Helicobacter pylori (H. pylori) to routinely used antibiotics in Cambodia, an unexplored topic in the country, and assessed next-generation sequencing’s (NGS) potential to discover genetic resistance determinants. Fifty-five H. pylori strains were successfully cultured and screened for antibiotic susceptibility using agar dilution. Genotypic analysis was performed using NGS data with a CLC genomic workbench. PlasmidSeeker was used to detect plasmids. The correlation between resistant genotypes and phenotypes was evaluated statistically. Resistances to metronidazole (MTZ), levofloxacin (LVX), clarithromycin (CLR), and amoxicillin (AMX) were 96.4%, 67.3%, 25.5%, and 9.1%, respectively. No resistance to tetracycline (TET) was observed. Multi-drug resistance affected 76.4% of strains. No plasmids were found, but genetic determinants of resistance to CLR, LVX, and AMX were 23S rRNA (A2146G and A2147G), GyrA (N87K and D91Y/N/G), and pbp1 (P473L), respectively. No determinants were genetically linked to MTZ or TET resistance. There was high concordance between resistant genotypes and phenotypes for AMX, LVX, and CLR. We observed high antibiotic resistance rates of CLR, MTZ, and LVX, emphasizing the need for periodic evaluation and alternative therapies in Cambodia. NGS showed high capability for detecting genetic resistance determinants and potential for implementation in local treatment policies. We evaluated the primary resistance of  Drug resistance is recognized as one of the greatest public health threats, leading to 700,000 deaths per year worldwide. This figure is expected to reach to 300 million people, with a loss of up to US$100 trillion for the global economy by 2050, if not addressed [Helicobacter pylori (H. pylori) has emerged as an alarming bacterium due to its resistance rate, and it has been noted as one of 16 antibiotic-resistant pathogens that pose the most serious threat to human health, according to World Health Organization (WHO), since 2017 [H. pylori is also known to cause a wide range of gastroduodenal diseases, including gastric adenocarcinoma. H. pylori elimination is thus one of the most effective strategies to reduce the H. pylori-related disease burden [H. pylori has become a major roadblock to eradicating the infection and a challenge for clinicians, because of a dramatic decline in the efficacy of classic first-line therapy, which has reached an unacceptable threshold (<80%) compared to other bacteria [H. pylori treatment  have beH. pylori treatment, and the choice of eradication therapy is mainly based on empirical decisions made by clinicians. Thus, an antibiotic susceptibility profile is needed in Cambodia to establish effective local guidelines for H. pylori eradication therapy and allocate appropriate public health policies against H. pylori antibiotic resistance-caused threats.Cambodia is a developing country located in southeast Asia, a WHO region for which the standard triple therapy still remains effective because of a low clarithromycin (CLR) resistance rate . EpidemiH. pylori eradication regimen. However, this approach is not cost-effective, and is cumbersome and unfeasible in clinical practice. In fact, this method is not available for all clinical settings because of the special clinical microbiology laboratory requirements, like specific microaerobic culture conditions and a selective, nutrient-rich medium. Moreover, it is also time-consuming and laborious (about 10–14 days) because of H. pylori’s slow growth. As a result, it is hard to routinely perform H. pylori sensitivity testing in a clinic [Antibiotic susceptibility testing is a reliable assay that provides useful information for clinicians to prescribe an appropriate a clinic .H. pylori treatment, which is essential for risk stratification of patients and controlling drug resistance. The advent of molecular biology techniques , Sanger sequencing) has enabled the identification of molecular mechanisms underlying the observed phenotypic drug susceptibility testing (DST) to various antimicrobial agents. These conventional methods focused on specific mutations in a small region of the target gene. Some mechanisms of drug resistance are heterogeneous and some remain unclear and are still being explored, so these approaches limited the discovery of novel or rare resistance mechanisms [Mycobacterium tuberculosis with an accuracy of up to 93%, and the cost of NGS screenings were 7% cheaper than routine clinical workflows . The results from NGS were also obtained faster than routine diagnosis (9 days vs. 21 days) [H. pylori that have limited the use of these approaches for this bacterium. First, while other bacterial species have well-established comprehensive antibiotic resistance data like Antibiotic Resistance Gene-ANNOTation (ARG-ANNOT) [H. pylori data. Second, its enormous genetic diversity, due to high mutation and recombination rates, made it difficult to apply and interpret the results using approaches commonly employed in other bacteria to H. pylori. Here, we used a fast, computationally efficient, and accurate NGS-based approach employing a user-friendly graphical interface from the commercial software CLC genomic workbench that could circumvent these limitations. To deal with the drawbacks, a molecular method allowing identification of drug-resistant markers to promptly and accurately diagnose drug resistance is needed to improve chanisms . Recentlchanisms ,18. For 21 days) . HoweverG-ANNOT) , ComprehG-ANNOT) , and ResG-ANNOT) , these dH. pylori clinical isolates in Cambodia. We also determined the corresponding genotypic molecular mechanism of each antibiotic using an NGS approach, and evaluated the correlation between genotypic and phenotypic DST. In this study, we aimed to characterize the primary resistance rate of five clinically used antibiotics including amoxicillin (AMX), CLR, metronidazole (MTZ), levofloxacin (LVX), and tetracycline (TET) from H. pylori previously. Patients had an esophagogastroduodenoscopic examination at the Endoscopy Department of Cho Ray Phnom Penh Hospital in Phnom Penh, Cambodia in March 2015. During the endoscopic procedure, gastric biopsy specimens were sampled from the antrum of each participant and used to isolate H. pylori using a standard previously-established culture [H. pylori-selective plate  before being incubated for up to 10 days. The H. pylori-like colonies growing on the selective plates  were identified using the biochemical reactions of catalase, oxidase, and urease, and by microscopic examination after Gram staining. They were sub-cultured for 3 to 4 days on Mueller Hinton II Agar plates  and supplemented with 10% horse blood . All procedures were performed in microaerophilic conditions  and incubated at 37 °C. A total of 55 H. pylori clinical strains from 55 separate patients  were successfully isolated from the gastric biopsy specimens. This cross-sectional study included 206 consecutive outpatients  who had not been treated in relation to  culture . BrieflyThe study protocol was approved by the ethical committees of Oita University, Japan and Cho Ray Phnom Penh Hospital, Cambodia. All patients recruited signed informed consent.H. pylori strain 26695 was used as the control strain. Clinical breakpoints were used for discriminating between susceptible and resistant phenotypes following the guidelines of the European Committee on Antimicrobial Susceptibility Testing . The resistance to antibiotics was defined as MICs exceeding 0.125 mg/L for AMX, 0.25 mg/L for CLR, 8 mg/L for MTZ, and 1 mg/L for TET and LVX. The multidrug-resistant (MDR) phenotype was defined as the phenotype that was resistant to two or more antibiotic classes while single-drug resistance (SDR) was resistance to only one of five antibiotics used in the assay. The antibiotic susceptibility phenotype was defined using the agar dilution assay to determine the minimum inhibitory concentrations (MICs) of antibiotics following the protocols of the Clinical and Laboratory Standards Institute  . A set oThe total genomic DNA of isolates was extracted using the QIAamp DNA Mini Kit , quantified using the Quantus Fluorometer , and sequenced using the Miseq platform . DNA libraries were prepared using the Nextera XT DNA sample kit , allowing paired-end sequencing of 300-bp reads.H. pylori clinical isolates were used to investigate the known plasmids encoding antibiotic resistance using PlasmidSeeker with 26695 (NC_000915.1) as the reference genome [http://bioinfo.ut.ee/plasmidseeker/) was analyzed, and the k-mer abundance was used to distinguish between plasmid and bacterial sequences. A plasmid was considered present with at least 80% of k-mers found.Raw short read data from To reduce the false discovery rate of single nucleotide polymorphisms (SNPs), the low-quality bases  and adapters were trimmed using Trimmomatic v0.30 . The filH. pylori strains, a core SNP alignment from draft genome mapping against reference strain 26695 was generated by using Snippy v.3.2 [To assess the genetic relatedness between py v.3.2 . Recombipy v.3.2 . A maximpy v.3.2 . H. pylori resistance-related genes using the CLC genomic Workbench v8.5.1 . Using the map read to reference function, the trimmed reads were first directly mapped against a custom database of target gene sequences that likely play a biological role in antibiotic resistance in H. pylori species. These targeted genes were retrieved from the 26695 reference genome, including gyrA (HP0701) for LVX, rdxA (HP0954) for MTZ, pbp1 (HP0597) for AMX, 23S rRNA for CLR, and 16S rRNA for TET resistance. Variant identification between each isolate sequence and reference gene was performed using the CLC basic variant detection function with default options. These variants were summarized and assessed with statistical approaches for their eventual association to the resistance phenotype using the CLC Fisher exact test function. The nomenclature describing genetic variants was followed as previously reported [The antibiotic susceptibility genotypes were assessed in reported . For insreported .U test. Genotype-phenotype agreements were analyzed using Cohen’s kappa statistics. A kappa coefficient (k) value of < 0.4, 0.4–0.6, 0.61–0.8, and 0.81–1.0 indicated low, moderate, substantial, and perfect agreement, respectively. A significance of p < 0.05 using a two-tailed test was established as statistically significant. All statistics and data visualization were performed in R Environment version 3.5.1. The proportions of phenotypic DST and its 95% confidence intervals (CIs) were calculated using the Clopper-Pearson exact method from ‘DescTools’ package . The assThe antibiogram of 55 isolates from Cambodia is shown in p = 0.01) and CLR . In addition, MTZ (81.2 mg/L vs. 69.3 mg/L) and AMX (0.18 mg/L vs. 0.06 mg/L) also showed higher mean MICs in MDR compared to SDR.Notably, 21.8%  were SDR, and all of them had MTZ resistance. The rate of MDR was high, up to 76.4% . Among all clinical isolates, dual drug resistance to LVX + MTZ and MTZ + CLR was found in 40%  and 7.3%  of isolates, respectively. Triple resistance to AMX + LVX + MTZ and CLR + LVX + MTZ accounted for 9.1%  and 18.2% , respectively. The average MDR MIC was significantly higher than SDR MIC in LVX  and A2147G (p = 0.000001) were significantly associated with CLR resistance (p < 0.05). The average MICs were not significantly different between strains with mutations at position 2146 or 2147  mapped to either nucleotide position G-2146 or G-2147, indicating that the mutations occurred in both copies of 23S rRNA. Only two CLR-resistant strains had heterogeneous alleles between two copies of 23S rRNA, indicating that the mutations occurred in one of two copies of 23S rRNA, namely KH0097 (29.6% reads mapped to G at nucleotide position 2146) and KH0113 (13.9% reads mapped to G at nucleotide position 2147). H. pylori LVX resistance via point mutations in the quinolones resistance-determining region (QRDR) [p = 0.005) and D91Y/N/G (p = 0.00004) were significantly associated with LVX resistance  . The gensistance . Interesp < 0.05). Interestingly, strains with double mutations at codon 87 and 91 showed significantly higher MICs than those with single mutations at either codon 87 or 91 (p < 0.05)  strains had mutations at codon 87 or 91. On the other hand, six strains did not show any mutations at these codons, but they were resistant. Overall, strains harboring mutations at either codon 87 or 91 showed significantly higher MICs than those without mutations  B.pbp1. Mutations in the gene are thought to be the major factor for AMX resistance [p = 0.005), P473L (p = 0.0004), and T593A/G/K (p = 0.005), were significantly associated with AMX resistance  in the gene result in protein mistranslation or truncation, which is known to play a crucial role in MTZ resistance [rdxA truncation, including 15 (50%) frameshift and 15 (50%) nonsense mutations. Both MTZ-susceptible strains had an intact rdxA encoding a full-length functional rdxA (n = 51) and MTZ-susceptible strains (n = 2).We investigated sistance . We founnal rdxA . HoweverWe examined the 16S rRNA gene to discover mutations reported to confer TET resistance, specifically A926G and A928C . No straTo determine the utility of whole genome sequencing (WGS) in predicting phenotypic drug susceptibility to different antibiotics, we compared the antibiotic resistance phenotypes with their corresponding genotypes. As shown in Despite an increasing worldwide trend in antibiotic resistance, a recent meta-analysis showed that only southeast Asia (10%) and the Americas (10%) appeared to have low CLR resistance rates compared to all WHO regions . In addiH. pylori in the Association of Southeast Asian Nations (ASEAN) and declared that the resistant patterns in ASEAN countries were characterized by high CLR and MTZ resistance, as observed in this study with 40% strains being CLR + MTZ dual resistant [A survey collected expert opinions about esistant . In agreesistant . De Franesistant . LikewisLVX has recently been prescribed as a salvage therapy in patients with first-line therapy failure . HoweverH. pylori is mainly based on the former. Unlike the other bacteria, an approach based on point mutations in the genomes to identify genetic determinants of H. pylori resistance is useful and less complex [Bacteria use two main genetic strategies to respond to antibiotic attack. One is the result of mutations in genes associated with the mechanism of action of the drug, and the second is due to the acquisition of external genetic resistance elements via horizontal gene transfer . In the  complex .H. pylori population structures or mutational effects on fitness  that do not confer resistance [Because of greater speed and accuracy, molecular susceptibility testing has become a promising alternative choice to replace phenotypic DST . The mossistance . Therefosistance . This toH. pylori has two copies of the gene [Streptomyces ambofaciens, where one of four rRNA alleles caused macrolide resistance, it was suggested that changes in one of the two alleles could be sufficient to confer H. pylori CLR resistance [Additionally, our approach also considered the number of mutated gene copies like in the case of the 23S rRNA. the gene . It is ssistance .pbp1A gene [368–371, SLN433–435, SNN559–561, KTG555–557) using experimental study [The mechanism of AMX resistance is unclear, but it was believed to be caused by a point mutation in the p1A gene . Some gep1A gene  concludeal study . P473L ial study . InteresrdxA gene encoding an oxygen-insensitive nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) nitroreductase that catalyzes the reduction of MTZ [rdxA is responsible for 58.8% of MTZ-resistant isolates in Cambodia. Chua EG et al. recently reported that a R16H/C substitution of rdxA is significantly associated with MTZ-resistant strains [Resistance to MTZ primarily involves the inactivation of the n of MTZ . Our stu strains . HoweverH. pylori antibiotic-resistant patterns in Cambodia to provide an overview of antibiotic resistance conditions in the country. This could provide direction for national guidelines or protocols for H. pylori eradication. However, our study also had several limitations. First, this is a hospital-based study, so the result does not reflect the resistance patterns in the general population. Nevertheless, our study also revealed reliable, accurate resistance markers to predict phenotypic DST, especially in AMX, CLR, and LVX. This could be applied as rapid alternative investigation without culture in a clinic. Second, the sample size is relatively small for some antibiotics. Therefore, there were only few (AMX) or no resistant strains (TET) that might affect the strength of association. In addition, we only determined the genetic determinants for antibiotic resistance within well-known genes in this study. However, we provided high-quality genomes with clear clinical information, so future studies at genome level, like bacterial genome-wide association studies (GWAS), can provide further insights into the mechanism of drug resistance together with the other H. pylori genomes [This is the first study to report  genomes . FinallyH. pylori primary resistance rates to CLR, MTZ, and LVX were high in Cambodia, warning that the standard triple therapy as a first-line treatment may no longer be effective in southeast Asia, particularly in Cambodia. A periodic evaluation is essential to closely monitor and control the antibiotic resistance status in this area. Our results suggest reliable genetic markers that can be used as a rapid and accurate detection test for H. pylori resistance, especially for CLR, LVX, and AMX resistance. Additionally, our data suggested that a next-generation sequencing based approach is a useful, reliable, and reproducible method to detect antibiotic resistance-related genetic determinants. We also noted that future studies using a bacterial GWAS approach with careful preparation of the dataset might provide further insights about the mechanism of drug resistance in H. pylori.In conclusion,


Staphylococcus. Bacteria belonging to this genus are some of the most influential commensals that reside on the skin. For example, colonization by Staphylococcus aureus, a well-known pathogen, increases inflammatory responses within the skin. Conversely, colonization by Staphylococcus epidermis, a coagulase-negative staphylococcal species that are prevalent throughout the skin, can be innocuous or beneficial. Thus, manipulating the abundance of these two bacterial species likely alters the skin microbiome and modulates the cutaneous immune response, with potential implications for various inflammation-associated skin diseases. Importantly, before researchers can begin manipulating the skin microbiome to prevent and treat disease, they must first fully understand how these two species can modulate the cutaneous immune response. In this review, we discuss the nature of the interactions between these two bacterial species and immune cells within the skin, discussing their immunogenicity within the context of skin disorders.As the outermost layer of the body, the skin harbors innumerable and varied microorganisms. These microorganisms interact with the host, and these interactions contribute to host immunity. One of the most abundant genera of skin commensals is  The skin is the largest organ of the body and is inhabited by millions of commensals, including bacteria, fungi, and viruses. Despite their abundance, whether these microorganisms benefit the host is unclear. Recent technological advances such as genome sequencing have expanded our understanding of the composition and function of the skin microbiome. We now know that the microorganisms which reside in our body fall on a spectrum from ‘good’ to ‘troublesome’ residents. Specifically, beneficial microflora (the ‘good’ residents) usually cause no secondary infections, but cannot be eliminated as causes of illness . AlternaStaphylococcus aureus. These bacteria have long been notorious pathogens. S. aureus belong to the genus Staphylococcus, which is comprised of fifteen unique species that are differentiated based on molecular data [S. aureus should be considered a commensal is controversial; however, approximately 30% of the human population carries asymptomatic S. aureus in their nose [S. aureus, indicating that the presence of S. aureus is not always directly associated with disease [Staphylococcus has shown that species other than S. aureus can also interact with hosts. One such species that has been the subject of recent research is Staphylococcus epidermidis. As members of the same genus, S. aureus and S. epidermidis share key phenotypic properties [S. epidermidis belongs to a group known as coagulase-negative staphylococci (CoNS). As the name suggests, S. epidermidis do not possess coagulase, a blood-clotting enzyme, which distinguishes these bacteria from S. aureus. In addition, unlike S. aureus, S. epidermidis are generally good residents. They can also interact with the environment, and these interactions often benefit host immunity. It is interesting that although these two bacteria belong to the same genus, their effects are antagonistic. Several proteins expressed by these microorganisms, and their immunogenicity, have been uncovered. However, more research is needed regarding the biochemical characteristics and functions of these microorganisms. In this review, we provide an overview of what is currently known regarding the effects of skin colonization by S. epidermidis and S. aureus on protein production and host cutaneous immunity.In the skin, one of the most studied troublesome residents is lar data . Whether disease . Comprehoperties . HoweverS. aureus and local immunity, we must first note that the interactions as a member of the skin microbiome differ from deep or systemic staphylococcal infections. Indeed, interactions as a resident microorganism are confined to only a minor breach of the cutaneous barrier. Soft tissue staphylococcal infections and biofilm infections on implanted medical devices are beyond the scope of this review.Before addressing the interactions between S. aureus colonization is associated with various skin diseases, including atopic dermatitis (AD). Even before DNA sequencing technology emerged, S. aureus was known to be more frequent in AD patients [S. aureus in the skin lesions of AD patients [S. aureus, inflammation, and Th2 responses has been brought to the attention of those who study S. aureus and AD . Spls are a group of secreted bacterial proteases that are currently known for their allergenic properties in mice and humans. When stimulated with Spls, human peripheral blood T cells produce significant amounts of Th2, but not Th1/Th17, cytokines [S. aureus colonization and Th2 responses in AD.Importantly, nd TNF-α . Upon en peptide . This peytokines . One sucytokines . Thus, SS. epidermidis, S. aureus secrete toxins. One such toxin, δ-toxin, is known to promote Th2 responses [S. aureus, but not with a mutant deficient in δ-toxin, promoted immunoglobulin-E (IgE) and interleukin-4 production, as well as inflammatory skin disease. δ-toxin induces degranulation of dermal mast cells and increases IgE, which further enhances mast cell degranulation. This cycle may also contribute to Th2 skewing.Unlike esponses . Skin coS. aureus membrane components and diacylated lipopeptide induce keratinocyte production of thymic stromal lymphopoietin (TSLP), which is an important initiator of AD [S. aureus cell wall components also downregulate IP-10, trigger activation of MAPK, p38, and ERK, and inhibit STAT1 signaling in monocytes, all of which may contribute to the abrogation of Th1 cell-recruiting chemokines [Furthermore, or of AD . S. aureS. aureus also express superantigens (SAgs), including toxic shock syndrome toxin-1 and staphylococcal enterotoxin, that cross-bridge major histocompatibility class II molecules on antigen presenting cells and the T cell receptor on T cells. This binding strongly promotes T cell proliferation the release of Th1 cytokines. When applied to human skin, staphylococcal enterotoxin B (SEB) promotes cutaneous T cell accumulation and an AD-like response [response . Whetherresponse . IL-31 iresponse ,15.S. aureus colonization may also contribute to psoriasis and new-onset pediatric AD, both of which are associated with a Th17 rather than a Th2 response [S. aureus exposure for one week induces skin inflammation. S. aureus produces phenol-soluble modulin α (PSMα), which triggers IL-36R/MyD88 signaling. Consequently, IL-36 from epidermal keratinocytes stimulates T cells, which generate IL-17 [S. aureus may also be responsible for Th17-polarized skin inflammation.Finally, recent reports indicate that response . In theste IL-17 . As humate IL-17 ,20, exceS. aureus PSMs, nonetheless, may be of advantage to the host. PSM derivatives from PSMα1 and α2 display antimicrobial activity against M. luteus and S. pyogenes, a component of the normal flora and a highly pathogenic bacterium, respectively. Contrary to expected results, lantibiotics (lanthionine-containing peptide antibiotics produced by bacteria) did not show antimicrobial activity in the study [he study .S. aureus strain isolated from human perinasal skin demonstrated the ability of fermenting glycerol to release short-chain fatty acids (SCFAs), which are known for their bactericidal activity. USA300, a representative methicillin-resistant strain of S. aureus, does not possess such ability. When commensal S. aureus and methicillin-resistant S. aureus (MRSA) with/without 2% glycerol were applied onto the skin of wounded mice, the number of MRSA was significantly less in wounds inoculated with bacteria and glycerol than in those inoculated without glycerol. Furthermore, immunization of mice with recombinant α-hemolysin from commensal S. aureus yielded high antibody titers , which provided protection against MRSA skin infection. These data suggest that commensal S. aureus bacteria may provide benefits to the host by maintaining host probiotic activity, whereas this characteristic has not been evolutionarily conserved in MRSA [Recently, a commensal  in MRSA .S. aureus, S. epidermidis is less pathogenic, and colonization by S. epidermidis may even benefit the host local immune system  2. Exposure of cultured human keratinocytes to a sterile nontoxic small molecule (<10 kDa) isolated from S. epidermidis-conditioned culture medium (SECM) increases mRNA expression of antimicrobial peptides . It also enhances the capacity of cell lysates to inhibit group A Streptococcus (GAS) and promotes S. aureus growth [S. epidermidis and recognized by TLR2, has proven to be effective in suppressing inflammation. Following skin injury, LTA-induced TLR2 signaling inhibits TLR3-mediated skin inflammation. Specifically, TLR3 signaling is suppressed by induction of TNF receptor–associated factor-1 (TRAF1), a negative regulatory factor that suppresses keratinocyte cytokines  released via the TLR3 signaling cascade [Compared to s growth . In mice cascade . Lipopep cascade .S. epidermidis also produces PSMs (PSMγ and PSMδ), which directly induce lipid vesicle leakage and antimicrobial action against skin pathogens such as S. aureus [S. epidermidis growth. Finally, synthetic PSMδ not only binds to and increases the killing capacity of neutrophil extracellular traps, it also binds to host-derived AMPS  and enhances their endogenous antimicrobial activity [. aureus . PSMs reactivity .S. epidermidis also produce 6-N-hydroxyaminopurine (6-HAP), a molecule that inhibits DNA polymerase activity. In the context of cancer, 6-HAP inhibits tumor cell proliferation in vitro and B16F10 melanoma growth in vivo [mARC with small interfering RNA (siRNA) partially inhibits sensitivity to 6-HAP in non-transformed human keratinocytes. Alternatively, colonization with a 6-HAP-producing S. epidermidis strain reduces the incidence of ultraviolet-induced tumors in mice. Thus, further study is needed to examine whether the loss of specific S. epidermidis strains increases skin cancer risk in humans, or if increasing S. epidermidis skin colonization prevents cancer. Ongoing research is exploring how dysbiosis affects cancer. It has been demonstrated that inflammation induced by dysbiosis promotes carcinogenesis [S. epidermidis or other skin commensals may interact with the tumor environment.The Gallo group recently discovered that some strains of  in vivo . This anogenesis ,30. Otheogenesis ,31. ThisS. epidermidis colonization also affects adaptive cutaneous immunity  to the skin of non-human primates increases the absolute number of Tc17 cells, but not CD4+ T cells. Hence, the ability of S. epidermidis to promote skin CD8+ T cell responses is conserved in non-human primates and likely exists in humans. Furthermore, the researchers specified the characteristics of these CD8+ T cells. Using MHC class I KbDb−/− dendritic cells from H2-M3−/− mice that lack MHC class Ia alleles, they demonstrated that S. epidermidis-specific CD8+ T cells are confined to MHC class Ib, particularly H2-M3 MHC class Ib. H2-M3-resticted CD8+ T cells are specific to S. epidermidis-derived fMet peptide ligands. RNA sequencing also revealed the immunoregulatory and tissue-repair signatures of these CD8+ T cells. In a skin wounding model, S. epidermidis-induced CD8+ T cells promoted more rapid epidermal keratinocyte progression, leading to accelerated wound healing.A recent study by the Belkaid group also showed that a specific clade (A20) of S. epidermidis is also involved in immune tolerance. Tolerance to commensals is crucial for maintaining immune homeostasis at barrier sites. Scharschmidt et al. has created an S. epidermidis strain that expresses a model T cell antigen [ antigen . Using tS. epidermidis (epi-LTA), the ratio of IL-12p70 to IL-10 was close to 1 , whereas LTA preparations from S. aureus (aureus-LTA) induced DCs to produce much higher levels of IL12-p70 (ratio >3). Naïve CD4+ T cells primed by epi-LTA produced significantly lower amounts of IFN-γ and IL-17 compared with those primed by aureus-LTA. The IL-10-balanced immune profile of DCs exposed to epi-LTA was preserved in the presence of the Th2 hallmark cytokine IL-4. Although this should be tested in vivo, we can assume that S. epidermidis LTA does not contribute to enhanced cutaneous inflammation in an IL-4-rich environment, but rather regulates the inflammatory response [S. epidermidis can be used to prevent skin diseases.When murine dendritic cells (DCs) were exposed to LTA preparations from response . Taken tS. epidermidis, it remains a major nosocomial pathogen [S. epidermidis is also a predominant pathogen of sepsis in preterm infants. Setting aside nosocomial environmental factors, S. epidermidis alone is capable of inducing pro-inflammatory cytokine production . Data concerning S. epidermidis-induced inflammatory mediators are predominantly based on in vitro experiments and should be studied further in vivo [Despite the favorable results that have been reported for pathogen . Risk fapathogen ,37. S. e in vivo .S. epidermidis isolated from infections belong to a subset of those found on the skin surface [S. epidermidis appears to represent a pathogenic sub-population. These organisms are likely to have acquired genetic elements and related phenotypes that promote infection [Multiple studies have reported that the  surface . Using anfection .S. epidermidis was present in severely AD-affected skin lesions, with a positive association to SCORing Atopic Dermatitis (SCORAD) [S. epidermidis detected in flares, and that those with severe disease were predominantly colonized by clonal S. aureus strains [S. epidermidis and its effects on local inflammation and immune priming are needed to determine the role of S. epidermidis in inflammatory skin diseases.Unfortunately, identifying the specific factor or event that drives the transition from harmless commensal to pathogenic factor in skin disease, especially those other than infection, is difficult. Hon et al. examined 100 adolescents with AD and found that (SCORAD) . Alterna strains . Thus, fS. aureus and S. epidermidis are not the only Staphylococcal species present in the skin, these bacteria are particularly important because they are the most recognized in pathogenic and healthy skin, respectively. Manipulation of these bacteria is likely to significantly affect skin microbiota-host interactions. One way of manipulating the skin microbiota is to introduce specific strains to subjects by topical application. A study has shown that such introduction successfully decreased colonization by S. aureus. The result was due to the antimicrobial peptides produced from CoNS [Although rom CoNS .S. aureus and/or S. epidermidis affect cutaneous immunity and the homeostatic response through these T cells. Studies evaluating how manipulating these microbes affects various subsets of T cells and long-term local immunity should be performed to discover novel therapeutic strategies. As such, numerous studies are required to understand the complex dialogue between cutaneous microorganisms and host immunity. For now, detailed characterization of the immunogenicity of S. aureus and S. epidermidis is essential.Although these two bacteria share substantial phenotypic properties, their effects on the skin microbiota are contradictory. These differential effects are likely related to different structural adaptations, but those are currently unknown. Additionally unknown are the detailed mechanisms underlying how a specific subset of antigen presenting cells and their epitopes stimulate conventional T cells. Importantly, the skin is a reservoir for tissue-resident memory T cells, which are distinct from recirculating central and effector memory T cells and mucosal-associated invariant T cells. Thus, it is plausible that


Riptortus pedestris is a major agricultural pest on leguminous plants in South Korea and Japan. Recent studies have revealed that R. pedestris can form beneficial symbiosis with bacteria belonging to genus Burkholderia acquired from soil newly for every generation. Although their physiological interactions are relatively well-understood, infection rate and abundance of the Burkholderia in overwintering natural populations of R. pedestris remain unknown. Therefore, the objective of this study was to characterize Burkholderia infection ratio and clade composition of overwintering R. pedestris populations as well as prevalence and diversity of the genus Burkholderia in soil by conducting a two-year field survey. From the field survey, we found 29 overwintering R. pedestris adults in forested areas nearby soybean fields. Diagnostic PCR analysis revealed that overall infection rate of the symbiotic Burkholderia was 93.1% from overwintering adults. Among the Burkholderia-infected R. pedestris, 70.4% of individuals harbored unclassified Burkholderia clades whereas 22.2% and 7.4% of R. pedestris harbor stinkbug-associated beneficial and environmental (SBE) group and Burkholderia cepacia and complex (BCC), respectively. All R. pedestris were infected with a single clade of Burkholderia. In soil, 56.2% of soil samples were Burkholderia positive, and unlike R. pedestris, multiple Burkholderia clades were detected from 62.2% of those samples. Clade composition of the genus Burkholderia in the samples with the bacteria was 91.1%, 60.0%, 31.1% and 8.8% for plant-associated beneficial and environment (PBE), BCC, SBE and unclassified clade, respectively. During winter season, insects face thermally stressful environments. They have developed various strategies to successfully withstand and survive inhospitable environments through evolutionary interactions with organisms and abiotic conditions ,2. TheirAphis fabae Scolpoli (Hemiptera: Aphididae) [Rhopalosiphum padi Linnaeus (Hemiptera: Aphididae) [Understanding overwintering ecology is particularly important for pest species because levels of survived winter populations can serve as a barometer for estimating pest pressures in succeeding active seasons ,3,4. Indhididae) ,8,9 and hididae) .Although overall density levels of overwintering pest populations are undoubtedly foundational information to forecast pest pressures in the spring, biological variations at individual level are also worthwhile to consider because these variances could affect the survival rate of overwintering insects themselves as well as their performance after emergence ,11. PhysRiptortus pedestris (Fabricius) (Hemiptera: Alydidae), is a polyphagous insect pest in East Asia including South Korea, Japan, and Taiwan [R. pedestris attack leguminous plants, especially soybeans [R. pedestris possess symbiotic bacteria of the genus Burkholderia in the midgut crypts [Burkholderia are not transmitted vertically from parents to offspring. Instead, R. pedestris acquire their symbionts newly at every generation from soil environments mainly during the 2nd instar period [The bean bug, d Taiwan –19. Bothsoybeans ,20–22. Rt crypts ,24. Inter period . Formingr period ,27.Burkholderia infection on the biology of active, non-diapausing R. pedestris is relatively well understood, interaction between symbionts and overwintering R. pedestris remains unknown. It is likely that R. pedestris with Burkholderia would impose a higher risk level against pest management compared to aposymbiotic individuals because symbiotic individuals are known to exhibit enhanced life history parameters such as fecundity and developmental rate. Moreover, Kikuchi et al. [R. pedestris acquire fenitrothion-degrading Burkholderia strain, these insects can have resistance to fenitrothion, a widely used insecticide against R. pedestris. Therefore, the infection rate of Burkholderia in overwintering R. pedestris population could serve as a basic information to accurately assess the risk level of overwintering population to crops in the spring.Although the effect of i et al.  have fouRiptortus pedestris are known to enter facultative diapause, as opposed to preprogramed obligate diapause, in adult stage with response to abiotic changes especially for short-day photoperiods [R. pedestris can use leaf litter as a primary overwintering structure and that overwintering individuals are randomly distributed when forested areas adjacent to crop fields are surveyed [Burkholderia in overwintering R. pedestris and prevalence and diversity of the genus Burkholderia in soil environments by conducting a two-year field survey in South Korea. We collected leaf litter from forested areas nearby soybean fields from eight provinces in South Korea to locate overwintering R. pedestris. We also collected soil samples at the same locations from which leaf litter were collected. All overwintering R. pedestris found and soil samples collected were individually subjected to molecular analysis to assess the prevalence of Burkholderia in both insects and soil samples.operiods –31. In asurveyed . The objBurkholderia in overwintering R. pedestris populations as well as prevalence and diversity of Burkholderia in soil samples of South Korea. For the survey, a representative soybean field was selected in each province in South Korea based on the Korean Statistical Information Service (KOSIS) [A field survey was conducted over two winter seasons from January to February in 2017 and 2018 to characterize infection rate and clade composition of genus  (KOSIS) : Paju (3 (KOSIS) . After cR. pedestris. When live overwintering R. pedestris or other live overwintering hemipteran species were detected, insects were individually kept in 1.5-ml conical tubes  with 90% ethanol after species and sex identification. Dominant leaf types in sampling areas were confirmed based on Forest Geospatial Information System (FGIS) [Collected leaf litter samples were brought to the laboratory and kept in an indoor area (temperature ca. 4°C) until samples were examined to locate overwintering m (FGIS) . After sR. pedestris and soil samples were individually subjected to DNA extraction and diagnostic PCR. The abdomen part of R. pedestris was used for DNA extraction using MagListoTM 5M Genomic DNA Extraction Kit  according to the manufacturer’s instructions. DNA extractions for soil samples were performed with 25 mg of soil per sampling point using DNeasy PowerSoil kit .Collected overwintering Burkholderia clades in total DNA extracts from insect and soil samples. First, all samples were subjected to PCR analysis to detect the presence of genus Burkholderia using Burkholderia-specific 16s rRNA primers  of overwintering R. pedestris was found in Paju and Muan over two years. Percentages of leaf litter samples containing R. pedestris ranged from 0 to 80% in both 2017 and 2018 across provinces surveyed. In general, overwintering R. pedestris were present solitarily in leaf litter samples . Over the two-year survey, a total of 12 males and 17 females were found. The sex ratio was not significantly deviated from 1:1 . In addition, a total of 22 hemipteran species in families of Coreidae and Pentatomidae were collected over two years, including Cletus punctiger Dallas (Hemiptera: Coreidae), winter cherry bug, Acanthocoris sordidus Thunberg (Hemiptera: Coreidae), and brown marmorated stink bug, Halyomorpha halys Stål (Hemiptera: Pentatomidae).From the two-year field survey, a total of 11 and 18 overwintering ectively . Among tR. pedestris found . There was no significant difference in depth  or moisture content  between R. pedestris-detected and non-detected samples collected either. Based on FGIS [R. pedestris were almost exclusively detected from deciduous leaf leaves especially from Family Fagaceae whereas only one individual was found from a forest mainly with Family Pinaceae.Characteristics of leaf litter samples were compared between the two groups with and without overwintering is found . There wR. pedestris collected over two years, 93.1% of individuals were infected with genus Burkholderia Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Active sensing is crucial for navigation. It is characterized by self-generated motor action controlling the accessibility and processing of sensory information. In rodents, active sensing is commonly studied in the whisker system. As rats and mice modulate their whisking contextually, they employ frequency and amplitude modulation. Understanding the development, mechanisms, and plasticity of adaptive motor control will require precise behavioral measurements of whisker position.Advances in high-speed videography and analytical methods now permit collection and systematic analysis of large datasets. Here, we provide 6,642 videos as freely moving juvenile  and adult rodents explore a stationary object on the gap-crossing task. The dataset includes sensory exploration with single- or multi-whiskers in wild-type animals, serotonin transporter knockout rats, rats received pharmacological intervention targeting serotonergic signaling. The dataset includes varying background illumination conditions and signal-to-noise ratios (SNRs), ranging from homogenous/high contrast to non-homogenous/low contrast. A subset of videos has been whisker and nose tracked and are provided as reference for image processing algorithms.The recorded behavioral data can be directly used to study development of sensorimotor computation, top-down mechanisms that control sensory navigation and whisker position, and cross-species comparison of active sensing. It could also help to address contextual modulation of active sensing during touch-induced whisking in head-fixed vs freely behaving animals. Finally, it provides the necessary data for machine learning approaches for automated analysis of sensory and motion parameters across a wide variety of signal-to-noise ratios with accompanying human observer-determined ground-truth. Whiskers, or mystacial vibrissae, are sensory hairs that are densely organized as a grid on the snout. Rats and mice actively move their whiskers in an oscillatory motion to explore their environment as they integrate sensory information spatiotemporally across whiskers and whisk cycles . The motAll experiments have been performed according to the Dutch law concerning animal welfare and the guidelines for the care and use of laboratory animals upon institutional ethical committee approval. All efforts have been made to minimize animal suffering and discomfort, and all precautions were taken to reduce the number of animals used.RRID:MGI:5315557). Parvalbumin neurons in this line express Cre-recombinase, but the mice were otherwise not genetically or pharmacologically altered. The founder line was outcrossed to C57Bl6 for 20+ generations before the start of experiments. All mice were studied when between 2 and 4 months of age.The experiments were performed on 38 male rats and 10 male mice. Rats were either genetically engineered or pharmacologically treated to alter serotonergic neurotransmission, a neuromodulatory neurotransmitter that contributes to motor control , stimulu−/−) rats that were outcrossed for 12+ generations with wild-type Wistar rats obtained from Harlan Laboratories . Ear punches were taken at the age of 21 days after weaning for genotyping; 5HTT−/− and 5HTT+/+ rats were randomly assigned to SERT KO (N = 14 rats) and wild-type groups (N = 14 rats), respectively.SERT knockout (KO) rats (Slc6a41Hubr) were generated on a Wistar background by N-ethyl-N-nitrosurea-induced mutagenesis as described before . ExperimThe 5-HT transporter deletion alters neural function starting from embryonic brain development . Thus, iAnimal behavior was studied as they located (or attempted to locate) a tactile target under infrared light between postnatal days (P) 21-P30, i.e., as juveniles, and/or after they reached sexual maturity  whisker or single (C) row bilaterally; the other half received “sham plucking” during which they were handled similarly to the whisker-deprived animals; however, their whiskers were left unplucked. Whisker regrowth was assessed every day; if needed, whisker plucking was repeated.We observed animals, under infrared light, as they shuttled between two elevated platforms with a variable gap-distance in between them. The animals were not food deprived, nor did they receive any reward for successful task execution. In this, so-called spontaneous gap-crossing task , 8, 13, The experimental setup consists of two elevated platforms and a high-speed camera that are mobilized by linear actuators Fig. . The aniEach session starts with the experimenter positioning the animal on one of the two platforms. The task of the animal in any given trial is to locate the other platform, if it is within tactile reach. The success on the task is defined as the animal traveling between the two far ends of the platforms, as assessed by motion sensors in real time. If an animal starts and returns to the same starting position without interrupting the middle motion sensor on the other platform, the trial is classified as a failure. Animals are allowed to visit the gap as many times as they require before making a decision on whether or not to gap-cross. Upon decision, the door attached to the only access point of the platform that the animal is located upon is closed, and the target platform is positioned in its new position as described above.Animals’ sensorimotor behavior as they attempt to locate the target is recorded using a high-speed camera. The camera is mobilized using a linear actuator to ensure a comparable field of view across trials. An infrared backlight is positioned below the gap to provide the necessary contrast for imaging Fig. .The videography data can be used to track body and whisker position in high spatiotemporal resolution. To provide the ground-truth data for future machine learning approaches for whisker tracking, three human observers tracked whisker and nose position in a nonoverlapping subset of videos (>150 tracked frames/video). Corresponding raw data are provided in .mat (MATLAB) format; see Fig. All video files are stored as four-dimensional matrices in .mat files as well as .mp4 files for streamlined navigation in standard browsers. The .mat formatted data can be visualized using the “implay” function in the Image Processing Toolbox or using the standard “movie” function in MATLAB. Movies can be converted to other formats using built-in functions “movie2avi” or “videowriter.” The videos can be manually or automatically segmented using open-source software  were manually tracked by human observers using a custom-written interface in MATLAB [This database will help to address numerous fundamental questions in systems neuroscience, including but not limited to, development of sensorimotor computation, top-down mechanisms that control sensory navigation and whisker position, and cross-species comparison of active sensing. By comparing the sensorimotor exploration across wild-type juvenile and adult animals, one could address how adaptive control of body and whisker position develop. Because adaptive motor control of whiskers is likely to be an outcome of a vector computation that ensures spatial constancy despite the coupled changes in the body , developThe data provided could serve the ongoing machine learning efforts that will ultimately allow automated segmentation of whiskers in near real time, i.e., in temporal resolution shorter than the duration of a whisk cycle. To ensure the usability of the database as a training set, we have included ground-truth data from a subset of video recordings. Understanding the principles of active sensing in biological systems might help to instruct adaptive solutions for artificial systems to adapt sensory navigation to the ever-changing motor demands of the navigating agent.Freely behaving animal experiments are often burdened by high dimensionality and the associated sampling limitations. Even if animals execute behavior in a constrained environment, e.g., exploring a stationary target while standing on an elevated platform, as in the behavioral experiments described herein, animals could change their approach angle, kinematics of whisking, duration of exploration, number of whiskers used to sample the target, head angle, and head elevation, among other variables across different trials. Previous studies quantifying the sensory, motor, and perceptual behavior during whisker-based object localization showed that both rats and mice perform spontaneous gap-crossing in a stereotypical manner and that ∼100 trials  is sufficient to gather reproducible statistics of sensory and motor behaviors , 15, 22.Project name: MATLAB Whisker Trackerhttps://github.com/DepartmentofNeurophysiology/Matlab-Whisker-TrackerProject home page: Operating system(s): Platform independentProgramming language: MATLABOther requirements: MATLAB 2017a or higherLicense: GNU GPLRRID:SCR_016538GigaScience repository, GigaDB [Snapshots of the database and code, including further supporting data, are available in the , GigaDB .Azarfar_Metadata_Supplemental Table1.xlsxKO: knockout; SERT: serotonin transporter.The authors declare that they have no competing interests.This work was supported by the European Commission , European Regional Development Fund , and the Netherlands Organisation for Scientific Research .Investigation: A.A., Y.Z., A.r.A.,S.M., L.K., D.v.d.W., M.v.d.M.. Formal analysis and data curation: A.A.. Software: A.A., R.P., T.C.. Validation: A.A., Y.Z., A.r.A., S.M., L.K., D.v.d.W., M.v.d.M.. Resources: J.H., D.S., T.C.. Visualization and Writing : A.A., T.C.. Writing (review and editing):A.A., Y.Z., A.r.A., S.M., L.K., D.v.d.W., M.v.d.M., J.H., D.S., R.P., T.C.. Conceptualization, Supervision, Project Administration, Methodology, Funding Acquisition: T.C.GIGA-D-18-00333_Original_Submission.pdfClick here for additional data file.GIGA-D-18-00333_Revision_1.pdfClick here for additional data file.Response_to_Reviewer_Comments_Original_Submission.pdfClick here for additional data file.Reviewer_1_Report_.pdfClick here for additional data file.Supplement FileClick here for additional data file.


Anaplasma capra is an emerging zoonotic tick-borne pathogen with a broad host range, including many mammals. Dogs have close physical interactions with humans and regular contact with the external environment. Moreover, they have been previously reported to be hosts of Anaplasma phagocytophilum, A. platys, A. ovis, and A. bovis. To confirm whether dogs are also hosts of A. capra, pathogen DNA was extracted from blood samples of 521 dogs, followed by PCR amplification of the citrate synthase (gltA) gene, heat shock protein (groEL) gene, and major surface protein 4 (msp4) gene of the A. capra. A total of 12.1% (63/521) of blood samples were shown to be A. capra-positive by PCR screening. No significant differences were observed between genders (P = 0.578) or types (P = 0.154) of dogs with A. capra infections. However, significantly higher A. capra infections occurred in dogs with regular contact with vegetation (P = 0.002), those aged over 10 years (P = 0.040), and during the summer season (P = 0.006). Phylogenetic analysis based on gltA, groEL, and msp4 sequences demonstrated that the isolates obtained in this study were clustered within the A. capra clade, and were distinct from other Anaplasma species. In conclusion, dogs were shown to be a host of the human pathogenic A. capra. Considering the affinity between dogs and humans and the zoonotic tick-borne nature of A. capra, dogs should be carefully monitored for the presence of A. capra. Vector-borne diseases are major causes of morbidity and mortality in dogs and are potentially of great public health importance because of their zoonotic nature and the role of pets as reservoirs  in the USA in 1994  and three rescue centers for stray dogs . Blood samples from pet clinics were obtained during outpatient testing, and detailed medical records were also collected. Blood samples from stray dog rescue centers were collected from a random proportion (5–10%) of dogs with the assistance of an experienced veterinarian. All blood samples were collected from the anterior tibial vein of the dogs with the help of a pet doctor or local veterinarian.A total of 521 EDTA-K2 whole blood samples from different types of dogs were collected. Information about pet dogs, including age, gender, and clinical features, was obtained from sampling records, descriptions of chief complaints, and veterinarian diagnoses. However, few documents about stray dogs were available, and a review of their clinical symptoms suggested that they were healthy.Pathogen genomic DNA was extracted from 200 μL blood samples using a Blood DNA Kit  according to the manufacturer's protocol. The quantity and quality of the extracted DNA were evaluated using a NanoDrop™ 2000 spectrophotometer , and it was stored at −20°C before use.A. capra by PCR amplification of the citrate synthase gene (gltA), heat shock protein gene (groEL), and major surface protein 4 gene (msp4) using previously described primers and PCR conditions  was used as a positive control. PCR reactions were performed in an ABI 2720 thermal cycler . The products were examined by agarose gel electrophoresis and visualized after staining with GelRed .DNA samples were screened for the presence of nditions . Each DNhttps://www.ncbi.nlm.nih.gov/) using ClustalX 2.1  to determine new variant strains of A. capra.Positive PCR products were purified using Montage PCR filters  and sequenced using a BigDye Terminator v 3.1 cycle sequencing kit  on an ABI 3730 DNA analyzer (Applied Biosystems). Nucleotide sequences were confirmed by bidirectional sequencing and by sequencing a new PCR product if necessary. They were then compared with reference sequences downloaded from the National Center for Biotechnology Information (http://mrbayes.sourceforge.net/). FigTree v 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/) was used to visualize and edit the maximum clade credibility tree generated by these analyses. Posterior probability values were estimated based on 1,000,000 generations with four simultaneous tree building chains, with trees being saved every 100th generation. A 50% majority rule consensus tree for each analysis was constructed based on the final 75% of trees generated by BI.Phylogenetic trees were conducted by Bayesian inference (BI) and Monte Carlo Markov Chain methods in MrBayes v 3.2.6 (https://www.ncbi.nlm.nih.gov/)  to evaluated the homology of the sequences obtained in the present with the sequences downloaded from the National Center for Biotechnology Information (ih.gov/) .A. capra infection of dogs at different locations, seasons, age groups, genders, clinical signs, and use of vermifuge were analyzed by Fisher's exact test with SPSS (version 22.0) software. Differences were considered statistically significant if P < 0.05. Odds ratios (ORs) and their 95% confidence intervals (95% CIs) were estimated to explore the strength of the association between A. capra-positivity and the conditions tested. Then, the significant different correlation factors were used to process the canonical correspondence analysis by canoco5 (http://www.canoco5.com) (Variations in co5.com) .gltA (MK838608 and MK838609), groEL (MK862099), and msp4 (MK838605– MK838607).The representative sequences obtained in this study have been submitted and deposited in the GenBank database with the following accession numbers: This study was carried out in accordance with the Chinese Laboratory Animal Administration Act (1988) after it was reviewed and its protocol was approved by the Research Ethics Committee of Henan Agricultural University. Appropriate permission was gained from the dog owners before the collection of blood specimens.A. capra-positive by PCR screening . The infection rates of A. capra in pet dogs and stray dogs were 12.9%  and 6.3% , respectively, There were no significant differences in the infection ratio of A. capra in pet dogs and stray dogs (P = 0.154)  .A. capra infections in dogs with different types of samples were analyzed . Additionally, higher infection rates were observed in dogs older than 10 years , and lower rates were seen in dogs aged 3–6 months compared with other age groups  (P = 0.040). As expected, the highest infections were documented in the summer months  (P = 0.006), and in dogs with anemic symptoms such as pale mucous membranes (P = 0.019). However, there were no differences in infection rates between dogs of different genders (P = 0.578), nor in those with influenza-like illness symptoms including fever, cough, malaise, and depression (P = 0.370), nor in those administered vermifuge in the last month (P = 0.651).analyzed . A. caprA. capra-positive specimens, 26 were PCR-positive based on gltA, 17 based on groEL, and 61 based on msp4. Sequence analysis showed that gltA sequences were divided into two distinct sequence types: one  shared 100% identity with A. capra isolates from China, with previous hosts including humans (KM206274), goats (KM869310), sheep (KM869280), and ticks (MG940871 and MH029895). The others  showed 99.7% identity with A. capra isolates from humans (KM206274)  . Phylogees clade . These i species .groEL gene sequences obtained in this study (MK862099) were 100% identical to each other. Findings showed that the sequence had two single nucleotide polymorphisms  compared with the human isolate sequence (KM206275), and shared 99.9% identity with sequences isolated from sheep (KX417341), goats (KJ700629), and ticks (KX987393)  . Phyloge species .msp4 gene sequences, we acquired three different sequences (MK838605–MK838607) from dogs in the present study. One (KM838607) shared 100% identity with those of A. capra isolates from humans (KM206277), and the others had one (T→ C at position 320) or two  SNPs compared with the human sequence (KM206277), and shared 99.7–99.8% similarity with sequences of Anaplasma spp. from humans (KM206277), goats (KJ700631), sheep (KX417357), and ticks (KR261641)  . Phyloge species .Anaplasma species is dependent upon the presence of tick vectors and infected vertebrate reservoir hosts .Host and environmental factors are thought to play an important role in the epidemiology of TBD in dogs . Likewise, A. ovis, A. bovis, and A. phagocytophilum have been identified in dogs in Henan with prevalences of 6.2%, 4.1%, and 0.4%, respectively , which is located in a suburban area of the city surrounded by rich vegetation. Similar findings were previously observed for the tick-borne fever with thrombocytopenia syndrome which was also significantly associated with vegetation-rich regions  obtained in this study shared 100% identity with an A. capra isolate (KM206274) from humans, while the other sequence (MK838609) shared 99.7% identity with the same isolates from humans  shared 99.9% identity with the sequence from humans (KM206275), and three different msp4 sequences obtained in the present study shared 99.7–100% similarity with sequences of Anaplasma species from humans (KM206277) , in view of the affinity between dogs and humans, indicating that a high level of attention should be paid to A. capra infection in dogs for public health reasons.Phylogenetic analysis of A. capra sequences (20/26) obtained from dogs in this study showed 100% identity with isolates from ticks, sheep, goats, and humans , groEL (MK862099), and msp4 (MK838605–MK838607).The datasets generated for this study can be found in the GenBank database with the following accession numbers: gltA ; Research Ethics Committee of Henan Agricultural University. Written informed consent was obtained from the owners for the participation of their animals in this study.CN conceived the study. CN and LZ designed the experiments. KS, YY, QC, KW, YZ, and YP performed the experiments. KS, DL, YC, and FY performed data analysis. KS, JL, and CN wrote the manuscript. All authors approved the final version of the manuscript.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 steadily increasing demand on transportation fuels calls for renewable fuel replacements. This has attracted a growing amount of research to develop advanced biofuels that have similar physical, chemical, and combustion properties with petroleum-derived fossil fuels. Early generations of biofuels, such as ethanol, butanol, and straight-chain fatty acid-derived esters or hydrocarbons suffer from various undesirable properties and can only be blended in limited amounts. Recent research has shifted to the production of branched-chain biofuels that, compared to straight-chain fuels, have higher octane values, better cold flow, and lower cloud points, making them more suitable for existing engines, particularly for diesel and jet engines. This review focuses on several types of branched-chain biofuels and their immediate precursors, including branched short-chain (C4–C8) and long-chain (C15–C19)-alcohols, alkanes, and esters. We discuss their biosynthesis, regulation, and recent efforts in their overproduction by engineered microbes. High petroleum prices and increasing concerns over energy security and climate change are driving the development of renewable biofuels in recent years , 2. BioeWhile production of straight-chain biofuels has been extensively reviewed , 16, thiEscherichia coli . How. HowB. soduction . To solv207 mg/L . Incorpo207 mg/L .Fig. 3EnB. subtilis alsS and the E. coli ilvCD  in the range of C12 to C18 are more suitable as diesel fuel replacements than their straight-chain counterparts. Jiang et al. [E. coli. A modular engineering approach was then used to balance the flux between α-keto acid synthesis, acyl-ACP generation, and alcohol formation. The best performing strain produced BLFLs at 350 mg/L, where 75% of the produced fatty alcohols were BLFLs. In addition, Tao et al. [E. coli, and produced LCFA esters containing methyl branches at both acid and alcohol moieties at 35 mg/L with a yield of 1.9 mg/g glycerol.Besides the overproduction of terminally branched LCFAs, some metabolic engineering works on the production of branched alkanes, alcohols, and esters have also been reported , 8. Brang et al.  construco et al.  combinedRubrobacter xylanophilus produces 12-methylhexadecanoic acid and 14-methyloctadecanoic acid as the major fatty acid species in its lipids [Leptogorgia piccolo [Scytonema sp. [Mycobacterium tuberculosis and related species [E. coli, S. typhimurium, and M. tuberculosis. They are also found in the seed oils of some higher plants including Malvales, Fabales, and Sapindales [Compared to straight and terminally branched LCFAs, internally branched LCFAs are relatively rare in nature. Thermophilic bacterium s lipids . 13-Meth piccolo , and 9- nema sp. . The bes species . TSBA is species . Similarpindales . The pospindales . For exapindales . TherefoM. tuberculosis and related species, TSBA has been found in multiple phospholipids such as phosphatidylethanolamine (PE), phosphatidylinositol (PI), and diphosphatidylglycerol (DPG), and glycolipids such as phosphatidylinositol mannosides and lipoarabinomannans [M. tuberculosis and are likely involved in subverting the immune system [In omannans . These ge system . Only sme system , indicatl-methionine (SAM) as the methyl donor. The second step is the reduction of the intermediates, which are believed to be 10-methylene-octadecanoyl phospholipids, by a reductase using NADPH as the cofactor [umaA from M. tuberculosis H37Rv is capable of converting the oleoyl-phosphatidylcholine (PC) to TSBA-PC in vitro. Meena et al. [ufaA1 can also catalyze the formation of TSBA. Purified recombinant UfaA1 protein can convert oleoyl-phosphatidylcholine and oleoyl-PE to TSBA-PC and TSBA-PE in the presence of SAM and NADPH. However, neither umaA nor ufaA1 has any functional domain associated with redox reactions. Machida et al. [Mycobacterium chlorophenolicum that is responsible for TSBA production. In this cluster, the bfaB gene encodes a SAM-dependent methyltransferase, and the bfaA gene encodes a FAD-dependent oxidoreductase. Heterologous expression of these two genes in E. coli produced TSBA from oleic acid, confirming their functions. These two enzymes, BfaA and BfaB, were found to have surprisingly high regioselectivity, only converting 18:1Δ9, but not 16:1Δ9 nor 18:1Δ11 to branched fatty acids when tested in E. coli. Furthermore, it is not clear which phospholipids can be the substrates of the TSBA enzymes. Additionally, because unsaturated fatty acids (UFAs) are usually located at the sn-2 position of phospholipids in E. coli, TSBAs produced in E. coli are likely located at sn-2 position [sn-1 position of phospholipids. These results suggest that BfaA and BfaB might be able to convert UFAs at both sn-1 and sn-2 positions of phospholipids [The mechanism for TSBA synthesis is still not fully understood. It was hypothesized that the biosynthesis of TSBA involves two reaction steps Fig. b. The ficofactor . Meena ecofactor  demonstra et al.  also shoa et al.  later idposition . Meanwhiholipids , 78. FurE. coli after exposure to pH 4 for 16 h [P. putida results in decreased cell tolerance to organic solvents [M. tuberculosis pcaA gene, which encodes a CFA synthase and acts on α-mycolates, inhibits M. tuberculosis from killing infected mice [Similar to other branched LCFAs, CFAs are also believed to modulate the fluidity and stability of cell membranes. CFAs are important to improve cell survival when microbes are subjected to environmental stresses such as high osmotic pressure , high , cardiolipin (CL), and PC. In addition, the E. coli Cfa was reported to prefer preferentially act the sn-2 position of phospholipids [Sterculia foetida acts on sn-1 position [Biosynthesis of CFAs is catalyzed by cyclopropane fatty acid synthase (Cfa), which transfers a methylene group from SAM to the double bond of UFAs, creating a cyclopropane ring on the alkyl chain Fig. . CFAs arway Fig. a. Therefguration . An in vholipids , whereasposition .Fig. 5Pacis-UFAs of the membrane phospholipid bilayers are converted into CFAs. This growth phase-depended regulation of CFA synthesis is caused by a RpoS-dependent promoter, which is activated only when cells enter stationary phase. In addition, the activity of the Cfa protein is also believed to be controlled by an unidentified energy-independent protease that is transcribed by a RpoH-dependent promoter [The formation of CFAs occurs at the onset of the stationary phase in the cell growth cycle and continues until all of the promoter .cfa gene from E. coli into cyanobacterium Synechocystis sp. PCC 6803, CFAs were produced in engineered cells with up to 30% of total fatty acids [cfa in C. acetobutylicum ATCC 824 showed an increased butanol tolerance [cfa-deficient E. coli [S. typhimurium [E. coli cfa gene in the seeds of Arabidopsis and resulted the accumulation of CFAs up to 9.1%. By coexpressing a Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT), the CFA content was further increased to 35%.Although CFAs has not been currently used as biofuels, their lower melting temperature compared to straight LCFAs and improved stability against oxidation compared to unsaturated FAs have made them promising biofuel targets as recently explored . By intrty acids . In addiolerance , while a E. coli  or S. tyhimurium  was veryhimurium  expresseWe reviewed the progress of branched-chain biofuels, including branched short- and long-chain alcohols, alkanes, and esters. As we understand more about the biochemistry and regulation of lipid biosynthesis, novel metabolic pathways have been continuously developed, leading to new generations of biofuels with structures and properties highly similar to or identical with existing petroleum-derived fuels. The reviewed studies have proved the concept of producing advanced biofuels from engineered microorganisms; however, current titers and yields are still too low for economically viable production. These challenges demand efficient metabolic engineering tools to improve pathway yields, titers, and production stability of engineered microbial strains. Fortunately, some tools are already emerging and used for biofuel production, for example, modeling-guided pathway optimization , metabol


Acinetobacter baumannii and Pseudomonas aeruginosa. Their clinical use was abandoned in the 1960s because of nephrotoxicity and because better-tolerated drugs belonging to other antibiotic classes were introduced. Now, due to the global dissemination of extremely-drug resistant Gram-negative bacterial strains, polymyxins have resurged as the last-line drugs against those strains. Novel derivatives that are less toxic and/or more effective at tolerable doses are currently under preclinical development and their properties have recently been described in several extensive reviews. Other derivatives lack any direct bactericidal activity but damage the outermost permeability barrier, the outer membrane, of the target bacteria and make it more permeable to many other antibiotics. This review describes the properties of three thus far best-characterized “permeabilizer” derivatives, i.e., the classic permeabilizer polymyxin B nonapeptide (PMBN), NAB7061, and SPR741/NAB741, a compound that recently successfully passed the clinical phase 1. Also, a few other permeabilizer compounds are brought up.Polymyxins (polymyxin B (PMB) and polymyxin E (colistin)) are cyclic lipodecapeptide antibiotics, highly basic due to five free amino groups, and rapidly bactericidal against Gram-negative bacteria, such as the majority of Enterobacteriaceae as well as  Acinetobacter baumannii and Pseudomonas aeruginosa. All members of the polymyxin class contain a cyclic heptapeptide core, linked to a linear tripeptide “panhandle” with an N-terminal fatty acyl moiety (Polymyxins (polymyxin B (PMB) and polymyxin E (colistin)) are cyclic lipodecapeptide antibiotics, highly basic due to five free amino groups, and quite effective against Gram-negative bacteria such as the majority of Enterobacteriaceae as well as l moiety . The disThe clinical use of polymyxins was abandoned in the 1960s because of nephrotoxicity and because better-tolerated drugs belonging to other antibiotic classes were introduced. Now, due to the global dissemination of extremely-drug-resistant Gram-negative bacterial strains, polymyxins have resurged as the last-line drugs against those strains ,6,7,8,9.mcr) genes has elicited lots of alarms and publicity, but strains resistant to polymyxins by other mechanisms, such as those with altered mgrB, display high-level resistance and are truly worrisome [K. pneumoniae (n = 3753) only 0.4% and in E. coli (n = 7397) only 0.3% [K. pneumoniae (n = 1703) and E. coli (n = 407) collected during the SMART program in 2015–2016, the prevalence of colistin resistance was 21% and 14%, respectively [E. coli or K. pneumoniae and 0.5% by colistin-resistant A. baumannii or P. aeruginosa [However, even the new role of polymyxins is now in jeopardy. The recent appearance of mobile colistin resistance  molecules, exclusively present in Gram-negative bacteria and located in the outer leaflet of their outer membrane (OM) ,18. The Polymyxin B and colistin are directly antibacterial. Novel derivatives of them , all under preclinical development, might offer advantages such as enhanced efficacy in experimental infections, as recently reviewed by Vaara . Other rOn the other hand, certain derivatives of polymyxins have lost their direct bactericidal activity but still damage the OM, restructure it, and weaken its function as a permeability barrier to many noxious agents, including antibiotics . AccordiThis review sums up the properties of polymyxin derivatives that lack any notably direct antibacterial activity but sensitize the bacteria to other antibiotics.E. coli and K. pneumoniae. On the other hand, they still possess the OM-permeabilizing activity and, hence, act as permeabilizers that allow enhanced entry of other antibiotics across the OM [Polymyxin derivatives that lack a fatty acid tail  are significantly less active than polymyxin B against species such as s the OM .N-terminal diaminobutyryl (Dab) residue and is devoid of any direct antibacterial activity against enterobacterial species [E. coli, K. pneumoniae, several other enterobacterial species, and P. aeruginosa.The classic permeabilizer, polymyxin B nonapeptide PMBN,  was disc species ,25. Howe species . Target K. pneumoniae or P. aeruginosa in conditions where none of the agents alone was effective [The combination of PMBN with erythromycin, as well as with novobiocin, administered intraperitoneally, protected mice infected with ffective .PMBN was shown to be 15 times less toxic than polymyxin B in an acute-toxicity assay in mice, 25 times less active in releasing histamine from rat mast cells, approximately 100 times less toxic in a eukaryotic cytotoxicity assay and approximately 150 times less active in causing neuromuscular blockade . PMBN elPMBN has been widely exploited as a useful tool to increase the permeability of the OM in various in vitro studies, including those dealing with the discovery and development of novel antibacterial drugs. A selected list of such studies conducted in years before 1992 can be found in the review by Vaara published in 1992 . ThereafE. coli (11 strains), other polymyxin-susceptible Enterobacteriaceae (12 strains), and A. baumannii (three strains) by factors of 85–750, 10–2,000, and 25–125, respectively [K. pneumoniae strain CL5762B by factors of 24 and 12, respectively [E. coli peritoneal infection model in mice [NAB7061 possesses the same heptapeptide core as polymyxin B, but its side chain consists of octanoyl-threonyl-aminobutyryl . As a coectively . With clectively . The pot in mice . When co in mice ,59. NAB7 in mice .NAB741, discovered at Northern Antibiotics Ltd.  by Vaara et al. and published in 2010 ,60, possE. coli [3(5)) [Atomic force microscopy revealed that SPR741 causes undulations (approx. 10 nm) and breaks (approx. 20 nm) in the OM of E. coli . No subsi [3(5)) . In conti [3(5)) . Two elei [3(5)) ,65. Thesi [3(5)) .E. coli, K. pneumoniae, E. cloacae, and A. baumannii. The potentiation factors against these bacteria, as defined as the fold reduction in the MIC in the presence of 4 mg/L of SPR741 (relative to the MIC in the absence of SPR741) ranged from >2000 to 16 for rifampin and from 340 to 6 for clarithromycin. Marked synergism was also found with azithromycin, mupirocin, fusidic acid, and vancomycin against most of these bacterial species [As shown by Vaara et al., in 2010 , NAB741  species .E. coli and K. pneumoniae in the presence of SPR741. The antibiotics were rifampin, four macrolides/ketolides , the pleuromutilin antibiotic retapamulin, fusidic acid, and mupirocin. Corbett et al. showed that the potentiation factor against E. coli ATCC 25922 in the presence of 8 mg/L of SPR741 was 8000 for rifampin and 4000 for clarithromycin. Furthermore, SPR741 reduced the MIC of rifampin, clarithromycin, erythromycin, retapamulin, and fusidic acid for A. baumannii by >32 fold [These findings were later corroborated by Corbett et al., in 2017 . Out of >32 fold . Amongst>32 fold , an “ant>32 fold .of E. coli, K. pneumoniae, and A. baumannii [A. baumannii, including strains displaying the MDR phenotype [K. pneumoniae. Their subsequent study revealed that the MIC90 values (the MICs for ≥90% of the strains) of clarithromycin in the presence of 8 mg/L of SPR741 were for E. coli (n = 178), K. pneumoniae (n = 115) and E. cloacae (n = 111), 2, 8, and 1 mg/L, respectively [E. coli and K. pneumoniae, four strains carried acquired resistance elements such as ermB, efflux pump element mrs, or the macrolide phosphorylase gene mphA [Other studies give further support to the original studies by Vaara et al., too. Hackel et al. showed strong synergism between SPR741 and rifampin, as well as with clarithromycin against recent clinical isolates aumannii . Mendes henotype . The potectively . Both inene mphA . These sA. baumannii, Zurawski et al. showed that amongst the 29 extensively drug-resistant strains, 28 strains were susceptible to the combination of SPR741 and rifampin [A. baumannii, the addition of SPR741 at 8 mg/L reduced the MIC90 from 16 mg/L to 2 mg/L [In a study exclusively focused on rifampin . In the o 2 mg/L .rpoB, could be expected to develop as easily in Gram-negative bacteria as has been experienced in Gram-positive bacteria. Furthermore, many MDR strains of Gram-negative bacteria have already been shown to possess resistant determinants that confer resistance to macrolide antibiotics (see above). Accordingly, these two antibiotics may not prove to be the partners of choice in clinical use.Because of the impressive magnitude of potentiation exhibited by rifampin and clarithromycin in combination with polymyxin derivatives, most of the studies have focused on them as potential partner antibiotics to SPR741. However, chromosomal resistance to rifampicin, mediated by E. coli, K. pneumoniae, and E. cloacae by a factor of 4–48 and that of TZP-resistant strains by a factor of ≥16 [Bacteroides spp., Fusobacterium spp., Porphyromonas spp., and Prevotella spp. [Regarding potential partnering with betalactams, the MDR strains are generally resistant to piperacillin-tazobactam (TZP), ceftazidime and other betalactams. At the fixed concentration of 8 mg/L, SPR741 decreased the MICs of TZP-susceptible strains of r of ≥16 ,76,77. Wr of ≥16 . No synella spp. .gyrB gyrase and the parE topoisomerase IV. The ureabenzimidazole compounds SPR719 and SPR720 (in-licensed by Spero from Vertex in 2016) as well as the pyridylurea compounds SPR750 and SPR751 (in-licensed by Spero from Biota in 2016) are inactive against Gram-negative bacteria but in the presence of SPR741 at 8 mg/L their MICs for Enterobacteriaceae and A. baumannii are notably low  [A. baumannii [Amongst the newer drugs under development against Gram-positive bacteria, two groups of non-quinolone-structured gyrase inhibitors are notably synergistic with SPR741. Both groups inhibit the  1 mg/L) ,81,82,83aumannii .E. coli by a factor of 32 and at 4 mg/L by a factor of 16 [Other partner candidates will certainly include the tetracycline derivative minocycline. PMBN has long ago been shown to increase its entry to the cell . Howeveror of 16 .In screening for any novel drugs under development that might be potential partners for SPR741, one should note that the desirable pharmaceutical formulation should be intended for intravenous (IV) use, because SPR741 is administered intravenously. Naturally, if the potential partner is very well absorbed orally, it might be useful as well. The entire concept of a sensitizer compound and its partner antibiotic has lots of challenges on its road to become clinically relevant (see Concluding Remarks below).K. pneumoniae murine model of urinary tract infection, a combination of SPR741 at the dose of 10 mg/kg every 8 h  and rifampin at the dose of 4 mg/kg TID was very effective in decreasing the bacterial load in the kidneys, whereas monotherapies with either agent alone were ineffective [K. pneumoniae while both agents alone lacked any efficacy [In the ffective . SPR741 efficacy .A. baumannii murine lung model, the combination of SPR741 at 10 mg/kg TID plus clarithromycin at 100 mg/kg once daily decreased the bacterial load by 1.15 log10 from the pretreatment [E. cloacae expressing blaKPC [A. baumannii pneumonia model, whereas rifampin alone had only a modest efficacy [In the reatment . This isg blaKPC . In lineefficacy .mphA, mphE, msr, and ermB [E. coli, K. pneumoniae and A. baumannii. Against E. cloacae, the combination of SPR741 and rifampin was effective (clarithomycin was not tested) [Strains of Enterobacteriaceae seldomly cause complicated skin and soft tissue infections (cSSI). However, the neutropenic murine thigh infection model (representing cSSI) is widely used in pharmacokinetic/pharmacodynamic (PK/PD) evaluations. Therefore, five publications describe the performance of SPR741 in the thigh infection model. The study by Stainton et al.,  employedand ermB . The papand ermB ,90,91 sh10 from the vehicle control [The neutropenic murine thigh infection studies have also included one in which the combined efficacy of SPR741 and SPR719 (see above) was assessed. Whereas both agents alone were ineffective, the combination of SPR741 at the daily dose of 180 mg/kg and SPR719 at the daily dose of 150 mg/kg decreased the bacterial load by approx. 4 log control . HoweverAfter a single intravenous dose of 1 mg/kg to rats, the urinary recovery rate for NAB741 was 51% . When coThe cytotoxicity of SPR741 towards proximal tubular kidney cell lines has previously been shown to be much lower than that of the old polymyxins . PolymyxAs discussed previously , agents A non-GLP 7-day cynomolgus monkey study indicated that SPR741 was at the dose of 60 mg/kg/day less nephrotoxic than polymyxin B at the dose of 12 mg/kg/day . A GLP  . SubjectA. baumannii to rifampin, clarithromycin, fusidic acid, vancomycin, and meropenem [Polymyxins increase the permeability of the OM to noxious agents but this action is in most instances masked by their direct bactericidal action which takes place at the very same concentrations. However, at subinhibitory concentrations many novel polymyxin derivatives do sensitize the target to antibiotics, in most studies to the model antibiotic rifampin ,105,106.eropenem .Furthermore, polymyxin-like novel compounds, such as those under development by Rabanal et al., may act as sensitizers ,108. SenLinear peptides that carry cationic amino acyl residues integrated to a sequence of hydrophobic amino acyl residues  act synergistically with several antibiotics . They maThe “permeabilizer” concept, where a permeabilizer (such as SPR741) makes the OM of the target bacteria permeable to other agents (partner antibiotics), is scientifically meaningful. Further studies are needed to evaluate the efficacy and tolerability of the combination of SPR741 with other antibiotics in clinically relevant conditions. The existence and further development of chromosomal and acquired resistance against many of the potential partner antibiotics makes the choice of the final partner very difficult. Furthermore, if both the permeabilizer and the partner completely lack any direct antibacterial activity by themselves, Phase 2 clinical studies may be quite problematic to justify in regulatory terms. One of the concrete alternatives for a partner antibiotic to SPR741 in a potential Phase 2 or Phase 2/3 clinical study would be a wide-spectrum betalactam . They have notable activity even against strains that have been labelled as nonsusceptible by the laboratory standards. As presented above, SPR41 would potentiate their action against the target. Whether Spero is going to take SPR741 into the clinical Phase 2 has not been announced.Klebsiella pneumoniae that produce carbapenemase [mcr strains [mcr and other PMR strains [Regarding the emerging polymyxin-resistant (PMR) strains (see Introduction), Tascini et al. showed in 2013 that there is notable synergism with colistin and rifampin against the PMR strains of penemase . Very re strains . Brennan strains . Interes strains ,20,55 is strains . Accordi


Camellia is an economically, ecologically and phylogenetically valuable genus in the family Theaceae. The frequent interspecific hybridization and polyploidization makes this genus phylogenetically and taxonomically under controversial and require detailed investigation. Chloroplast (cp) genome sequences have been used for cpDNA marker development and genetic diversity evaluation. Our research newly sequenced the chloroplast genome of Camellia japonica using Illumina HiSeq X Ten platform, and retrieved five other chloroplast genomes of Camellia previously published for comparative analyses, thereby shedding lights on a deeper understanding of the applicability of chloroplast information. The chloroplast genome sizes ranged in length from 156,607 to 157,166 bp, and their gene structure resembled those of other higher plants. There were four categories of SSRs detected in six Camellia cpDNA sequences, with the lengths ranging from 10 to 17bp. The Camellia species exhibited different evolutionary routes that lhbA and orf188, followed by orf42 and psbZ, were readily lost during evolution. Obvious codon preferences were also shown in almost all protein-coding cpDNA and amino acid sequences. Selection pressure analysis revealed the influence of different environmental pressures on different Camellia chloroplast genomes during long-term evolution. All Camellia species, except C. crapnelliana, presented the identical rate of amplification in the IR region. The datasets obtained from the chloroplast genomes are highly supportive in inferring the phylogenetic relationships of the Camellia taxa, indicating that chloroplast genome can be used for classifying interspecific relationships in this genus. Camellia, containing about 280 species, is a genus with high economic, ecological and phylogenetic values in the family Theaceae [C. sinensis var. sinensis and C. sinensis var. assamica have the highest economic value in Camellia. Tea leaves have been proven to be beneficial for human health as they contain over 700 chemical constituents [Camellia is also known as ornamental trees for urban gardening. The cultivation history of Camellia has been at least 1300 years in China [Camellia, e.g. C. chrysantha, are grown for ornamental purposes, with thirteen to sixteen petals of a flower and blooming several times in a year. Many other Camellia species, e.g. C. japonica, also had local uses. The other most economically valuable species, C. oleifera and C. reticulata, are used for edible oil and cooking in China [Camellia oil production, and the yield of the Camellia nearly 164,000 tons of edible oil [Camellia is native to Asia, because of its variety use, the cultivated species are now found all over the world [Camellia is phylogenetically and taxonomically under controversial that detailed investigation is required, as a result of frequent interspecific hybridization and polyploidization. Whereas classification of the genus Camellia is traditionally based on morphology [Camellia.Theaceae , 2. It iTheaceae . Besidestituents , 4. Camein China , 6. At phe world . Howeverrphology –16, the Camellia. A number of studies focus on the taxonomy, species identification and phylogenetics of the Camellia, but still have not get a satisfied resolution. A recent study used complete chloroplast genomes in several Camellia species [Camellia japonica has been present in Qingdao, Shandong province since the tertiary, it has evolved in dependently after that. A recent research shows that the early flower development sequence placed C. japonica (Naidong) in a most primitive branch of the phylogenetic tree compared to other species [C. japonica with other Camellia species is in dispute.Being relatively stable and not easily affected by the environment, molecular methods can provide useful information for taxonomic classification and phylogenetic. Molecular methods, e.g. DNA and RNA sequences , 17–24,  species –31 and g species . The taxIn plant cells, the chloroplast is not only the most important and universal organelle, but also one of the major genetic systems (the other two are nucleus and mitochondria). It is involved in photosynthesis and associated with metabolism, such as fatty acid and amino acid synthetic pathways –35. As aC. japonica using next generation sequencing technology and genomic comparative analysis with other five published chloroplast genome sequence download from the NCBI. This study aims to deeply analyze the chloroplast genomes of six Camellia species and to determine their  phylogenetic positions.Here, we report newly sequenced complete chloroplast genomes of http://ly.qingdao.gov.cn/) to collect the sample. This research was carried out in compliance with the laws of People’s Republic of China.College of Landscape Architecture and Forestry, Qingdao Agricultural University has had a permit from local forestry authorities  . The leaves were dried immediately with silicagel. Total genomic DNA was extracted according to Wiland-Szymańska [We collected fresh leaves from an adult C. sinensis JQ975030) and get the relative location of the contig sequences and the structure diagram of cp genomes were obtained. The software Gap Closer 1.12 were used to fill the gaps. Finally, the complete cp genome sequence were obtained. The chloroplast genome sequences were annotated with CpGAVAS software and DOGMA software, and then manually corrected.We used an ultrasonicator to randomly fragment the extracted genomic DNA into 400–600 bp. The NEBNext Ultra DNA Library Prep Kit was used to construct an Illumina paired-end cpDNA library. Paired-end sequencing (2 × 150 bp) was run on an Illumina HiSeq X Ten platform. After filtering the raw data, the cp genomes were assembled according to the following steps. Frist the clean reads were used to assembled into contigs using SOAPdenovo 2.017. Then, the contigs were aligned to the relative species  software to calculate the nucleotide diversity (Pi) of the six complete  genomes . We set For different lengths of SSRs, including mono-, di-, tri-, tetra-, penta-, and hexa-nucleotides, minimum numbers (thresholds) were 10, 5, 4, 3, 3, and 3, respectively. We manually verified all the repeats found, and removed unwanted results.japonica to other five published chloroplast genomes of the genus Camellia, i.e., C. huana (KY_626042), C. crapnelliana (KF_753632), C. azalea (KY_856741), (KY_626042), C. liberofilamenta (KY_626041) with the shuffle-LAGAN mode [The mVISTA program GAN mode , so thatGAN mode  to deterDnaSP software was used for manual detection of insertions/deletions. To estimate selection pressures, the 78 protein coding genes in the chloroplast genomes were combined. We used PAML with the yn00 program to calculate the Ka and Ks rates of the combined sequences .Ka and Ks values of genes containing SNP variations, so that we can analyze how different Camellia have evolved under different environmental pressures. We also analyzed the codon preference, and mapped them by R software.We used KaKs_Calculator 2.0  to deterCamellia species sequences from the NCBI Organelle Genome and Nucleotide Resources database: C. crapnelliana (KF_753632), C. azalea (KY_856741), C. luteoflora (KY_626042), C. huana (KY_626042), C. liberofilamenta (KY_626041), C. oleifera (JQ_975031), C. taliensis (KF_156836), C. yunnanensis (KF_156838), C. cuspidate (KF_156833), C. reticulate (KJ_806278), C. pitardii (KF_156837), C. danzaiensis (KF_156837), C. petelotii (KJ_806276), C. leptophylla (KJ_806275), C. impressinervis (KF_156835), C. grandibracteata (KJ_806274), C. sinensis (JQ_975030), C. pubicosta (KJ_806277). We implemented maximum likelihood (ML) analyses on the CIPRES cluster1[Phylogenies were constructed using the 19 cp genome of the iana KF_7632, C. a cluster1. This moWe also used PAUP v4b10 to analyze maximum parsimony (MP). We treated gaps as missing, and character states as unordered. We selected MULPARS option when performing Heuristic search. Further steps include tree bisection-reconnection branch swapping, and random stepwise addition with 1,000 replications.Camellia species ranged from 156,607 bp (C. japonica) to 157,166 bp (C. luteoflora). The structure of all chloroplast genomes is quadripartite, which is typical of angiosperm cpDNA. Each chloroplast genome consists of a large single copy region  and a small single copy region , separated by two inverted repeat regions   was 37.32% and the others were 37.30%. The average of GC content is almost similar among the six Camellia species. The well-conserved genomic structure resembled those of other higher plants, including gene number and gene order  . The GC ne order . The comC.azalea) to 74 (C.huana) among the six camellia taxa. The number of nucleotide repeats had no significant difference among the six camellia taxa (Chloroplast simple sequence repeats (cpSSRs) play a crucial role in studying phylogeny and population genetics. We anallia taxa .Camellia chloroplast genomes [C. huana and C. luteoflora, while the highest value (58) of nucleotide substitutions was observed between C. huana and C. liberofilamenta, showing a wider range of variability according to the sequence alignment of the six chloroplast genomes . Only a minor portion are located in the SSC regions  and IR regions . Same as previously reported the SSR loci exhibited a significantly variable distribution among all regions in each of the six  genomes , 56. The genomes . The valilamenta . The resCamellia species  as a relative intuitionistic to measure the extent of codon bias . The synCamellia species, the 20 amino acids were encoded by 64 codons, in which most of the amino acids had codon preferences except tryptophan Click here for additional data file.S2 Table(DOCX)Click here for additional data file.