Cryptococcus gattii is a less frequent cause of cryptococcosis than Cryptococcus neoformans in South Africa. We performed multilocus sequence typing (MLST) and fluconazole susceptibility testing of 146 isolates randomly selected from 750 South African patients with C. gattii disease identified through enhanced laboratory surveillance, 2005 to 2013. The dominant molecular type was VGIV , followed by VGI , VGII  and VGIII . Among the 146 C. gattii isolates, 99 different sequence types (STs) were identified, with ST294  and ST155  being most commonly observed. The fluconazole MIC50 and MIC90 values of 105 (of 146) randomly selected C. gattii isolates were 4 μg/ml and 16 μg/ml, respectively. VGIV isolates had a lower MIC50 value compared to non-VGIV isolates, but these values were within one double-dilution of each other. HIV-seropositive patients had a ten-fold increased adjusted odds of a VGIV infection compared to HIV-seronegative patients, though with small numbers , the confidence interval (CI) was wide . Whole genome phylogeny of 98 isolates of South Africa’s most prevalent molecular type, VGIV, identified that this molecular type is highly diverse, with two interesting clusters of ten and six closely related isolates being identified, respectively. One of these clusters consisted only of patients from the Mpumalanga Province in South Africa, suggesting a similar environmental source. This study contributed new insights into the global population structure of this important human pathogen.As is the case globally,  Cryptococcus is the most common cause of meningitis among adults in South Africa. Most human disease is caused by the members of two species complexes within the genus, Cryptococcus neoformans and Cryptococcus gattii. The environmental range of these species complexes, both found in soil, overlaps in southern Africa though C. gattii is a less common human pathogen. C. gattii is divided into six molecular types: VGI, VGII, VGIII, VGIV, VGV and VGVI. In earlier molecular epidemiology studies including relatively few isolates, most southern African isolates were confirmed as molecular type VGIV. We aimed to determine the molecular diversity of C. gattii in South Africa by genotyping patient isolates obtained through laboratory surveillance, 2005–2013. We confirmed that VGIV was the dominant molecular type and that HIV-seropositive patients were more likely to be infected with VGIV compared to those HIV-seronegative. Analysis of the genomes of South African VGIV isolates revealed that they spanned the whole VGIV clade and confirmed that most isolates did not cluster specifically. However, we observed two interesting clusters of closely related isolates, consisting of patients from three neighbouring provinces in South Africa, suggesting a similar environmental source. Further studies of clinical and environmental African C. gattii isolates are needed to gain a better understanding of this pathogen. Cryptococcus are the most common cause of meningitis among HIV-seropositive adults in southern Africa [Cryptococcus neoformans and Cryptococcus gattii [C. gattii causing fewer than 1% of cases of cryptococcal disease globally [C. gattii also causes disease in immunocompromised people [C. gattii appear to present with pulmonary cryptococcosis more often than cryptococcal meningitis, with the opposite pattern being observed for disease caused by C. neoformans [C. gattii disease (meningitis with or without fungaemia) has been reported to be indistinguishable from disease caused by C. neoformans [Basidiomycetous fungi within the genus n Africa . Human ds gattii , with C.globally . While md people –5. Peoploformans ,6. Amongoformans . Regardloformans .C. gattii was historically thought to be confined to tropical and subtropical regions and was found less frequently in temperate regions [C. gattii is a less common pathogen [C. gattii is mainly associated with decomposing plant matter and certain tree species, such as eucalyptus in Australia and California [Ficus spp.) [C. gattii recently described after its isolation from hyrax faeces in an environmental study carried out in Zambia [ regions . The envpathogen . C. gattlifornia ,12, firslifornia  and numeus spp.) , with a C. gattii is mainly restricted to sedentary trees, it is not as widely distributed as C. neoformans, which is hypothesized to have a global distribution owing partially to its ability to metabolise pigeon droppings [C. gattii has also demonstrated the ability to adapt to new temperate niches, causing a geographically restricted outbreak in North America [C. gattii in North America. A recently-published hypothesis suggests that C. gattii was transported by ships in contaminated ballast water tanks from South America to North America in 1914 [C. gattii into the coastal forests. It adapted subsequently to a new environmental niche (i.e. land/forest) causing human infections about three decades later [C. neoformans has been hypothesized to originate from Africa, C. gattii could have originated from South America considering the high genetic diversity of South American C. gattii isolates [Since roppings . C. gatt America ,15–17. T in 1914 . This orisolates ,20.C. gattii is divided into six molecular types: VGI, VGII, VGIII, VGIV, VGV and VGVI [and VGVI ,22, of wand VGVI , with thand VGVI . VGII isand VGVI . This moand VGVI , Brazil and VGVI . VGIII hand VGVI , Mexico,and VGVI . The moland VGVI , Colombiand VGVI , Botswanand VGVI  and Zimband VGVI . VGVI haand VGVI . In earland VGVI ,32, withand VGVI .C. gattii in South Africa by genotyping a subset of clinical isolates from enhanced laboratory-based surveillance conducted between 2005 and 2013; (2) describe the fluconazole susceptibility of these isolates, and (3) determine if there was an association between patient clinical characteristics and the infecting molecular type, and the effect of molecular type on in-hospital mortality. In addition, we analysed the genomes of 98 isolates of the most common South African C. gattii molecular type VGIV to place them into a global context.To expand on these earlier findings, we aimed to: (1) determine the molecular diversity of Ethics clearance for this study was obtained from the Human Research Ethics Committee , University of the Witwatersrand with clearance certificate numbers: M160375 and M1809107. All data analysed was anonymized.Cryptococcus from any clinical specimen. Recurrent isolates from the same patients were excluded and only cases with cultured and viable isolates were included [C. neoformans or other Cryptococcus species. Following these inclusion and exclusion criteria, 750 cases of C. gattii infection in total were available for analysis. HIV infection status was recorded for 387 of these patients at ESS; we excluded all other patients with an unknown HIV infection status. Among these 387 patients, 374 were HIV-seropositive and 13 were HIV-seronegative. One hundred and thirty-six C. gattii isolates were randomly selected from the 374 HIV-seropositive cases using a random-integer generator (https://www.random.org/integers/) and all 13 isolates from HIV-seronegative persons were selected but only ten were viable. In total, 146 unique cases were selected from 387 cases of C. gattii disease between 2005 and 2013 and the corresponding isolates were genotyped . The surveillance methodology changed from 1 July 2008 to 31 December 2013 when only enhanced surveillance sites (ESS) , National Health Laboratory Service (NHLS) laboratories in KwaZulu-Natal Province, and pathology laboratories in the private, mining, and military sectors were required to submit isolates. A case was defined as a person diagnosed with cryptococcal disease by any one of the following positive tests during a 30-day period: (1) India ink microscopy on cerebrospinal fluid (CSF) or (2) a positive cryptococcal antigen test on blood or CSF or (3) culture of included . We alsoenotyped .Cryptococcus species at diagnostic pathology laboratories, a sweep of the culture plate was inoculated onto Dorset medium (Diagnostic Media Products (DMP), NHLS, Johannesburg, South Africa) and transported to a reference laboratory at the NICD in Johannesburg, where the isolates were identified by phenotypic methods [C. gattii isolates were retrieved from -70°C storage and sub-cultured onto Sabouraud dextrose agar  to check for purity and viability [Following primary isolation of  methods  and storiability . We pheniability .C. neoformans and C. gattii containing six housekeeping genes: CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and the intergenic spacer region IGS1 was used as previously published [http://www.genecodes.com). Allele types (ATs) and sequence types (STs) were assigned using the online C. gattii MLST database (http://mlst.mycologylab.org). The concatenated DNA sequences for the seven loci  following the manufacturer’s instructions. The only modification from the manufacturer’s instructions was the starting material for DNA extractions, since single yeast colonies grown on Sabouraud agar  at 30°C after 48h were used. The International Society for Human and Animal Mycology (ISHAM) MLST consensus scheme for ublished  for genoublished . Amplicoven loci  were aliMating type identification was performed by conventional PCR amplification using primer sets and cycling conditions from previous studies ,37, whicC. gattii isolates, fluconazole susceptibility testing was performed for 105 isolates: 60 randomly selected VGIV , 40 VGI, three VGII and two VGIII. We determined the fluconazole minimum inhibitory concentration (MIC) values (range: 0.125 μg/ml to 64 μg/ml) using custom-made broth microdilution (BMD) panels  prepared, inoculated and read according to susceptibility testing methods published previously and the Clinical and Laboratory Standards Institute (CLSI) M27-A3 and M60 recommendations [Of the 146 ndations –40. We indations , and thendations .de-novo using SPAdes (https://cab.spbu.ru/software/spades/) in careful mode and then used as references for the cluster trees. A cluster was defined as a group of more than two isolates that shared a common ancestor with fewer than 1100 SNPs between isolates. All WGS data were deposited in the NCBI SRA under the BioProject accession number PRJNA804139.Whole genome sequencing (WGS) was performed on 98 molecular type VGIV isolates using Illumina MiSeq sequencing technology . Previously extracted DNA samples (extraction procedure described above) were prepared for paired-end sequencing using the NEBNext Ultra II DNA Library Prep Kit for Illumina followed by 2 × 300 bp sequencing on a MiSeq instrument at the Translational Genomics Research Institute . Single nucleotide polymorphisms (SNPs) were detected from raw read data using the publically available Northern Arizona SNP pipeline (NASP) . The pipC. gattii and C. neoformans disease to determine if there were any differences between these two species complexes. For C. gattii infection, we used logistic regression models to determine associations between patient characteristics and molecular type. Patient case data were collected by nurse surveillance officers at ESS who either interviewed patients or reviewed their medical charts [50 values of the VGIV isolates and non-VGIV isolates using a Wilcoxon rank-sum test. We also compared the association between MIC value (≤8 μg/ml vs >8 μg/ml) and outcome data of patients infected with C. gattii. Statistical analyses were performed using Stata statistical software ver. 14.1 .For the 2005–2013 surveillance period, we compared demographic characteristics of patients with l charts . We defil charts . We divil charts , into moCryptococcus isolates were collected from 24,286 cases of cryptococcal disease. Only 3% of the viable isolates  were identified as C. gattii originating from 750 patients compared to 97%  of viable C. neoformans isolates originating from 23,512 patients. Owing in part to changes in surveillance methodology, the number of culture-confirmed C. gattii cases varied by year: 2005: 114 , 2006: 133 (18%), 2007: 91 (12%), 2008: 61 (8%), 2009: 75 (10%), 2010: 65 (9%), 2011: 58 (8%), 2012: 56 (7%) and 2013: 97 (13%). The median age for patients infected with C. gattii or C. neoformans was 35 years, though the inter-quartile ranges (IQR) differed slightly . In terms of gender differences of cases infected by the two species complexes, 57% (426/748) of patients infected with C. gattii were male while 48%  infected with C. neoformans were male (p = 0.0001). More patients diagnosed with either C. neoformans or C. gattii disease were from the mostly temperate provinces of South Africa  compared to the mostly arid provinces . For both C. gattii and C. neoformans infections, CSF was the most common specimen type from which the pathogen was cultured  followed by blood culture .From 2005 to 2013, 25,676 viable C. gattii isolates  and the p-value was large (0.28)  Tables. C. gattii isolates of the VGIV molecular type versus 25% (11/44) for patients infected with a non-VGIV molecular type  . The case-fatality ratio was 30% (29/98) for patients infected with  = 0.57) . On mult = 0.57) .50 and MIC90 values for all 105 tested C. gattii isolates  were 4 μg/ml and 16 μg/ml, respectively, with a geometric mean of 4.53 μg/ml , though these differed by only one double-dilution (4 μg/ml for VGIV isolates vs. 8 μg/ml for non-VGIV isolates) (The average fluconazole MIC53 μg/ml . There wsolates) . Most ofin-silico using a custom kraken database indicated that none of our isolates belonged genetically to the new molecular type VGV identified recently in Zambia [In order to place our South African VGIV isolates into a global context, we compared the WGS data between South African VGIV isolates and global VGIV isolates. n Zambia . Scattern Zambia . There wn Zambia  and SoutThere were two clusters that included South African VGIV isolates from our study and some of the VGIV isolates from other countries. The minimum, median and maximum number of SNPs were 0, 6 and 478 SNPs, respectively, between four Colombian isolates , a publiWe then analysed the Gauteng/Limpopo cluster and Mpumalanga cluster separately using the draft assembly of one of our isolates  as a reference in NASP. There were 1600 SNPs called within the Gauteng/Limpopo cluster of ten isolates . The minThe Mpumalanga cluster of six isolates from our study and two from a previously published South African study  containeC. gattii. Only 3% of viable isolates were identified as belonging to the C. gattii species complex with the remainder identified as belonging to the C. neoformans species complex. Previous clinical studies from neighbouring southern African countries have reported a much higher proportion of C. gattii infections, such as 13% in Botswana and Malawi [Laboratory-based surveillance for cryptococcosis, a common HIV-associated opportunistic infection in South Africa, provided a platform to determine the molecular epidemiology of d Malawi  or 17% id Malawi . HoweverC. gattii isolates, indicating a high genetic diversity, with VGIV being the dominant molecular type in South Africa. This confirms the results of studies undertaken in Zimbabwe, Botswana and Malawi [C. gattii isolates recovered from four patients and four isolates each had the molecular type VGI and VGIV, respectively [C. gattii in South African patients. Twenty-seven per cent of the clinical isolates in our study had the VGI molecular type; this molecular type has been described in a previous study as being dominant among Kenyan clinical and environmental isolates [C. gattii isolates from Africa, Asia, Europe, North America and South America, VGII was the dominant molecular type followed by VGI [We observed 99 STs among our clinical d Malawi , 31. In ectively . In our isolates . In a std by VGI . There wd by VGI . However50 values, MIC90 values and ranges overlapped for isolates with all four molecular types in our single-centre study. Our study therefore does not provide sufficient evidence for any meaningful difference in fluconazole susceptibility in relation to molecular type. Compared to the fluconazole MIC data for C. neoformans isolates from a previous South African study using the same surveillance platform, the MIC50 and MIC90 values for C. gattii differed by two fold higher dilutions [C. gattii than in C. neoformans [C. gattii isolates were also more resistant to xenobiotics than C. neoformans which could be due to the ecological niche C. gattii inhabits since tree hollows may harbour various ubiquitous saprophytic microorganisms that produce xenobiotics [C. gattii isolates showed heteroresistance in this one study; therefore, this is an intrinsic property of C. gattii that the authors speculate reflect an acquisition from its ecological niche but this needs to be studied further [The fluconazole MICilutions . A previoformans . These Cobiotics . All C.  further .C. gattii meningitis in HIV-seropositive patients [C. gattii molecular type, sequence type, fungal population genetics and environmental distribution “probably interact to mediate infection in patients with varying degrees of immune competence” [C. neoformans whereby the VNB genotype was associated with a worse survival outcome [Cryptococcus species [Galleria mellonella larvae were infected with the four C. gattii molecular types and highly virulent isolates were observed from all molecular types, suggesting that virulence may be related to strain-specific characteristics regardless of the molecular type [We found incidentally that HIV-seropositive patients had an increased odds of a VGIV infection compared to those HIV-seronegative, after adjusting for age, sex and climatic region. With a small sample size, our estimate of this effect is likely to be imprecise. However, previous studies in southern Africa have also shown that the VGIV molecular type is a major cause of patients ,31. In gpatients ,5,54,55.petence” . Based o outcome . Recent  outcome . These a outcome . VGIV al species . There i species ,60. In olar type .C. gattii molecular types before the continental drift [C. neoformans and C. gattii shared a common Pangean ancestor somewhere in between South America and Africa since these two continents were next to each other on Pangea [C. gattii species complex [In our WGS analysis, we observed a total of ten closely-related clusters and we zoomed in on two clusters specifically which we called the Gauteng/Limpopo cluster and Mpumalanga cluster consisting of ten and six isolates, respectively. The median number of SNPs for each of these two clusters were 426 and 598, respectively. A previous study described nine VGI isolates isolated from patients living in the south-eastern states of the USA that clustered together with 41,000 SNPs and 4558 SNPs between any two isolates . Firacatal drift ,62. Accon Pangea . This coAspergillus fumigatus with the TR/L98H substitution [C. gattii in a recent study done in Zambia [C. gattii was mostly associated with the Zambian Central Miombo woodlands that is dry and tropical [C. gattii. We did not find any VGV isolates in our collection of clinical C. gattii isolates. As Farrer and colleagues conclude, it is possible that this molecular type is strictly environmental and yet to cause clinical infection [C. gattii isolates from southern Africa should be performed to gain a better understanding of this pathogenic species complex [Our surveillance data also consisted of the patients’ residential addresses and healthcare facility at the time of clinical diagnosis. However, this does not take into account the long latency period of cryptococcosis in some cases and relocation of residence between provinces. The Gauteng, Limpopo, North West and Mpumalanga Provinces are located in the northern parts of South Africa. These provinces are inland. However, climatic differences between these provinces do exist. The Gauteng Province is elevated and temperate, whereas parts of the Mpumalanga Province are low-lying, subtropical to tropical and humid but also dry in some areas. The North West Province and the Limpopo Province are hot and dry. There was a cluster of six isolates from patients living in the Lowveld region of Mpumalanga Province in South Africa, suggesting a common environmental source. Based on our findings, we can speculate that the VGIV molecular type may be widely distributed in this subtropical environment. The reason for its widespread environmental distribution could be due to this genotype’s high fitness in the environment. This has been observed with triazole-resistant titution  as well n Zambia . In the tropical . The gentropical . This monfection . Further complex .C. gattii isolates, collected through enhanced laboratory-based surveillance from 2005–2013, were highly diverse, the VGIV molecular type dominated, and these isolates also mostly had low fluconazole MIC values. HIV-seropositive patients were more likely to be infected with VGIV isolates. We observed two interesting clusters of closely related VGIV isolates, with one cluster consisting of patients from the Mpumalanga Province in South Africa, suggesting a similar environmental source. Our large study provides a broader baseline for more extensive WGS studies in southern Africa, to enable us to gain a more complete picture of C. gattii, the lesser known cause of HIV-associated cryptococcal meningitis in this part of the world.In conclusion, our study showed that South African clinical S1 FigNote: NHLS–National Health Laboratory Service, CSF–Cerebrospinal fluid.(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S1 Supporting Information(XLSX)Click here for additional data file.S2 Supporting Information(DOCX)Click here for additional data file.


Clostridium difficile (C.difficile) is an exclusively anaerobic, spore-forming, and Gram-positive pathogen that is the most common cause of nosocomial diarrhea and is becoming increasingly prevalent in the community. Because C. difficile is strictly anaerobic, spores that can survive for months in the external environment contribute to the persistence and diffusion of C. difficile within the healthcare environment and community. Antimicrobial therapy disrupts the natural intestinal flora, allowing spores to develop into propagules that colonize the colon and produce toxins, thus leading to antibiotic-associated diarrhea and pseudomembranous enteritis. However, there is no licensed vaccine to prevent Clostridium difficile infection (CDI). In this study, a multi-epitope vaccine was designed using modern computer methods. Two target proteins, CdeC, affecting spore germination, and fliD, affecting propagule colonization, were chosen to construct the vaccine so that it could simultaneously induce the immune response against two different forms (spore and propagule) of C. difficile. We obtained the protein sequences from the National Center for Biotechnology Information (NCBI) database. After the layers of filtration, 5 cytotoxic T-cell lymphocyte (CTL) epitopes, 5 helper T lymphocyte (HTL) epitopes, and 7 B-cell linear epitopes were finally selected for vaccine construction. Then, to enhance the immunogenicity of the designed vaccine, an adjuvant was added to construct the vaccine. The Prabi and RaptorX servers were used to predict the vaccine’s two- and three-dimensional (3D) structures, respectively. Additionally, we refined and validated the structures of the vaccine construct. Molecular docking and molecular dynamics (MD) simulation were performed to check the interaction model of the vaccine–Toll-like receptor (TLR) complexes, vaccine–major histocompatibility complex (MHC) complexes, and vaccine–B-cell receptor (BCR) complex. Furthermore, immune stimulation, population coverage, and in silico molecular cloning were also conducted. The foregoing findings suggest that the final formulated vaccine is promising against the pathogen, but more researchers are needed to verify it. Clostridium difficile (C. difficile) was isolated by Hall and O’Toole in 1935 and recently reclassified as Clostridioides difficile (C. difficile as a pathogen related to antibiotic-associated diarrhea. Clostridium difficile infection (CDI) not only places a heavy burden on the healthcare system but also has a significant negative impact on the community population, causing nearly 230,000 CDIs in the United States each year, resulting in over 12,000 deaths and more than $1 billion in economic burden (Clostridium difficile infection (rCDI)  . To bettC. difficile colonization or spore spreading from the host to the environment but may lead to an increased number of asymptomatic carriers  but also the protein sequence of adjuvant LT-IIb (ID: 5G3L_H). The protein structure of TLR2 (ID: 2Z7X), TLR4 (ID:4G8A), TLR5 (ID:3J0A), B-cell receptor (BCR) (5DRW), major histocompatibility complex (MHC) I (ID:4U6Y), and MHC II (ID:5JLZ) molecules were downloaded from the Protein Data Bank using accession numbers (https://www.rcsb.org/).Not only the amino acid sequences of CdeC (CBE03081.1) and fliD (CBE01872.1) in Fasta format were retrieved from the protein database of the National Center for Biotechnology Information (NCBI) (http://www.cbs.dtu.dk/services/NetCTL/) that integrates the prediction of peptide MHC class I binding, proteasomal C terminal cleavage, and Transporter associated with Antigen processing (TAP) transport efficiency across 12 MHC class I supertypes  in the human population (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) (http://tools.iedb.org/immunogenicity/) (https://www.ddg-pharmfac.net/AllerTOP/index.html) (https://webs.iiitd.edu.in/raghava/toxinpred/index.html) (via Tepitool (http://tools.iedb.org/tepitool/) (The CTL epitopes were predicted by the NetCTL 1.2 server (pulation . The anten.html) , and epien.html) . To enhaex.html)  and Toxiex.html)  were emppitool/) , and allhttp://www.cbs.dtu.dk/services/2.5NetMHCIIpan/) (http://crdd.osdd.net/raghava/ifnepitope/) (https://webs.iiitd.edu.in/raghava/il4pre) (http://metagenomics.iiserb.ac.in/proinflam/index.html) (https://webs.iiitd.edu.in/raghava/il2pred/predict.php).The NetMHC II pan 3.2 server (CIIpan/)  was utilCIIpan/) , 45. Thupitope/)  and the /il4pre) , respect/il4pre) . Therefoex.html) , which pex.html) . Therefohttps://webs.iiitd.edu.in/raghava/abcpred/) (We used the ABCpred server (bcpred/)  to predihttps://blast.ncbi.nlm.nih.gov/Blast.cgi)  .via the EAAAK linker.The ultimately selected epitopes screened from CdeC and fliD in the previous steps were used for constructing the multi-epitope vaccine. The CTL and HTL epitopes were connected through KK linkers, while B-cell epitopes were linked together by GPGPG linkers. LT-IIb, a type II heat-labile enterotoxin (HLT), enables the formation of a greater proportion of effector memory cells by slowing CD8+ T-cell expansion and contraction dynamics . Thus, thttps://web.expasy.org/protparam/) (We employed the ProtParam server (tparam/)  to preditparam/) , a multitparam/) , transmetparam/) , and a mtparam/)  were chohttp://scratch.proteomics.ics.uci.edu/), and Secret-AAR servers (http://microbiomics.ibt.unam.mx/tools/aar/)  and AllerTOP v2.0 were used to check the allergenicity of the vaccine, and the toxicity of the vaccine was predicted by the ToxinPred server.Antigenicity prediction of the final vaccine construct was performed using the Vaxijen v2.0, ANTIGENpro (ls/aar/) . Accordils/aar/) . The Sechttp://scratch.proteomics.ics.uci.edu/) predicted the propensity of a protein to be soluble upon overexpression in E. coli using a two-stage support vector machine (SVM) architecture with a precision of more than 74%    was emplC. difficile is an intestinal pathogen, and intestinal mucosal immunity is very important for protection against CDI (https://webs.iiitd.edu.in/raghava/igpred/index.html#)   was usedhttps://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa.gor4.html) to predict the secondary structure of the vaccine sequences based on the Garnier-Osguthorpe-Robson 4 (GOR4) method with an average accuracy of 64.4% , which was more suitable for the prediction of targets without close homologs in the protein structure, using an ultradeep convolutional residual neural network from a primary sequence or multiple sequence alignments , which could refine the non-conserved and less reliable zones of the protein’s 3D structure and further generate five refined models of each primary 3D model (https://saves.mbi.ucla.edu/), and ProSA Web servers (https://prosa.services.came.sbg.ac.at/prosa.php) to verify its quality. The Ramachandran plot generated by PROCHECK reflects the stereochemical quality of a protein structure, and a superior model has more residues in the Ramachandran-favored region. ERRAT  (https://cluspro.org/tutantibody.php) was selected to conduct the molecular docking for vaccine and BCR in this study. Meanwhile, to further verify vaccine affinity between vaccine and receptors, the docking between vaccine and receptors  was also performed by Rosetta software   to perfoock2.4/)  has an asoftware . The TLRMolecular dynamics (MD) simulation was conducted by Amber18 software , 81 to bhttp://tools.iedb.org/population/) to predict the population coverage of the designed vaccine in different areas with an algorithm based on HLA allele binding and HLA allele frequencies    that utiE. coli is the most commonly used prokaryotic expression system (E. coli (K12 strains) was selected as the host organism in this study. To achieve better expression of the vaccine in E. coli, the JCAT online server (http://jcat.de/)   performecat.de/) , is goodthe host , 88. Thethe host . Meanwhithe host . Additiothe host . Howeverthe host  discoverits best ; both deits best , which mCTLs, HTLs, and B cells are important participants in the human protective immune response. CTLs indirectly orchestrate human adaptive immune responses by secreting and aggregating a variety of cytokines, in addition to directly mediating the death of target cells . HTLs noE. coli. The physicochemical parameters of the final vaccine construct were compared to those of positive controls  but also B-cell epitopes. Cytokines play an important role in the host immune response .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Campylobacter remains the most prevalent foodborne pathogen bacterium responsible for causing gastroenteritis worldwide. Specifically, this pathogen colonises a ubiquitous range of environments, from poultry, companion pets and livestock animals to humans. The bacterium is uniquely adaptable to various niches, leading to complicated gastroenteritis and, in some cases, difficult to treat due to elevated resistance to certain antibiotics. This increased resistance is currently detected via genomic, clinical or epidemiological studies, with the results highlighting worrying multi-drug resistant (MDR) profiles in many food and clinical isolates. The Campylobacter genome encodes a rich inventory of virulence factors offering the bacterium the ability to influence host immune defences, survive antimicrobials, form biofilms and ultimately boost its infection-inducing potential. The virulence traits responsible for inducing clinical signs are not sufficiently defined because several populations have ample virulence genes with physiological functions that reflect their pathogenicity differences as well as a complement of antimicrobial resistance (AMR) systems. Therefore, exhaustive knowledge of the virulence factors associated with Campylobacter is crucial for collecting molecular insights into the infectivity processes, which could pave the way for new therapeutical targets to combat and control the infection and mitigate the spread of MDR bacteria. This review provides an overview of the spread and prevalence of genetic determinants associated with virulence and antibiotic resistance from studies performed on livestock animals. In addition, we have investigated the relevant coincidental associations between the prevalence of the genes responsible for pathogenic virulence, horizontal gene transfer (HGT) and transmissibility of highly pathogenic Campylobacter strains. Virulence and antibiotic resistance frequently arise at the same time, but the relationship between the two at the genetic level has been largely overlooked. Studies have associated virulence with multi-drug resistance in multiple animal infection models , but theAcinetobacter baumannii augments its virulence, altering its normally low virulence into one that induces lethal disease [Hospital-acquired diseases caused by antibiotic-resistant bacteria derive from complex interactions between various dynamic factors, including fitness costs, bacterial pathogenicity and selective pressures from antibiotic therapy. The rise and aftermath of mutations that confer antibiotic resistance are shaped by these interactions. Mutations that increase antibiotic resistance can either cause decreased or increased pathogenic potential. Further research is needed to elucidate the origin of altered virulence potential in pathogens displaying antibiotic resistance and to better understand the modulating implications of resistance traits on disease outcomes. Additionally, it is important to note that the disease process itself generates selective pressures that constrain the resistance spectrum . For exa disease . This ab disease . Researc disease ,7,8.The development of “new-generation” antibiotics, which are mostly derivatives of already existing classes, has been followed by the rise of antibiotic-resistant bacterial strains . The sprConcerns are rising regarding the presence of antibiotic-resistance genes and virulence-associated genes in food-producing animals and humans. These genes, considered environmental pollutants, can cause severe complications for animals and humans. Misuse and overuse of antibiotics are accelerating the problem of increasing antimicrobial resistance and posing a threat to all inhabitants on earth . Thus, tCampylobacter genus can exchange genetic material with one another, as well as with other bacterial genera [Campylobacter and allow it to withstand exposure to antibiotics. This bacterial genus is widely present in multiple hosts, including humans, animals, food products and the environment. The interconnected nature of these factors presents challenges for effectively addressing the potential negative consequences for human and animal health. Exposure to the recent relevant discoveries concerning virulence factors and emphasising the genomic diversity and plasticity of Campylobacter makes it inconceivable to control its spread and evolution. However, monitoring the molecular changes could pave the way to a better understanding of the pathogenicity of evolution and virulence in humans, animals and the environment. Clearly, our review is not aiming to identify a genetic relationship between antibiotic resistance and virulence since it has been shown that there is no linkage between virulence and antibiotic resistance at either the chromosomal or plasmid levels [Campylobacter species belonging to different sources and hosts. We also showed associations between the prevalence of the genes responsible for pathogenic virulence, horizontal gene transfer (HGT) and transmissibility of highly pathogenic Campylobacter strains.It has been observed that various species within the l genera ,18,19,20d levels , but ratCampylobacter genus represents a well-known group of foodborne pathogens responsible for causing self-limited gastroenteritis in humans, which sometimes requires antimicrobial treatment due to the potential for serious complications [Campylobacter spp. being detected in all major animal categories, including pigs, broilers, small ruminants, cattle, cats and dogs [Staphylococcus aureus, Clostridium difficile and Escherichia coli, besides Campylobacter and Salmonella, with an increased impact on human health. Mitigation measures, such as increased biosecurity or vaccines, are proposed as solutions to reduce antimicrobial use in livestock [Campylobacter spp. poultry gut colonisation and virulence, both in vitro and in vivo [Staphylococcus aureus and Clostridium difficile [Campylobacter spp. spreading in livestock [Campylobacter spp. from chickens and pigs to macrolides suggesting that continuous surveillance is required to reduce the antibiotic-resistant campylobacteriosis in livestock and humans [The ications . In the and dogs . Howeverivestock . Natural in vivo . The posifficile  and coulifficile . These nifficile . Measureifficile ; howeverivestock . The avad humans .Campylobacter spp. can have various clinical consequences in animals as well, including reproductive failures in ruminants. For example, C. fetus subsp. venerealis induces cattle infertility, C. fetus subsp. fetus causes abortions in goats, sheep and cattle, and C. hepaticus causes spotty liver disease in layer hens [C. hepaticus in chickens may have an environmental origin [Campylobacter to β-lactams, tetracyclines, fluoroquinolones and macrolides emphasises the importance of ongoing susceptibility testing. The global crisis of multi-drug resistance (MDR) among bacteria is of significant public health concern, prompting efforts to restrict or eliminate antibiotic use across sectors, including their use in animal feed and for metaphylaxis. In 2019, the European Union implemented a regulation addressing the use of antibiotics in metaphylaxis [Infections with yer hens ,32. The l origin ,34. The phylaxis . Resistaphylaxis .Campylobacter can be acquired through various mechanisms, including drug inactivation, target alteration, modification of the antibiotic efflux pumps and decreasing membrane permeability [cmeABC operon encoding for multidrug efflux pump [tetO gene, which encodes a protective ribosomal protein, is generally associated with resistance to tetracycline [gyrA) gene within the quinolone resistance-determining region (QRDR). Other genes associated with Campylobacter MDR are the aadE or sat4 (streptomycin/streptothricin resistance), ermB (erythromycin resistance), aphA-3 (aminoglycosides resistance) and blaOXA-61 (b-lactams resistance) genes [Campylobacter species [Antibiotic resistance in eability . Macrolilux pump ,38. The acycline . Resistae) genes . Ultimat species . All theThe dissemination of these genes between host bacterial cells occurs through horizontal gene transfer (HGT) or vertical gene transfer (VGT). The HGT process relies on the transfer of genetic material via mobile genetic elements (MGEs) such as phages, plasmids, transposons and integrons ,42. MDR The persistence of plasmids in bacterial populations has been studied due to their potential negative impact on host fitness. Empirical data suggests that plasmids can persist for extended periods, even in the absence of positive selection for their traits, which presents a challenge to understanding the mechanisms of plasmid persistence . They alE. coli can migrate within plants and transfer ARGs through HGT. The use of antimicrobials in healthcare settings also leads to the occurrence of antibiotic-resistant bacteria in domestic wastewater, which can be a source of horizontal gene transfer of ARGs in municipal wastewater treatment facilities [E. coli strains isolated from wastewater successfully transfer resistance genes and all together give rise to antibiotic resistance phenotypes [Plasmid-encoded antibiotic resistance is a well-studied trait and is becoming increasingly important in clinical settings . Consequcilities . Moreoveenotypes .Campylobacter resides in the diversity of its virulence factors, including invasion of epithelial cells, adhesion to the intestinal mucosa, toxicity, motility and chemotaxis, as well as surface structure [cadF gene, encoding the Campylobacter adhesion protein to fibronectin (CadF), the flaA gene, encoding the major flagellin protein FlaA and the docA gene, encoding a periplasmic cytochrome C peroxidase. Other adhesion-associated factors include the FlpA, PEB1, MOMP, HtrA, CapA and JlpA [Campylobacter invasion antigen conferred by the ciaB gene, phospholipase A encoded by the pldA gene, invasion-associated marker (encrypted by the iam gene) and the virB11 gene [Campylobacter spp. consist of capsular polysaccharide (CPS), lipooligosaccharide (LOS) and S-layer proteins. CPS participates in the invasion, adherence, intestinal colonisation and systemic infection, withstanding complement-mediated killing, while LOS is a mediator of invasion and adherence. LOS activates Toll-like receptor 4-mediated innate immunity and resists killing via cationic antimicrobial peptides. The S-layer proteins are relevant to immune evasion and resist complement-mediated killing [Campylobacter spp., the most characterised is the cytolethal distending toxin (CDT) encoded by the cdtABC operon. Some genes have been associated with GBS occurrence, namely cgtB and wlaN genes, which both encode a β-1,3-galactosyltransferase enzyme involved in the production of sialylated lipooligosaccharide (LOSSIAL) [The pathogenic character of tructure ,49. Adheand JlpA . Among sB11 gene ,50. The  killing . Of all Campylobacter spp. was initially identified as a cause of animal disease in 1909, but it was not until 1980 that it was discovered that it also induces human infection [Campylobacter spp. is capable of surviving in food products for long periods of time. These bacteria are usually sensitive to environmental stress but have developed various mechanisms for survival in the environment and the food chain, which can lead to human infection [Campylobacter spp. are widely distributed in the environment, which ultimately serves as an intermediate link between different hosts and habitats in the transmission of this bacterial pathogen [Campylobacter spp. recovered from various animal and human hosts, including the environment.nfection . This genfection . Despitenfection . Thermoppathogen ,54. Detepathogen ,55. ConsCampylobacter spp. colonises the mucosa of the cecum and cloaca crypts in infected chickens and may also be present in other organs such as the spleen and liver [C. jejuni isolates recovered from avian and human samples in Egypt revealed the presence of the flaA gene in all C. jejuni isolates, while the cdtA, cdtB and cdtC genes were present together in only ≈58.54% of the strains. None of the isolates displayed a concomitant presence of all the virulence genes screened  in this study. Moreover, the cdtC gene revealed high similarity among C. jejuni isolates investigated, while the iam and cdtB genes showed the lowest similarity. Notably, the analysed strains unveiled 13 novel alleles. The C. jejuni strains obtained from the same host showed high heterogeneity, although the isolates recovered from chicken strains displayed the highest diversity [C. jejuni isolates obtained from poultry, and human sources were investigated by Ammar et al. (2021) in Egypt. All tested isolates unveiled the presence of the flaA gene, while 52% displayed the presence of the virB11 gene, and 36% encoded the wlaN gene [Given the large spectrum of available data from poultry and because of the different approaches taken in these studies, connecting increased/decreased virulence with the levels of antibiotic resistance is difficult. nd liver . A genomiversity . SimilarlaN gene .Campylobacter chicken isolates (C. jejuni 200605 and C. coli 200606), it was revealed that strain 200605 is more virulent than strain 200606. This observation was based on the identification of cadF, pebA, jlpA (adhesion), ciaBC (invasion), cdtABC (toxin production), flgSR, as well as eptC (biofilm formation) genes only in the C. jejuni strain 200605. Similarities we only identified in regard to genes cheAVW (chemotaxis), gmh, waa, kps (capsule formation), kpsDEFCST (capsular genes) and hldE (LPS-associated gene). Notably, genes expressing capsular biosynthesis were detected in C. coli strain 200606, namely Cj1416c, Cj1417c, Cj1419c and Cj1420c, suggesting potential genetic exchange between C. coli and C. jejuni species [Campylobacter (C. jejuni and C. coli) isolates originating from a layer farm in South Korea. The presence of the cdtB, cstII, cadF, dnaJ and flaA genes was reported in all of the isolates, while no strain was positive for the ggt gene. The prevalence of csrA, pldA and ciaB genes varied between 33.3 and 95.9% [C. jejuni and C. coli strains, respectively. Furthermore, all tested isolates showed susceptibility to sitafloxacin, as well as low erythromycin and gentamicin resistance and the presence of both gyrA gene point mutation and tetO gene was reported [Also, based on genome analysis of two fluoroquinolones (FQs) resistant  species . The samnd 95.9% . All 55 reported .C. jejuni were also investigated in Brazilian slaughterhouse isolates from two different time periods . The antimicrobial-resistant isolates, including those displaying multidrug resilience, were significantly more prevalent in 2011–2012, except for tetracycline. More prevalence rates were observed regarding C. jejuni isolation. However, the virulence genes revealed higher prevalence among the isolates from 2015 to 2016. All isolates were also tested for seven virulence-associated genes, namely ciaB, flaA, pldA, cadF and cdtABC genes. Approximately 83.6% of the 2011–2012 C. jejuni isolates harboured at least one of the screened virulence genes, whereas the 2015–2016 isolates revealed a significantly higher prevalence (97.7%). Despite the low prevalence rate of 2015–2016 C. jejuni isolates, these strains revealed increased virulence potential compared to the 2011–2012 group [C. jejuni isolates obtained from chicken carcasses and 20 C. jejuni clinical strains. Regardless of origin, 43.7% of all tested isolates harboured the flaA, pldA, cadF, luxS, dnaJ, cbrA, ciaB, cdtABC, htrA, cstII, neuA and hcp genes, while none of the analysed genomes displayed the presence of pVir genes. Their results indicated that the chicken isolates were more pathogenic than the human strains. The cadF, pldA and ciaB genes showed higher prevalence among the isolates obtained from chicken carcasses. Moreover, the ctdABC and luxS genes showed low prevalence among the clinical isolates, showing their presence in only 15% and 45% of human C. jejuni genomes [The antibiotic resistance and virulence factors of 12 group . More re genomes .C. jejuni chicken isolates, 26.92% of the tested isolates had more than two virulence-associated genes. For example, the ciaB, cdtA, cdtB, cdtC, pldA and cadF genes revealed 23.1%, 26.9%, 30.8%, 42.3%, 34.6% and 23.1% prevalence among the C. jejuni isolates, respectively. Integrons, particularly class I and II, play a crucial role in the transmission of antibiotic resistance and are frequently associated with the Tn7 transposon family [Campylobacter, but neither class II nor class III integrons have been detected in these species [C. jejuni isolates displayed the presence of Class I integrons, whereas 65.4% of the tested isolates harboured Class II integrons [The genetic elements of Gram-negative bacteria, such as integrons, may play an important role in the spread of resistance genes, given their presence in mobile genetic elements such as plasmids and transposons . In Soutn family ,95,96. C species . Howeverntegrons .C. jejuni isolates derived from chicken and duck meat showed that virulence factors were more prevalent in the C. jejuni strains obtained from retail chicken meat, while the duck meat isolates displayed a more increased prevalence of antibiotic resistance. Notably, the findings of this study were the first ones to present aerotolerant duck meat C. jejuni isolates. Particularly, the isolates obtained from duck meat displayed a more inferior prevalence for ciaB and cdtB genes than chicken isolates, whereas the virB11 gene showed low prevalence in both cases. The iam gene was harboured by 97.8% of the chicken isolates, while the C. jejuni isolates obtained from duck meat revealed its presence in 88.9% of the tested isolates. PldA gene was present in 94.4% of the poultry isolates and 91.1% of the tested duck isolates. Both aerotolerant and hyper-aerotolerant C. jejuni strains isolated from duck meat revealed higher prevalence for the cadF, peb1, pldA and docA genes in contrast to the oxygen-sensitive strains [Campylobacter isolates from ducks in southwestern China revealed that the most prevalent virulence-associated genes were cadF, cheY and cdtB, present in 100%, 92.7% and 92.3% of the isolates. Other highly prevalent virulence determinants consisted of flaA, iamA, cdtA, cdtC and ciaB, harbouring 77.2%, 71.8%, 60%, 54.1% and 42.7% of the isolates of interest. The virB11 gene was detected in only 7.7% of the isolates. Collectively, the C. jejuni strains displayed a higher number of virulence genes when compared to C. coli [An earlier comparative analysis regarding the aerotolerance, virulence-associated genes and antimicrobial resistance of  strains . The pre C. coli .Campylobacter spp., either directly or indirectly, due to their high mobility. Many free-living and migratory birds serve as reservoirs for pathogenic Campylobacter species, which can cause illness in humans. While the risk of human infection from zoonotic agents in wild birds is low, it is considered a growing concern [Campylobacter spp. The flaA, racR, docA, cadF and dnaJ genes were present in all white-fronted geese and bean geese isolates, while a high prevalence (66.7–100%) was observed among both graylag geese and mallards. The ciaB gene displayed high prevalence rates among bean geese, greylag geese, white-fronted geese and mallards isolates. Likewise, the pldA gene was also positively prevalent in the bean, white-fronted geese and mallards isolates. Only the strains originating from mallards sheltered the virB11 and iam genes, showing 40% and 10% frequency, respectively. All isolates obtained from graylag, bean and white-fronted geese harboured the cdtB and cdtC genes, while only 55% and 75% of the mallard isolates carried these genes. The Campylobacter isolates originating from white-fronted geese, mallards, and greylag geese displayed the presence of the cdtA gene in a proportion of 80%, 60% and 33.3% of the investigated strains, respectively. The cgtB gene was carried by 20%, 33.3% and 20% of white-fronted geese, graylag geese isolates and mallards, respectively, while the wlaN gene was only found in 5% of Campylobacter isolates originating from mallards. The results underlined that adhesion-associated genes  were highly prevalent among all tested isolates [Wild birds can serve as vectors for the transmission of  concern . In a reisolates .cdtABC genes among Campylobacter isolates originating from humans, wild birds and broiler chickens reveals a high diversity of the cdtABC operon from the wild bird C. jejuni isolates, while both broiler and human isolates displayed high conservation of cdtABC. The cdtA gene showed a higher number of nucleotide substitutions and was less prevalent among the tested Campylobacter isolates compared to both cdtB and cdtC genes. Moreover, non-functional cdtABC operons were detected among the wild bird isolates. This study’s findings indicate that the cdtABC operon plays a key role in host-specific colonisation efficiency [Campylobacter isolates originating from 12 species of wild birds from South Korea. All wild bird-derived Campylobacter isolates showed a high occurrence of all 11 tested virulence genes, while no correlation between any particular sequence type and a specific virulence profile was observed. The cadF gene was present in 100% of the Campylobacter spp. isolates. All C. coli isolates harboured the cdtB and cdtC genes, while C. jejuni strains carried them in 98.1% and 94.3% of the tested isolates, respectively. The wlaN gene was present in six C. jejuni isolates, while the virB11 gene was carried by 11.3% of C. jejuni and 28.6% of C. coli strains [The variation of the ficiency . Others  strains .C. jejuni isolates from Lithuania enabled the identification of virulent genes involved in adhesion (cadF and peb1), cytotoxicity  and invasion (yidC and yidD). In addition to this, two C. jejuni strains unveiled the presence of nine phage-specific genes. Two of the isolates revealed the presence of czc (Zn resistance), chr (Cr resistance), ncc (Ni resistance) and mer (Hg resistance) genes. Also, both cationic antimicrobial peptide system and platinum drug resistance (ctpA) genes were found in three of the isolates. The genomes of three C. jejuni isolates revealed the presence of virB2, virB4, virB8 and virB9 genes responsible for the type IV-secretion system T4SS). Trg, flgE (chemotaxis and flagellar motility) and bdlA (biofilm dispersion) genes were also identified [A whole genome sequencing (WGS) analysis recently performed by Aksomaitiene et al. (2021) on seven (wild birds and cattle) entified .Campylobacter spp. also colonises the intestines of cattle and can cause miscarriages in cows and sheep, although they are usually asymptomatic. Campylobacter spp. can also be found in lymphatic nodes, on the surface of hooves, and in the bristles of ruminants. Both meat and dairy products can be contaminated with Campylobacter spp., with raw milk being especially susceptible to infection [virB11 gene. Moreover, the iam gene proved to be a trusted invasion-associated marker, and the gene pattern virB11-iam was ascribed to the isolates that presented the highest invasion level. This study showed a high prevalence for adherence  and invasion  associated genes in Campylobacter spp. isolates from Poland [nfection ,98. Therm Poland .Campylobacter isolates obtained from Pernambuco, Brazil. Out of the 40 tested strains, 27.5% belonged to C. jejuni, 30% to C. fetus subsp. fetus, and 42.5% of them were C. coli strains [cdtA gene was found in 92.5% of all tested isolates, while cdtB and cdtC genes were harboured by ≈75% and 70% of the Campylobacter strains, respectively. The cadF and racR genes also revealed high prevalence, namely 95% and 80%, while the dnaJ and ciaB genes were present in only 47.5% and 20% of the isolates, respectively. The pldA gene was found in only one C. jejuni isolate, while none of the strains carried the wlaN and virB11 genes. Additionally, the C257T point mutation of the gyrA gene was present in all Campylobacter strains, while the A256G mutation and point mutations A2074G and A2075G of 23S rRNA were absent [The virulence and the prevalence of antimicrobial determinants were described for the first time in sheep on  strains . The cdte absent .Campylobacter spp. isolates from pork, beef, unpasteurised dairy products and raw milk, all isolates sheltered the iam, racR, cdtB, sodB, csrA and virB11 genes. The wlaN gene was present in all isolates, regardless of source origin, except C. coli isolates originating from pork meat. The highest prevalence rate was observed for the cdtB, sodB and csrA genes [Campylobacter spp. identified an overall 83.3% prevalence in pigs and 16.1% in calves. The most frequent species detected were C. jejuni, except in pig samples, where C. coli was more prevalent. C. jejuni isolates carried the cdtABC, peb3 (major antigenic peptide), Cj0020c (cytochrome C551 peroxidase) and pglG (N-linked glycosylation gene) genes, while the C. coli isolates only harboured the cdtB and cdtC genes [In a separate study performed on 98 rA genes . A similtC genes .Campylobacter by analysing 5 isolates recovered from preputial bull mucosa originating from a beef herd with a reproductive failure medical history. The isolates displayed microaerophilic, catalase-negative, oxidase-positive, non-motile and Gram-negative characteristics. The identified bacteria were proposed as a novel Campylobacter species via hsp60 and 16S rRNA genes analysis. The novel-defined Campylobacter portucalensis displays similarity to other Campylobacter species in terms of certain coding sequences, including genes encoding for adhesion and invasion, a type IV secretion system (T4SS) and antimicrobial resistance. The genome of C. portucalensis also revealed a high degree of homology with the T4SS of C. fetus subsp. venerealis. The pathogenic potential of C. portucalensis is currently unknown [Interestingly, researchers from Portugal have identified a previously undescribed species of  unknown .Campylobacter spp. related to aminoglycosides, fluoroquinolones, β-lactams, lincosamides, phenicols, macrolides and tetracyclines. Compared to other Campylobacter species, C. coli genomes harboured both the highest number of antimicrobial resistance factors and a higher prevalence of multidrug genotypes. C. coli revealed the presence of 1 to 4 plasmids in 86.4% of the isolates, while C. jejuni isolates harboured 1 or 2 plasmids in only 14.1% of their genomes. Most antimicrobial resistance genes of C. coli were detected on plasmids, whereas other Campylobacter isolates revealed chromosomally encoded resistance. Collectively, only C. jejuni/C. coli isolates had antimicrobial resistance genes located on plasmids [Recent data identified the presence of 18 distinct genetic determinants associated with antimicrobial resistance on either chromosomes or plasmids in plasmids .C. jejuni isolate. Moreover, regardless of meat origin, gentamicin resistance was found in all tested isolates [Campylobacter isolates obtained from chicken samples originating from various stages of the poultry production chain showed different patterns of virulence gene profiles between the C. jejuni and C. coli isolates. The C. jejuni isolates revealed the presence of 8 out of 9 investigated virulence factors, namely cadF, cdtABC, iamA, flaA, ciaB and cheY genes, while only three virulence-associated genes were detected in the C. coli isolates, including virB11, flaA and cheY genes. Aminoglycoside resistance was highly prevalent among the tested isolates, revealing the presence of aph(3′)-III, aph(2″)-If, ant(6)-Ia, aac(6′)-aph(2″) and aadE-Cc genes. Genes encoding for β-lactamases resistance were diverse, including blaOXA−456, blaOXA−184, blaOXA−460 and blaOXA−193. All tested Campylobacter strains carried the catA gene, while the fexA gene was reported in only two C. coli ST830 isolates. The lnuC gene was reported in 95% of the C. jejuni isolates, while the emrB gene was detected in only one C. coli ST872 isolate. The tetO gene was highly prevalent among both strains, while the tetL gene was present in 85% of the C. jejuni isolates and in only 15% of the C. coli isolates [In poultry, fluoroquinolones and tetracycline resistance rates were higher among duck meat isolates compared to chicken isolates, while resistance to macrolides was detected in only a single duck isolates . Earlierisolates .Campylobacter strains from Santiago, Chile, revealed that C. jejuni and C. coli have 13 previously undescribed sequence types and alleles. All Campylobacter strains analysed showed the presence of the cmeABC operon, while a QRDR point mutation of the gyrA gene was present in only 53.1% of the isolates. The analysed strains maintained the tetO gene and a 23S rRNA mutation in 22.2% and 4.94% of the analysed strains. Notably, none of the strains unveiled the presence of the ermB gene, while 79% of the isolates displayed the presence of the blaOXA-61 gene [C. fetus isolates displayed resilience to streptomycin and sheltered aminoglycoside nucleotidyltransferase ant(6)-Ib. One of the isolates also revealed resistance to tetracycline and ciprofloxacin, conferred by the presence of the ribosomal protection protein tet44 and a mutation of the gyrA gene [Campylobacter isolates presented a high genetic diversity among C. jejuni, and C. coli isolates derived from various ruminants  from northern Spain. The C. coli isolates harboured various resistance encoding genes, such as aph(2″)-Ic, aph(3′)-III, ant(6)-Ia, aadE-Cc, aad9, tetO, tetO/M/O, blaOXA-489 and blaOXA-193 genes. The antimicrobial resistance genes detected in the C. jejuni isolates were ant(6)-Ia, blaOXA-184, blaOXA-193, blaOXA-461, blaOXA-61, tetO/32/O and tetO genes, as well as an rpsL K43R point mutation in only one C. jejuni isolate. Both C. jejuni and C. coli isolates showed the presence of aminoglycoside resistance clusters; however, none maintained the aadE-Cc gene. Eight out of twelve C. jejuni plasmids showed high similarity with pTet plasmids, carrying the tetO and T4SS-associated genes. One C. jejuni isolate included a plasmid-encoded tetO/32/O gene, while eleven of them acquired tetracycline resistance via the plasmid-encoded tetO gene. Notably, evenly distributed pTet plasmids were observed among all tested Campylobacter isolates, regardless of host origin. Quinolone resistance was encrypted by gyrA gene point mutations, namely C257T and T86I, which conferred resistance to ciprofloxacin and nalidixic acid. The A2075G point mutation of the 23S rRNA gene provided erythromycin resistance to seven C. coli isolates, while none of the tested isolates displayed the presence of the ermB gene. Moreover, no Campylobacter isolate presented to have cmeR and cmeABC operons [Likewise, the antimicrobial resistance determinants of 81 clinical -61 gene . Two cliyrA gene . A recen operons .C. jejuni isolates, the highest antimicrobial resistance was detected to nalidixic acid (96.1%), tetracycline (80.7%) and erythromycin (84.6%), while resistance to ciprofloxacin was present in only 19.2%. The prevalence of the catI, catII, catIII, catIV, floR, ermB, tetO, tetA, mcr-4 and ampC genes were present in 84.2%, 78.9%, 52.6%, 10.5%, 52.6%, 73.7%, 68.4%, 47.4%, 42.1% and 10.5% of the tested isolates [Campylobacter (C. jejuni and C. coli) poultry isolates were favourably prevalent, ranging from 80 to 100% for the cmeB, tetO and blaOXA-61 genes among all tested isolates, while the aphA-3 gene was not detected in any of the strains. The Thr-86-Ile mutation in the gyrA gene was detected in 80% of C. coli and 90% of C. jejuni strains. Moreover, the 23S rRNA A2075G point mutation was identified in 61% of C. coli and 86% of C. jejuni isolates, while the A2074C point mutation showed lower prevalence, holding by only 27% of C. coli and 14% of C. jejuni isolates. Notably, both A2074C and A2075G mutations were detected in 12% of the C. coli isolates [In South African broilers, in 26 isolates . Similarisolates .Campylobacter genus, regardless of the source of the isolates. C. jejuni/C. coli strains isolated from both clinical and animal sources.There have been numerous reports of multidrug resistance within the C. coli strains isolated from humans and swine between 2008 and 2014 in Osaka, Japan, identified that antimicrobial resistance (AMR) to erythromycin in the swine-derived strains was the highest. The swine strains that displayed erythromycin resistance harboured, besides a point mutation in the 23S rRNA, also the presence of the tetracycline resistance gene (tetO). The ST-1562 swine strain presented the highest frequency of multiple AMR, although this strain was not found in humans. Most of the human, poultry and cattle strains that displayed tetracycline resistance revealed the presence of the tetO gene on plasmids, yet swine strains displayed it on chromosomes [C. coli isolate [Previously investigated antimicrobial profiles and phylogenetic diversities of omosomes . The res isolate .C. jejuni isolates obtained from Egypt’s poultry, and human sources also demonstrated a multidrug-resistant pattern, particularly to erythromycin and ampicillin. Approximately 96.5% of the isolates revealed resistance to five or more antibiotics [Campylobacter isolates were classified as multidrug-resistant. C. jejuni showed relatively low resistance rates regarding gentamycin (15.6%), ciprofloxacin (13.8%) and nalidixic acid (25.7%), whereas the C. coli isolates displayed higher resistance rates to these antibiotics, namely 67.0%, 60.2% and 62.1%, respectively [gyrA gene or in the V domain of the 23S rRNA. Also, the presence of either the blaOXA-61 gene or the cmeABC efflux pump was also linked to the effect [The prevalence, virulence factors and antibiotic resistance profiles of 113 ibiotics . Resistaibiotics . In a Chectively . The gene effect .Campylobacter isolates originating from various livestock at different stages of the production chain in North Carolina concluded that 90.4% of the tested isolates encoded antimicrobial resistance genes, while 43% revealed resistance to multiple antibiotics. Approximately 121 gyrA mutations were identified among poultry isolates, while only 9.7% belonged to the swine and cattle strains. All isolates showing high resistance to clindamycin, azithromycin and erythromycin also held the A2075G point mutation of 23S rRNA. Moreover, all Campylobacter isolates carrying cmeABC and cmeR operons also carried the 50S L22 point mutation, while C. coli isolates had resistance genes related to aminoglycoside resistance [Phenotypic and genotypic characterisation of 541 sistance .C. jejuni isolates from broiler chickens in Brazil. The cmeABC operon and the cmeG gene revealed high prevalence among the tested isolates, with 66.7% of the C. jejuni isolates showing the presence of tetO and blaOXA-61 genes, while only 30.43% of the strains carried the aphA-3 gene. All of the C. jejuni isolates harboured the Thr-86-Ile mutation in the gyrA gene and the A2075G point mutation in the 23S rRNA gene. Also, 85.7% of the C. jejuni strains included at least one plasmid [Campylobacter isolates. All the chicken isolates were resistant to tetracycline, while β-lactam, fluoroquinolone and macrolide resistance were detected in 66.66%, 25% and 33.33% of the tested isolates. Moreover, the isolates displaying resistance to macrolide and ampicillin carried the blaOXA-61 and 23SrRNA genes in 50% and 25% of the isolates [C. jejuni isolates, respectively, while C. coli isolates displayed 100%, 86.7%, 20%, 80% and 80% resistance rates to the same antimicrobials. All screened antimicrobial resistance genes, namely tetO, tetA, tetL, tetB, cmeB, ermB, aphA-3 and blaOXA-61 genes, were detected among the tested Campylobacter isolates. Moreover, the C257T point mutation in the gyrA gene and the A2075G point mutation in the 23SrRNA gene were identified [Multi-drug resistance to antimicrobials was also identified in all tested  plasmid . Likewisisolates . Also, aentified .Campylobacter isolated from broilers, calves, pigs and humans in Latvia. The C. jejuni isolates unveiled high resistance to nalidixic acid (94%) and ciprofloxacin (93.6%), while C. coli strains presented the highest resistance to streptomycin (73.5%). Low resistance to erythromycin and gentamicin was observed in C. coli and C. jejuni isolates. Circa 9.5% of the C. jejuni isolates obtained from calves displayed multi-drug resistance, while the human isolates showed a 16.7% prevalence of multi-drug resistance. Moreover, the C. coli isolates revealed multi-drug resistance in 29.6% of the pig isolates and in one human isolate and three calves isolates. All 30 quinolones-resistant Campylobacter strains possessed a gyrA gene point mutation, while all tetracycline-resistant bacteria showed the presence of the tetO gene. Aminoglycoside resistance was conferred by the ant(6)-Ib and aph(3′)-IIIa genes. The cmeABC operon was detected in all sequenced isolates. Notably, the blaOXA-184 gene was detected in five clinical isolates and one pig isolate. Further analyses linked these two strains by clustering them together [C. coli isolates. Some of the C. coli isolates that displayed erythromycin-resistance carried the ermB gene, point mutations in the rplV and rplD ribosomal genes, 23S rRNA A2074G point mutation and the cmeABC operon [C. jejuni or C. coli isolates originating from poultry is most often associated with gene mutations or the presence of plasmids encoding for such mutations indicating that surveillance and close monitoring of AMR in such isolates is necessary.A similar study investigated the antimicrobial resistance genes of together . A similC operon . It is iCampylobacter genus can exchange genetic material with one another, as well as with other bacterial genera. This facilitates the transfer of genes that enhance the virulence of Campylobacter and allow it to withstand exposure to antibiotics. This bacterial genus is widely present in multiple hosts, including humans, animals, food products and the environment. The interconnected nature of these factors presents challenges for effectively addressing the potential negative consequences for human and animal health. Altogether the findings of this study emphasise the need for monitoring the relationship between virulence factors and antimicrobial resistance in Campylobacter spp. regardless of host origin.It has been observed that various species within the


Helicobacter pylori infection can cause gastritis, gastric or duodenal ulcers, mucosa-associated lymphoid tissue lymphoma, gastric cancer, and extra-gastrointestinal manifestations. Ideal treatment should be guided by antibiotic susceptibility testing. However, this is not feasible in many regions, so the treatment generally relies on clinical experience and regional culture sensitivity profiles. We aimed to integrate the treatment of pediatric H. pylori infection through a systematic literature review. Databases including PubMed, Cochrane Library, EMBASE, and Scholar were searched using terms containing (Helicobacter OR Helicobacter pylori OR H. pylori) AND (child OR pediatric) for all relevant manuscripts and guidelines, published from January 2011 to December 2021. The eradication rate for pediatric H. pylori infection was not satisfactory using triple therapy, sequential therapy, concomitant therapy, bismuth-based quadruple therapy, or adjuvant therapy with probiotics as the first-line therapy. Most therapies could not achieve the recommended eradication rate of >90%, which may be attributed to varying regional antibiotic resistance and possible poor children’s compliance. More studies are required to establish a best practice for pediatric H. pylori infection treatment. Helicobacter pylori (H. pylori) is a gram-negative bacterium, first isolated by Warren and Marshall in 1983 on the surface of the stomach [H. pylori colonize gastric mucosa and could produce urease-dependent ammonia locally, raising the stomach pH, which promotes bacterial survival and solubilizes the mucous gel to facilitate bacterial motility [H. pylori infection can cause gastritis, gastric or duodenal ulcers, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer especially adenocarcinoma [H. pylori infection and inflammatory bowel disease [ stomach . H. pyloarcinoma . In addiarcinoma , chronicarcinoma . An inve disease .H. pylori infection in adults was significantly higher than in children , after an analysis of 410,879 participants from 73 countries on 6 continents [H. pylori infection in Taiwan has varied in the past decades and in different areas. The declining prevalence rate between 1993 and 2012 was shown in a study conducted by Yeung et al., explained by urbanization and public health education regarding proper sanitation and food handling [The global prevalence of H. pylori infection in children is not recommended [H. pylori infection in children [H. pylori is suggested in children with gastric or duodenal ulcers and when investigating causes of chronic immune thrombocytopenic purpura. Still, it is not recommended in children with functional abdominal pain, short stature, or as part of the initial investigation in children with iron deficiency anemia before other causes have been excluded [H. pylori infection with non-ulcer dyspepsia [H. pylori in infants and young children has been pointed out in some studies [“Test and treat” strategy for ommended ,10 sincechildren . Testingyspepsia . The pos studies .H. pylori after treatment can be assessed using the 13C-urea breath test (UBT) or stool antigen test at least 4 weeks after therapy [The preferable diagnosis was performed by endoscopy with biopsy-based methods. The cure of  therapy ,14.H. pylori infection in children if the strain is known to be susceptible to clarithromycin [H. pylori are not available, the treatment may rely on clinical experience and regional antimicrobial susceptibility profiles. When the first-line treatment fails, culture with antibiotic sensitivity testing or polymerase chain reaction (PCR) or fluorescence in situ hybridization (FISH) on previously obtained paraffin-embedded biopsies should be performed to guide the subsequent therapy [The Joint ESPGHAN and NASPGHAN 2016 guidelines recommend a triple combination of PPI-CA  for 14 days as the preferred regimen for hromycin ,14. Howe therapy . Yet, loH. pylori infection is an ordeal due to limited therapeutic options. The antibiotic resistance rates also vary with regions, so whether the first-line therapy in the Western world is suitable for Asian children is uncertain.In addition, the available literature was sparse about the treatment choice for those who were non-responders to the first- or second-line regimen. Levofloxacin-based triple therapy, including levofloxacin, lansoprazole, and metronidazole, seemed safe and effective as third-line rescue therapy if lansoprazole, clarithromycin, and amoxicillin were used as the first-line therapy and bismuth subcitrate, lansoprazole, metronidazole, and doxycycline as second-line therapy in a prospective study . HoweverH. pylori infection through a systematic literature review.Therefore, we aimed to integrate the treatment of pediatric Helicobacter OR Helicobacter pylori OR H. pylori) AND (child OR pediatric) for all relevant abstracts, manuscripts, and guidelines, published from January 2011 to December 2021. The non-human studies and manuscripts that did not involve children or adolescents were excluded. The association and management eligibility were initially screened based on the title and the abstract. Then a full-text assessment was performed with the evaluation of the references in the included studies to identify additional information. The flow chart of literature selection was shown in Databases including PubMed, Cochrane Library, EMBASE, and Scholar were searched using terms containing  [The eradication rates with standard sequential therapy for 10 days were 80.4–91.2% ,30,34,39ed above . Only on > 0.05) .Bontems et al., tailored the therapy according to the antimicrobial susceptibility testing. Subgroup analysis indicated that eradication rates tended to be higher using the sequential treatment except for children harboring CLA-resistant strains with a per-protocol eradication rate of 80% in triple therapy and 64% in sequential therapy .The eradication rate of concomitant therapy regimen using omeprazole, amoxicillin, clarithromycin, and metronidazole for 14 days was 84.6% in one recent study . The eraBismuth-based quadruple therapy, including a proton pump inhibitor, amoxicillin, metronidazole, and bismuth citrate, has been reported in one study with an eradication rate of 83.9%, significantly higher than 67.7% in standard triple therapy . The eraH. pylori by immunological and non-immunological mechanisms, including regulating pro-inflammatory cytokines in the gastric mucosa, increasing local IgA concentration, and secreting antibacterial substances such as lactic acid, short-chain fatty acids, hydrogen peroxide and bacteriocin [H. pylori IgG antibodies was found with probiotics use in an animal model, yet not to a statistically significant level [Some studies have proposed that probiotics can inhibit teriocin . A tendent level .A review included eight studies comparing the group treated with antibiotics to those using with same antibiotics therapy plus probiotics. Most of them imply a benefit of the probiotics, but only two studies reached a significant difference . One ranStandard sequential therapy includes a proton pump inhibitor with amoxicillin for 5 days followed by a proton pump inhibitor with clarithromycin and metronidazole for 5 days.H. pylori culture and antibiotic susceptibility tests to tailor the subsequent therapy [H. pylori infection in Korea is the same as that in the Joint ESPGHAN and NASPGHAN 2016 guidelines . Among the 38 children who remained infected, 30 children received second-line treatment, with 24 of them using a tailored triple therapy according to antimicrobial susceptibility test and 6 of them using a repeated sequential therapy; however, eradication rates of both therapies were low (65% and 40%). There were no sufficient details described in the latter. However, the overall eradication rate was only 67.3% after second-line treatment with sequential treatment or triple therapy.There was scarce data concerning the efficacy of second-line therapy for the treatment of pediatric bstracts ,48 were H. pylori eradication therapy [Adverse events were mentioned in some studies, and most were resolved once the treatment was stopped. Bontems et al. reported abdominal pain found in 20% , diarrhea in 14% (12% vs. 16%), nausea in 6% (8% vs. 5%) and vomiting in 2% (4% vs. 0%) in pediatric patients while receiving  therapy . Another therapy . The ove therapy . Black tH. pylori infection depends on the knowledge of H. pylori susceptibility to antibiotics and adherence to treatment [H. pylori infection.Successful eradication of pediatric reatment . The desreatment ,49. HoweH. pylori resistance rates as two of the articles were performed in Turkey. Another estimated reason is the risk of increasing resistance rate to sequential therapy in recent years as the study conducted by Zhou et al. was performed from 2017 to 2018. Whether prioritizing sequential therapy to triple therapy when antibiotic susceptibility testing is not available is worthy of consideration.If the strain is known to be susceptible to clarithromycin, both the Joint ESPGHAN and NASPGHAN 2016 guidelines and the JSPGHAN 2020 guidelines recommend the triple combination PPI-CA  for 14 days as the preferred regimen ,10. HoweH. pylori infection should be chosen according to the regional H. pylori resistance rate to clarithromycin and metronidazole. H. pylori resistance to antibiotics varies geographically and throughout the decades. A study in Zagreb showed H. pylori resistance to antibiotics in treatment-naïve pediatric patients: clarithromycin (11.9%), metronidazole (10.1%) and amoxicillin (0.6%) [H. pylori strains were clarithromycin-resistant from 2005 to 2016 [H. pylori in World Health Organization (WHO) regions in 2018, including 178 studies and 65 countries, reported resistance rates to clarithromycin, metronidazole, and levofloxacin were 15% in all WHO regions [H. pylori isolated from children in southern Taiwan in the past two decades, and the overall antimicrobial resistance rates of clarithromycin and metronidazole were 22.9% and 21.4%, respectively; the dual resistance rate of clarithromycin and metronidazole was 10%, and the resistance rates of levofloxacin and amoxicillin were 8.3% and 2.9%, respectively [An alternative first-line therapy for pediatric n (0.6%) . A studyn (0.6%) . In Swed to 2016 . In a me to 2016 . Much hi to 2016 . Overall regions . In Taiwectively . Increasectively . TherefoH. pylori strains are clarithromycin-resistant, triple therapy with metronidazole and amoxicillin (PPI-MA) is recommended as first-line therapy in the Joint ESPGHAN and NASPGHAN 2016 guidelines [Ideal first-line treatment should be guided by antibiotic susceptibility testing. If idelines  and the idelines . Howeveridelines . The Joiidelines . The Koridelines ,31. Concidelines . The assidelines .H. pylori infection is not satisfactory, whether using first-line or second-line therapies. Standard 10-day sequential therapy seemed more effective (88.3–91.2%) than standard 14-day triple therapy (54.2–74.1%), especially if the strains are susceptible to clarithromycin [The eradication rate for pediatric hromycin . Howeverhromycin ,40. Somehromycin ,44,45. SH. pylori transmission [H. pylori treatment could partially increase the eradication rate in children and reduce recurrence over a single-infected patient treatment strategy [Intrafamilial spreading is an important route of smission . Zhao etstrategy . HoweverH. pylori infection, compared to adult populations, are not satisfactory, no matter using triple, sequential, concomitant, or bismuth-based quadruple therapies, and most studies could not achieve the goal of 90% or above. In addition, the efficacy data of second-line therapy is sparse. Otherwise, retreatment as double resistance strains using high dose amoxicillin plus metronidazole, bismuth-based therapy, or concomitant therapy for 14 days is recommended in current guidelines modified from adult guidelines. However, the treatment protocol still varies by area and is practiced by clinical experience without consensus. The regimen may show superior results compared to other regimens in some studies, but the contrary results may be disclosed in other studies. More studies are required to improve the eradication therapy for pediatric H. pylori infection. The best strategy is still tailored treatment guided by culture with antibiotic sensitivity test and regional data about the eradication rates of different therapies need to be created for the best policy.The eradication rates for pediatric


Helicobacter pylori (H. pylori) is one of the main risk factors for this type of neoplasia. Carcinogenetic mechanisms associated with H. pylori are based, on the one hand, on the onset of chronic inflammation and, on the other hand, on bacterial-specific virulence factors that can damage the DNA of gastric epithelial cells and promote genomic instability. Here, we review and discuss the major pathogenetic mechanisms by which H. pylori infection contributes to the onset and development of gastric cancer.Gastric cancer is the sixth most commonly diagnosed cancer and the fourth leading cause of cancer death worldwide.  There were over 1,000,000 new cases of gastric cancer in 2020 and it was responsible for approximately 768,000 deaths, making it the sixth most frequently diagnosed cancer and the fourth leading cause of cancer death [Helicobacter pylori (H. pylori) is the main risk factor for gastric cancer, along with alcohol consumption and smoking. Other dietary habits associated with an increased risk of developing the disease are the consumption of salt-preserved foods and a low-fruit diet. [H. pylori is a gram-negative, helix-shaped, microaerophilic bacterium that specifically colonizes the gastric epithelium causing the most common bacterial infection in the world [H. pylori infection are asymptomatic, all will sooner or later develop gastritis which, in the long run, may result in the emergence of gastroduodenal ulcers and eventually gastric carcinoma and lymphoma of the lymphoid tissue associated with the mucosa [it diet. . H. pylohe world . Althouge mucosa . Althouge mucosa   and vacuolating cytotoxin A (VacA), play a fundamental role in causing damage to host cell DNA and in the activation of specific pathways that sustain cell survival, with both processes often supporting each other . H. pyloA damage . Such anA damage ,7. H. pylori infection with gastric carcinogenesis. The studies summarized here were retrieved from the biomedical literature using ”H. pylori” AND “gastric cancer” and ”H. pylori” AND “carcinogenesis” from PubMed, Scopus, and Web of Science bibliographic databases. Studies were selected after applying the following inclusion and exclusion criteria. The inclusion criteria were: (1) articles published in the English language and within the past 25 years; and (2) articles describing the main mechanisms linking H. pylori with gastric carcinogenesis with a high citation rate. The exclusion criteria were: (1) articles published in languages other than English; and (2) articles not available in full-text format.In this article, we review and discuss the major pathogenetic mechanisms that link H. pylori-related gastric tumorigenesis [H. pylori infection leads to inflammatory reactions through a variety of molecular pathways induced in both gastric epithelial cells, which the bacterium first contacts, and in circulating immune cells recruited to the site of infection, such as neutrophils, macrophages, and lymphocytes [Chronic inflammation contributes to the pathogenesis of several types of malignant diseases  and is pigenesis . H. pylophocytes .H. pylori, neither ROS or RNS eliminate the bacterium. Indeed, H. pylori may survive oxidative stress due to defense mechanisms related to the production of antioxidant enzymes  [H. pylori may contribute to increased oxidative stress in gastric epithelial cells [Among the immune cells that participate in the innate immune response, neutrophils actively seek, ingest, and destroy pathogenic microorganisms, this latter action realized through the production of substances with antimicrobial activity  . In partatalase) . Interesal cells  (Table 1H. pylori-derived CagA induces the expression of spermine oxidase (SMOX), an enzyme for the back-conversion of spermine to spermidine. This reaction leads to the production of H2O2 as a by-product. Increased H2O2 may, in turn, cause ROS accumulation through mitochondrial membrane depolarization, as well as activation of caspase-mediated apoptosis [poptosis .H. pylori infection can differently regulate APE1 function. While the H. pylori-driven increase in oxidative stress upregulates APE1 levels, helping repair DNA damage, a chronic H. pylori infection can eventually inhibit the expression of APE1 leading to genetic instability [Overproduction of ROS and RNS induces various types of DNA damage, including point mutations, DNA adducts, and single- or double-strand DNA breaks (DSB) . Among ttability .H. pylori are complex, but locally induced cytokines seem to play a key role in maintaining the ongoing inflammation. H. pylori infection is associated with marked mucosal induction of T helper (Th) type 1  and Th17-type  cytokines that are governed by specific antigen-presenting cell-derived molecules, such as interleukin IL-12 and IL-23 [The molecular pathways controlling adaptive immune responses driven by H. pylori IgA and IgG and, in particular, for a specific response of Th1 effectors—resulting in increased synthesis of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and IL-12—in the gastric antrum of H. pylori-infected patients, which can play a role in the genesis of either peptic ulcer or H. pylori-related gastric B cell lymphoma [H. pylori IgG and IgA may indicate a widespread immune response caused by bacterial infection and serve as a highly accurate, simple, and non-invasive method for monitoring H. pylori infection status in patients with gastric atrophy, intestinal metaplasia, and dysplasia [D’ Elios and colleagues provided evidence for a local production of anti-lymphoma . Notablyysplasia .H. pylori-infected patients, as compared with specimens/cells isolated from the stomach of patients without H. pylori infection [H. pylori-infected patients reduced Smad7 expression. Overall, these data suggest that, in the gastric mucosa infected with H. pylori, IFN-γ triggers the upregulation of Smad7 that, in turn, prevents endogenous TGF-β1 from dampening the ongoing tissue-damaging Th1 response [H. pylori-infected patients compared to those taken from normal patients and disease controls [H. pylori-infected gastric explants with an antibody against IL-21 decreased the production of epithelial cell-derived MMP-2 and MMP-9 [H. pylori-infected gastric mucosa is IL-17A [H. pylori infection, IL-17A is produced by T lymphocytes of the lamina propria and non-T cells through a process regulated by IL-1β, IL-21, and IL-23 [Transforming growth factor (TGF)-β1 constitutes a key negative regulator of Th1-type immune responses . In Crohnfection . In the response . T cell-controls . The humnd MMP-9 . Anothers IL-17A , which is IL-17A ,27. Durind IL-23 . IL-17A H. pylori can persist in the gastric mucosa for decades. Recent studies have shown that H. pylori’s expression of cholesterol-α-glucosyltransferase reduces cholesterol levels in infected gastric epithelial cells by blocking the IFN-γ signaling pathways, allowing bacteria to escape the host′s inflammatory response [Despite inducing an inflammatory response, response .H. pylori to establish successful colonization and persistent infection: (i) survival in the acidic stomach environment; (ii) movement towards epithelium cells through flagella-mediated motility; (iii) attachment to host cells through interaction of microbial adhesins with host cell receptors; and (iv) inducing tissue damage by toxins release [Once in the host stomach, four steps are critical for  release  , an acid-gated urea channel (ureI), and accessory assembly proteins (ureE-H) [H. pylori, and this activity is regulated by the UreI channel with proton urea, which allows entry of urea only under acidic conditions to prevent lethal alkalinization during periods of relative neutrality. Extracellular urease breaks down urea into carbon dioxide and ammonia, which, when combined with water, determines the production of ammonium hydroxide. Therefore, H. pylori can pass through gastric juice safely as ammonium hydroxide neutralizes the acidic microenvironment close to bacteria [H. pylori moves to the basal layer of the gastric epithelium, where the pH value is close to 7.0, per 4–7 polar flagella action. Kao et al. reported that patients infected with H. pylori strains with increased motility showed a higher bacterial density, resulting in a more severe inflammatory response in the upper part of the stomach as well as pathological results. In these respects, the flagellum can be considered a colonization and virulence factor in the initial phase [H. pylori are known to adapt to different hosts/tissues, including in particular neutrophil activating protein (NAP) and heat shock protein 60 (Hsp60) [H. pylori infection [H. pylori-infected patients and the titers are associated with the progression of gastritis or gastric cancer [(ureE-H) . The intbacteria . H. pyloal phase . The int (Hsp60) . As alrenfection . Hsp60 hnfection . In addic cancer .H. pylori virulence factors have been identified so far [H. pylori  as well as in the development of gastric cancer [H. pylori positive for CagA in Western countries is almost 60% and around 90% in Asian countries [cagPAI consists of 40 kb chromosomal DNA and contains more than 30 genes. Among them, cagPAI carries at least six genes that are homologous to the type IV secretion system (T4SS), acting as a molecular syringe that injects the bacterial protein CagA into the cytoplasm of the host gastric cell [Several d so far . Among tc cancer . The epiountries . The cagric cell . The traPhosphorylated CagA binds to the phosphatase SHP-2 and affects cell adhesion, spread, and migration . MoreoveCagA also exerts phosphorylation-independent actions, many of which remain unclear. Suzuki and colleagues identified a conserved motif in the C-terminus of nonphosphorylated CagA designated as CRPIA (conserved repeat responsible for phosphorylation-independent activity) . The autH. pylori CagA increased tumor suppressor gene hypermethylation by upregulating the expression of DNMT1, a methyltransferase involved in the malignant transformation of various cancers [Aberrant hypermethylation of the promoter CpG islands of tumor suppressor genes, leading to their inactivation, occurs at high levels during gastric inflammation and carcinogenesis . In this cancers , via the cancers . Mechani cancers .H. pylori-mediated delivery of CagA into cultured gastric epithelial cells promotes the oncogenic YAP pathway, resulting in the downregulation of E-cadherin and an increase in the epithelial mesenchymal transition program, further promoting gastric carcinogenesis [The mammalian Hippo tumor suppressor signaling pathway is crucial in maintaining the size and homeostasis of developing organs . Yes-Assogenesis .H. pylori-related virulence factor is the VacA, considered a multifunctional toxin responsible for eliciting multiple effects on host cells, including vacuolization, necrosis, and apoptosis. The VacA complex can also be incorporated into the host cell membrane and present the characteristic of an anionic selection channel. Acting as a channel, VacA can promote the release of bicarbonate and organic anions into the host cytoplasm, thus helping H. pylori colonization through the outflow of potential metabolic substrates for bacterial growth [H. pylori-mediated induction of apoptosis in gastric epithelial cells may play a role not only in gastric injury but also in the development of gastric atrophy and cancer.Another key l growth . Previoul growth  and extrl growth . Furtherl growth ,54. H. pH. pylori infection causes inflammation and leads both directly and indirectly to DNA damage, such as oxidative damage and DSBs, in host cells. Furthermore, the resulting genetic and/or epigenetic disturbances alter the choice of DNA repair paths. These changes cause inaccurate DNA repair, genomic instability, and chromosomal aberration, which may ultimately promote gastric carcinogenesis. DNA damage repair can be achieved through a large number of proteins, which include several important DNA repair pathways, such as mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), homologous recombination (HR), non-homologous end join (NHEJ), and alternative end join (A-EJ) [H. pylori alters the expression of DNA repair genes and/or interferes with the activity of DNA repair [We have seen that n (A-EJ) . Such DNA repair .The MMR pathway is one of the most studied DNA repair mechanisms. The MMR machinery maintains the stability of the genome by correcting errors in newly synthesized strains during DNA replication. Diseases caused by MMR defects include brain tumors and hereditary non-polyposis colorectal cancer ,58,59.Several human MMR proteins have been identified in human cells. Base–base mismatches and small insertion–deletion loops are preferentially recognized by the hMSH2–hMSH6 (hMutSα) complex; whereas MutSβ, combining the MSH2 and MSH3 proteins, the hMSH2–hMSH3 (hMutSβ) complex, mainly binds to larger insertion–deletion loops . Both coH. pylori infections than those with MSI-negative ones, suggesting that H. pylori itself may affect the DNA MMR system during stomach carcinogenesis [In eukaryotic cells, DNA MMR defects can be detected as instability in simple sequence repeats or short tandem repeats called microsatellites. Therefore, microsatellite instability (MSI) is considered a hallmark of MMR deficiencies ,60. MSI ogenesis   is another important repair pathway that is key to ensuring genome stability, as it repairs a number of endogenously generated lesions in the DNA double-helix structure, such as oxidation, deamination, methylation, depurination, and hydroxylation. BER is initiated by recognition and cleavage of the damaged base by specific DNA glycosylases, resulting in the creation of apurinic/apyrimidinic (AP) sites ,74. The H. pylori [H. pylori infection causes an imbalance between the generation and repair of AP sites, which is highly mutagenic [H. pylori infection [H. pylori protein extracts instead of live bacteria [H. pylori-positive gastritis, APE1 levels were also upregulated, but decreased after bacterial eradication [Machado et al. showed that APE1, but not OGG1 expression, was downregulated in gastric cells infected with . pylori . These rutagenic . In appanfection ,69. Thesbacteria . In patidication .H. pylori infection may differently affect the BER pathway through different mechanisms, leading to different outcomes of DNA damage in the host cells.Overall, these results indicate that H. pylori. In particular, DSBs appeared at mitosis and required direct contact between living bacteria and host cells, but not the H. pylori virulence factor VacA or ROS-mediated DNA damage [H. pylori showed that bacterial infection downregulated some components of the HR machinery , thus suggesting a shift in the choice of the DNA repair pathway away from HR [H. pylori infection, DSBs are introduced by NER via the endonucleases XPG and XPF in a T4SS-dependent fashion [H. pylori-induced DSBs were repaired via error-prone NHEJ rather than HR, possibly due to the upregulation of NHEJ-related genes and the downregulation of HR-related genes [A combination of decreased expression of other repair genes together with an increased level of genotoxic factors could enhance the accumulation of somatic mutations in gastric epithelial cells, thus favoring a malignant transformation. Toller and colleagues showed that DSBs accumulate in a time- and dose-dependent manner in both gastric cell lines and primary gastric epithelial cells that were incubated with A damage . RNA-seq from HR ,79. Hart fashion . Interesed genes .H. pylori infection and significantly increased the percentage of DSBs. Nuclear SNHG17-mediated recruitment of NONO, an RNA- and DNA-binding nuclear factor involved in the NHEJ pathway, together with the role of cytoplasmic SNHG17 as a decoy for miR-3909, which regulates RAD51 expression, shifted the DSB repair balance from HR toward NHEJ. Notably, the elimination of SNHG17 in gastric cancer cells inhibited chromosomal aberrations after H. pylori infection and overexpression of SNHG17 was correlated with a poor prognosis in patients with gastric cancer [In line with this latter result is a recent work by Han and co-workers . The autc cancer .H. pylori-related gastric cancers.Taken together, these findings indicate that various modifications of DNA repair functions are closely associated with DSB formation and chromosomal abnormalities during the development of H. pylori infection has been shown to call for multiple known mechanisms (and probably others still to be discovered) to induce the onset and progression of gastric cancer, making it the most important risk factor in the pathogenesis of this neoplasia. In addition to efforts to discover and validate new biomarkers [H. pylori eradication therapy could confer long-term protection against gastric cancer in high-risk populations, especially for infected individuals without advanced gastric lesions at baseline [The etiology of gastric cancer is complex and multifactorial, involving environmental and host-related factors as well as genetic and epigenetic alterations. omarkers , and chabaseline .