Plasmodium infection in the Anopheline mosquitoes of the Western Highland Fringe of Madagascar.The Madagascar National Strategic Plan for Malaria Control 2018 (NSP) outlines malaria control pre-elimination strategies that include detailed goals for mosquito control. Primary surveillance protocols and mosquito control interventions focus on indoor vectors of malaria, while many potential vectors feed and rest outdoors. Here we describe the application of tools that advance our understanding of diversity, host choice, and Anopheles mosquitoes (10 species), all of which were subjected to BLOODART analysis. QUEST collection captured a heterogenous distribution of mosquito density, diversity, host choice, and Plasmodium infection. Concordance between Anopheles morphology and BLOODART species identifications ranged from 93–99%. Mosquito feeding behavior in this collection frequently exhibited multiple blood meal hosts . The overall percentage of human positive bloodmeals increased between the December 2017 and the April 2018 timepoints (27% to 44%). Plasmodium positivity was frequently observed in the abdomens of vectors considered to be of secondary importance, with an overall prevalence of 6%.We employed a modified barrier screen trap, the QUadrant Enabled Screen Trap (QUEST), in conjunction with the recently developed multiplex BLOOdmeal Detection Assay for Regional Transmission (BLOODART). We captured a total of 1252 female Plasmodium DNA in their abdomens, indicating a need to account for these species in routine surveillance efforts. Mosquitoes exhibited multiple blood feeding behavior within a gonotrophic cycle, with predominantly non-human hosts in the bloodmeal. Taken together, this complex feeding behavior could enhance the role of multiple Anopheline species in malaria transmission, possibly tempered by zoophilic feeding tendencies.The QUEST was an efficient tool for sampling exophilic Anopheline mosquitoes. Vectors considered to be of secondary importance were commonly found with  Plasmodium infection across traps placed within and between two nearby villages at two timepoints; indicating a strong, small-scale spatial component to disease transmission that warrants further investigation. Many of the mosquitoes in this sample (46.4%) fed on two or three host species, indicating complex feeding behaviors that could influence malaria transmission. Further, Plasmodium DNA was detected in the abdomens of numerous vectors of supposed secondary importance, indicating a neglected parasite reservoir and an increased need to account for these species in routine surveillance efforts.Malaria continues to be a significant threat to public health in Madagascar. Elimination of this disease is impeded by numerous factors, such as vector surveillance that does little to account for the potential role of secondary malaria vectors, which tend to rest and feed outdoors. In this study, we designed a low cost, modified barrier screen trap, called the QUadrant Enabled Screen Trap (QUEST). We used this in conjunction with the recently developed BLOOdmeal Detection Assay for Regional Transmission (BLOODART) to assess mosquito feeding behavior in the Western Highland Fringe of Madagascar. Our analysis revealed significant variability in mosquito density, diversity, host choice, and  Gambusia sp.) to cisterns and irrigation ditches, and 4.) drainage works to limit mosquito breeding sites [Anopheles funestus) from the highlands by 1952 [Plasmodium positive An. funestus in the highlands [The Madagascar National Strategic Plan for Malaria Control 2018 (NSP), developed in coordination with the Madagascar National Malaria Control Program (NMCP), outlines pre-elimination strategies and a plan of action for malaria control in Madagascar [ng sites . The firng sites . Much of by 1952 . Believi by 1952 –10. A plighlands ,12.Anopheles funestus populations from the highlands are genetically similar to populations on the east coast [An. funestus may have come from the east. Furthermore, it is likely that additional Anopheline vector species contributed to the malaria outbreak [Anopheles thought to play a lesser role in transmission than the principal vector; capable of maintaining malaria transmission at a reduced level [Today, an evolved perspective of the entomological side of the 1980s epidemic has emerged. Spraying campaigns are never truly comprehensive, leaving reservoirs that facilitate future recolonization events . Anophelst coast , suggestoutbreak ,15. Secoed level .” LooselPlasmodium sporozoites by dissection of the salivary glands: An. coustani [An. mascarensis [An. squamosus [An. pharoensis [An. rufipes [An. maculipalpis [An. coustani [An. mascarensis [An. squamosus [Plasmodium infection status of potential malaria vectors in the Western Highland Fringe of Madagascar to achieve a more comprehensive picture of mosquito behavior in the region. To accomplish this goal, we utilized the recently developed Bloodmeal Detection Assay for Regional Transmission (BLOODART), designed to simultaneously assess Anopheles mosquito species, mammalian hosts, and Plasmodium parasites from an excised mosquito abdomen or abdomen squash [Numerous secondary vectors, present in Madagascar, have been documented with coustani ,17, An. carensis , An. sququamosus , An. pha rufipes , and An.lipalpis . Circumscoustani ,20,21, Acarensis ,18,22, aquamosus  as malarquamosus , most ofquamosus ,22,25. Dn squash .Plasmodium positivity with spatial resolution, demonstrating the ability of these tools to contribute to the entomological surveillance goals of the NSP [In conjunction with BLOODART, we employed a modified barrier screen trap, the Quadrant Enabled Screen Trap (QUEST). Barrier screens represent a relatively recent collection method, designed to address the paucity of effective and unbiased collection tools for exophilic mosquitoes . Outdoor the NSP .Wild-caught mosquitoes were collected by Case Western Reserve University and Madagascar NMCP entomologists in December 2017 (six nights) and April 2018 (five nights), corresponding to the beginning and end of the rainy season (December to March ), observAnopheles mosquitoes were collected. Mosquitoes were aspirated on a quadrant-by-quadrant basis and deposited live into pre-labeled paper cups at the beginning of each collection timepoint. Collected mosquitoes were incapacitated by ether and keyed to species using local unpublished keys from Institut Pasteur de Madagascar. Mosquitoes were preserved in individual 2 ml tubes containing 90% ethanol with a label including a unique specimen voucher code containing the prefix “APY.” A subset of mosquitoes in April 2018 were collected on filter paper (n = 115) after ethanol supplies were exhausted.Mosquitoes were collected using QUEST, modified from a previously described barrier screen design . One rouQUESTs were designed to improve yields and increase our understanding of mosquito mobility within the study site . Poles fHigh-resolution images of preserved specimens (taken ~60 days post collection) were captured using a Passport Storm system , including: a Stackshot z-stepper, a Canon 5D SLR, a MP-E 65 mm macro lens, three Speedlight 580EX II flash units, and a computer running Canon utility and Adobe Lightroom 3.6 software. The z-stepper was controlled through Zerene Stacker 1.04 and images were stacked using the P-Max protocol. To prepare for photography, the specimen was removed from EtOH, allowed to dry for several minutes, and temporarily affixed to a pin with a small dab of K-Y Personal Lubricant. Images were captured over an 18% gray card background and processed in Adobe Photoshop CC 2018 to adjust lighting and sharpness, and to add scale bars. Minor adjustments were made using the stamp tool to correct background and stacking aberrations.Plasmodium parasites were carried out as previously described [DNA extraction and PCR amplification of targets for mosquito species, mammalian host, and escribed .An. squamosus, two probes that distinguish An. gambiae sensu stricto from An. arabiensis, ringtail lemur (Lemur catta), Coquerel’s sifaka (Propithecus coquereli), and the house mouse (Mus musculus). A list of probes and fluorescent microspheres for the mammalian and mosquito probes is located in Plasmodium species are described elsewhere [We utilized the Bloodmeal Detection Assay for Regional Transmission  with thelsewhere .Contingency tables and plots were generated in R Version 3.4.0  using thAn. flavicosta, An. gambiae, and An. pretoriensis due to insufficient sample size) , with An. maculipalpis and An. rufipes being more active at the 21:00 hrs timepoint, while An. mascarensis activity peaked at 03:00 hrs. The mosquito activity pattern for individual nights was variable. No mosquitoes were captured at 18:00 hrs. Only eight mosquitoes were captured by pyrethroid spray catch across 17 dwellings, with all mosquitoes identified as An. funestus.We captured a total of 1252 mosquitoes, 501 in December 2017, and 751 in April 2018. The QUEST captured 1143 mosquitoes over 33 trap-nights  for 34.6 mosquitoes per trap/night. Overall, there was a greater concentration of mosquitoes on the traps at the April 2018 timepoint. The distribution of mosquitoes within each trap differed, with some QUESTs showing a homogenous distribution of mosquitoes across the four quadrants while others had a clear concentration of samples on a subset of the four quadrants . The staAn. gambiae s.s. and An. arabiensis were considered as the An. gambiae sensu lato complex in our comparison of morphological versus BLOODART identifications. Further data analyses here preferentially used the species determination of BLOODART over morphology. Where BLOODART identifications were inconclusive, we used the original morphological identification. Of the 64 mosquitoes designated as “inconclusive” by BLOODART, 16 produced a clean PCR band, 24 produced either a smear or several bands of unexpected size, five produced faint bands, and 19 produced no band at all. Concordance for individual probes ranged from 93–99% (An. mascarensis (22/34), and will be considered in this manuscript as the An. mascarensis probe. Probes for An. pretoriensis, An. flavicosta, and An. fuscicolor were not included. We provide several photographs , An. maculipalpis (n = 263), An. rufipes (n = 147), and An. squamosus (n = 228). Species captured in lower numbers were An. arabiensis (n = 64), An. mascarensis (n = 35), An. funestus (n = 26), An. gambiae s.s. (n = 14), An. pretoriensis (n = 5), and An. flavicosta (n = 1). The abundance of particular species shifted on either side of the rainy season, with an even mix of An. arabiensis and An. gambiae s.s. in December 2017 shifting to exclusively An. arabiensis in April 2018. The proportion , An. coustani = 94.9% (445/469), An. funestus = 96.2% (25/26), An. flavicosta = 100% (1/1), An. gambiae = 100% (14/14), An. maculipalpis = 93.9% (247/263), An. mascarensis = 97.1% (34/35), An. pretoriensis = 100% (5/5), An. rufipes = 96.6% (142/147), and An. squamosus = 97.8% (223/228).We collected 1035 visibly blood fed, and 217 visibly unfed mosquitoes. We were able to detect a mammalian host in 1195 specimens, including 160/217 visibly unfed mosquitoes. We were unable to detect a host in 57 specimens. Blood feeding rates were calculated based on detection of host DNA rather than visible engorgement. Blood feeding rates were as follows: 2 = 42.3, 2 DOF, p < 0.0001) from December 2017  to April 2018 . The most anthropophilic species were An. coustani and An. mascarensis. The propensity for human feeding increased significantly from the December 2017 (27% human positive) to the April 2018 (44% human positive) collection , with anthropophilic shifts observed for An. coustani (32% to 60%), An. funestus (9% to 50%), An. rufipes (23% to 44%), and An. arabiensis (38% to 53%) , followed by human (39.5%) and porcine (15.3%) blood . Canine  to 53%) .2 = 23.5, 2 DOF, p < 0.005, AMB vs AMP in 2018: χ2 = 25, 2 DOF, p < 0.005) and timepoint   between village (AMB vs AMP in 2017: χ< 0.005) . The prePlasmodium species  were detected in our assay  and P. vivax (n = 19), followed by P. malariae (n = 10) and P. ovale (n = 8). There was a total of 12 mixed Plasmodium species infections, including the combinations P. falciparum/P. vivax (n = 9), P. falciparum/P. malariae (n = 1), P. falciparum/P. ovale (n = 1), and P. malariae/P. ovale (n = 1). This parasite profile is consistent with human malaria surveys in the region [Plasmodium-infected bloodmeals were commonly observed across Anopheles species that the Madagascar NMCP typically consider to be secondary or non-vectors, such as An. coustani , An. maculipalpis , An. squamosus , and An. rufipes . These secondary vector infection rates (70/1078) are not significantly different from infection rates in primary vectors (5/99) : An. funestus , An. arabiensis , and An. gambiae . Human positive bloodmeals were not significantly more likely to be associated with Plasmodium parasites . However, Plasmodium prevalence was higher in human positive  than human negative  bloodmeals.All four common human ur assay  with a te region . PlasmodP. vivax infected mosquitoes. The abundance of positive samples, and diversity of Plasmodium spp. within them, shifted in these two villages, with P. falciparum comprising nearly all of the Plasmodium positive bloodmeals in April 2018. The village of Amparihy had no Plasmodium-positive mosquitoes in December 2017, while similar numbers of Plasmodium-positive mosquitoes were observed between the villages in April 2018 . Apart from this difference in Plasmodium positive mosquito distributions between Amparihy and Ambolodina, no specific pattern of Plasmodium positive mosquitoes was observed across QUESTs , we could obtain further insight into the behavior of these medically important mosquitoes. The data produced by a BLOODART analysis of captured mosquitoes augments the utility of QUESTs (or any other mosquito trap strategy), efficiently monitoring additional important vector indicators. By analyzing all of our captured Anopheline mosquitoes, as opposed to focusing only on the predetermined important vectors, we detected a significant parasite reservoir in the abdomens of secondary vectors. In the context of limited time and resources in outbreak or surveillance scenarios, secondary vectors may be passed over for assessment of vector indicators, or never collected at all. This highlights the problematic nomenclature surrounding “primary” or “secondary” vectors, in that they may constrain a thorough investigation of complex disease transmission networks. Further studies should seek to characterize the role of these secondary vectors in malaria transmission.This study demonstrates the ability of QUESTs and BLOODART to refine perspectives on mosquito collection and assessment. QUESTs collect a diverse and substantial sample of As a result of this study, and training missions carried out at the Madagascar NMCP, the skillset necessary to use both QUEST and BLOODART is currently in place. This study provides a framework for how these tools can be deployed to enhance Madagascar’s capacity for entomological surveillance and malaria elimination.Anopheles mosquitoes. Placing the QUESTs in the same locations in December 2017 and April 2018 allowed for comparison of QUEST data across the two timepoints. The QUESTs AMB1, AMB3, and AMP1 had significantly different mosquito distributions across the four quadrants between the December 2017 and April 2018 timepoints. In December 2017, the traps AMB3 and AMB4 had non-homogenous mosquito distributions across the QUEST quadrants. In April 2018, the traps AMB1 and AMP1 had non-homogenous mosquito distributions across the QUEST quadrants , deviating from the general trend of a 00:00 hrs peak activity time. Alternately, An. mascarensis activity peaked later in the evening (03:00 hrs). More frequent collections throughout the night would provide greater resolution for these mosquito activity patterns.Two mosquito species, Anopheles ITS2 sequence probe [An. mascarensis, facilitating the first online deposition of genetic data for this common Malagasy species (Genbank accession: MH560267). By vouchering, photographing, and depositing our mosquito data on VectorBase, we have secured the future utility of our mosquito collection for the purpose of further morphological and molecular investigations.Concordance between mosquito morphological and BLOODART identifications ranged from 93–99%. Unique specimen vouchers enabled us to refer back to discordant specimens, which revealed that morphological misidentification was responsible for many of the discrepancies between morphological and molecular identifications. Field conditions (such as poor lighting through the microscope) likely contributed to less than optimal species identifications. Additionally, vouchered specimens allowed us to associate a novel ce probe  with theAnopheles coustani, comprising 37% of our mosquito collection, was recently implicated as a vector of malaria [An. maculipalpis, An. rufipes, An. squamosus, and An. mascarensis have all been documented with Plasmodium parasites [The predominant mosquito species in this collection have been considered vectors of secondary importance. However, their abundance relative to primary vectors necessitates closer examination of their potential impact on human health.  malaria . Despite malaria . Similararasites –22. The Anopheles gambiae s.s. is uncommon in the highlands of Madagascar [An. arabiensis being the predominant representative of the An. gambiae s.l. complex in this region. Permanent shifts in species dominance from An. gambiae s.s. to An. arabiensis have been reported in continental Africa [An. gambiae s.s., with negligible impacts on the exophilic/exophagic An. arabiensis [An. gambiae s.s. preferring a more humid climate, while An. arabiensis is found in both humid and arid environments [dagascar , with Anl Africa ,43. Typiabiensis ,43. The ronments ,20,44,45ronments ), and thronments ,47, we mronments .There are several phenomena that limit direct comparison of mosquito data between villages. Due to logistical issues ranging from inclement weather to security, the number of nights sampled in each village differed. Further, villages were sampled consecutively as opposed to concurrently, introducing a unique mixture of irretrievable ecological factors into each trapping night across the overall sampling period. Nevertheless, there are common observations, namely a diversity of mosquitoes, host choice, and multiple blood feeding behavior, between the villages.The distribution of mosquitoes collected from the QUEST is potentially influenced by wind direction, precipitation, temperature, lunar cycle, proximity to breeding sites, and distance to viable hosts ,49–51. SAn. gambiae sibling species, sampling site (indoors vs outdoors) is more closely associated with human positive bloodmeals than mosquito species [An. gambiae s.s. and An. funestus. The BLOODART analysis revealed multiple blood feeding behavior (46.4%) in our mosquito sample. PCR has the capacity to detect blood consumed 36–48 hrs after ingestion [An. coustani, An. funestus, An. rufipes, and An. arabiensis. An increase in human feeding could lead to an increase in the presence of Plasmodium positive mosquitoes, though evidence of such from this study was variable between villages  likely reflects our unbiased approach to mosquito collection and processing, as we did not limit our molecular analysis to predetermined primary vector species. However, we should consider that Plasmodium positivity in this assay is observed in the abdomen of the mosquito, and does not indicate the status of sporozoites in the mosquito’s salivary glands. An infectious mosquito is typically defined as a mosquito with sporozoites in the head/thorax [An. coustani, was recently implicated as an important vector of Plasmodium parasites [An. coustani and the remaining secondary vectors may be responsible for sustained malaria transmission in these villages, which have low primary vector density. Consequently, we have identified a potential reservoir of Plasmodium parasites outside the realm of many current vector interventions. Therefore, targeted efforts focused on the suppression of An. funestus and An. gambiae s.l., which primarily include indoor interventions, may not be sufficient to effectively reduce malaria transmission.The incidence of d/thorax , which, d/thorax , will ond/thorax . We chosd/thorax ) for indd/thorax ,14,17–23arasites  in the rPlasmodium positive bloodmeals indicates that the mosquito acquired the Plasmodium parasite from a human, digested the human blood, yet still retains the Plasmodium DNA, likely as an oocyst-stage parasite.We assume that the observation of human negative/Plasmodium spp. positivity across space and time. This may reflect the seasonality of P. falciparum and P. vivax malaria, which peaks in the study region around the time of the second round of surveys described here [P. vivax positivity restricted to the beginning of the rainy season, and a predominance of P. falciparum toward the end of the rainy season. The distribution of Plasmodium positive mosquitoes on the nets was fairly homogeneous, with the exception of the village of Amparihy in December 2017, which had no positive mosquitoes. The observation of a statistically significant pattern is limited by the total number of positive mosquitoes (n = 75) distributed across 48 possible quadrants.We revealed differences in the occurrence of bed here . Our mosPlasmodium parasites. Substantial parasite reservoirs in neglected vector species adds to the series of gaps that expose hard-earned malaria-free districts to the perpetual threat of recrudescence.To achieve the goal of malaria elimination in this country, the role of neglected secondary vector species must be considered. The lack of comprehensive surveillance, and the absence of adequate distribution of artemisinin combination therapy drugs, insecticide treated nets, and rapid diagnostic tests in remote areas of the country provide a safe refuge for S1 FigAnopheles coustani (APY1071), (B) Anopheles maculipalpis (APY684), (C) Anopheles rufipes (APY1016), (D) Anopheles squamosus (APY702).(A) (TIF)Click here for additional data file.S2 FigAnopheles arabiensis (APY689), (B) Anopheles funestus (APY237), (C) Anopheles mascarensis (APY437), (D) Anopheles mascarensis (APY733).(A) (TIF)Click here for additional data file.S3 FigP. falciparum, V = P. vivax, M = P. malariae, O = P. ovale. Letter combinations indicate the presence of two or three species in the bloodmeal. Quadrant numbers refer to the direction of their orientation: 1-Northwest, 2-Northeast, 3-Southwest, 4-Southeast. (A) Ambolodina December 2017, (B) Ambolodina April 2018, (C) Amparihy December 2017, (D) Amparihy April 2018. Numerical version available in F = (PNG)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(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.


Breast cancer is the most frequent cancer among women worldwide. Therapeutic strategies to prevent or treat metastatic disease are still inadequate although great progress has been made in treating early-stage breast cancer. Cancer stem-like cells (CSCs) that are endowed with high plasticity and self-renewal properties have been shown to play a key role in breast cancer development, progression, and metastasis. A subpopulation of CSCs that combines tumor-initiating capacity and a dormant/quiescent/slow cycling status is present throughout the clinical history of breast cancer patients. Dormant/quiescent/slow cycling CSCs are a key component of tumor heterogeneity and they are responsible for chemoresistance, tumor migration, and metastatic dormancy, defined as the ability of CSCs to survive in target organs and generate metastasis up to two decades after diagnosis. Understanding the strategies that are used by CSCs to resist conventional and targeted therapies, to interact with their niche, to escape immune surveillance, and finally to awaken from dormancy is of key importance to prevent and treat metastatic cancer. This review summarizes the current understanding of mechanisms involved in CSCs chemoresistance, dissemination, and metastasis in breast cancer, with a particular focus on dormant cells. Finally, we discuss how advancements in the detection, molecular understanding, and targeting of dormant CSCs will likely open new therapeutic avenues for breast cancer treatment. Breast cancer (BC) is the most common cancer in women and the second cause of cancer-related death among women worldwide . Current+CD24−/lowLin− fraction was significantly enriched for cells with tumor forming ability as compared to the CD44+CD24+Lin− population. Moreover, tumors that formed by CD44+CD24−/lowLin− cells could be serially passaged and reproduced the cellular heterogeneity observed in the tumor of origin [+CD29+ cells and CD24+CD49f+ cells were isolated from BRCA1-mutated mammary tumors and displayed self-renewal and tumor-initiating capacity in vivo [+ BC) often results in disappearance of the therapeutic target or acquisition of resistance through alternative signalling pathways [−CD44+ phenotype [Breast Cancer Stem Cells (BCSCs) were initially described in 2003 by Al-Hajj and colleagues, who found that the CD44f origin . Subsequ in vivo . Several in vivo ,19,20,21 in vivo ,24,25,26 in vivo ,28,29,30 in vivo . Such a  in vivo  and obse in vivo . From a pathways . The SWHpathways . In linepathways . A key ppathways . These opathways . Intereshenotype . More rehenotype . Translahenotype . The abihenotype . In summ+ and CD44+/CD24low population inhibiting tumor growth and metastatic ability [hiE-cadherinlo mesenchymal phenotype. These cells are present in the early phases of tumor formation and they possess the metastasis-initiating capacity typical of BCSCs [hi/ERlo/IL6hi BCSCs [+ luminal BC and its expression has been associated with poor prognosis in this subtype but not in other BC subtypes [+ BC. Ectopic expression of DAXX reduced stemness gene expression, NOTCH signaling, and BCSCs survival upon endocrine therapy, which suggests that a combination of hormone therapy and DAXX-stabilizing agents may inhibit ER+ tumor recurrence [Analogously to what happens in normal stem cells, the self-renewal of BCSCs is regulated by the activation and reciprocal interaction of pathways that are important for embryonic development, such as WNT/β-catenin, Hedgehog, NOTCH, and HIPPO pathways. As a number of reviews examine the role of each pathway in BC in detail, we will limit our discussion to some aspects particularly relevant for BCSCs biology. The role of WNT signalling in the generation of BCSCs was recognized as early as in 2003 by observing that exogenous expression of WNT pathway components expands a population of bipotent epithelial progenitors and it generates mixed-lineage mammary tumours that are composed of basal and luminal cell subtypes . Interes ability . Two stuof BCSCs . Secondlof BCSCs . Notablyof BCSCs . These oof BCSCs . In lineof BCSCs . Hh signof BCSCs . In TNBCof BCSCs . Interesof BCSCs . Recent of BCSCs . TSPAN8 of BCSCs . The NOTof BCSCs . NOTCH4 of BCSCs . The actof BCSCs . NOTCH3 hi BCSCs . Trastuzhi BCSCs . Recentlhi BCSCs . DLL1 wasubtypes , highligcurrence . The HIPcurrence ,65. YAP/currence . Consistcurrence . A stroncurrence . Finallycurrence . Interfehi CD44+ BC cells are responsible for both chemotherapy and radiation resistance and ALDHs inhibition sensitizes BCSCs to chemotherapy [Increasing evidence suggests that BCSCs execute multiple drug resistance mechanisms, including the overexpression of ATP-binding cassette (ABC) transporters, increased ALDH activity, enhanced DNA repair mechanisms, reinforced reactive oxygen species (ROS) scavenging, cell death escape, induction of dormancy, autophagy, and possibly other resistance mechanisms that are yet to be characterized. ABC transporters utilize the energy of ATP binding and hydrolysis to transport various substrates across cellular membranes. They play a crucial role in multidrug resistance by regulating the efflux of a wide variety of anticancer agents , but areotherapy . Severalotherapy . Enhanceotherapy . Subsequotherapy . The nucotherapy . Besidesotherapy . BCSCs potherapy . In the otherapy . Subsequotherapy . Howeverotherapy . In BC, otherapy  and to rotherapy . Autophaotherapy . The rolotherapy . During otherapy . The mecotherapy  or by inotherapy . Recentlotherapy . Furtherotherapy . In addiotherapy . On the otherapy . This dootherapy . Finallyotherapy . In summ+ BC, were dependent on N2RF1-mediated dormancy [−/CD44+/epithelial surface antigen (ESA)+ expression and spheroid forming capacity [BCSCs in a slow cycling state or in a non-proliferating state are present throughout the clinical history of BC patients and crucially influence disease outcome . Therefodormancy . Interesdormancy . The intcapacity . Other icapacity . Interescapacity . Althougcapacity .A population of drug-tolerant cells has been identified in several tumors upon treatment with chemotherapeutic or targeted agents ,110,111.The tumor microenvironment (TME) is a critical driver of the malignant phenotype and it has been shown to be specifically implicated in promoting CSCs functions, such as self-renewal, plasticity, and chemoresistance in breast and other cancers . TME com+GPR77+ CAF subset was linked to chemoresistance and poor prognosis in BC and lung cancer patients and a neutralizing anti-GPR77 antibody abolished tumor formation in PDX models, which indicated CAFs targeting as a therapeutic strategy that is able to affect the BCSCs compartment [In this regard, hypoxic microenvironments were recently shown to generate dormant chemoresistant BC cells and enhance the stem cell phenotype in BC xenografts ,124,125.partment . An intepartment . Finallypartment . Metabolpartment . Adipocypartment . Lipid mpartment . Furtherpartment . Adipsinpartment . In addipartment . Howeverpartment . BM-MSCspartment . Other fpartment . Besidespartment . More repartment . In the partment . Interacpartment . In summpartment . Therefopartment ,153,154,+ BC patients, indicating that stratifying patients according to MSK1 expression could improve prognosis [In contrast to the traditional notion that metastasis is a late event in tumor progression, increasing evidences indicate that neoplastic cells can disseminate from early tumors, which results in the parallel progression of primary tumor and metastases . The conrognosis . Collectrognosis  will lik+ BC patients have a risk of late relapse until twenty years from primary tumor removal [+ tumor cells carrying such a signature were more likely to undergo prolonged dormancy before resuming metastatic growth. Moreover, the suppression of dormancy-associated genes basic helix-loop-helix family member 41 (BHLHE41) and NR2F1 resulted in increased in vivo growth of ER+ MCF7 cells, highlighting the importance of dormancy regulation for BC progression and relapse [+ BC [Understanding the biology of dormant CSCs has a great clinical relevance for the treatment of BC patients. In fact, while more aggressive forms of BC, such as TNBC, have a peak of distant recurrence within three years of diagnosis , ER+ BC  removal . Such a  relapse . Therape relapse . Sleepinse [+ BC ,181.+ BC [Adjuvant anti-estrogen therapies given for up to ten years after diagnosis have significantly improved the survival of patient with ER+ BC . The mai+ BC , DYRK1A + BC ,183, and+ BC ,178. Sle+ BC . Secondl+ BC ,185. The+ BC . However+ BC . Selecti+ BC . Recentl+ BC ,188. How+ BC . Besides+ BC . Moreove+ BC ,168,169.+ BC . These o+ BC . Besides+ BC . Obesity+ BC ,194,195,+ BC ,197.Although great progress has been made over the past twenty years in BC diagnosis and treatment, BC remains a potentially deadly disease due to the ability of BCSCs to resist therapies, disseminate, and metastasize. The ongoing advancements of basic and preclinical research in understanding BCSCs biology must be readily translated into clinical practice to improve the effectiveness of current cancer therapies. Moreover, the emerging network existing between BCSCs, the immune system, and the micro/macroenvironment will likely provide essential information regarding how to integrate anticancer therapies and lifestyle-related factors for improved management of BC patients.


Mycobacterium abscessus is the most pathogenic and drug resistant opportunistic germ, responsible for disease manifestations ranging from “curable” skin infections to only “manageable” pulmonary disease. Challenges in M. abscessus treatment stem from the bacteria’s high-level innate resistance and comprise long, costly and non-standardized administration of antimicrobial agents, poor treatment outcomes often related to adverse effects and drug toxicities, and high relapse rates. Drug resistance in M. abscessus is conferred by an assortment of mechanisms. Clinically acquired drug resistance is normally conferred by mutations in the target genes. Intrinsic resistance is attributed to low permeability of M. abscessus cell envelope as well as to (multi)drug export systems. However, expression of numerous enzymes by M. abscessus, which can modify either the drug-target or the drug itself, is the key factor for the pathogen’s phenomenal resistance to most classes of antibiotics used for treatment of other moderate to severe infectious diseases, like macrolides, aminoglycosides, rifamycins, β-lactams and tetracyclines. In 2009, when M. abscessus genome sequence became available, several research groups worldwide started studying M. abscessus antibiotic resistance mechanisms. At first, lack of tools for M. abscessus genetic manipulation severely delayed research endeavors. Nevertheless, the last 5 years, significant progress has been made towards the development of conditional expression and homologous recombination systems for M. abscessus. As a result of recent research efforts, an erythromycin ribosome methyltransferase, two aminoglycoside acetyltransferases, an aminoglycoside phosphotransferase, a rifamycin ADP-ribosyltransferase, a β-lactamase and a monooxygenase were identified to frame the complex and multifaceted intrinsic resistome of M. abscessus, which clearly contributes to complications in treatment of this highly resistant pathogen. Better knowledge of the underlying mechanisms of drug resistance in M. abscessus could improve selection of more effective chemotherapeutic regimen and promote development of novel antimicrobials which can overwhelm the existing resistance mechanisms. This article reviews the currently elucidated molecular mechanisms of antibiotic resistance in M. abscessus, with a focus on its drug-target-modifying and drug-modifying enzymes.The incidence and prevalence of non-tuberculous mycobacterial (NTM) infections have been increasing worldwide and lately led to an emerging public health problem. Among rapidly growing NTM,  Mycobacterium avium complex  or Mycobacterium abscessus, accentuating the medical importance of these emerging pathogens  encompass all species of mycobacteria that do not cause tuberculosis (TB) or leprosy . NTM areathogens . For a lmination .Mycobacterium abscessus is an opportunistic pathogen, ubiquitous in the environment, that often causes infections in humans with compromised natural defenses such as patients with cystic fibrosis or other chronic lung diseases  which can serve as donors of novel drug resistance or virulence genes  as well as some second-line agents (such as capreomycin) are ineffective against this pathogen (Table 1). Thus, the term “incurable nightmare” is often used to describe M. abscessus  for clinical isolates susceptible to macrolides, during the initial treatment phase. For the continuation phase of treatment, nebulised amikacin and an oral macrolide combined with one to three of the following oral antibiotics: linezolid, clofazimine, minocycline, co-trimoxazole, and moxifloxacin are generally recommended  in the corresponding 23S (rrl) and 16S (rrs) rRNA genes . In this article, we provide an up-to-date overview of the main molecular mechanisms of antibiotic resistance in M. abscessus with a focus on the drug-target-modifying or drug-modifying enzymes and discuss the potential impact on clinical treatment. We also discuss how this knowledge could facilitate the discovery and development of new improved antimicrobial agents or help rescue the function of currently available antibiotics against M. abscessus and provide an update on the latest research underway to combat multi-drug resistant M. abscessus infections.Mechanisms underpinning intrinsic drug resistance of e target . For lonM. abscessus infections almost always includes macrolides, particularly clarithromycin or azithromycin. Macrolides target the nascent polypeptide exit tunnel within the 50S ribosomal subunit, which accommodates the newly synthesized polypeptide chain as it emerges from the ribosome. Following macrolide binding, the elongation of polypeptide chain continues up to a few amino acids before the peptidyl-tRNA dissociates from the ribosome thereby arresting protein synthesis . Interestingly, the erm(41) gene, first described by M. abscessus complex. The subspecies M. massiliense harbors deletions in its erm(41) gene copy which render the Erm(41) enzyme inactive and the bacterium susceptible to macrolides. This may explain why patients with M. massiliense infections have better treatment outcomes than patients with M. abscessus or M. bolletii infections. To further complicate matters, a T/C polymorphism at position 28 of the erm(41) sequence determines the appearance of inducible macrolide resistance in M. bolletii and M. abscessus subspecies. Only those isolates that harbor a T28 erm(41) sequevar develop inducible macrolide resistance. Phenotypic detection of inducible macrolide resistance in the M. abscessus complex by drug susceptibility testing requires extended incubation (14 days rather than 3 days) in the presence of the drug  in inducible resistance to macrolides has been confirmed by genetic studies. Deletion of erm(41) in M. abscessus lowered the minimal inhibitory concentration (MIC) of clarithromycin by 128-fold (Table 1), while introduction of a functional M. abscessus erm(41) gene into M. massiliense led to a 64-fold increase in the clarithromycin MIC on day 14 , are a frequent cause of constitutive macrolide resistance in M. abscessus ], encoded by MAB_4395 gene. This enzyme is capable of acetylating several aminoglycosides bearing a 2′ amino group including gentamicin C, dibekacin, tobramycin and kanamycin B . This was evident upon deletion of MAB_4395 in M. abscessus ATCC 19977 strain which led to a 4-64-fold reduction in the MICs of these aminoglycosides (Table 1)  protein . Eis proteins are widespread in mycobacteria as well as other Gram-positive bacteria. Eis protein in Mycobacterium tuberculosis has been shown to enhance intracellular survival within macrophages by acetylating and activating dual-specificity protein phosphatase 16/mitogen-activated protein kinase phosphatase-7 (DUSP16/MPK-7), a JNK-specific phosphatase that inhibits inflammation, autophagy and subsequent death of infected macrophages  . Interesrophages . Furtherocations . More reycosides .M. abscessus genome revealed two genes namely, MAB_4124 (eis1) and MAB_4532c (eis2), which show homology to eis from M. tuberculosis (Rv2416c) and A.variabilis (Ava_4977), respectively. Drug susceptibility testing could confirm a functional role for Eis2 but not Eis1, in intrinsic aminoglycoside resistance. Of note, deletion of MAB_4532c enhanced M. abscessus susceptibility towards a heterogenous group of aminoglycosides including the cornerstone drug amikacin as well as a non-aminoglycoside antibiotic capreomycin (Table 1) , M. smegmatis (Eis_Msm), and A. variabilis (Eis_Ava) revealed the presence of an unusually large and complex active site unlike in other aminoglycoside acetyltransferases. The unique structure equips Eis with a regio-versatile multi-acetylating acetyltransferase activity towards a broad range of substrates   . Furthertibility . Capreomnt of TB . Althougbstrates . HoweverTable 1) .in vitro activity against M. abscessus (MIC: 32 mg/L) . Twelve putative aminoglycoside phosphotransferases are encoded in the genome of M. abscessus (aph(3″)-Ic, an aminoglycoside 3″-O-phosphotransferase gene which was previously shown to confer streptomycin resistance in Mycobacterium fortuitum (M. abscessus towards streptomycin by 16-fold (Table 1). Furthermore, MAB_2385 was able to confer streptomycin resistance when heterologously expressed in M. smegmatis (Aminoglycoside acetyltransferases Eis2 and AAC(2′) do not appear to modify streptomycin, an aminoglycoside which exhibits moderate 32 mg/L) . Insteadbscessus . Among tortuitum . Deletiomegmatis . To the M. abscessus. WhiB7, an autoregulatory transcriptional activator, is a determinant of innate antibiotic resistance in mycobacteria. In M. tuberculosis, genes conferring antibiotic resistance including ermMT (Rv1988), eis (Rv2416c) and tap (Rv1258c) are induced in a whiB7-dependent manner, upon exposure to antibiotics. Similarly, the erm(41) and eis2 genes in M. abscessus are included in the whiB7 regulon  and clarithromycin (eightfold), while complementation with an intact gene copy restored resistance to these antibiotics, suggesting a role of whiB7 in M. abscessus amikacin and clarithromycin resistance. Interestingly, pre-exposure to clarithromycin, which is a potent inducer of M. abscessus whiB7, enhanced the resistance of M. abscessus wild-type strain towards amikacin by fourfold but had no effect on amikacin susceptibility of a ΔwhiB7 mutant. In addition, pre-treatment with clarithromycin markedly increased resistance to itself in the wild-type strain but did not alter clarithromycin sensitivity of a ΔwhiB7 mutant. Furthermore, a quantitative reverse transcription-PCR assay revealed a whiB7-dependent upregulation of eis2 and erm(41) genes following pre-exposure to clarithromycin. These observations support the following conclusions: (i) in M. abscessus, whiB7 (MAB_3508c) activates the expression of genes which confer amikacin and clarithromycin resistance, i.e., eis2 and erm(41), (ii) strong induction of whiB7 following exposure to clarithromycin confers cross-resistance to amikacin in addition to activating inducible macrolide resistance  found in human pathogen Legionella longbeachae, the causative agent of Legionnaires’ disease and Pontiac fever . Deletion of MAB_1496c (encoding a putative FAD binding monooxygenase) enhanced the susceptibility of M. abscessus towards tetracycline and doxycycline by 15–20-fold (Table 1), while a complemented ΔMAB_1496c strain exhibited even higher levels of resistance towards both antibiotics compared to the wild-type strain, supporting the notion that MAB_1496c is a primary determinant for M. abscessus intrinsic tetracycline resistance. Furthermore, UV-Visible and mass spectrometry assays with purified MAB_1496c protein provided evidence for hydroxylation and subsequent degradation of tetracycline and doxycycline by MabTetX  which represses the MAB_1497c-MAB_1496c operon by binding to a 35bp operator sequence tetO. This repression is relieved in the presence of tetracycline and doxycycline which can bind the repressor protein thereby preventing its interaction with the tetO operator -dependent monooxygenases in both benign and pathogenic bacteria has been recently documented. Few well characterized examples of such tetracycline-modifying enzymes include TetX from the obligate anaerobe ac fever . Whether MabTetX . Interesbscessus . In factoperator .Bacteroides TetX, which was recently also identified in numerous clinically relevant pathogens , a degradation product of tetracycline with poor antibiotic activity which was recently shown to be a competitive inhibitor of TetX and Tet(56) flavin monooxygenases, to rescue antibiotic activities of tetracycline and doxycycline against  MabTetX . However complex .D-alanyl-D-alanine dipeptide of peptidoglycan, a substrate of penicillin binding proteins  that catalyze the final cross-linking step of peptidoglycan synthesis. Penicilloylation of the D, D-transpeptidase active site following treatment with β-lactams, disables the enzyme and prevents the formation of peptidoglycan cross-links, thereby interfering with cell wall biosynthesis  with L, D-transpeptidases for instance, may reduce the activity of penicillins and cephalosporins that have lower affinity for L, D-transpeptidase  which renders it highly resistant towards most β-lactams . The M. abscessus β-lactamase Bla_Mab, encoded by MAB_2875 is endowed with an exceptional broad-spectrum activity and can effectively hydrolyse several members of first- and second-generation cephalosporins, carbapenems, and penams  M. tuberculosis strains raised a renewed interest in β-lactams for use in TB therapy  as well as non-β-lactam-based inhibitors (avibactam and vaborbactam) were evaluated for synergistic effects with a large panel of β-lactams. Importantly, all observed synergy combinations exclusively involved non-β-lactam-based inhibitors, either avibactam or vaborbactam. Combinations of avibactam with each of the following drugs including ampicillin, amoxicillin, tebipenem, and panipenem displayed synergistic effects against M. abscessus. Furthermore, the carbapenems, tebipenem, and panipenem also exhibited synergy with a novel boronic acid β-lactamase inhibitor, vaborbactam  (Table 1). Since rifamycins inhibit transcription by binding with high affinity to rpoB encoded β-subunit of bacterial RNA polymerase, resistance towards this group of antibiotics is most commonly conferred by point mutations within the target gene rpoB, especially in M. tuberculosis . Deletion of MAB_0591 enhanced the susceptibility of M. abscessus towards three tested rifamycins including rifaximin, rifapentine and rifampicin by 64–512-fold, respectively  suggests its increased accumulation within the bacterial cells. This may be due to differences in membrane penetration or specificity of efflux systems towards different rifamycins . Based on the findings reported by M. abscessus pulmonary disease should be further explored.Surprisingly, a rifampicin derivative, rifabutin showed up as an attractive hit (MIC: 2.5 mg/L), when a set of 2,700 FDA-approved drugs were screened against a clinical isolate of fections . The higM. abscessus to exhibit resistance to a wide array of antibiotics has long been recognized, our knowledge of the prodigious diversity of mechanisms involved has improved immensely in recent years, owing to the development of tools for genetic manipulation of M. abscessus. A better understanding of the genetic basis of innate antibiotic resistance is a prerequisite for the discovery and development of synergistic drug combinations, where one agent serves to salvage the antibacterial activity of a failing antibiotic by inhibiting an intrinsic resistance mechanism. This is exemplified by the recent discovery that avibactam is a potent inhibitor of M. abscessus β-lactamase Bla_Mab. When used in combination, avibactam lowered the MICs of several β-lactams in M. abscessus by 4–32-fold , are induced in a whiB7-dependent manner upon exposure to ribosomal antibiotics (eis2 and erm(41) resistance genes in M. abscessus by modulating the synthesis and/or activity of WhiB7, should be sought.Since the discovery that mutations within an cells . Remarkariabilis . These fng drugs . Recent ibiotics . Thus, wM. abscessus is a daunting and time-consuming task, an immediate and relatively simple solution to improve treatment efficacy may involve repurposing and repositioning of currently available antibiotics. A number of candidate drugs suited for this purpose have been reviewed in detail elsewhere (in vitro activity against M. abscessus (MIC: 2.5 mg/L) (M. abscessus. In addition to promising in vitro activity (MIC ≤ 1 mg/L), clofazimine displayed adequate clinical efficacy and safety when evaluated in patients with M. abscessus pulmonary infections  , as well as second generation thiacetazone derivatives like D6, D15, and D17 (Since the discovery and development of compounds that can circumvent existing resistance mechanisms in lsewhere  and are .5 mg/L) . Similarfections . Likewisme class . Moreove 1 mg/L) . Besides and D17 .in vitro synergies between novel combinations of existing antibiotic classes. For example, vancomycin, an inhibitor of cell wall synthesis, exhibited synergy with clarithromycin, an inhibitor of protein synthesis, in M. abscessus  compounds that can rescue current antibiotics by neutralizing intrinsic resistance (termed antibiotic resistance breakers), (ii) new drugs with one or more improved properties like ability to resist enzymatic modification and/or degradation, high affinity for modified or mutated targets, better solubility/uptake and low propensity for efflux , RNA synthesis (rifamycins) and protein synthesis . The impressive resistance diversity shown by M. abscessus is expected given its natural habitat is shared by many antibiotic producers, primarily the soil dwelling actinomycetes which are also reservoirs of extensively diverse resistance elements   .Staphylococcus aureus, P. aeruginosa and E. coli , fmtA, mprF, SAOUHSC_01025, and SAOUHSC_01050 which if inhibited, can greatly improve the efficacy of existing antibiotics including daptomycin, vancomycin, gentamicin, ciprofloxacin, oxacillin, and linezolid and extend their utility for treating S. aureus infections (The utility of TIS in elucidating novel antibiotic resistance functions is highlighted by its recent application to the discovery of a repertoire of previously unknown intrinsic factors impacting resistance to antibiotics of different classes including oxazolidinones, fluoroquinolones, and aminoglycosides in pathogens of high clinical concern such as  E. coli . Screenifections .M. abscessus and other emerging NTM pathogens. Such breakthrough technologies will greatly impact the kinds of questions which can be addressed in this field, for example, which resistance elements unique to M. abscessus render it unusually more drug resistant than other NTM species? or does M. abscessus already carry genes which confer resistance to novel NTM drugs in development?In the coming years, high-throughput next-generation strategies like TIS will undoubtedly rise to prominence in the NTM drug resistance field and radically advance our understanding of resistance mechanisms in All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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. The reviewer UG and handling Editor declared their shared affiliation at time of review.


Australopithecus africanus and Paranthropus robustus exploited and—in part—partitioned their environment. Specifically, we explore the extent to which first maxillary molar roots (M1) are oriented and thus, by proxy, estimate the direction of loads habitually exerted on the chewing surface. Landmark-based shape analysis of M1 root reconstructions of 26 South African hominins and three East African Paranthropus boisei suggest that A. africanus may have been able to dissipate the widest range of laterally directed loads. Paranthropus robustus and P. boisei, despite having overlapping morphologies, differ in aspects of root shape/size, dento-cranial morphologies, microwear textures and C4 food consumption. Hence, while Paranthropus monophyly cannot be excluded, equivalence of dietary niche can. The South African hominins occupied distinct ecological niches, whereby P. robustus appears uniquely adapted to dissipate antero-posteriorly directed loads.Plio-Pleistocene hominins from South Africa remain poorly understood. Here, we focus on how  Despite species  or shoul species . Nor do  species ,5. To unnchronic –9 and apnchronic –12. SpecGorilla and Pan, for example [A. africanus may have been slightly larger than P. robustus [A. africanus and P. robustus that attest to different feeding strategies. Interpreting these morphological differences is difficult however. First, A. africanus and P. robustus overlapped for only a short period of time, and different habitat specificities through time might be expected. Second, despite its assumed shared ancestry with East African P. boisei, P. robustus is not only unique in cranial morphology but, surprisingly, has no (known) modern or extinct analogue as regards enamel microstructure and microwear textures ; this m1) to jaw kinematics in South African A. africanus and P. robustus using landmark-based geometric morphometrics  whether P. robustus and P. boisei from East Africa are likely to have shared an ecological niche.Tooth root orientation and splay promises to provide novel insights into South African hominin dietary ecology. Masticatory forces generated at the occlusal surface must be dissipated into the jaw through the tooth roots. This is most efficiently achieved by aligning the force vector parallel with the long axis of the tooth, thus preventing potentially damaging bending and shear forces along the roots ,24. Withometrics . We enqu2.2.1.1s of a sample of 21 West African extant great apes ; Pan troglodytes ) as well as of 26 fossil hominins from South Africa (14 A. africanus from Makapansgat and Sterkfontein and 12 P. robustus from Kromdraai and Swartkrans)1 and, for comparison, of three P. boisei from Kenya (West Turkana and Chesowanja) and Tanzania (Olduvai) to indirectly address issues of phylogenetic constraints in P. robustus were segmented from computed tomography scans (CTs) in Avizo 9.01   were collected in Avizo for each molar around the neck of the tooth (cervix) as well as on the furcation of the roots and the tips of the three roots  by generalized least squares Procrustes superimposition. Principal components analysis (PCA) of Procrustes coordinates was used to generate a morphospace of M1 root shape variation. All analyses were done in PAST v. 3.14 [A. africanus, P. robustus, P. boisei, Pan troglodytes and G. gorilla) were compared using canonical variates analysis (CVA) and the statistical significance was assessed with a permutation test (10 000 rounds) in MorphoJ [p = 0.66) and chimpanzees , respectively, we did not take into account sexual dimorphism in any of the subsequent analyses.Centroid sizes of the fossils and great apes by species were compared using the Mann–Whitney  v. 3.14 . In orde v. 3.14  in R [52 MorphoJ . Allomet MorphoJ  and its 3.1 root shape variance  and reflects variation in bucco-palatal root splay , mesio-distal tilting of the roots  and mesio-distal expansion of the root base . PC 2  accounts for differences in the relative proportion of the palatal and the buccal roots  and the orientation of the distobuccal root , while PC3  reflects differences in the height of the interradicular furcation  and the mesio-distal spread of the buccal roots .The first PC explains 39% of the MA. africanus and P. robustus are more distinct from one another than are the sympatric great apes, i.e. there is less overlap (a). Australopithecus africanus exhibits roots that are splayed bucco-palatally and aligned vertically in buccal view . Although fossils within the A. africanus cluster have been attributed to a second species, A. prometheus, the overall variation observed for A. africanus does not exceed that seen in extant great apes. Hence, we did not explore the possibility of a second species further in the present study. The majority of the P. robustus and the three P. boisei fossils have both negative PC1 and PC2 scores which are associated with equally proportioned buccal and palatal roots with a moderate bucco-palatal root splay. The overall distally inclined roots, when viewed buccally, distinguishes them from A. africanus . When PC3 is considered, P. boisei forms a separate cluster outside the P. robustus shape space and this is associated with a lower furcation and more diverging buccal roots . As regards the great apes, Pan troglodytes shows a low degree of bucco-lingual divergence and both the palatal and buccal roots are tilted distally, while G. gorilla has a moderate bucco-palatal root splay and mesio-distally diverging buccal roots . A CVA based on Procrustes distances confirms the observed grouping pattern in the PCA, i.e. mean shapes are significantly different within and between the two South African fossil hominins and the great apes; there is no statistical difference for P. boisei, probably due to the group's small sample size  and between the great apes (G. gorilla > Pan troglodytes) (p = 0.006) as revealed by a multivariate regression. Large M1 roots as in P. boisei and some G. gorilla correspond to more evenly sized buccal and palatal root portions, while small roots—as seen in Pan troglodytes—have a narrower root splay and a relatively large palatal root portion .Although there are significant M4.A. africanus and P. robustus could initially have been driven by niche partitioning or may—perhaps—be due to phylogenetic constraints in the latter taxon. To do so, we focused on tooth root form (shape and size) and—in particular—tooth root splay, a functionally important trait that may inform whether a species habitually consumes foods that need to be broken down in crush  or in shear . Analyses reveal clear differences between all extant and extinct primates, indicative of distinct dietary adaptations, whereby A. africanus has the most bucco-palatally splayed M1 roots compared to the other species  and slender, supero-inferiorly oriented tooth roots of Pan troglodytes  suggests that this species engaged in an increased lateral stroke of the mandible to consume more ductile (tougher) foods than P. robustus, albeit—presumably—at a lower bite force, as judged by the smaller roots  seem to imply more vertical  loading at, perhaps, higher bite forces [Extrapolating from great ape ecology and their relative positions within the morphospace , the larots (see ). By cone forces ,63,64. W1 crown base areas in A. africanus and P. robustus are similar [P. robustus teeth were subjected to higher occlusal loads in vivo. Such an interpretation is in accord with a higher occurrence of three-rooted maxillary fourth premolars in P. robustus when compared with A. africanus [P. robustus, like A. africanus but unlike P. boisei, displays a strengthening of the anterior face, i.e. an anterior pillar, which is probably an adaptation to higher bite forces, too [P. robustus are also implicated by the apically oriented enamel prisms in relation to phase I and phase II wear facets [P. robustus from A. africanus [P. boisei exhibits relatively flatly worn molars, too [P. robustus and P. boisei. Such an interpretation seems highly unlikely, however, not least because of the different ecological settings in which P. robustus and P. boisei lived [The relationship between tooth crown size and root size reflects dietary preferences among anthropoid primates ,65, such similar , whereasfricanus ; this woces, too ,67. Highr facets , which wfricanus ,68 . Jaw kinematics and the mechanism by which these two taxa achieved their flat molar wear most certainly differed. In contrast with P. robustus, P. boisei apparently engaged in horizontally directed repetitive chewing [P. boisei are most probably an adaptation to high rates of dental wear [P. boisei lacks substantial amounts of enamel prism decussation, thus making the dental tissue vulnerable to tensile forces; this argues against substantial amounts of laterally directed loads that are necessary when masticating tough foods [P. robustus teeth [P. boisei, P. robustus displays overall distally facing M1 roots and converging buccal roots . This unusual tooth root morphology explains the apparent contradiction and raises questions about the foods consumed by P. robustus.Although both and size , as well chewing ,72,73, a chewing . Such a  chewing ; such anus teeth , suggestal roots a,c. ThisParanthropus robustus has a unique microwear texture pattern among primates [P. boisei [. Overall, P. robustus' microwear texture seems perhaps closest to Papio cynocephalus [P. robustus. Yet, as regards the carbon isotope composition of dental tissue, P. robustus is not distinct from either A. africanus or the papionins but from P. boisei [P. robustus. As it is unlikely that P. robustus occupied a dietary niche to the exclusion of all other primates (both extant and extinct), the possibility must therefore be entertained that P. robustus simply comminuted food in a different manner, e.g. through an increased antero-posterior component to the mandibular stroke. Such a suggestion is reasonable on the grounds of P. robustus’ mandibular morphology [P. boisei TMJ morphology in this taxon [P. robustus  that all masticatory strokes have a slight antero-posterior component and (ii) that the TMJ, which links the mandible with the cranium, has six degrees of freedom, thus allowing for mandibular movement (rotation and translation) in all directions. Such a morphological set-up also implies that the masticatory apparatus is a redundant system, whereby the same outcomes, i.e. comminution of foods, can be achieved through different mechanisms, i.e. mandibular strokes. This compounds dietary reconstructions and highlights that the answer to the question about P. robustus diet may not so much lie in what they were eating, but in how. Only a multi-disciplinary approach has the potential to throw light on the ‘what’, although some general conclusions are nonetheless possible.primates ,58,76 wh. boisei . Overallcephalus ,76, but . boisei . This marphology  combinedis taxon . To whatH 7, see ) compareP. robustus. Whatever the habitual (as yet unidentified) foods of this species may turn out to have been, it is clear from its tooth root morphology that this hominin occupied a dietary niche distinct from that of P. boisei and probably from A. africanus, too. Importantly, however, the foods habitually consumed not only required a different mechanism for breakdown, but were most likely of relatively high quality and abundant. The latter is suggested on the basis that P. robustus had absolutely and relatively larger brains than either A. africanus or P. boisei [P. robustus the opportunity to adjust to fluctuating environmental conditions and to survive well after the other taxa had become extinct, i.e. into the mid-Pleistocene Climate Transition.All primates including anthropoids are behaviourally flexible in their food choices , and may. boisei ,83. Buil


AbstractRhynchotermesarmatussp. nov. is described from soldiers and workers collected in the Magdalena River Valley of Colombia. Both castes of this new termite are superficially similar to R.perarmatus (Snyder) but the former are smaller, head capsules yellowish instead of reddish, and among additional characters, the soldier has narrower mandibles and marginal teeth. Rhynchotermes Holmgren constitutes a peculiar group of neotropical termites ranging from Belize to Argentina. They feed openly on surface litter during crepuscular or nocturnal forays. They nest underground or in shallow epigeal nests. Soldiers are either monomorphic or weakly dimorphic and workers are monomorphic. A thorough revision of Rhynchotermes by R.amazonensis Constantini & Cancello, R.bulbinasus Scheffrahn, R.diphyes Mathews, R.matraga Constantini & Cancello, R.nasutissimus (Silvestri), R.perarmatus (Snyder), and R.piauy Cancello. I herein describe an eight species, R.armatus sp. nov., from Colombia.The genus EVA) was prepared for photography by removing the entire worker P2 section in ethanol. Food particles were expelled from the P2 tube by pressure manipulation. The tube was quickly submerged in a droplet of PVA medium (BioQuip Products Inc.) which eased muscle detachment by further manipulation. The remaining EVA cuticle was longitudinally cut, splayed open, and mounted on a microscope slide using the PVA medium. The EVA was photographed with a Leica CTR 5500 compound microscope with bright field optics using the same montage software.Live specimens were preserved in 85% ethanol. External and internal worker morphology was depicted using two different methods. In the first method, soldiers and workers were suspended in Purell Instant Hand Sanitizer in a plastic Petri dish. This allowed for transparent posturing and support during photography using a Leica M205C stereomicroscope controlled by Leica Application Suite version 4.0 montage software. The worker enteric valve armature , 23JAN96, col. J. Krecek. Three colonies: CO879, holotype soldier  and those with mandibles shorter than the head . Rhynchotermesarmatus falls into the former group and is closest R.perarmatus, each having a tubular nasus and large mandibles. R.perarmatus and 3.0–3.2 mm for its junior synonym, R.major  mandibles, including the marginal teeth  (https://www.termitediversity.org/sandsi-sinocapri?lightbox=dataItem-jy00jfyy).The next closest species, R.perarmatus. Although R.perarmatus, he gave an apt analogy for the gestalt of all Rhynchotermes soldiers: “It is a thoroughly armed species and runs about audaciously with its nasus or beak elevated at an angle of 45°, reminding one of an antiaircraft gun”.The species name is a truncated derivative of its closest congener, Rhynchotermes key by R.armatus:A new couplet (3) for the R.perarmatus, R.bulbinasus, and R.armatus) are restricted to Central America and northern Colombia . No doubt further exploration will yield more new taxa.The Magdalena River Valley lies between the central and eastern ranges of the Colombian Andes and is host to various endemic faunas including birds , fishes  Miocene . This ti Miocene  which mae region . Additio


Salmonella enterica poses a growing threat to public health. Here, we studied the genome-wide evolution of 90 S. enterica AMR isolates, representing one host adapted serotype (S. Dublin) and two broad host range serotypes (S. Newport and S. Typhimurium).The emergence of antimicrobial-resistant (AMR) strains of the important human and animal pathogen S. Typhimurium had a large effective population size, a large and diverse genome, AMR profiles with high diversity, and frequent positive selection and homologous recombination. AMR S. Newport showed a relatively low level of diversity and a relatively clonal population structure. AMR S. Dublin showed evidence for a recent population bottleneck, and the genomes were characterized by a larger number of genes and gene ontology terms specifically absent from this serotype and a significantly higher number of pseudogenes as compared to other two serotypes. Approximately 50% of accessory genes, including specific AMR and putative prophage genes, were significantly over- or under-represented in a given serotype. Approximately 65% of the core genes showed phylogenetic clustering by serotype, including the AMR gene aac (6′)-Iaa. While cell surface proteins were shown to be the main target of positive selection, some proteins with possible functions in AMR and virulence also showed evidence for positive selection. Homologous recombination mainly acted on prophage-associated proteins.AMR S. enterica and serotype. Evolutionary patterns observed in S. Typhimurium are consistent with multiple emergence events of AMR strains and/or ecological success of this serotype in different hosts or habitats. Evolutionary patterns of S. Newport suggested that antimicrobial resistance emerged in one single lineage, Lineage IIC. A recent population bottleneck and genome decay observed in AMR S. Dublin are congruent with its narrow host range. Finally, our results suggest the potentially important role of positive selection in the evolution of antimicrobial resistance, host adaptation and serotype diversification in S. enterica.Our data indicates a strong association between genome content of The online version of this article (10.1186/s12862-019-1457-5) contains supplementary material, which is available to authorized users. Salmonella enterica, the causative agent of salmonellosis, is a human and animal pathogen that causes substantial economic losses and major public health concerns worldwide [S. enterica have further raised the public concern, as AMR S. enterica compromise the ability to treat infections in humans and animals [S. enterica may be more virulent than susceptible ones [orldwide , 2. Salmorldwide . In the orldwide  and mediorldwide . The eme animals , 7. In able ones .S. enterica contains > 2500 recognized serotypes, which display a broad range of epidemiological and ecological characteristics. Host-adapted serotypes typically induce systemic disease in a limited number of host species, while non-host-adapted serotypes usually cause self-limiting gastroenteritis and less commonly systemic disease in a wide range of hosts [S. enterica serotype differences in host ranges and virulence characteristics are associated with genomic characteristics . These genomic characteristics are consequences of a variety of evolutionary and population genetics processes, such as gene acquisition and deletion, positive selection, homologous recombination and changes in population size. In particular, acquisition of non-homologous novel genes  by plasmid- or phage-mediated horizontal gene transfer, has been demonstrated to play a critical role in the evolution of Salmonella [Salmonella serotypes [S. Typhi and S. Paratyphi A (human), and S. Gallinarum (fowl) [Salmonella as well [Salmonella genes reported by [S. Typhi and S. Paratyphi appear to have experienced a burst of recombination involving a quarter of their genes during the course of their adaptation to a highly virulent and human-specific lifestyle [Ne) can also have important impacts on emergence and diversification of bacterial lineages [S. Typhi in Asia and Africa [of hosts . Previouof hosts –12 have lmonella , 13. Preerotypes . Loss ofm (fowl) –16. Besi as well , 17, 18.orted by  showed eifestyle . Changeslineages . For exad Africa .Salmonella, many of these studies have focused on a few specific serotypes (often S. Typhi) [Salmonella, especially the potential influence of processes such as positive selection on the evolution of its antibiotic resistance. A few specific Salmonella serotypes are of a particular concern with regard to emergence and spread of MDR strains, including, but not limited to, serotypes Typhimurium, Newport, and Dublin, which represent the three serotypes this study focused on. As a pathogen infecting a wide range of hosts, S. Typhimurium is one of the leading causes of gastroenteritis in humans, and it is able to produce systemic infection in calves, sheep, goats, and pigs [S. Typhimurium phage type DT 104 represents one of the first MDR Salmonella clonal groups that was considered as a particular public health concern. This clonal group is characterized by a typical pattern of penta-resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide, and tetracycline, and appears to have emerged in the United Kingdom as early as 1980 with global dispersal since the 1990s [S. Newport is a non-host-adapted pathogen that has been linked to many human gastroenteritis cases in both the United States and Europe in the past decades [S. Newport has been shown to be polyphyletic and S. Newport populations have been reported to have a geographic structure [S. Newport strains isolated from Europe were more likely to belong to Lineage I, while strains from North America were more likely to belong to Lineages II and III [Salmonella resistant to expanded-spectrum cephalosporins between 1998 and 2001 [S. Dublin is highly adapted to cattle and rarely infects humans [S. Dublin has drastically increased after the acquisition of a resistance (R)-plasmid in the early 1980s; this plasmid confers resistance to multiple antibiotics, including ampicillin, kanamycin, and nalidixic acid. [S. Dublin increased more than that for infection with other serotypes, and a higher proportion of isolates were resistant to > 7 classes of antibiotics during 2005–2013 than during 1996–2004 [S. Dublin, previous studies suggested that at least some antimicrobial resistance traits in S. Dublin, such as resistance to nalidixic acid, were acquired within the bovine reservoirs [S. Typhimurium, S. Newport, and S. Dublin strains, emergence of AMR and MDR has also been described in other serotypes including Heidelberg and Paratyphi B [While a number of studies have explored the evolution of . Typhi) , 21, and. Typhi) , 16, 22 and pigs , 23, 24.he 1990s , 26. In he 1990s . Most DThe 1990s . S. Newp decades , 28. S.  and III . A widesand 2001 . As a hos humans . In Japaic acid. . In the 996–2004 . Consistservoirs , 32. Whiatyphi B .S. enterica, we employed comparative genomic, evolutionary and phylogenetic approaches to further analyze previously reported [S. Dublin, S. Newport, and S. Typhimurium isolates from dairy cattle and humans from Washington state and New York state. The specific aims of this study were i) to characterize the evolutionary history of AMR S. Dublin, S. Newport, and S. Typhimurium; ii) to assess the relationship between genome content, including AMR genes and putative prophage genes, and serotype; iii) to identify genomic characteristics associated with the narrower host range of S. Dublin compared to other two serotypes; iv) to explore the influence of population dynamics, homologous recombination, and positive selection on the evolution of AMR S. enterica.In order to better understand the serotype-specific microevolution patterns of AMR reported  genome sS. Typhimurium was larger (by > 1000 orthologous genes) than that of S. Dublin and S. Newport, and the core genome of AMR S. Typhimurium was smaller (by approximately 300 orthologous genes) than that of the other two serotypes  and genetic characteristics  , these gion Fig. . The genS. enterica [S. Typhimurium isolates exhibited a large range in the number of AMR genes with 3 to 22 annotated AMR genes in a given isolate . A total of 732 prophage protein groups (PPGs) were identified, including 44 PPGs present in all 90 genomes  population models, built based on single nucleotide polymorphism (SNP) data, were compared. Core SNPs were identified within each of the three serotypes including 2725 in S. Dublin, S. Newport and S. Typhimurium isolates, including 126, 80, and 83 genes present in all AMR S. Dublin, S. Newport and S. Typhimurium, respectively  as well as the largest number of genes specifically absent (including genes encoding some cytoplasmic proteins and transporters) . A total of 445, 360, and 427 genes were significantly over-represented in AMR S. Dublin, S. Newport and S. Typhimurium isolates. aadB and cmlA were significantly enriched in AMR S. Dublin, while blaCARB, sulI, aadA, tetRG, and tetG were significantly enriched in AMR S. Typhimurium  in S. Dublin, S. Newport, and S. Typhimurium, respectively. Specifically present GO terms of particular interest included Type I site-specific deoxyribonuclease activity (GO:0009035), protein N-linked glycosylation via asparagine (GO:0018279), and antimonite transport (GO:0015699) in S. Newport, and inositol catabolic process (GO:0019310), response to hydrostatic pressure (GO:0051599), sphingolipid metabolic process (GO:0006665), and regulation of autophagy (GO:0010506) in S. Typhimurium. Specifically absent GO terms of particular interest included arginine transport  and ribulokinase activity  in S. Dublin.At the gene ontology level, 4229 gene ontology (GO) terms  and 945 enzyme commission (EC) numbers were assigned to genes present in the 90 genomes  as compared to AMR S. Newport and S. Typhimurium genomes   and Lineage III [S. Newport isolates included in this study belong to Lineage IIC , while 2 isolates have the same ST as the DT 193 reference isolate (ST-34) -Iaa, which encodes an enzyme that acetylates aminoglycosides, displayed clustering by serotype, supported by high bootstrap values , indicating that a large fraction of core genes 65%) are serotype-clustered  showed evidence for diversifying selection across all serotypes, as detected by the site model in PAML (FDR <  0.05) . Genes encoding a phage tail protein, an anion permease, and a flagellin showed evidence for strong positive selection, indicated by an overall dN/dS (ω) value > 500  ; the strongest selection pressure was found in the core gene encoding a DNA-binding transcriptional regulator, with ω ~∞, p = 4.00%  and the gene encoding the outer membrane protein assembly factor BamA  AMR S. Dublin, S. Newport and S. Typhimurium exhibited distinct genomic characteristics and evolutionary patterns; (ii) genome content of AMR S. enterica, including AMR genes, was strongly associated with serotype; (iii) positive selection mainly targeted genes encoding cell surface proteins and genes likely to function in virulence and antimicrobial resistance, while homologous recombination mainly acted on prophage-associated genes. Overall, our data suggest that evolution of AMR characteristics in S. enterica shows serotype-specific patterns and may involve positive selection in antimicrobial resistance-related genes, in addition to acquisition of AMR genes through horizontal gene transfer.While a number of S. Dublin isolates included in this study displayed a relatively large core genome and small pan genome, showed low diversity of AMR genes , and encoded a few prophages including Salmon_ST64B, Entero_Fels_2 and Entero_P22, which appeared to be associated with AMR S. Dublin isolates. These genomic characteristics might be explained by the relatively small Ne, a recent population bottleneck, clonal population structure , and limited diversifying selection observed here in AMR S. Dublin. This observation is consistent with the evolution being mainly driven by genetic drift, coupled with negative selection in bacteria with small Ne [S. Newport and S. Typhimurium, S. Dublin genomes contained significantly more pseudogenes, and more orthologous genes and gene ontologies specifically absent from this serotype, suggesting genome decay, which are documented characteristics in host-adapted pathogens [S. Dublin  may not be required for growth and survival in the host niches typically occupied by S. Dublin. For example, a previous study reported that the arginine-ornithine antiporter is crucial for supplying external arginine as substrate to the Arginine Deiminase System (ADS), which contributes to acid resistance through production of ammonia. Consequently, for many pathogens the ADS system has been linked to virulence and fitness in the host [Staphylococcus epidermidis and Streptococcus suis [S. Dublin seems to be adapted to cattle, which have a rumen pH (ranging between 5.7 and 7.3) [S. Dublin, resulting in the loss of genes participating in the ADS-dependent arginine catabolism. In addition, L-ribulokinase participates in one of the two L-arabinose catabolism pathways to generate L-ribulose 5-phosphate [S. enterica [S. Dublin.AMR small Ne , 20. Comathogens , 14, 35.the host . Specificus suis , 37. As and 7.3)  much higand 7.3) , acid tohosphate . As L-arenterica , loss ofS. Dublin, AMR S. Newport genomes displayed a relatively low level of diversity, which might be a result of the likely-emergence of antimicrobial resistance in one single lineage, Lineage IIC, with all isolates tested here classified into this lineage. Also, the low genomic diversity may be due to a recent population size reduction, a relatively clonal population structure, and the low frequency of positive selection detected in AMR S. Newport in this study. In addition, geographic barriers might contribute to the low level of diversity observed in our data set; this hypothesis is supported by the association between geographic location and different Newport lineages observed in previous MLST and whole genome sequencing (WGS) studies [S. Newport. These GO terms may be particularly important for the growth and survival of S. Newport in diverse hosts and environments. For example, restriction enzymes are the primary bacterial defense against lytic phages. Therefore, the presence of type I site-specific deoxyribonuclease complex and activity specifically in AMR S. Newport may represent a specific defense system against lytic phages in this serotype. In addition, glycans resulting from asparagine-linked glycosylation play a crucial role in various biological processes, such as protein folding, cellular targeting and motility, and immune response, in all three domains of life [S. Newport in host niches. Another interesting GO term specifically detected in AMR S. Newport is the biological process of antimonite transport. Antimonite is the salt of antimony (Sb(III)) and is toxic to cells [S. Newport suggest that AMR S. Newport may have the ability to pump antimonite out of a cell, consequently conferring antimonite resistance. Interestingly, it has been suggested that microorganisms with multiple heavy metal resistances (including antimonite resistance) may also show resistance to some antibiotics due to co-location of heavy metal and antimicrobial resistance genes on the same mobile elements [Similar to AMR  studies , 42, 43. of life . Hence, to cells . Existenelements –48.S. Typhimurium exhibited a high level of genetic diversity, indicated by a relative open pan genome, a small core genome, a high diversity of AMR genes, and large variation in the number of AMR genes and prophage genes per genome. The high genetic diversity of AMR S. Typhimurium might result from its large Ne, relatively stable Ne over time, panmictic population structure , and relatively frequent positive selection observed in this study. These observations are consistent with the hypothesis that in large populations, selection overpowers genetic drift [S. Typhimurium may be associated with the emergence of antimicrobial resistance in multiple S. Typhimurium lineages, unlike the other broad-host serotype S. Newport, where antimicrobial resistance seems to have emerged in a single lineage (IIC). Specifically, at least two MDR S. Typhimurium groups have emerged in the past decades, DT 104 and DT 193 [S. Typhimurium. The functions associated with these gene ontologies might help S. Typhimurium to successfully compete in different niches and hosts. Notably, the GO term “regulation of autophagy” was also found to be specifically present in AMR S. Typhimurium. Autophagy is induced in the host to combat infection with various pathogenic bacteria, in which a double-membrane structure – autophagosome – engulfs invading pathogens and brings them to the lysosome for degradation [S. Typhimurium has been shown to exploit eukaryotic autophagy machinery during its intracellular life style in the host by inducing autophagic response to enter the host cell and suppressing autophagy within 3 h of infection [Salmonella serotypes may differ in their ability to suppress autophagy as genes involved in regulation of autophagy function were only found in S. Typhimurium.AMR ic drift . In addid DT 193 . Based od DT 193 , 50), weradation , 52. Hownfection ; our datS. enterica genome content and serotype could be explained by the periods of elevated serotype diversification  followed by long time of relatively lower but constant diversification observed in Salmonella [Even though serotypes within a bacterial species are defined based on cell surface antigens, genome content is typically correlated to serotype , 11. Ourlmonella .Salmonella serotypes. The association between AMR genes and AMR S. Typhimurium was especially strong, indicated by 5 AMR genes  significantly enriched and 6 AMR genes  significantly under-represented in this serotype. blaCARB, sulI, aadA, tetRG, and tetG have been previously reported to be significantly associated with plasmid replicon IncFII(S) [S. Typhimurium strains [blaCMY, sulII, tetA, tetR, strA, and strB were previously reported to be significantly associated with the plasmid replicon IncA/C2 [S. Newport [S. Newport isolates analyzed here. Interestingly, the only AMR gene present in all 90 genomes, aac (6′)-Iaa, displayed a robust clustering by serotypes in the gene tree. AAC (6′)-Iaa is a chromosomal-encoded aminoglycoside acetyltransferase, which effectively acetylates tobramycin, kanamycin, and amikacin [aac (6′)-Iaa might have been introduced into an ancestral strain of S. Dublin, S. Newport and S. Typhimurium before serotype diversification and subsequently transmitted vertically, through chromosome replication.Consistent with a previous analysis of the genomes studied here , differencFII(S) ; IncFII  strains . blaCMY, IncA/C2 . These r Newport  and wereamikacin . This reSalmonella [Salmonella, a number of genes showed evidence for homologous recombination or positive selection. Most of these genes were serotype-associated, suggesting the contribution of homologous recombination and positive selection in serotype diversification.Homologous recombination and positive selection have been shown to play critical roles in the evolution of bacteria including lmonella , 58, 59.Salmonella’s limited ability to acquire foreign DNA through transformation [Most of the serotype-associated accessory genes showing evidence for homologous recombination encode prophage-associated proteins, such as phage tail proteins, capsid proteins, the protein NinG, a baseplate assembly protein, and recombinases. This result supports phage-mediated homologous recombination as one important mechanism in the remodeling of the bacterial genome . Moreoveormation  or the lormation .Salmonella to antibiotics. Porins also may play a role in virulence and have been reported to inhibit leukocyte phagocytosis by activating the adenylate cyclase system in S. Typhimurium [Congruent with previous observations that cell surface proteins are main targets of positive selection in both eukaryotes and prokaryotes , 62–66, himurium .S. Cerro, which appears to show reduced human virulence [Escherichia coli to the ribosome binding antibiotic viomycin [S. enterica against some ribosome-targeted antibiotics. Overall, these findings suggest that positive selection may not only contribute to adaptation to stresses encountered in hosts and environmental niches, but also may play a role in the evolution of antimicrobial resistance.Genes that were found to have evidence for positive selection, but do not encode cell surface proteins, include genes encoding proteins that appear to contribute to bacterial virulence  or bacteria-bacteria interactions  –79. Inteirulence . In addiirulence . While iviomycin . Hence, S. Dublin, S. Newport and S. Typhimurium exhibited distinct evolutionary patterns and genomic characteristics. AMR S. Typhimurium showed a large and diverse genome and frequent positive selection and homologous recombination, consistent with multiple emergence events of AMR strains and/or effective dispersal and ecological success of this serotype in different niches. AMR S. Dublin showed evidence for experiencing a recent population bottleneck . For example, while positive selection in target proteins of antibiotics  is well documented [Salmonella serotypes and other human and zoonotic pathogens with different host ranges and geographical locations are thus needed to characterize a variety of evolutionary mechanisms , including their relative roles and dependencies. For example, some horizontally transferred plasmids may lead to intermediate- or low-level resistance that is not clinically relevant but enhances the risk of subsequent emergence of fully resistant subtypes via selection of mutations that lead to high-level resistance, as recently proposed for quinolone resistance [By performing a comprehensive genome-wide analysis, we show that antimicrobial-resistant a pestis  and Mycorculosis ) and gencumented , our stusistance . In addisistance . While iS. enterica isolates [n = 49) and the fecal samples of bovine (n = 41) between 2008 and 2012 was used for this study, 45 of which were from New York state and 45 were from Washington state , Newport (n = 32) and Typhimurium (n = 37)), as determined using the White-Kauffmann-Le Minor scheme [A previously reported set of genome sequences for 90 isolates  collecteine n = 4 between r scheme  and had ium n = 3), as detMethods of whole genome sequencing and genome assembly were detailed in . The seqS. Newport isolates, genome assemblies of S. Newport str. CVM 19443 (GenBank Accession AHUB00000000), S. Newport str. CVM 19567 (AHTR00000000), S. Newport str. SL254 (CP001113), S. Newport str. SL317 (ABEW00000000) were downloaded from GenBank as reference genomes of S. Newport Lineage IIA, IIB, IIC, and III, respectively. An ML tree of 32 S. Newport genomes and 4 reference genomes was constructed following the same approach as for the ML tree of 90 genomes.The reference-free variant calling among all 90 genomes and phylogenetic tree construction followed the methods provided by . To confS. Typhimurium isolates, S. Typhimurium str. LT2 (RefSeq NC_003197.1) was used as a reference genome; for S. Newport isolates, S. Newport str. SL254 (GenBank Accession CP001113) was used as a reference; for S. Dublin isolates, the contigs of isolate BOV_DUBN_WA_10_R9_3233 in this study were used as a reference as detailed in [To maximize resolution, variant calling was performed within each serotype using the Cortex variant caller (cortex_var)  followinailed in , and SNPailed in .S. Typhimurium was the general time reversible (GTR) model [S. Newport and S. Dublin is the Kimura 2-parameter model [S. Typhimurium was set to 1 × 10− 6 /site/year [S. Newport and S. Dublin were set to the average rate of bacteria (2.1 × 10− 7 /site/year) since the literature lacks estimations of substitution rates for either serotype. An MCMC algorithm was run for 100 million generations, with sampling every 10,000 generations, to estimates the posterior probability distributions of the genealogical and demographic parameters of a sample. Marginal likelihood estimations were computed by path sampling [The resulting high-quality SNPs were used to infer population dynamics for each serotype using BEAUti version 1.8.4 and BEAST version 1.8.4 . The besR) model , while ter model . An asceer model  and baseite/year . S. Newpsampling , 97 and AMR genes in all 90 assembled genome sequences were determined by nucleotide BLAST version 2.4.0  and the The putative prophage-associated proteins were detected based on the sequence similarity of the protein-coding gene predicted by Prodigal version 2.60  to a curS. Typhimurium isolates analyzed in this study, two reference strains of phage type DT 104 , one reference strain of phage type DT 193 – str. SO4698–09 (NZ_LN999997), and all the 37 assembled Typhimurium genomes from our dataset were analyzed using the MLST online service provided by Center for Genomic Epidemiology (https://cge.cbs.dtu.dk/services/MLST/).To determine the sequence type of AMR https://rocaplab.ocean.washington.edu/tools/genbank_to_fasta/). Amino acid sequences of each orthologous group were aligned using MUSCLE version 3.7 [Assembled genomes were annotated by the NCBI Prokaryotic Genome Annotation Pipeline . OrthoMCsion 3.7 . Amino asion 3.7 . Gene trsion 3.7  under thsion 3.7 . Customision 3.7 .The Recombination Detection Program (RDP) and Pairwise Homoplasy Index (PHI) test were used to detect homologous recombination in individual orthologous genes. Cleaned-up alignments in which identical sequences were removed were used. The PHI test was performed in SplitsTree4 version 4.14.2 . Seven h0/L1), where L0 is the likelihood estimate for null model  and L1 is the likelihood estimate for alternative model (positive selection model) which has more free parameters. The degree of freedom (df), which is calculated as the difference in the number of free parameters between the null and alternative models, was set to 2 for site model and 1 for site-branch model. Statistical significance was determined by approximating the test statistic to a χ2 distribution. A false discovery rate (FDR) correction was applied and genes with FDR < 0.05 were considered significant.Nucleotide sequence alignments of protein coding genes containing isolates from all three serotypes and only isolates from each serotype were cleaned up by removing identical sequences. Alignments that had more than three non-identical sequences and contained at least one non-synonymous mutation were used for positive selection analyses using the PAML program version 4.8 . UnrooteThe presence/absence of orthologous gene content table was used to perform the rarefaction curve estimation of core genome size and accumulation curve of pan genome size  estimation for each serotype using an R software script provided by . The proAdditional file 1:Table S1. Distribution of the orthologous genes identified among all 90 genomes. Table S2. The pan and core genome size of all three serotypes and each serotype. Table S3. Basic information of 90 AMR S. enterica isolates. Table S4. Distribution of the AMR genes identified among all 90 genomes. Table S5. Distribution of the PPG genes identified among all 90 genomes. Table S6. Orthologous genes significantly over- and under- represented in S. Dublin, S. Newport, and S. Typhimurium. Table S7. Distributionof the GO and EC terms identified among all 90 genomes. Table S8. GO terms and EC numbers significantly over- and under- represented in S. Dublin, S. Newport, and S. Typhimurium. Table S9. Prophages and number of corresponding PPG genes significantly over- and under- represented in S. Dublin, S. Newport, and S. Typhimurium. Table S10. MLST sequence type of S. Typhimurium isolates. Table S11. Isolates identified by Gubbins showing recombinant regions. Table S12. Genes undergoing positive selection across all serotypes, within S. Newport and S. Typhimurium detected by site model using PAML. (XLSX 3780 kb)Additional file 2:Figure S1. The distribution of AMR gene numbers of AMR S. Dublin, AMR S. Newport, and AMR S. Typhimurium isolates. The AMR gene number of AMR S. Dublin is significantly different from that of AMR S. Newport and AMR S. Typhimurium. Figure S2. The distribution of PPG gene numbers of AMR S. Dublin, AMR S. Newport, and AMR S. Typhimurium isolates. Figure S3. The distribution of pseudogene numbers in S. Dublin, S. Newport, and S. Typhimurium. Figure S4. Gene tree of AAC (6′)-Iaa inferred by maximum likelihood method. Tree is rooted by midpoint. Bootstrap values > 70% are presented on the tree. S. Dublin is indicated by blue, S. Newport by blue, and S. Typhimurium by red. Figure S5. Maximum likelihood tree of AMR S. Newport isolates, and Lineage II  and Lineage III reference isolates. Tree is rooted by midpoint. Bootstrap values of major clades are presented on the tree. Reference isolates are indicated by red. (DOCX 681 kb)