Mycobacterium tuberculosis, is the leading cause of mortality worldwide due to a single infectious agent. The pathogen spreads primarily via aerosols and especially infects the alveolar macrophages in the lungs. The lung has evolved various biological mechanisms, including oxidative stress (OS) responses, to counteract TB infection. M. tuberculosis infection triggers the generation of reactive oxygen species by host phagocytic cells (primarily macrophages). The development of resistance to commonly prescribed antibiotics poses a challenge to treat TB; this commonly manifests as multidrug resistant tuberculosis (MDR-TB). OS and antioxidant defense mechanisms play key roles during TB infection and treatment. For instance, several established first-/second-line antitubercle antibiotics are administered in an inactive form and subsequently transformed into their active form by components of the OS responses of both host  and pathogen . Additionally, M. tuberculosis has developed mechanisms to survive high OS burden in the host, including the increased bacterial NADH/NAD+ ratio and enhanced intracellular survival (Eis) protein, peroxiredoxin, superoxide dismutases, and catalases. Here, we review the interplay between lung OS and its effects on both activation of antitubercle antibiotics and the strategies employed by M. tuberculosis that are essential for survival of both drug-susceptible and drug-resistant bacterial subtypes. We then outline potential new therapies that are based on combining standard antitubercular antibiotics with adjuvant agents that could limit the ability of M. tuberculosis to counter the host's OS response.Tuberculosis (TB), caused by the bacterium  Tuberculosis (TB) is the leading cause of mortality worldwide due to a single infectious agent, killing approximately 1.67 million people in 2016 alone ) and spirometric restriction (adjusted OR of 2.13 [95% CI: 1.42–3.19]) [Oxidative stress (OS) arises due to an imbalance between the free reactive oxygen species (ROS) and the antioxidant mechanisms , 21. In There are two distinct groups of antioxidant processes: enzymatic and nonenzymatic systems. Enzymatic antioxidant processes present in the lung include superoxide dismutases (SOD), glutathione peroxidase, and catalase, whereas nonenzymatic processes involve ferritin, ascorbic acid ceruloplasmin, and carotene –26. Togeα1-antitrypsin [Excess formation of ROS can initiate series of chemical reactions and cause damage to cellular components, including proteins, lipids, and nucleic acids , 24, 25.itrypsin .It is noteworthy that OS is a double-edged sword and proceeds in an indiscriminate chemical manner. Although ROS can damage the host cells, it also kills infectious agents, including invading pathogens within the host. At the molecular level, excess OS causes damage to foreign pathogens via direct binding of ROS to amino-acid side chains like arginine, threonine, lysine, and proline and/or histidine oxidation or modification of tyrosine to nitrotyrosine, affecting the structural and functional properties of proteins . ROS is Candida albicans [Furthermore, excess ROS production can trigger a series of oxidative reactions on polyunsaturated fatty acids present on the cell membrane of microorganism(s), collectively known as lipid peroxidation . These ralbicans .M. tuberculosis invades and replicates within the host macrophages. As an immune response, the infected macrophage initiates a respiratory burst and produces high levels of ROS to counteract and kill the mycobacteria [M. tuberculosis disease compared to healthy controls [M. tuberculosis is highly dependent on the levels of ROS produced by the host immune cells [M. tuberculosis antioxidant systems, then the pathogen will continue to survive and replicate in the host [bacteria . Upregulcontrols . It is ine cells . If the the host .M. tuberculosis [M. tuberculosis possesses bacterial SODs and catalases that are known to degrade superoxide and hydrogen peroxide, respectively, there is no countermechanism for hydroxyl radicals [M. tuberculosis responds to ROS in a concentration-dependent manner. Exposure of M. tuberculosis to a low concentration of ROS (0–5.0 mM hydrogen peroxide) had no effect on the bacterial cell viability, but it initiated the expression of responsive genes which are sensitive to ROS [M. tuberculosis cells [M. tuberculosis triggers the release of DNA damage-responsive genes that initiate DNA repair mechanisms within the bacteria to counteract the damage done by ROS [M. tuberculosis do not protect against high levels of ROS [Mechanistically, among all the ROS, nitric oxide (NO) is known to be one of the major contributors as an anti-TB agent. NO is synthesized by NO synthase in macrophages, and NO synthase-deficient mice have been shown to exhibit increased susceptibility to rculosis . Patientrculosis . The accrculosis . Althougradicals . Interese to ROS , whereasis cells . Collecte by ROS , 38. Hows of ROS , 38.https://www.tballiance.org/).The standard therapeutic regimen for drug-susceptible pulmonary TB recommended by the WHO requires patients to take four drugs: isoniazid, rifampicin, pyrazinamide, and ethambutol for 2 months, followed by continuation with isoniazid and rifampicin for an additional 4 months (2HRZE/4HR) . This isAnother driver for the development of new regimens to manage TB is the emergence of MDR-TB that are resistant to the key first-line drugs, isoniazid and rifampicin. There was an approximate 95% increase in estimated MDR-TB cases from 250,000 per year in 2009 to 490,000 cases per year in 2016 . In addiM. tuberculosis, for a total treatment duration of 20 months [MDR-TB is difficult and costly to treat. The treatment completion success rate drops from 83% overall for TB to 54% for MDR-TB cases globally due to a higher frequency of treatment failure, loss to follow-up, and death of patients . Further0 months . In 20160 months . Here, t0 months . This neTB treatment is associated with levels of toxicity in patients, in some cases, requiring discontinuation of specific drugs. The first-line drugs isoniazid and pyrazinamide are associated with hepatotoxicity . Along wM. tuberculosis, KatG, to produce an isonicotinic acyl radical [+ and NADP+ to form a number of adducts which inhibit a 2-trans-enoyl-acyl carrier protein reductase, InhA, an essential component in mycolic acid biosynthesis [M. tuberculosis is particularly sensitive to OS and hence requires KatG for protection against ROS, including hydrogen peroxide. Paradoxically, then, in shielding the bacterial cell from the toxic activities of ROS, KatG also exposes the pathogen to the anti-infective action of isoniazid [Several of the TB drugs are administered to patients in an inactive form and must be activated inside the host. One of those is isoniazid (isonicotinylhydrazide). This prodrug is oxidatively activated by a catalase/peroxidase enzyme present in  radical . This coynthesis . Converssoniazid .M. tuberculosis cells [M. tuberculosis. The thioamides, ethionamide, and its analogue, prothionamide, are also prodrugs and are activated by an enzyme, EthA, encoded by M. tuberculosis. EthA is a flavoprotein monooxygenase that catalyzes the NADPH- and O2-dependent monooxygenation of thioamides [Pretomanid, a bicyclic nitroimidazole, is a prodrug that requires activation by a deazaflavin- (cofactor F420-) dependent nitroreductase (Ddn) to produce three primary metabolites, with des-nitroimidazole (des-nitro) being the major antimycobacterial component . The desis cells . Hence, ioamides . The metioamides .M. tuberculosis shows marked survival within the host despite the toxic effects of ROS, in particular due to its ability to effectively monitor and counter redox signals through a number of mechanisms. The interplay between oxidative stress and TB is depicted in M. tuberculosis [M. tuberculosis with the host cells [It is now known that rculosis . The prest cells .M. tuberculosis has also developed several cellular mechanisms to counter the redox imbalance that may also adversely affect the efficacy of frontline antitubercle antibiotics. For instance, an increase in the mycobacterial NADH/NAD+ ratio, through mutation of ndh which encodes a type II NADH dehydrogenase, confers coresistance to isoniazid and ethionamide [M. tuberculosis strains that exhibit resistance to isoniazid and pretomanid also demonstrate higher resistance to ROS compared to wild-type M. tuberculosis (CDC1551 and CB3.3) [M. tuberculosis are equipped to produce a specific protein, namely, enhanced intracellular survival (Eis), which can detect ROS and respond in a counteractive manner [M. tuberculosis possesses proteasome (peroxiredoxin) that can recognize, repair, and remove oxidative altered or damaged proteins [M. tuberculosis against ROS stress [M. tuberculosis utilizes the thioredoxin (Trx) system for defense against OS through its disulfide reductase activity regulating proteins' dithiol/disulfide balance [M. tuberculosis under nutrient-limiting conditions exhibit arrested growth, reduced respiration rate, and increased tolerance against isoniazid, rifampicin, and metronidazole [Furthermore, ionamide . M. tubed CB3.3) , 36, 58.d CB3.3) , 58. Seve manner . Moreoveproteins . More reS stress . M. tube balance . Betts enidazole .M. tuberculosis also expresses alternative peroxidase systems to maintain its virulence, including the expression of alkyl hydroperoxide reductase subunit C (AhpC) [M. tuberculosis exhibiting resistance to isoniazid [M. tuberculosis and/or upon transmission of M. tuberculosis to a new host [C (AhpC) . AhpC resoniazid . Mycobacnew host .M. tuberculosis also generates and secretes antioxidant enzymes to persist in the abnormal redox environment; these enzymes include SOD and KatG. Notably, M. tuberculosis harbours one catalase (KatG) gene that accounts for catalase, peroxidase, and peroxinitritase activity [M. tuberculosis as it aids bacterium to persist within the infected host tissues [M. tuberculosis also contains two metalloenzymes, an iron-containing SOD (SodA or FeSOD) and a copper- and zinc-containing SOD (SodC or CuZnSOD) [M. tuberculosis, which has been shown to impart protection against superoxide in in vitro experiments [activity . KatG is tissues . In addiCuZnSOD) , 69. Soderiments . The experiments . SodC coeriments .M. tuberculosis survives an oxidative burden in the host. For instance, Nambi et al. performed genome-wide genetic interaction assays and demonstrated that SodA forms a membrane-associated oxidoreductase complex with DoxX (a membrane protein) and SseA (thiol oxidoreductase). This SodA-DoxX-SseA complex links radical detoxification with cytosolic thiol homeostasis, and any abnormality in this complex results in defective recycling of mycothiol and accumulation of cellular oxidative damage [M. tuberculosis to encounter and overcome the oxidative environment in the host.Other recent advances have been made to understand how e damage . OverallM. tuberculosis, which has been reviewed elsewhere [3+) form, whereas DosT exists in the O2-bound (oxy) form [2, NO, and CO act as ligands for both DosS and DosT [M. tuberculosis and potentially leads to latent infection [M. tuberculosis in the oxygen-depleted microenvironment (during anaerobic dormancy), as well as rapid recovery of bacterial reproducibility once the favorable growth conditions prevail [M. tuberculosis to shift to dormancy under hypoxia [Genomic analysis has revealed the presence of heme-based redox sensors (DosS and DosT) in lsewhere . Kumar exy) form . Notablyand DosT . The autand DosT . It has nfection . DosR re prevail . Mechani hypoxia .M. tuberculosis to counter the host's OS, simultaneously enhancing the activation of prodrugs and elimination of bacterium.As mentioned, several front-line TB drugs need to be activated first to demonstrate their complete antitubercle properties , 62. TheM. tuberculosis has also been proposed as the source of a potential new class of drugs for treating MDR-TB [Interestingly, targeting cytochrome P450 enzymes in g MDR-TB . Similarg MDR-TB . NotablyM. tuberculosis than isoniazid alone [In vitro experiments revealed that cysteine (when added within 1 day of isoniazid treatment) caused increasing delays in the growth of isoniazid-resistant mutants, which was completely inhibited at highest concentration (4 mM). Interestingly, both isoniazid and the combination of isoniazid and cysteine resulted in similar bacterial killing in a strain-independent manner  for the first 7 days; however, only isoniazid/cysteine combination yielded approximately 6-log killing of the initial cultures after 3 weeks, without development of isoniazid-resistant strains. Thus, the use of supplements in combination with antitubercle drugs may prevent the formation of “persister” bacteria, as well as producing more effective killing of M. tuberculosis in shorter time periods [Treatment with cysteine (in a concentration- and time-dependent manner with increased oxygen consumption) has recently been shown to prevent the emergence of “persisters” and isoniazid resistance by increasing the respiration rates and thus promoting both OS and metabolism in the pathogen . Similarid alone . In vitr periods .in vitro cultures of M. tuberculosis, including drug-susceptible (H37Rv) and MDR-resistant (mc24997) and extensively drug-resistant (TF275) variants of bacillus. The transcriptional profile of vitamin C-treated M. tuberculosis revealed downregulation in the mbtD gene (biosynthesis of siderophore) [M. tuberculosis in anaerobic conditions (oxygen level < 0.001%), suggesting a prooxidant effect of vitamin C [M. tuberculosis (ΔmshA), hypersensitive to OS, showed rapid growth reduction in the presence of vitamin C, confirming the role of the oxidative process in its bactericidal activity [M. tuberculosis most likely due to its prooxidant activity, at least in in vitro cultures [Several other potential combinatorial therapeutic strategies are now being investigated. Vilchèze et al. reported that vitamin C (4 mM) specifically sterilizes rophore) . The antrophore) . The effitamin C . In addiactivity . Vitamincultures .in vitro cultures of drug-susceptible M. tuberculosis more rapidly than the drugs alone (~1 week). Similar effects were observed with even second-line TB drugs, namely, fluoroquinolone ofloxacin (OF), the injectable kanamycin (Km), and ETH [M. tuberculosis over a shorter time frame than isoniazid and rifampin combination only [M. tuberculosis (H37Rv) via the aerosol route. The mice receiving lower infective dose and treated with the combination of isoniazid/rifampicin/vitamin C exhibited lower lung bacterial burden (~one log) compared to mice treated with isoniazid/rifampicin in average at four weeks post-infection. Post-6-week treatment, M. tuberculosis was recovered from the lungs of one mouse in both isoniazid/rifampicin/vitamin C and isoniazid/rifampicin groups. The mice receiving higher infective dose showed a significantly lower bacterial recovery from lung only after 6 weeks of treatment in the isoniazid/rifampicin/vitamin C group [Recently, Vilchèze et al. have also shown that addition of vitamin C (1 mM) to isoniazid/rifampicin sterilized the  and ETH , where a and ETH . Moreoveion only . Briefly C group . The stu C group .M. tuberculosis by directly depleting the amount of free thiols, especially at acidic pH (5.7), and to enhance the bactericidal activity of commonly used antitubercle drugs, including isoniazid and clofazimine [Another compound, AC2P36 (5-chloro-N-(3-chloro-4-methoxyphenyl)-2-methylsulfonylpyrimidine-4-carboxamide), has been shown to kill fazimine . The antfazimine .M. tuberculosis infections. The utilization of relatively safe and inexpensive therapeutic supplements, such as vitamin C or cysteine, may aid in bacterial killing through increasing ROS burden in the bacterium and alterations in the lipid/thiol biosynthesis in a shorter time frame and without the formation of a drug-resistant “persister” population in the host. Future studies should also focus on developing novel strategies to target the DosS/DosT mechanisms as well as mycolic acid biosynthesis for M. tuberculosis survival, in combination with existing antibiotics that may pave the way for improved therapies to treat the disease.Collectively, these studies highlight the crucial roles of oxidant : antioxidant balance in limiting and treating M. tuberculosis. However, certain strains of M. tuberculosis are known to be resistant against the inhibitory effects of the oxidative burst produced by macrophages, primarily via the presence of lipids, proteins, enzymes, and antioxidant defense systems, like SOD, KatG, and DosR regulon, within the mycobacteria. These act in a counteractive mode against OS. These antioxidative mechanisms also affect the antibiotic drug treatment. Various research findings have led to a greater understanding of the effects of both drug-susceptible and MDR-TB strains and the generation of novel therapeutic interventions to tackle the response of mycobacteria towards oxidative burden. The research has just only begun to assess the effects of a combination of therapies, including antitubercle antibiotics and agents targeting the oxidant : antioxidant axis, which may prove beneficial in eliminating the pathogen from the host in a shorter time frame.Oxidative stress mediated by host cells, especially macrophages, plays a pivotal role to prevent the vicious cycle of


Clessinia Doering, 1875 are typical inhabitants of this ecoregion. The inclusion of their distribution areas into Spixia range, their shell shape similarities, and a former molecular study raised doubts on the monophyly of this genus. The present study review the species of Clessinia, under a morphological, geometric morphometrics, and molecular combined approach.Land gastropods of the Dry Chaco merit special attention because they comprise a highly diverse but barely studied group. Clessinia and Spixia species from GenBank were performed.Adults were collected, photographed, measured, and dissected for anatomical studies. Shell ultrastructure was studied with scanning electron microscope. Geometric morphometric analyses on shells were performed testing if they gave complementary information to anatomy. Two mitochondrial genes, and a nuclear region were studied. Phylogenetic reconstructions to explore the relationships of DNA sequences here obtained to those of Clessinia cordovana striata to species rank, naming it as Clessinia tulumbensis sp. nov. The periostracum, consisting of hairs and lamellae, has taxonomic importance for species identification. Shell morphometric analyses, inner sculpture of penis and proportion of the epiphallus and penis, were useful tools to species identification. Nuclear markers do not exhibit enough genetic variation to determine species relationships. Based on the mitochondrial markers, genetic distances among Clessinia species were greater than 10%, and while C. cordovana, C. nattkemperi, and C. pagoda were recognized as distinct evolutionary genetic species, the distinction between C. stelzneri and C. tulumbensis sp. nov. was not evident. Clessinia and Spixia were paraphyletic in the molecular phylogenetic analyses. Species of Clessinia here treated have narrow distributional areas and are endemic to the Chaco Serrano subecoregion, restricted to small patches within the Dry Chaco. Clessinia and Spixia are synonymous, and the valid name of the taxon should be Clessinia Doering, 1875 which has priority over Spixia Pilsbry & Vanatta, 1894.Species description on shell, periostracal ornamentation and anatomy is provided. We raised former C. cordovana complex by three species, C. cordovana, C. stelzneri, and C. tulumbensis sp. nov. The low genetic divergence between C. stelzneri and C. tulumbensis sp. nov. suggests that they have evolved relatively recently. The former Spixia and Clessinia are externally distinguished because Clessinia has a detached aperture from the body whorl forming a cornet, periostracal microsculpture extended over dorsal portion of the peristome, five inner teeth on the shell aperture instead of three–four found in Spixia. Morphological similarities exists between both genera in shell shape, type of periostracum microsculpture, reproductive anatomy, besides the overlap in geographic ranges.Our results support the composition of  Taxonomy is a crucial discipline in biology if practiced within an evolutionary framework . The taxThe combination of morphological and ecological information with different molecular markers can be a good method of species identification, because it can provide an accurate perspective on evolutionary history of an organism and its taxonomic relationships . In thissensuThe purpose and framework of our work is to study and identify endemic species of the Dry Chaco ecoregion in Argentina sensu as a firAnctus von Martens, 1860 and some Cyclodontina Beck, 1837 have no apertural teeth. Odontostomidae is understudied, most of the genera shows a lack of clear-cut diagnostic characters, and the species composition of each genus is still a matter of controversy. The last revised family nomenclator  is dominated by deciduous forests over extensive lowland plains and mountains  with arid and semiarid climates   is one of the largest remaining patches of forest/savanna ecosystems in Latin America . The Dryainfall) . This ecainfall) . The Semainfall) .http://qgis.osgeo.org). Shapefiles layers corresponding to administrative areas of Argentina were obtained from DIVA resources (http://www.diva-gis.org/gdata) and Instituto Geográfico Nacional (http://www.ign.gob.ar/sig). Classification of Argentinean ecoregions follows http://siga.proyungas.org.ar/recursos).Species distribution is based on point records  of species occurrences obtained through field work in the Córdoba and Catamarca provinces, Argentina between 2006 and 2017. All specimens collected were deposited in the Instituto de Biodiversidad Neotropical (IBN), Instituto-Fundación Miguel Lillo (IFML-MOLL), Tucumán, Argentina and the malacological collection at the Instituto de Biología Subtropical, Misiones, Argentina (IBS-Ma). Additionally, we examined other specimens and obtained records from the following collections: IBN; IFML-Moll; MACN-In, Museo Argentino de Ciencias Naturales “Bernardino Rivadavia,” Buenos Aires, Argentina . This inClessinia were carried out on rocky outcrop in xerophytic areas of Córdoba and Catamarca provinces, Argentina. Specimens collected were photographed alive, then drawn in water for relaxation previous to fixation in 96% ethanol, body preservation was done using 75% ethanol. Several specimens were fixed directly in absolute ethanol, without relaxation in water, for molecular studies. Special attention was paid to the shells to preserve the periostracum structures. Shells were cleaned in an ultrasonic cleaner, air dried, and then photographed in ventral, dorsal and lateral positions, and kept in plastic boxes separated from the bodies. Photographs were taken using a Zeiss Stemi 508 with ActionCam, and measured using the software ImageJ 1.49 ; IBN 878, C. nattkemperi; IBN 880, S. cuezzoaeS. holmbergi ; IBN 882, C. stelzneri; IBN 883, C. tulumbensis sp. nov.; IBN 885, Plagiodontes daedaleus ; IBN 886, C. cordovana; IBN 890, C. pagoda.Hand collection of live adult specimens and dry shells of geJ 1.49 –1D. Voucordovana ; IBN 882telzneri ; IBN 883tkemperi . Voucherhttp://www.iczn.org).The different zones in which the shell aperture is divided: basal, palatal  and parietal, are the same as used by Traditional linear shell measurements were taken from specimens of each species according to availability . The numClessinia, testing if they gave complementary information to our anatomical observations or differed from them. On these grounds, the geometric morphometric analysis was performed with 15–61 specimens per species according to their availability, totalizing 144 specimens used  genes, and a nuclear region including the 3′ end of the 5.8S-rRNA gene, the complete ITS-2 region, and the 5′ end of 28S-rRNA gene (hereafter referred to as ITS-2) were amplified by means of the primers 16SF-104 (5′-GAC TGT GCT AAG GTA GCA TAA T-3′) and 16SR-472 (5′-TCG TAG TCC AAC ATC GAG GTC A-3′) for 16S-rRNA . The amplification was conducted in a T18 thermocycler as follows: after an initial denaturing for 3 min at 94 °C; 35 cycles of 1 min at 94 °C, 1 min at 50 °C, 1 min at 72 °C were performed; followed by a final extension at 72 °C for 5 min. The success of polymerase chain reactions (PCRs) was verified by agarose gel electrophoresis. The PCR products were purified by means of an AccuPrep PCR Purification Kit . Following purification, both DNA strands for each gene were then directly cycle-sequenced . The resulting sequences were trimmed to remove the primers, and the consensus sequences between forward and reverse sequencing were obtained by means of the BioEdit 7.2.5 software  and the Kimura’s two-parameter (K2P) substitution model. Phylogenetic analyses were performed using maximum likelihood (ML), and Bayesian inference (BI). For both analyses, the COI and 16S-rRNA datasets were concatenated to improve the resolution of phylogenetic reconstructions. The total length of the analyzed matrix was 992 bp. In addition, COI-based phylogenetic reconstructions were performed to explore the phylogenetic relationships of the DNA sequences here obtained to those of other Clessinia and Spixia species from various locations available in GenBank  used as an additional outgroup for the mitochondrial DNA sequence data. Sequences of C. incanum were extracted from the complete mitochondrial genome for the species  with the Nearest-Neighbor Interchange branch swapping algorithm. Substitution models were selected using the SMS program  were analyzed via the ABGD web-server (http://wwwabi.snv.jussieu.fr/public/abgd/) using the K2P model (p) from 0.001 to 0.1 was used. In addition, the K/θ method was used to assess the status of Clessinia species under the evolutionary genetic species concept (EGSC) (K), in order to identify clades that are diverged enough to be considered separate species (K/θ method was performed on the concatenated dataset following K/θ ratios ≥4 were considered to represent sequences that come from different evolutionary species with probability ≥0.95 (The Automatic Barcode Gap Discovery (ABGD) method, which clusters sequences in putative species based on differences between intraspecific and interspecific distance variation  was used2P model . The mint (EGSC) . This me species . The K/θhttp://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:8DB0CC34-AE26-44BA-B7F8-A5F17254BD13. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central, and CLOCKSS.The electronic version of this article in portable document format will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix Clessinia, consisting in hairs of different lengths and densities, spines and rounded to quadrate lamellae. These structures were useful tools for species recognition. Both C. cordovana and C. stelzneri show periostracal hairs on the teleoconch surface, being notably longer among C. cordovana specimens, with more prominent hairs in specimens from Sierra de Pocho area in Córdoba. Periostracal hairs are shorter and more densely arranged in C. stelzneri. Teleoconch surface is traversed by periostracal spiral rows in the three species of the cordovana-group, with a greater number of minor spiral rows between the major hair bearing rows in C. stelzneri. In C. tulumbensis sp. nov. the periostracal hairs are absent and the spiral rows are more scatter. In Clessinia pagoda periostracal structures consist of spiral rows bearing rounded to quadrate lamellae slightly imposed over each other. In Clessinia nattkemperi lamellae are spine-shaped with wider bases almost as a triangle. All Clessinia species have an interesting pattern of periostracal microsculpture in the space between spiral rows which is traversed by axial irregular microfolds cut by spiral or diagonal microribs forming an irregular net.Periostracal structures are particularly well developed in Anatomical information obtained on the pallial, digestive and reproductive systems of each species is described in the taxonomic section.Clessinia species (p < 0.001). The shell shape variation among the five taxa considered was successfully discriminated using CVA of the residuals from the regression of shape on centroid size. On the canonical axis 1 (CV1) , the main changes in shell shape are associated with the expansion of the base of spire and body whorl. Specimens of C. cordovana with high scores on CV1 have thinner whorls, whereas specimens of C. nattkemperi and C. pagoda with low scores, have both spire and body whorl more expanded. It also indicates that when shells are thinner they are also taller while shells more expanded are less tall. On CV2 axis  the main shell shape variation referred to the shape of the aperture and the degree of inclination of the suture before the aperture . Residuals from the regression of shape on centroid size were used in the analysis. On the CV1 , the main changes in shell shape are associated with the expansion of the base of spire and body whorl. Specimens of C. cordovana from Cerro de la Cruz and the area surrounding San Marcos Sierras, plus specimens of C. tulumbensis sp. nov. from Virgen de Fatima, Route 16 and Cerro Colorado have high scores on CV1, showing thinner whorls. Specimens of C. stelzneri from Cerro San Vicente, C. tulumbensis sp. nov. from route between Dean Funes and Tulumba, Tulumba and San José de la Dormida and specimens of C. cordovana from Sierra de Pocho have low scores, showing whorls of the spire and body whorl more expanded. On CV2 axis  the main shell shape variation is related with the shape of the aperture and the expansion of the first whorl of the shell. High scores in specimens of C. tulumbensis sp. nov. from all localities considered, except Cerro Colorado, indicate wider first whorls of the shell and a thinner aperture. Specimens of C. stelzneri and C. cordovana, with low scores, exhibit thinner fist whorl of the shell and more expanded central portion of the aperture. C. tulumbensis sp. nov. from Cerro Colorado have intermediate forms between both previous described groups. Analysis using landmarks in shell lateral views did not show significative differences among the species  or S. cuezzoae and C. pagoda , S. holmbergi, S. pervarians , C. cordovana, and C. gracilisBy using the concatenated dataset, the ABGD approach recovered six candidate species based on the distribution of the pairwise genetic distances with a maximum prior of intraspecific divergence of 0.035938 . The sam species , except . popana  were cluClessinia and Spixia and according to the principle of priority  the valid name of the taxon should be Clessinia Doering, 1875 which has priority over Spixia Pilsbry & Vanatta, 1894. In the following, we provide the taxonomic description and new systematic arrangement of the treated species.As a result of the anatomical studies performed, shell periostracum observations, shell geometric morphometrics and genetic analyses, and based on previous findings , we hereSuperfamily Orthalicoidea Martens, 1860Family Odontostomidae Pilsbry & Vanatta, 1898Clessinia Genus Bulimus “Clessinia”Bulimus “Macrodontes”–Scalarinella Doering–in Odontostomus –Cyclodontina (Clessinia)–Clessinia Doering–Odontostomus (Spixia) Pilsbry & Vanatta, 1898: 57 [new synonymy]Type species.Clessinia stelzneri .Definition. Shell fusiform to turritelliform. Protoconch with delicate axial ribs and spiral bands delimited by thin grooves. Shell with periostracal complex structures consisting on spiral rows bearing “hairs” or triangular, rectangular to quadrate lamellae. Few species lacking periostracal ornamentation. Last portion of body whorl with aperture with slightly reflexed peristome, some species forming a cornet detached from rest of shell body whorl with peristome thin and expanded. Body whorl microsculpture complex, consisting in microfolds forming an irregular net, which in some cases is expanded dorsally over shell cornet. Fourth to five inner apertural teeth forming a complex apertural barrier, except for one species with three teeth. Dorsal portion of shell body whorl with a medial marked notch corresponding to the basal lamella. Columellar lamella undulating, in some species L-shaped. Presence of a short penial sheath overlapping distal portion of the penis. Insertion of penial muscle at proximal penis or distal epiphallus. Vas deferens thin, running freely along penis and attached to penial retractor muscle.Diagnosis.Clessinia is one of the odontostomid groups showing most complex apertural teeth arrangements. Number of apertural teeth/lamellae ranges from three to five. Together with Plagiodontes  has a similar protoconch sculpture consisting on axial ribs and transversal grooves between ribs. It differs from Plagiodontes in general shell shape, showing thinner and taller spires and larger numbers of whorls plus strong differences in number of apertural teeth/lamellae. Species here redescribed plus the new species show a shell aperture detached from the body whorl and this character is not observed in the remaining species of Clessinia (former Spixia) and in no other odontostomid genus. Shell aperture shape varies from subcircular to subquadrate.Clessinia is found in dry habitats with its distribution area ranging from Argentina, Uruguay to Bolivia and Paraguay. Clessinia differs from Cyclodontina  , in the general shell shape because Pilsbrylia species have a fusiform, broader shell shape, and the presence of only two apertural teeth. On the contrary to Clessinia, Pilsbrylia species inhabit in humid forest.x, 1827)  in havinHabitat preferences. Species of Clessinia inhabit dry areas where rocky formations are frequently found among low xerophytic vegetation. Few species occur in Yungas ecoregion, but in transition zones with dryer forests. They usually live below rocks in contact to the ground, in rock crevices, or buried in soil under shrubs. Some species can be found glued to leaves in bushes. Clessinia nattkemperi is usually found attached to the surface of cactuses or under dead cactus in contact with soil.Species distribution  have a wider area of distribution in the Dry Chaco, Espinal and Monte ecoregions in Argentina  cordovanus–Odontostomus (Macrodontes) cordovanus–Odontostomus  cordovanus–Cyclodontina  cordovana–Cyclodontina (Clessinia) cordovanus–Cyclodontina (Clessinia) gracilisClessinia cordovana–Clessinia gracilis–Cyclodontina (Clessinia) gracilis–Type material. Lectotype SMF 10417a ; paralectotype SMF 10417b ; holotype Cyclodontina (Clessinia) gracilisType locality. Argentina, Córdoba province. “Pendiente Oeste de la Sierra de Aconjigasta, en las quebradas húmedas como la de la Mermela, de Jatan, del Nieve y más al sud cerca de Aguas de los Oscuros”  intercalated with two to three spiral rows, one of each bearing hairs wider at base and less tall Bulimus (Clessinia) stelzneriBulimus “Macrodontes” cordovanus var. stelzneri–Odontostomus  cordovanus var. stelzneri–Odontostomus  cordovanus stelzneri–Scalarinella  cordovana stelzneri–Cyclodontina  cordovanus stelzneri–Scalarinella  cordovana stelzneri–Clessinia cordovana stelzneri–Clessinia stelzneri–Type material. Lectotype SMF 10417/3a; paralectotypes SMF 26582 (1), SMF 26583 (2), SMF 325584 (4).Type locality. “… quebrada de Yatan .” According to DescriptionExternal features  cordovanus striatusClessinia cordovana–Cuezzo, Ovando & Miranda, 2013: 162 [partim].Type material. Holotype: IBN 883 (preserved in ethanol 96%) . Paratypes: IBS-Ma 311 (3 specimens); IBN 571 ; IBN 558  . Holotype Clessinia cordovana striata, MACN-In 9127.Type locality. Holotype: Córdoba, Tulumba department, Route 16, between Villa Tulumba and San José de la Dormida , October 21, 2017, Cuezzo MG and Dominguez E collectors. Paratypes: IBS-Ma 311, idem to holotype; IBN 571, Córdoba, Tulumba, R 16 , November 25, 2018, Cuezzo MG collector; IBN 558, Córdoba, Tulumba, Route 16 before reaching the town of Tulumba , November 24, 2008, Cuezzo MG collector.Etymology. The specific name, “tulumbensis,” is given in reference to Tulumba, the political department of Córdoba Province, Argentina, where the type of the new species was collected.Definition. Shell with marked wide axial ribs narrowly separated at regular spaces, traversed by major periostracal spiral rows with shallow continuous lamellae. Periostracal hairs absent. Lower columellar lamella in shell aperture deeply undulating. Proximal penis inner sculpture with undulating thin folds in a reticulated disposition and a short thick, medium pilaster. Medial portion with a pilaster of ⅔ the length of penis. Vagina almost the same length of the distal portion of penis.DescriptionExternal features  have been deposited in GenBank with accession numbers: MG963436 and MG963437 for COI; MG963462 and MG963463 for 16S-rRNA, and MH789460 and MH789461 for ITS-2.Remarks. The new species, Clessinia tulumbensis sp. nov. include Clessinia cordovana striata , Córdoba, Quilpo, 2-3/IV/1967. Cichero-Biraben Leg. Hylton Scott det., H: 22.2–19.8 (mean = 22.5); Hap: 7.4–6.6 (mean = 7.04); Dap: 5.9–4.4 (mean = 5); Dm: 8.4–6.5 (mean = 7.3); IFML-Moll 14239, Córdoba, Quilpo, 5/4/1967, Birabén-Cichero leg.; MLP-Ma 11077, Córdoba, Quilpo (paratype); SMF 220916/2a.Type locality. Argentina, Córdoba, Cruz del Eje department, Sierra Chica de Córdoba, Quilpo.DescriptionExternal features  nattkemperi–Clessinia nattkemperi–Type material. Holotype MACN-In 25713; Paratypes MACN-In 25713-1   .Type locality. Argentina, Catamarca, Esquiú department, Pomancillo, 23 km from San Fernando del Valle de Catamarca, F. Nattkemper leg. July 1943.DescriptionExternal features  and C. tucumanensis .Clessinia in 1875, he mentioned that the number of plaques of the jaw would be the best character to differentiate the new created genus from Odontostomus, Plagiodontes, and Spixia. However, the number of plaques in Clessinia overlaps with the ones present in species of other genera, so by itself this character is not enough for genera differentiation.Traditional used characters for taxonomic diagnosis in Odontostomidae, such as the shell morphology, provide important information for species identification, but due to their intraspecific variability they should be carefully considered. When Doering described cordovana-group is formed by three species, C. cordovana, C. stelzneri, and C. tulumbensis sp. nov. The shell in C. tulumbensis sp. nov. has prominent, well-marked ribs, more raised than in C. cordovana and C. stelzneri. C. cordovana and C. tulumbensis sp. nov. have thinner shells than the other species. Among the cordovana species complex, the shape of the lower columellar lamella is deeply undulating around the columellar axis in C. tulumbensis sp. nov. and straighter in C. cordovana and C. stelzneri, thus showing to be a good character for species identification. The periostracum is of special taxonomic interest because it bears distinct microscale architectures. However, where and how these structures are formed is yet unknown for the majority of the species  and 19.3% (K2P distance) and from C. tulumbensis sp. nov. by distances ranging between 17% (p distance) and 19.8% (K2P distance), thus suggesting that C. cordovana is a different species from C. stelzneri and C. tulumbensis sp. nov. Regarding pairwise interspecific divergence between C. stelzneri and C. tulumbensis sp. nov., the greatest genetic distances we found are less than 2% (1.1–1.8%). Similarly, based on the COI locus we found that C. cordovana differs from C. stelzneri by genetic distances ranging between 14.3% (p distance) and 17% (K2P distance), and from C. tulumbensis sp. nov. by divergences ranging between 14.9% (p distance) and 19% (K2P distance), while distances between C. stelzneri and C. tulumbensis sp. nov. ranged between 1.3% and 4.1%. As with the ribosomal marker, these COI-based values suggest that C. cordovana represent a different species from C. stelzneri and C. tulumbensis sp. nov.Molecular studies performed with the nuclear marker, revealed that the Clessinia satisfied the criteria to be considered different evolutionary genetic species, allowed us to recognize C. cordovana, C. nattkemperi, and C. pagoda as distinct evolutionary genetic species. However, the results from ABGD and K/θ approaches failed to recognize the morphological diversity between C. stelzneri and C. tulumbensis sp. nov., and the phylogenetic trees did not support the presence of separate species for both morphological species. The trees showed C. stelzneri as paraphyletic with respect to C. tulumbensis sp. nov., with DNA sequences of C. tulumbensis sp. nov. nested among those of C. stelzneri. Furthermore, the distance values obtained between C. stelzneri and C. tulumbensis sp. nov. are relatively low. This low genetic divergence suggest that the two species have evolved relatively recently, with C. tulumbensis sp. nov. having evolved within C. stelzneri, and would explain the reduced resolution of species boundaries detection using genetic data, as both ABGD and K/θ approaches are known to fail in cases of very recent speciation  is where C. cordovana is very abundant. Moreover, Quilpo is distant by a few kilometers from San Marcos Sierras where we have found specimens with more marked axial ribs. Based on COI sequence, C. gracilis groups together with C. cordovana in our phylogenetic trees, with genetic distances ranging between 7.1% (p distance) and 8.9% (K2P distance) from our C. cordovana specimens, and recognized as a different species by the ABGD analysis. Therefore, the identity of what C. gracilis should be tested using morphological information to confirm the correct taxonomic identification, whose morphological differentiation might imply the recognition of C. gracilis as a valid species. Regarding C. nattkemperi and C. pagoda, we found genetic distances greater than 10% in relation to the other Clessinia species, and both are recognized as different genetic species according to ABGD and K/θ approaches. However, a finding in need of further investigation is the close relationship we obtained in our phylogenetic analyses between C. pagoda and S. cuezzoae, which exhibits very different morphologic features (16S-rRNA locus and ranged from 4.8% (p distance) to 5.4% for the COI marker.and size . Nonethefeatures , but whoClessinia and Spixia. On the basis of morphological evidence Spixia and Clessinia are easily externally distinguished because former Clessinia species have the aperture detached from the body whorl forming a cornet, periostracal microsculpture extended over dorsal portion of the peristome, presence of five inner teeth on the shell aperture instead of the three–four in Spixia. However, there are several other morphological similarities among species of both genera in general shell shape, type of periostracum microsculpture, reproductive anatomy, presence of a pallial gland, besides the overlap in geographic ranges. Molecular evidence suggest that taxa placed in former genera Spixia and Clessinia belong to the same group, and both genera as currently known until now would be paraphyletic , is still only known by its shell description with no information on its internal anatomy and molecular identity. Nonetheless, based on the available evidence, Clessinia and Spixia are synonymous, and according to the principle of priority  the valid name of the taxon should be Clessinia Doering, 1875 which has priority over Spixia Pilsbry & Vanatta, 1894.The results obtained here have taxonomic implications at the genus level for phyletic . MoleculClessinia are endemic to the Chaco Serrano subecoregion, restricted to small patches between the Semiarid Chaco and Yungas ecoregion in north-central Argentina.Studied species of Clessinia provided useful information for species identification. The aperture, with a continuous peristome, is detached from the body whorl. The apertural teeth are five, always located in the interior of the cornet mostly with no relation to the peristome. The periostracum, a layer that produces a conspicuous ornamentation in Clessinia species has taxonomic importance for the identification of the species. Each species has a particular periostracal structure of hairs of different length and densities and lamellas of different shape and sizes.The shell in Clessinia cordovana-complex by three species, Clessinia cordovana, Clessinia stelzneri, and Clessinia tulumbensis sp. nov. Shell geometric morphometrics support our results on the species composition of the cordovana-complex but cannot be used alone as a single source of information for species identification.Morphological and molecular evidence support the composition of The genitalia and jaw are also useful sources of characters for species identification. Inner penial sculpture, comparative length of flagellum, proportion of epiphallus respect to penis, the attachment of the vas deferens to a thick penis muscular retractor are the most conspicuous characters.Clessinia and Spixia are synonymous, and according to the principle of priority  the valid name of the taxon should be Clessinia Doering, 1875 which has priority over Spixia Pilsbry & Vanatta, 1894.Based on our current analyses, and on previous findings  Clessini10.7717/peerj.5986/supp-1Supplemental Information 1Clessinia .Coordinates of the landmarks in two dimensions, digitized with Tpsdig 2, selected in lateral view of the shell of the species of Click here for additional data file.10.7717/peerj.5986/supp-2Supplemental Information 2The posterior-probability values for BI and bootstrap values for the ML tree are shown above the branches. Numbers within groups are GenBank accession numbers.Click here for additional data file.10.7717/peerj.5986/supp-3Supplemental Information 3Material from different malacological collections studied. Abbreviations: IBN, Instituto de Biodiversidad Neotropical, Tucumán, Argentina; IFML-Moll, Instituto-Fundación Miguel Lillo, Tucumán, Argentina; MACN-In, Museo Argentino de Ciencias Naturales, Buenos Aires, Argentina.Click here for additional data file.10.7717/peerj.5986/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.5986/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.5986/supp-6Supplemental Information 6Clessinia species considered in this study. Coordinates used in the Analysis of Canonical Variation (CVA) in MorphoJ.Coordinates of the 7 Landmarks in two dimensions, digitized with Tpsdig 2, selected in side view of the shell of all the Click here for additional data file.10.7717/peerj.5986/supp-7Supplemental Information 7Clessinia species considered in this study. Coordinates used in the Analysis of Canonical Variation (CVA) in MorphoJ.Coordinates of the 14 Landmarks in two dimensions, digitized with Tpsdig 2, selected in ventral view of the shell of all the Click here for additional data file.10.7717/peerj.5986/supp-8Supplemental Information 8Clessinia cordovana species complex , from different localities. Coordinates used in the Analysis of Canonical Variation (CVA) in MorphoJ.Coordinates of the 14 Landmarks in two dimensions, digitized with Tpsdig 2, selected in ventral view of the shell of the species of Click here for additional data file.


Background: Systemic lupus erythematosus (SLE) is a potentially fatal complex autoimmune disease, that is characterized by widespread inflammation manifesting tissue damage and comorbidities across the human body including heart, blood vessels, joints, skin, liver, kidneys, and periodontal tissues. The etiology of SLE is partially attributed to a deregulated inflammatory response to microbial dysbiosis and environmental changes. In the mouth, periodontal environment provides an optimal niche for local and systemic inflammation. Our aim was to evaluate the reciprocal impact of periodontal subgingival microbiome on SLE systemic inflammation.Methods: Ninety-one female subjects were recruited, including healthy (n = 31), SLE-inactive (n = 29), and SLE-active (n = 31). Patients were screened for probing depth, bleeding on probing, clinical attachment level, and classified according to CDC/AAP criteria with or without periodontal dysbiosis. Serum inflammatory cytokines were measured by human cytokine panel and a targeted pathogenic subgingival biofilm panel was examined by DNA-DNA checkerboard from subgingival plaque samples.Results: The results showed significant upregulation of serum proinflammatory cytokines in individuals with SLE when compared to controls. Stratification of subject's into SLE-inactive (I) and SLE-active (A) phenotypes or periodontitis and non-periodontitis groups provided new insights into SLE pathophysiology. Ten proinflammatory cytokines were upregulated in serum of SLE-I only and one in SLE-A only. Four molecules overlapped in SLE-A and SLE-I. Anti-inflammatory cytokines included IL-4 IL-10, which were upregulated in SLE-I sera (but not SLE-A), controlling clinical phenotypes. Out of 24 significant differential oral microbial abundances found in SLE, 14 unique subgingival bacteria profiles were found to be elevated in SLE. The most severe oral pathogens (Treponema denticola and Tannerella forsythia) showed increase abundances on SLE-A periodontal sites when compared to SLE-I and healthy controls. Inflammation as measured by cytokine-microbial correlations showed that periodontal pathogens dominating the environment increased proinflammatory cytokines systemically.Conclusions: Altogether, low-grade systemic inflammation that influenced SLE disease activity and severity was correlated to dysbiotic changes of the oral microbiota present in periodontal diseases. The human microbiome is in constant interaction with the host, modulating health and disease phenotypes. We now appreciate that oral bacteria plays indispensable roles in shaping the systemic host physiological landscape and in dysbiosis. As part of the upper digestive tract, the oral cavity presents specific niches, such as gingival sulcus and gingival crevicular fluid (GCF), which in turn harbor commensal and pathogenic bacteria with potential impact to oral and systemic health. Through local activation of inflammation, oral pathogens have shown to worsen the burden of chronic diseases through time including, type 2 diabetes, premature labor, rheumatoid arthritis, systemic lupus erythematosus (SLE), Alzheimer's, cardiovascular conditions, and cancer –4.Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola  is a multisystem autoimmune disease with increasing incidence worldwide . The LupStudies have suggested that specific cytokines  were increased while others are decreased  in either GCF or serum of SLE subjects –22, and In this study we have investigated SLE phenotypic differences, oral microbiota associations with systemic inflammation to unravel forces shaping oral and broader systemic health. As such, we compared the subgingival microbial signatures from SLE and healthy controls to investigate host-microbial relationships. Specifically, we first sought to establish if specific microbial compositions correlated to periodontal clinical phenotypes with systemic levels of inflammatory cytokines. The presence of periodontal disease was assessed using categories of local inflammation, clinical attachment levels and bone loss, while SLEDAI > or <2 was assessed to categorize the individuals into SLE-active, SEL-inactive. The results indicate that chronic periodontitis and SLE present low-grade inflammation modulated periodontal diseases and specific microbial signatures. The association of subgingival microbial profiles with SLE and its association with periodontal clinical status and inflammatory markers established novel links developing a new framework for oral-systemic studies.Subjects were recruited at the Center of Rheumatology at the University Hospital in Brasília from July 2013 to March 2014, and samples were obtained with informed consent of the subjects. The diagnosis of SLE was initially made by the primary care physician and confirmed by rheumatologist specialist following guidelines of the American Academy of Rheumatology (according to ACR 1982/1997 revised classification criteria) , 26. Thin = 29), and inactive . The control group was composed of 31 systemically healthy women recruited at the University Hospital in Brasília during the same time period. All patients answered a health questionnaire investigating medical and dental history. A visual investigation of the oral cavity was performed at enrollment to investigate the number of remaining teeth, other oral diseases and periodontal health.SLE activity and diagnosis was investigated according to SLEDAI classification . SLE actAfter recruitment of subjects, participants were referred to the Dental School of Brasília  for clinical periodontal examination and sample collection. The clinical periodontal examination was performed by a single trained examiner before the collection of subgingival plaque and blood samples prior to oral and non-surgical periodontal treatment. The following parameters were investigated by using a UNC-15 millimeter periodontal probe at 6 sites/tooth: pocket probing depth (PD), clinical attachment level (CAL), and gingival bleeding index . Plaque Subgingival plaque was collected prior to any treatment for all subjects, including non-surgical periodontal treatment. Subgingival plaque samples were collected from four sites showing the deepest probing depth in each patient. The area was isolated with cotton rolls to prevent contact with saliva and air dried. Supragingival plaque was removed with Gracey curettes previous to subgingival plaque collection. The subgingival plaque was retrieved by the introduction of PerioPaper strips into gingival sulcus for 30 s and stored in sterilized tubes at −80°C till processing. Blood samples were collected after routine examination for SLE monitoring. Control subjects were asked to perform similar examinations at the Hospital. PerioPaper strips were centrifuged at 3,000 × g for 15 min at 4°C in PBS elution for the dosage of the tissue destruction and bone resorption markers by Multiplex . The eluted samples were stored at −80°C up to processing.DNA-DNA checkerboard hybridization was performed using the method developed by Socransky et al. . Brieflyhttp://dorresteinappshub.ucsd.edu:3838/clusterMetaboApp0.9.1/) and visualized via EMPeror software  on the binary Jaccard distance metric using ClusterApp tool (available at software . Pairwishttp://itol.embl.de/) , and were aligned using ClustalW software within Molecular Evolutionary Genetics Analysis (MEGA) software version 7 . The phymbl.de/) .Whole blood serum collected from the subjects were immediately stored at −80°C. ProcartaPlex Immunoassays include a platform which allows for detection up to 100 proteins simultaneously through magnetic beads. A panel of 20 inflammatory cytokines were measured in duplicate by ProcartaPlex Human Inflammation Panel 20-plex  on a Luminex 200 System  as previously described  and adapp-values were set at 5% threshold. For binary abundances we dichotomized the relative abundances data into presence  and absence  binary values. We used a logistic regression model to analyze the relationship between the binary abundance data and two clinically important categorical covariates: SLE group and periodontal condition  ondition . For thep < 0.05. For visual simplicity, we showed only the significant correlations. Topological parameters were analyzed by importing the pairwise correlations using network analyzer algorithm within Cytoscape, 3.7.1. Hierarchical clustering was applied on the relative abundance data of species in each sample based on Spearman rank correlation and the heatmap was generated using ggplots package in R.Potential associations between microbial species and cytokine abundances were evaluated by Spearman's rank based correlations. Spearman's correlation coefficients were calculated using R package psych, and significant correlations were plotted with R package corrplot. Pairwise correlations were computed separately for control and SLE samples. To avoid false positives, we selected correlations with adjusted p = 0.045) than SLE-A patients which are considered acute cases, and were younger (32.58 ± 8.9 years) than controls (37.42 ± 8.72 years), as well. SLE-I individuals showed increased clinical attachment loss (CAL) and worsen periodontal clinical outcomes (8.31 ± 9.438) than SLE-I and healthy controls, but with no significant differences between groups, except for bleeding on probing, which was significantly reduced in both SLE-A and SLE-I compared to controls. All periodontal patients presented localized, chronic periodontitis, according to the classification proposed , SLE-inactive (n = 28), and SLE-active (n = 31) groups; we performed a collection of samples by probing the gingival site with PerioPaper strips, following elution. After hybridization with specific periodontal pathogen probes, DNA-checkerboard revealed pathogen signatures and abundances. A principal coordinate analysis (PCoA) on the Jaccard distances was generated taking into consideration of periodontal status stratification , purple complex , orange complex , and yellow complex . Additionally, Capnocytophaga ochraceae, C. gingivalis, Prevotella nigrescens, and Selenomonas noxia showed significant differences in their relative abundances among SLE and control subjects.We next sought to understand the underlying microbial associations among SLE subjects and their clinical phenotypes. To investigate the subgingival plaque bacterial composition, we thus applied Wilcoxon rank-sum test on two datasets related to SLE phenotypes. The first dataset was based on relative abundance and the second dataset was based on logistic regression model binary abundances (presence/absence) to evaluate prevalence. Based on the relative abundance profiles, and the hierarchical clustering using Spearman's rank correlation coefficients, we identified two distinct groups of bacterial species in SLE individuals with and without periodontitis when compared to their respective controls , 3A. Intp-values listed in C. ochracea, F. nucleatum, S. sanguinis, A. gerencseriae, T. denticola. When comparing SLE-A vs. SLE-I subjects, four signatures were significant including, C. ochracea, S. intermedia, A. odontolyticus, F. polymorphum. Whereas, in the periodontitis subjects, SLE-I vs. healthy controls showed three significant microbial changes A. gerencseriae, S. oralis, S. noxia. An increase to seven significant microbial abundance changes were observed on SLE-A vs. healthy controls comparison, including, C. ochracea, F. nucleatum, P. nigrescens, S. sanguinis, C. gingivalis, T. forsythia, S. gordonii. Finally, when comparing SLE-A vs. SLE-I subjects, five signatures were significantly different including, C. ochracea, P. nigrescens, A. gerencseriae, S. oralis, and T. forsythia. While the long-term chronically inflamed SLE-I subjects showed higher abundances of periodontal pathogens when compared to SLE-A, SLE-I only showed significance difference to healthy controls in periodontitis groups. Overall the results suggest that the presence of periodontitis drove most of significant microbial differences among the SLE systemic status comparisons .When further stratifying the SLE comparisons according to periodontal status, the results showed that systemic comparisons were significantly different according to periodontal status . A totalC. gingivalis, S. gordonii, P. nigrescens, C. ochracea, F. nucleatum, and S. sanguinis were significantly reduced on SLE subjects . While four unique bacterial species  were significantly abundant only in non-periodontitis subjects, six species were unique in periodontitis subjects  of ividuals . These fAt the center of autoimmune pathogenesis, this study provides evidence that inflammatory response specific to oral bacteria controls not only the disease severity, but also the magnitude of the SLE disease . The datp = 0.01, A more distinct host cytokine dysregulation was associated with SLE subjects when compared to healthy controls. The data presented herein show no evidence that SLE-active subjects had higher serum concentrations of pro- and anti-inflammatory cytokine. But, both SLE groups showed higher expressions than healthy control. Interestingly IL-10 which is a anti-inflammatory cytokine response was found increased in SLE-I inactive when compared to active and controls  in the SLE states, suggesting positive association among cytokines are an important driver of SLE pathogenesis . We did Functional inflammation data at the host and microbiota levels are necessary to make assumptions on ecological systems from the oral cavity and its influences on systemic disease phenotypes. The data presented here generate additional questions about the extent to which detrimental and protective microbiome-related features are innately acquired and significantly modulated by the host systemic conditions. Overall the results offer a new perspective on the host-microbial interactions in lupus disease and periodontal pathogenic environments. Consequently, these results favor the hypothesis of oral-systemic inflammation linking both conditions. Moreover, the realization that there remain high anti-inflammatory profiles on subjects with inactive disease when compared to active, offer a mechanistic hypothesis to the lack of disease comorbidities of this suppressed condition, but the host is still able to produce low-grade inflammation systemically. In future studies using next generation sequencing strategies of highly diverse SLE subjects, it will be possible to map the microbial ecology of multiple sites of the human body, including oral sites. As the science of host-microbes is advancing, highly individualized, rather than simple binary distinctions of healthy and disease or single-microbe etiology across populations, might be found .The results presented here show that oral-systemic pathogenic burden is evident in lupus subjects. These data are also consistent with complex ecological interactions among multiple human chronic diseases . Among tAll datasets generated for this study are included in the article/This research project was approved by the Bauru Dental School-USP Ethics Committee of Research (protocol # 111.718) and by the Ethics Committee of Research at the Forsyth Institute (IRB #037). The patients/participants provided their written informed consent to participate in this study.AS and MF conceived and designed the experiments. LP, LN, DN, and TY performed the experiments. YZ, WL, and GA analyzed the data. AS, MF, and KN contributed reagents, materials, and analysis tools. MF, AS, and SC wrote the paper.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.


Reticulitermes grassei and the data obtained was compared with sequence surveys of three other Reticulitermes species. Each Reticulitermes species had a significantly different bacterial gut microbiota (pairwise significance tests using the Kolmogorov-Smirnov test), but a similar pattern of distribution (P-test in weighted Unifrac). The core gut microbiota from the analyzed Reticulitermes species contained 16 bacterial operational taxonomic units. Enzymes (KO) were detected from 14 pathways related to carbohydrate metabolism. R. grassei and R. hesperus, based on relative abundance of KO, had the most similar carbohydrate pathway patterns. In addition, we described the gut microbiota and functionality pathways in R. grassei after a 7-day dietary shift and antibiotic (ciprofloxacin) treatment. Both factors, but above all the antibiotic, altered the relative abundance of certain microbial groups, although the changes were not statistically significant (P-test in weighted Unifrac). The cellulose diet enhanced the carbohydrate pathways related to propanoate, butanoate, ascorbate, and glyoxylate metabolism. The antibiotic treatment affected galactose metabolism, the citrate cycle and inositol phosphate metabolism. Those functional changes may be related to changes in the abundance of several bacterial groups. Our findings provide insights into the stability of the gut microbiota in R. grassei and a resilience response to dietary shift or antibiotic treatment disturbance after 7 days.Gut microbial structure in animals depends on the host, dietary habits and local environment. A random event, dietary change or antibiotic treatment may alter the gut environment with possible repercussions for the bacterial community composition and functionality and ultimately host fitness. The present study was focused on the composition, structure and functionality of gut microbiota in  Higher termites, the family Termitidae, have lost their gut protists, having only bacteria [Termites are unique among social insects because they undergo incomplete metamorphosis and display a diversified caste polyphenism . The cas period) . Six fambacteria . Termitebacteria . Gut micbacteria , 10.Reticulitermes grassei (a subterranean termite native to the Iberian Peninsula and southwest France) [Reticulitermes (Rhinotermitidae) is a Holarctic genus of subterranean termites widespread in Western Europe, North America (in the USA), and in Eastern Asia (mostly in Japan and China). Recent analysis  showed that diversification of the genus occurred during the early Miocene, about 18.4 Myr ago, later than the Reticulitermes fossils found in Baltic amber deposits with an estimated age of ~38 Myr (late Eocene) [Reticulitermes presents traits that can vary among the species, especially in two aspects: (i) social organization, such as reproductive systems and colony breeding structures [The present study focused on the microbial composition, structure and functionality of gut microbiota in  France) . Reticul Eocene) . The genructures , 13 and ructures , 15.R. grassei were compared with sequence surveys of other Reticulitermes species [R. grassei after a 7-day dietary shift and antibiotic (ciprofloxacin) treatment, after which the changes in microbiota were not significiant enough to cause dysbiosis.The data obtained from  species , 17 in oR. grassei during a 7-day ciprofloxacin (antibiotic) treatment. The findings reported herein provide insights into the dynamic stability of the gut microbiota in R. grassei and resilience response to diet and antibiotic perturbation.Diet is considered one of the most important environmental factors that influences the assembly of gut microbiota –20. Our Reticulitermes grassei workers were obtained from their natural environment, a forest composed mostly of pines and, to a lesser degree, holm oaks in the Serra del Corredor . Serra del Corredor is not a protected area. It is not necessary specific permission for work in the area, and the field studies did not involve endangered species. Samples were collected from a colony in a pine stump. In the laboratory, three groups of ca. 60 workers from the same colony were placed in 3.5-cm Petri dishes containing the respective diet and antibiotic treatment: (i) natural diet  (Rg_1); (ii) pieces of sterilized filter paper, size of 10 mm2 (Whatmann grade 4) (Rg_2); and (iii) pieces of sterilized wood + ciprofloxacin (antibiotic) (Rg_3). The antibiotic treatment consisted of moistening the wood pieces with 200 μl of ciprofloxacin at a concentration of 1 mg/ml, on alternate days. Termites in treatments 1 to 3 were maintained at room temperature for 7 days, after which 50 individuals from each treatment were dissected. Termite survival after the cellulose diet and ciprofloxacin treatment was similar to that of the control samples, although we have previously observed that a longer stay in the Petri dish causes termite death, probably due to the low humidity environment.For each group, DNA extraction was performed in triplicate on samples each containing 16 termite guts. Guts were removed using sterilized forceps and placed on TE buffer in Eppendorf tubes for DNA extraction. The whole guts were homogenized using a FastPrep system  with 0.1-mm glass beads. Bulk DNA was extracted by several washings with phenol-chloroform. All material and solutions were sterile. Dissection and extraction were carried out in an aseptic environment (laminar flow cabinet) under a laminar hood to avoid contamination . We prep5´-GAGTTTGATCCTGGCTCAG-3´ and 5´-TGCTGCCTCCCGTAGGAGT-3´). PCR conditions and purification were performed as in Berlanga et al. (2017). Pyrosequencing coverage (depth sequencing) resulted in 170,134 total raw reads that after quality control processing resulted in 30,751 reads  for the R. grassei samples. Data deposition: Bioproject PRJNA482509.We performed amplicon sequencing of the bacterial 16S rRNA gene. The primers used for multiplex Roche 454 GS FLX pyrosequencing were the universal bacterial nucleotide sequences for the region V1–V2, 8F-338R  [http://qiime.org/scripts/compute_core_microbiome.html) [Taxonomy was assigned by the Silva database (ilva.de) . Alpha ailva.de) . Core mime.html) . Core OThttp://www.microbiomeanalyst.ca) was used [Functionality was predicted from the 16S rRNA data using PICRUSt and Tax4Fun. PICRUSt analysis was performed by the predict_metagenomes.py script run against the functional database of KEGG Orthology . Functiowas used .R. grassei  (this work). In addition, previously published data were used to determine microbiota diversity and functionality of the intestinal tract of several species of Reticulitermes: R. flavipes_1  [R. flavipes_2 , R. hesperus  and R. virginicus (Florida) (Bioproject PRJNA238270) [Reticulitermes species  were not included in the comparative study because no database using the variable region V1-V3 and the same sequencing platform was found.Diversity and functionality analyses were performed in A172449) , R. flavR. grassei (Rg_1) and R. flavipes_1, the diet consisted of pine wood (Pinus sp.), although in R. flavipes_1 sterile wood diet experiments were also performed for six weeks in the laboratory. For the others, the diet was also wood from the pine family or other conifers, e.g., R. hesperus was sampled in Galiano Island (Canada), where forests of cedars (Cedrus sp.) and Douglas firs (Pseudotsuga menziesii) are predominant.For Reticulitermes species , the most abundant bacterial phyla were Spirochaetes , Elusimicrobia , Bacteroidetes , Firmicutes  and Proteobacteria   . Sequenchesperus .Reticulitermes species, including the two studied samples of R. flavipes. To determine which taxa groups differ from each pairwise Reticulitermes species, extended error bar plots were generated to indicate statistically significant features (P < 0.05) [R. grassei that were significantly different with respect to R. flavipes were Spirochaetaceae, Dethiosulfovibrionaceae, Porphyromonadaceae, unclassified Clostridiales, unclassified Bacteroidales and Synergistaceae, the first two being more abundant in R. grassei than R. flavipes. Differences in family proportion between R. grassei and R. hesperus were in Spirochaetaceae, Dethiosulfovibrionaceae, Ruminococcaceae. The comparison between R. grassei and R. virginicus showed similar microbiotas to R. hesperus with the addition of Endomicrobia. In the case of R. flavipes_1 and R. flavipes_2, the differences were significant due to the unassigned sequences of Endomicrobia, Porphyromonadaceae. Mycoplasmataceae and unclassified Rickettsiales (Pairwise significance tests using the Kolmogorov-Smirnov test based on representative OTUs showed significant differences between  < 0.05) . Relativttsiales .Reticulitermes species support a highly diverse community of bacteria, comparable with other wood- or herbivorous-feeding insects [Reticulitermes species except for R. flavipes_1   .Reticulitermes species, indicating meaningful relationships or associations between taxa. Elusimicrobia presented positive interactions with Firmicutes, Proteobacteria and several bacteria from the unclassified group. Bacteroidetes showed strong positive correlation with Fusobacteria. Spirochaetes showed strong negative correlation with Proteobacteria, negative correlation with Firmicutes, but positive correlation with Synergistetes.Reticulitermes species had different bacterial gut microbiota, but a similar pattern of distribution (beta-diversity). We were interested in finding the conserved or permanent microbiota in Reticulitermes by characterizing the core microbiota, which could reflect which microbiota coevolved with the Reticulitermes host before the species diversification. Core OTUs were determined by the shared OTUs at 90% in all samples. Core gut microbiota from Reticulitermes species contained 16 bacterial OTUs: unassigned bacteria (1 OTU), Verrucomicrobia (1 OTU), Plantomycetes (1 OTU), Tenericutes–Mycoplasmataceae (1 OTU), Betaproteobacteria (1 OTU), Bacteroidetes (1 OTU), Firmicutes–Clostridia (2 OTU), Alphaproteobacteria (2 OTU), Synergistetes (2 OTU), and Spirochaetes (4 OTU).As mentioned, every The nearest sequence taxon index (NSTI) value is a measure of how closely related the OTUs in each sample are to the reference genomes in the database. In our case, NSTI values per sample were in the range of 0.070–0.181. The taxonomic classification was accurate at the family level, and in several cases at genus level, but few OTUs could be classified at the species level. This result could explain the NSTI values obtained. The identified biological processes are essential for sustaining prokaryotic life in the environment, and they include genetic information functions , cellular processes such as cell motility (5.8–6.9%), genes related to membrane transport (16.45–18.8%) and metabolic functions (55.4–59.9%). Of all the metabolic process genes detected, those associated with carbohydrate metabolism were the most abundant . We focuReticulitermes species, extended error bar plots were generated to show statistically significant differences P < 0.05 (31)  . We dete pattern . Principrginicus . These rP-test in weighted Unifrac P > 0.05). (Alpha diversity (Shannon index) in the wood diet was slightly higher than in the cellulose diet and antibiotic treatment . Cellulo> 0.05). .R. grassei, we observed that the cellulose diet rendered an increased proportion of Actinobacteria, Firmicutes, Bacteroidetes, and Synergistetes and a reduced abundance of Spirochaetes (e.g. Treponema primitia), Endomicrobia and Delta protoeobacteria . The antibiotic treatment was associated with decreased proportions of Synergistetes (TG5) and Bacteroidetes , with a variable effect on Spirochaetes depending on the OTUs. For example, the proportion of Treponema primitia decreased, whereas other Treponemes increased. A significantly higher relative abundance was observed in Endomicrobia and Deltaproteobacteria (e.g. Desulfovibrio) (When comparing the pine and cellulose diet in ovibrio) .R. grassei consisted of 46 OTUs across 100% of samples  belonging to: Actinobacteria , Bacteroidetes (Candidatus Azobacteroides and Dysgonomonas), Elusimicrobia (Endomicrobia), Firmicutes (Lactococcus), Proteobacteria , Spirochaetes (Treponema), Synergistetes (TG5), Tenericutes (Mycoplasmataeae), and unclassified bacteria. Of these 46 OTUs, seven belonging to Spirochaetes, Synergistetes, Tenericutes, and unclassified bacteria were conserved in other Reticulitermes species.Core microbiota in The NSTI values per sample calculated by PICRUSt were in the range of 0.070–0.112. Results of the two functional assignment strategies showed differences in functional categories as determined by KO genes. Tax4Fun detected 15.3% more KO than PICRUSt. However, when we discarded the accounts contribution by sample < 0.0001, Tax4Fun identified 4.7% more KO than PICRUSt . It is iWe focused on carbohydrate metabolism (pathways associated with lignocellulose and cellulose feeding) , diet-reReticulitermes) harbors a complex microbial community consisting of protists and bacteria. Many protist species are not necessarily restricted to one termite species and their abundance can vary [Metanobrevibacter) [Reticulitermes is wood, a complex material composed mainly of cellulose, hemicellulose and lignin  along with other complex carbohydrates [Reticulitermes genus has a “foraging lifestyle”, leaving the nest to search for food [The host , diet and local environment shape the gut microbial structure , 37. Thecan vary . They macan vary , and endibacter)  and spiribacter) . Therefoibacter) , 17. Thehydrates . The lighydrates , and couhydrates . Wood dihydrates , and bachydrates . Reticulfor food , so expoReticulitermes species . Nevertheless, we observed that the four Reticulitermes species analyzed shared a similar distribution pattern (beta-diversity), and principal coordinates analysis and P-test in weighted Unifrac indicated that the microbial communities were not significantly different (P > 0.05)  from the Palearctic lineages  [Reticulitermes species from North America, R. virginicus is the most distant lineage from R. grassei (a representative Reticulitermes species from Western Europe), followed by R. flavipes. R. hesperus is the most related lineage to R. grassei [R. grassei being most closely related to that of R. hesperus  Figs , S1 Fig.05) Figs . As an ern Asia) . Among t grassei , which ihesperus , S1 Fig.Reticulitermes  were related to the host phylogeny and the lignin contents of the diet (pine or coniferous tree wood) collected from their natural habitat [The results indicate that the gut bacterial community profiles in  habitat . The tra2–H2 gradient, and intermediate metabolites, so shifts in the bacterial community are also expected [Different diets may change the gut environment, affecting factors such as the C/N ratio, Oexpected . Less coexpected . An increxpected , 51. ChaR. grassei, which in turn could affect the abundance of protist-associated bacteria. We observed a reduced abundance of Endomicrobia and Desulfovibrio (symbiont microorganisms associated with protists). The Endomicrobia class provides protists and the insect host with vitamins and amino acids [Desulfovibrio retains biosynthetic pathways for various amino acids and cofactors [R. grassei was fed with cellulose, the protists Microjoenia hexamitoides and Pyrsonympha sp. increased in abundance, suggesting they intervene in cellulose degradation and displace other protists populations such as Trichonympha agilis [Trichonympha agilis permanently hosts two symbiotic bacteria, ‘Candidatus Endomicrobium trichonymphae’ and ‘Candidatus Desulfovibrio trichonymphae’, which have a mutualistic relationship [Treponema), especially Treponema primitia. Treponema is responsible for the transformation of H2 and CO2 into acetate, the main source of carbon and energy for termites. Treponemes encode glycoside hydrolases, which are important enzymes in hemicellulose and lignin degradation [Our results confirm previous reports that microbial communities are sensitive to diet , 21, andno acids . Desulfoofactors . When R.a agilis . The protionship . Thereforadation .R. grassei (P-test in weighted Unifrac P > 0.05),  and Bacteroidetes such as Azobacteroides and Dysgonomonas, and an increasing relative abundance of Elusimicrobia (Endomicrobia) and Desulfovibrio  and metronidazole (antiprotozoan) [Ciprofloxacin is a fluoroquinolone with a broad antibacterial spectrum, although fluoroquinolones show limited activity against anaerobic bacteria . They in grassei  and the  grassei . However> 0.05), . Treatmefovibrio . A higheflavipes . We did otozoan) . Again, Azobacteroides pseudotrichonympha, which provide amino acids and cofactors for the nutrition of the protist and termite hosts [The functional effect of ciprofloxacin treatment may have induced changes in several bacterial groups, such as a decreased proportion of Bacteroidetes, Synergistetes and Spirochaetes. The antibiotic seemed to affect the xylene degradation, nitrogen fixation and the generic pathway for xenobiotic biodegradation –S7 Figs.te hosts . We obsete hosts .A random event (dietary change or antibiotic treatment) may alter the environment with possible repercussions on the community composition. Understanding the drivers of microbial community stability is important for predicting the community response to disturbance, which may include resistance  or resilience  or function (genes)) , 67. AltR. grassei was observed. A multiplicity of treatments over time can erode the resilience of the community [The 7-day period of dietary shift and antibiotic treatment was not long enough to cause a significant change in microbiota and general function, but a dynamic equilibrium in the bacterial gut community of ommunity . A distuThe work carried out provides insights into how antibiotic treatment and dietary changes may disturb the structure and function of a gut bacterial community and the relationship between its members (bacteria and protists). To further assess the impact of diet/antibiotics on the microbiome and its subsequent re-establishment, and understand which conditions lead to an irreversible change affecting host survival, tests need to be performed over a longer period. Establishing which factors cause permanent effects could result in gut microbiota modification becoming a strategy for pest control.S1 Table(PDF)Click here for additional data file.S1 FigSeveral representative OTUs could be classified at genus level.(TIF)Click here for additional data file.S2 FigP values are shown at right.Corrected (TIF)Click here for additional data file.S3 FigReticulitermes species. (B) PCA of carbohydrate pathways from Reticulitermes species.(A) Extended error bar plot identifying significant differences between mean proportions of carbohydrate pathways in pairwise (TIF)Click here for additional data file.S4 FigReticulitermes grassei treatments, wood-diet, cellulose-diet and ciprofloxacin treatment.Extended error bar plot identifying significant differences between mean proportions of bacterial taxa in pairwise (TIF)Click here for additional data file.S5 FigReticulitermes grassei treatments, wood-diet, cellulose-diet and ciprofloxacin treatment.Extended error bar plot identifying significant differences between mean proportions of carbohydrate pathways in pairwise (TIF)Click here for additional data file.S6 FigReticulitermes grassei treatments, wood-diet, cellulose-diet and ciprofloxacin treatment.Extended error bar plot identifying significant differences between mean proportions of xenobiotic pathways in pairwise (TIF)Click here for additional data file.S7 FigReticulitermes grassei treatments, wood-diet, cellulose-diet and ciprofloxacin treatment.Extended error bar plot identifying significant differences between mean proportions of energy pathways in pairwise (TIF)Click here for additional data file.S8 FigReticulitermes grassei treatments, wood-diet, cellulose-diet and ciprofloxacin treatment.Extended error bar plot identifying significant differences between mean proportions of amino acid, cofactors and vitamins metabolism in pairwise (TIF)Click here for additional data file.S9 Fig(A) Phyla contribution by percent of sample respect to K01784 . (B) Phyla contribution by percent of sample respect to K00446 (xylene degradation).(TIF)Click here for additional data file.


Helicobacter pylori (H. pylori) is a gram-negative bacterium that colonizes the human stomach. Infection with this microaerophilic bacterium causes gastric and duodenal ulcer. This study sought to isolate H. pylori, from gastric biopsy samples of dyspeptic patients in Ghana using a 2,3,5-triphenyltetrazolium chloride (TTC) dye incorporated medium method. This TTC dye method was further used in an antimicrobial susceptibility assay involving Dissotis rotundifolia extract (DRE). H. pylori were successfully isolated from gastric biopsy of dyspeptic patients. Pure cultures of H. pylori in 2,3,5-triphenyltetrazolium chloride (TTC) dye incorporated medium were seen as sparkling colonies. Isolates, identified as H. pylori, were gram-negative and urease, catalase, and oxidase positive and showed characteristic morphology as spiral-shaped bacteria under the microscope. The organisms were found to be susceptible to cephalothin and resistant to nalidixic acid. Above all, the observation that H. pylori grew only at 37°C and not 25°C or 42°C affirms that the bacterium is neither Helicobacter cinaedi nor Helicobacter fenneliae. The anti-H. pylori study depicts a statistically lower zone of inhibition for DRE compared to standard drugs [amoxicillin and clarithromycin] (p<0.05), whereas metronidazole showed no zone of inhibition. This study reports the first successful isolation and culturing of H. pylori in Ghana using TTC dye. It also shows that DRE possess an in vitro anti-H. pylori activity and that DRE has some therapeutic potential against H. pylori infection. Helicobacter pylori (H. pylori) in 1982  showed antimicrobial activity against 5 confirmed H. pylori isolates , it corroborates the reported ethnomedicinal use of the plant in managing gastric disturbances. The smaller zone of inhibition demonstrated by the flavonoid rich fraction of this plant in vitro, compared to standard antibiotic drugs, does not necessarily imply that the extract demonstrates weak antimicrobial effects in vivo. As with some orthodox drugs, the active ingredients in Dissotis rotundifolia may be pro-drugs; thus its potency could improve in vivo due to biotransformation [As part of this research, a flavonoid-rich fraction oformation . Dissotis rotundifolia [ Piper carpunya and are reported to possess pharmacological activity against growth of H. pylori [ Dissotis rotundifolia whole plant extract may be contributing to the inhibitory effects of the plant in this study. The observation that H. pylori clinical isolates in this study showed resistance to metronidazole is not contrary to what has been reported in South Africa, Egypt, and many other African countries [ H. pylori resistance to metronidazole in Ghana and thus calls for an in-depth study of the pattern of drug resistance by H. pylori in Ghanaian dyspeptic patients.C-glycosylflavones compounds, namely, vitexin and isovitexin, have been isolated from the methanolic extract of the whole plant ofndifolia . Vitexin. pylori . Impliciountries , 17, 29. H. pylori from Ghanaian dyspeptic patients. It also depicts the fact that Dissotis rotundifolia extract possesses inhibitory potential against growth of clinical isolates of Helicobacter pylori. This finding suggests that Dissotis rotundifolia extract has some therapeutic potential against H. pylori infection, which could be explored in managing gastrointestinal problems.In conclusion, this study reports on a first successful isolation of