken of the wounded areas using NIS-Elements BR4.13.04 imaging software. The cell migration distance was calculated by subtracting the distance at the lesion edge at 24 h from the distance measured at 0 h.Migration assays were performed as previously described , 146. CeT. denticola, P. gingivalis or F. nucleatum for 2 h then seeded (1 × 105) in the upper chambers of the transwells with 100 μl of serum free medium. Various concentrations of nisin ZP were placed in the lower chambers and cells were then incubated at 37°C for 24 h. Cells that invaded the matrigel-coated membrane and emerged on the underside surface of the membrane were labeled with Calcein AM fluorescent dye (BD Biosciences) as per the manufacturer's instructions. Fluorescence intensity was read at 485/520 in a fluorescence spectrophotometer  and reported as fold change in invasion relative to a media control.To evaluate invasion, transwell inserts with 8 μm pores (Becton Dickinson) were first coated with 50 μl matrigel . Cells grown overnight were challenged with T. denticola, P. gingivalis or F. nucleatum for 2 h, then tumorspheres were developed by maintaining cells under suspension conditions on poly-HEMA coated plates  in the presence or absence of nisin ZP for 24 h. In this assay, cells that survive anchorage withdrawal form multicellular aggregates or tumorsphere. A tumorsphere is defined as an aggregate of cells that is at least 50 μm in diameter. The total area occupied by tumorspheres in each well was quantified for each treatment condition using NIS-Elements BR4.13.04 imaging software.OSCC tumorspheres were prepared as previously reported , 148. InT.denticola, P. gingivalis or F. nucleatum for 2 h. After a two hour challenge, cells were washed three times with PBS and then treated with or without nisin for 24 h. After treatment, cells were stained with an EB/AO reagent containing 100 μg/ml of ethidium bromide and 100 μg/ml of acridine orange in PBS. The stained cells were visualized under fluorescence microscopy and images were captured using a microscope equipped with a digital imaging system . One hundred cells were counted in each group and classified as either vital, apoptotic or necrotic based on their color and morphology.To determine the effect of the bacterial pathogens and nisin on cell proliferation, the CyQUANT NF Cell Proliferation Assay Kit was used according to manufacturer’s instructions . To determine the effect of the bacterial pathogens and nisin on apoptosis Ethidium Bromide and Acridine Orange (EB/AO) staining was used to measure apoptosis as previously described , 149. EBWestern blot analyses were performed using an anti-alpha V antibody , phospho-FAK antibody  FAK antibody , TLR2 antibody , TLR4 antibody , or MyD88 antibody  followed by a horseradish peroxidase-conjugated anti-mouse antibody . Blots were then developed with the ECL-plus detection system . To evaluate the samples for equal protein loading, membranes were stripped and re-probed with an anti-β-actin antibody .UM-SCC-14A and HSC-3 cells were transduced with alpha V-shRNA  FAK shRNA , MyD88 shRNA  or scrambled-shRNA  lentiviral particles in 0.5 mL of serum-free media, then selected in 10 μg/mL puromycin  for an additional 10 days. Surviving cell colonies were picked and propagated before testing for alpha V and FAK expression using Western blot analyses.All mouse experiments were approved by the Animal Care and Use Committee at the University of Michigan and carried out according to the National Institutes of Health (NIH) guidelines for the ethical treatment of animals (UCUCA protocol #PRO00003743). These studies were performed at the University of Michigan when PK and YLK were affiliated with the University of Michigan.in vivo effects of the pathogenic bacteria and nisin ZP, an oral cancer floor-of-mouth mouse model was used as previously described [T. denticola, P. gingivalis or F. nucleatum for 2 h (50 MOI). After the 2 h challenge, cells were washed with PBS three times then suspended in DMEM, and mixed with an equal volume of growth factor-reduced Matrigel basement membrane matrix  to a final concentration of 1.0 x 106/0.05 mL for injection. Six-week-old athymic nude mice  were anesthetized by intraperitoneal injection with 100 mg/kg ketamine and 10 mg/kg xylazine. A total volume of 0.05 mL of OSCC cell/Matrigel suspension was injected submucosally into the floor of the mouth. Two weeks after tumor cell injections and upon confirmation that tumors were established, animals were equally distributed into several groups: a control group that was given water  and other treatment groups that were given nisin (800 mg/kg body weight per day) by oral gavage for 7 weeks. Oral gavage is the principal and common method used in toxicology, pharmacology and drug-development studies with rodent models. The main advantage of oral gavage over other methods of oral administration is that it is the most accurate and reliable method for administering substances into the oral and gastro-intestinal tract, as it eliminates risks of variability in intake between individual animals. Also, oral delivery is less invasive, and is a more physiological and clinically relevant option for testing the efficacy of drugs for oral cancer floor-of-mouth mouse models [To examine the escribed , 72, 73.e models , 151. Thin vitro experiments were repeated at least three times. For the in vivo studies, independent t-tests with unequal variance were used. The numerical data used in all figures are included in In general, values were expressed as means ± SD. Intergroup differences were analyzed by the analysis of variance (ANOVA) and Tukey-Kramer HSD test. All S1 TableTreponema denticola, Porphyromonas gingivalis or Fusobacterium nucleatum; MOI: 50) then treated with water (control) or nisin (800 mg/kg body weight/day) for 7 weeks.Time-course measurements of the body weights of mice injected with UM-SCC-14A cells challenged with control medium or media containing different periodontal pathogens ((DOCX)Click here for additional data file.S1 Fig(A-F) Graphs show the fold change in proliferation. Data represent mean ± SD from three independent experiments.(PDF)Click here for additional data file.S2 FigT. denticola, P. gingivalis or F. nucleatum for 2 h then treated with nisin (50 μg/ml) for 24 hours and evaluated for vital, apoptotic, and necrotic cells. (A-F) Graphs show changes in percentage of vital, apoptotic, and necrotic cells. Data represent mean ± SD from three independent experiments. *Comparison between groups relative to their media controls for apoptosis *p≤0.05.Cells (UM-SCC-14A and HSC-3) were challenged with control medium or media containing different MOIs of (PDF)Click here for additional data file.S3 FigT. denticola-LOS, P. gingivalis-LPS or F. nucleatum-LPS and (B) V. parvula-LPS for 2 h then evaluated for migration for 24 h. Graphs show the total migratory distance of cells from the edges of the wounds. Measurements were made after 24 h. Data represent mean ± SD from three independent experiments. *Comparison between groups relative to their media controls *p≤0.05.OSCC cells (HSC-3) were treated with control medium or media containing different concentrations of (A) (TIF)Click here for additional data file.S1 DataExcel spreadsheet containing, in separate sheets, the underlying numerical data and statistical analysis for Figure panels 1A-F, 2A-F, 3A-C, 4A-F, 6C-F, 6J and 7B.(XLSX)Click here for additional data file.