Comamonas thiooxydans. We first isolated a strain of an IMP-8-producing C. thiooxydans from a patient with urinary tract infection in China. Species identity was determined using MALDI-TOF MS analysis and carbapenemase-encoding genes were detected using PCR. The complete genomic sequence of C. thiooxydans was identified using Illumina Novaseq and Oxford Nanopore PromethION. Antimicrobial susceptibility analysis indicated that the C. thiooxydans strain ZDHYF418 was susceptible to imipenem, intermediate to meropenem, and was resistant to aztreonam, fluoroquinolones, and aminoglycosides. The blaIMP–8 gene was chromosomally located, and was part of a Tn402-like class 1 integron characterized by the following structure: IMP–8-intI1DDE-type integrase/transposase/recombinase-tniB-tniQ-recombinase family protein-aac(6′)-Ib-cr-bla. Phylogenetic analysis demonstrated that the closest relative of ZDHYF418 is C. thiooxydans QYY (accession number: CP053920.1). We detected 330 SNP differences between ZDHYF418 and C. thiooxydans QYY. Strain QYY was isolated from activated sludge in Jilin province, China in 2015. In summary, we isolated a strain of C. thiooxydans that is able to produce IMP-8 and a novel blaOXA. This is the first time that a CR gene has been identified in C. thiooxydans. The occurrence of the strain needs to be closely monitored.The emergence of carbapenem resistance (CR) caused by hydrolytic enzymes called carbapenemases has become a major concern worldwide. So far, CR genes have been widely detected in various bacteria. However, there is no report of CR gene harboring  Comamonas contains species that are Gram-negative, aerobic, non-pigmented, and rod-shaped bacteria that belong to β-Proteobacteria, which are motile through the use of at least one polar tuft of flagella, and has non-fermentative chemoorganotrophic metabolism. These are quite ubiquitous in the environment and have been isolated from soil, termite guts, activated sludge, humans, fresh water, sediments, and garden ponds. Comamonas strains have been found to be isolated from various clinical samples, as well as from the hospital environment. However, they are not seen as pathogenic to healthy humans  gene has been found in C. thiooxydans. Therefore, it is necessary to carry out further study on this strain, the genomic and phenotypic characteristics.The IMP-type metallo beta-lactamase (MBL) was first reported in Japan in 1991 when the  isolate . The IMP isolate , China , as well as average nucleotide identity (ANI). Carbapenemase-encoding genes were detected using PCR. 3 and annotated by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP)4 and RAST version 2.05  and sequenced using Illumina Novaseq  and Oxford Nanopore PromethION . Draft genomes were obtained using SPAdes version 3.9.1ion 2.05 . Acquireon 2.2.3 .C. thiooxydans ZDHYF418 and additional C. thiooxydans strains, we downloaded all 21 C. thiooxydans publicly available genomes in the NCBI Genome database8 as of October 2020 (C. thiooxydans CNB (accession number: CP001220.2) as reference strain. The phylogenetic tree was subsequently visualized and modified using iTOL version 59.In order to investigate the phylogenetic relationships between ber 2020 . Next, wber 2020  to creatC. thiooxydans, designated as ZDHYF418, from the mid-section urine specimen of a 60-year-old female patient that was admitted to a public hospital in Zhengzhou, China in 2019. The patient was admitted to the hospital for treatment of left kidney stones. During hospitalization, the patient was intermittently irritable, unconscious, and went into septic shock. Additionally, the patient experienced abdominal distension, nausea, and vomiting. The blood culture results indicated the presence of an E. coli infection. The patient was administered imipenem-cilastatin 1g ivgtt Q8H for 1 month to fight the infection. The doctor plans to perform transurethral ureteroscopy with lithotripsy when her condition became stable. C. thiooxydans was detected in the patient’s urine culture the day before the surgery. The patient’s stones suddenly recurred and the pain could not be relieved, so the doctor did not postpone the operation. The patient was treated for a week until her condition stabilized and she was discharged from the hospital.We isolated a novel strain of C. thiooxydans after the observation of ANIs analysis based on BLAST. In fact, the genomic sequences of ZDHYF418 are 96.830% identical by ANI to the genome of C. thiooxydans, with 86.2% coverage of the genome. This result reveals that a phylogenetic affiliation of strain ZDHYF418 belongs to the species C. thiooxydans.Strain ZDHYF418 was named in vitro susceptibility tests results indicated that ZDHYF418 is a multi-drug resistant strain (blaIMP–8 and aac(6′)-Ib-cr are all resistance genes contained in the sequence of strain ZDHYF418. In addition, gyrA, parC, and a novel class D beta-lactamase gene blaOXA were also found in the annotation results. The blaIMP–8 and blaOXA genes confer resistance to β-lactam antibiotics, β-lactamase production is the most common resistance mechanism. The aac(6′)-Ib-cr gene mainly modifies the amino group of aminoglycosides to inactivate aminoglycoside antibiotics, thereby conferring resistance to aminoglycosides. In addition, the resistance to fluoroquinolones is mainly caused by mutations in the coding regions of the gyrase subunit (gyrA) and DNA topoisomerase IV (parC)  . Usuallybe found . The effblaIMP–8 gene in ZDHYF418 and the sequences most similar to ZDHYF418 by BLAST are shown in blaIMP–8 in C. thiooxydans ZDHYF418 has a percent identity of 99.96 and 99.94% to the p447-IMP in K. pneumoniae (accession number: KY978631)  (blaIMP–8 gene in C. thiooxydans ZDHYF418 includes IMP–8-intI1DDE-type integrase/transposase/recombinase–tniB–tniQ-recombinase family protein-aac(6′)-Ib-cr-bla. p447-IMP and p16005813B both contain the In655 integron, which is an ancestral Tn402-associated integron. The differences among these sequences are that in ZDHYF418, the tniR module may be missing and replaced by a recombinase family protein. It has a 100% identity to MULTISPECIES: recombinase family protein (accession number: WP_003155741.1), it belongs to the serine recombinase (SR) family that can mediate site-specific recombination (WP_088244042.1). Both of the recombinase family protein and DDE-type integrase/transposase/recombinase are non-redundant protein sequences.We report here the genomic sequence of this strain contains 5,273,527 bp with a GC content of 61.4%. The genetic structure of the Y978631)  and p160K036884) . The genbination . The tniC. thiooxydans ZDHYF418 to the 21 C. thiooxydans are depicted in C. thiooxydans QYY (accession number: CP053920.1). There are 330 SNP differences between ZDHYF418 and C. thiooxydans QYY. Strain QYY was isolated from activated sludge in Jilin province in 2015. On the other hand, C. thiooxydans has been mainly isolated from the environment, especially soil (The phylogenetic relationship of lly soil , 3. AWTMC. thiooxydans strain from the urine of a patient with urinary tract infection caused by E. coli and C. thiooxydans. The patient ultimately developed septic shock. According to a previous case report of septic shock caused by bloodstream infection (C. thiooxydans.In this study, we describe a nfection , we specblaIMP–8-positive C. thiooxydans strain from a human urine sample. The isolation of C. thiooxydans from humans is very rare, and the strain we identified was clearly resistant to aztreonam, fluoroquinolones and aminoglycosides, and intermediate to meropenem. The increased resistance of bacteria to antibiotics is now starting to appear in less common bacteria, such as C. thiooxydans. This finding prompts us to standardize clinical medication and pay more attention to bacterial resistance monitoring of this species.In summary, we first identified a PRJNA623107, https://www.ncbi.nlm.nih.gov/bioproject/?term=623107.The datasets generated for this study can be found in NCBI under BioProject Written informed consent was obtained from the participant of this study. Ethical permission was granted by the Research Ethics Committee of The First Affiliated Hospital, College of Medicine, Zhejiang University, reference number 2018#752.XG and JG conceived and designed the experiments. QW, SL, and PW collected clinical samples and performed the experiments. HX, XH, and LG analyzed the data. QW wrote the manuscript. All authors contributed to the article and approved the submitted version.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.


Cyperus is one of the most promising health supplementing genera of the Cyperaceae family, housing ≈950 species, with Cyperus rotundus L. being the most reported species in pharmacological studies. The traditional uses of Cyperus spp. have been reported against various diseases, viz., gastrointestinal and respiratory affections, blood disorders, menstrual irregularities, and inflammatory diseases. Cyperus spp. are known to contain a plethora of bioactive compounds such as α-cyperone, α-corymbolol, α-pinene, caryophyllene oxide, cyperotundone, germacrene D, mustakone, and zierone, which impart pharmacological properties to its extract. Therefore, Cyperus sp. extracts were preclinically studied and reported to possess antioxidant, anti-inflammatory, antimicrobial, anticancer, neuroprotective, antidepressive, antiarthritic, antiobesity, vasodilator, spasmolytic, bronchodilator, and estrogenic biofunctionalities. Nonetheless, conclusive evidence is still sparse regarding its clinical applications on human diseases. Further studies focused on toxicity data and risk assessment are needed to elucidate its safe and effective application. Moreover, detailed structure-activity studies also need time to explore the candidature of Cyperus-derived phytochemicals as upcoming drugs in pharmaceuticals.Cyperaceae are a plant family of grass-like monocots, comprising 5600 species with a cosmopolitan distribution in temperate and tropical regions. Phytochemically,  Cyperus papyrus L.), a species of the Cyperaceae family [Cyperus, with ~950 species [Cyperus spp. are most commonly known as weeds, despite some cultures using them for medicinal purposes and as a source of food [Cyperus spp. predominantly exist in the wetlands throughout the globe in the tropical regions and act as source of primary productivity. The tubers, shoots, and fruits of this species are produced in larger quantities and act as a source of food for amphibians and aquatic animals [Immemorially, human societies have been using herbs and their products as sources of medicine, nutrition, and industrial applications . As an ee family . Cyperac species . Cyperus of food . CyperusCyperus plants has been reported from all over the world as a remedy against various human ailments [Cyperus including more than 950 species, the three most commonly reported species are purple nutsedge (Cyperus rotundus L.), yellow nutsedge (Cyperus esculentus L.), and C. papyrus. Cyperus rotundus is the most well-known species of Cyperus in South Asia, a perennial weed that grows best in high-moisture soil and reproduces easily through rhizomes and tubers [Cyperus rotundus rhizomes and tubers are mentioned in Oriental traditional medicine to treat fever, digestive disorders, and menstrual irregularities in several countries including China, India, Iran, and Japan [The traditional use of ailments  includinailments . Interesd tubers . This spd tubers . Cyperusnd Japan .Cyperus esculentus L. is an edible perennial grass-like plant native to the Old World. This species exists widely throughout tropics and subtropics of North America [Cyperus esculentus tubers were roasted and used as a sweetmeat in Egypt during the ancient times [Cyperus esculentus is widely cultivated for its edible tubers, called earth almonds or tigernuts [ America . The ear America . It has nt times . Cyperusigernuts , which aigernuts . Tigernuigernuts .Cyperus papyrus L. is an aquatic sedge mostly known for its use in the preparation of the paper by the traditional Egypt, Greek, and Roman civilizations. Paper made from dried, pressed, and woven strips of culm pith had been used since 3500 BC by ancient civilizations in the Egypt and the Mediterranean Basin. It was the only widespread recording medium until the 8th century in Europe [Cyperus family include Cyperus compressus L., Cyperus javanicus Houtt., and Cyperus monocephalus Roxb. . For instance, C. compressus is a grass-like plant and is widely distributed across the tropical and subtropical regions of the world. In India, the powdered roots of C. compressus have long been used in traditional medicine by the Santhal tribes to treat intestinal helminthic infections [Cyperus spp. reported from different parts of the world are briefly shown in n Europe . Other sfections . ExampleCyperus spp. were considered. This review is the first of its kind that gives a comprehensive discussion on the recent findings related to chemical composition, biological activities, and pharmacological effects of such promissory naturally occurring matrices. The safety and toxicity effects of the Cyperus sp. extracts are also considered in the scope of the manuscript. The diagram showing various components discussed in the review are presented in Taken together, the multiple potentialities reported so far for the most widely exploited Cyperus scariosus R.Br. essential oil, is present in various perfumes and medicines. In fact, cypriol's ambery, balsamic, spicy, warm, and woody features make it highly demanded in perfume industry [Cyperus, such as C. articulatus L., C. rotundus, and Cyperus maculatus Boeckeler [Cyperus spp. is also presented.The Cyperaceae family is one of the largest flowering plant families and is ranked the third largest monocot family after Orchidaceae and Poaceae . A risinindustry . In addioeckeler . SummariCyperus articulatus is a perennial herb with underground perennial rhizomes having scales which grade into culm leaves. They have exceptionally high photosynthesizing function compared to other plants and are also regarded as herbal switch plants as they are a reservoir of potentially useful drugs for the treatment metabolic disorders [C. articulatus include α-cyperone, α-corymbolol, α-pinene, caryophyllene oxide, cyperotundone, and mustakone. Researchers also identified articulone, myrtenal, and myrtenol from volatile oil of Nigerian C. articulatus [C. articulatus hexane extracts displayed the presence of isopatchoul-4(5)en-3-one, mandassidione, mustakone, and almost all sesquiterpene diketones [α-pinene (6.4%) [C. articulatus rhizomes showed the presence of α-pinene (3.5–25.2%), β-pinene (2.3–12.6%), trans-pinocarolol (2.2–5.5%), myrtenal + myrtenol (2.3–5.6%), α-copaene (1.3–2.6%), cyperene (0.7–1.6%), β-selinene (0.8–2.4%), lithol (0.9–5.1%), caryophyllene oxide (3.1–8.3%), mustakone (3.4–9.9%), cyperotundone (2.6–4.1%), and α-cyperone (3.2–8.8%) [isorders . Variousiculatus . Camerooiketones . Similare (6.4%) . Volatil.2–8.8%) . These r.2–8.8%) , 60.Cyperus conglomeratus is a perennial monocot with coarse rhizomes up to 12−16 cm long and 0.2–0.3 cm in width. It is native to India but also grows in temperate, tropical, and subtropical regions [C. conglomeratus showed the presence of palmitic, linoleic, heptadecanoic, oleic, myristic, arachidonic, lignoceric, and stearic acid. The unsaponifiable fractions of C. conglomeratus constituted two other bioactive compounds (β-sitosterol and α-amyrin). regions . It is a regions . The the regions , such as regions –71. The Cyperus distans is an annual herb of about 0.6–1.5 m tall, mostly found in humid areas along roadsides and rivers and as weeds [C. distans revealed the presence of scabequinone with antifeeding effects [α-cyperone (9.1%), humulene epoxide II (3.8%), cyperene (3.2%), endesma-2,4,11-triene (2.9%), nor-copernone (2.9%), and germacrene D (2.8%) [as weeds . The phy effects . From 80D (2.8%) –77.Cyperus esculentus, also known as yellow nutsedge, is a perennial herb growing in tropical and temperate regions of the world. Naturally, it is found as a weed in farming areas and in wastelands [C. esculentus are chufas, earth almond, nutsedge, and rush nut [stelands . The tasstelands . The comrush nut . Cyproturush nut , and vanrush nut , with inrush nut .Cyperus longus is an Egyptian plant, distributed throughout Africa and Europe and to Indian subcontinent and is used as herbal tonic and diuretic [C. longus essential oil were also identified such as β-himachalene (46.6%), α-humulene (16.7%), and γ-himachalene (10.1) as main components [diuretic . Compoundiuretic –88. Othemponents . In anotmponents .Cyperus rotundus is popularly known as Nagarmotha or purple nutsedge or nut grass [C. rotundus were determined using HPLC, and it was reported to contain two bioactive phenolics, i.e., quercetin and chlorogenic acid [Cyperus spp. are shown in ut grass . This isnic acid . StructuCyperus spp. is attributed to the plethora of phytochemicals present. Phenolic compounds, specifically flavonoids, tannins, and coumarins, are present in this species. The presence of these phytochemicals is directly correlated with antioxidant effects [Cyperus capitatus Vand. and C. conglomeratus Rottb.) and 3 from the Poaceae family (Elymus farctus (Viv.) Runemark ex Melderis, Lasiurus scindicus Henrard, and Panicum turgidum Forssk.) collected from the Egyptian coastal desert (Mediterranean coast of the Delta) and the interior desert (Wadi Hagoul). Strong radical scavenging activity with EC50 < 1 mg/ml assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was reported from the extracts of C. conglomeratus and C. capitatus [Cyperus tegetum Roxb. demonstrated significant DPPH radical, superoxide anion, and hydrogen peroxide scavenging activities compared to the standards, viz., hydroxybutylanisol, butylhydroxytoluene, and ascorbic acid, respectively [C. esculentus tubers, commonly known as tigernut, was utilized in a study on rats to assess its effect on preventing acetaminophen-induced liver damage (APAP). Its presence led to an increased activity of antioxidant enzyme superoxide dismutase (SOD), while malondialdehyde concentrations were lower than the control group, thus demonstrating a good antioxidant activity [C. rotundus tubers was found to have protective effects on liver and kidney function caused by exposure to heavy metals (cadmium chloride) in rats, through scavenging of free radicals [C. articulatus rhizome encapsulated in chitosan nanoparticles revealed a high potential to eliminate free radicals [Antioxidants are substances which remove reactive species or free radicals from cells and play a crucial role in maintaining the health and by preventing the diseases. The antioxidant capacity of  effects . For exaapitatus . An expeectively . In addiactivity . The impradicals . Also, cradicals . The encCyperus plant extracts have proven to have a neuroprotective effect caused due to reactive oxygen species (ROS). The deposition of beta-amyloid in the hippocampus promotes oxidative stress, reactive ROS formation, reduction of the antioxidant enzymes activity, and consequently, neuronal death. Previous studies have shown that flavonoids can modulate the function of immune cells, exerting a direct effect against inflammation and oxidative stress [C. rotundus extracts explains the increase in hippocampal neurogenesis of beta-amyloid in rat models and consequently improves the memory [C. esculentus, decreased oxidative stress generating a neuroprotective effect against cerebral ischemia/reperfusion injury in Sprague-Dawley rats through the middle cerebral artery occlusion method [e stress . Thus, te memory . Orientin method .Cyperus genus [C. rotundus rhizome was first described in 1971 [C. rotundus. The compound α-cyperone, one of the main phytochemicals found in C. rotundus oil, was found to inhibit lipopolysaccharide- (LPS-) stimulated inflammatory response in a murine BV-2 microglial cell line, by activating Akt (protein kinase B)/nuclear factor-E2-related factor (Nrf)-2/heme oxygenase- (HO-) 1 and suppressing the nuclear factor kappa light chain enhancer of the activated B cell (NF-κB) pathway [α-cyperone exerts a neuroprotective activity by attenuating the production of inflammatory cytokines in BV-2 cells through activating Akt/Nrf2/HO-1 and suppressing the NF-κB pathway.Numerous studies have proven the potent anti-inflammatory activity of extracts obtained from various plant parts of the us genus . The ant in 1971 , and sin pathway . A studyC. rotundus rhizomes revealed that cyperalin A has high anti-inflammatory activity through inhibition of prostaglandin E2 (PGE-2), cyclooxygenase-2 (COX-2), and arachidonate 5-lipoxygenase (LOX-5) and that sugetriol triacetate, another compound of biological interest in C. rotundus, presented a similar effect on PGE-2, COX-2, and LOX-5 enzymes in peripheral blood mononuclear cell (PBMC) lines [α-Cyperone revealed to suppress the inflammatory response in lipopolysaccharide- (LPS-) induced acute lung injury in mice, through inhibiting the growth of inflammatory cells along with cytokines and downregulating the NF-κB and NLR family pyrin domain containing 3 (NLRP3) signalling pathways [. rotundus rhizome extract in a rat model with chronic and acute dermatitis leads to a reduction in ear oedema and inflammatory cell infiltration generated by exposure to 12-O-tetradecanoylforbol-acetate (TPA). This ultimately suggested that the extract could be a potential new therapeutic tool for the treatment of inflammatory skin disorders [Another study using methanol extracts from C) lines . α-Cyperpathways . Moreoveisorders .C. rotundus extract has been shown in numerous studies [Cyperus extracts than Gram-negative bacteria. However, direct comparison of different studies was difficult due to variety of microbiological tests, microbial genera and species, presence of saccharides, herb cultivation conditions, extraction methods, and so on [C. articulatus essential oils revealed interesting inhibitory effects on Staphylococcus aureus and Escherichia coli [C. rotundus tubers on cultures of Streptococcus mutans, Aggregatibacter actinomycetemcomitans, and Candida albicans were investigated. Alcoholic extracts displayed marked inhibition of S. mutans and A. actinomycetemcomitans growth, making these extracts future candidates for both treatment and prevention of periodontitis and oral cavity affections [The antimicrobial activity of  studies , 105–109nd so on . For exahia coli . Traditihia coli . The effC. conglomerates, orally administered to mice, aiming to determine the degree of consumption toxicity. There was no damage to the liver or kidney at the doses used, and a negative impact on the growth of C. albicans, C. dubliniensis, C. famata, C. glabrata and C. inconspicua was listed. β-Sitosterol and α-amyrin were the most abundant components identified and were the chemicals responsible for the significant effect on the growth of C. famata and C. albicans, respectively [C. conglomeratus chloroform extracts were also assessed for their antibacterial activity [S. aureus, Enterococcus aureus, Bacillus subtilis, E. coli, and Pseudomonas aeruginosa, while butanol and ethyl acetate extracts showed a moderate activity. The antimicrobial activity of Cyperus sp. extracts is mainly contributed by the essential oil components. The essential oil components from Cyperus spp. easily penetrate inside and create pores in bacterial cells which results in leakage of intracellular components resulting to cell death.Another study used chloroform extracts of ectively . C. congactivity . ExtractC. rotundus extracts, evaluated on multidrug-resistant strains of Mycobacterium tuberculosis, also revealed to be prominent [S. aureus. The extract revealed a synergistic activity when in combination with ampicillin at the lowest inhibitory concentration. Using electron microscopy, it was observed that the combined treatment induced damages to the peptidoglycans and cell membrane, generating an increase in membrane permeability and revealing an inhibitory activity against β-lactamase [C. rotundus inhibited the growth of P. aeruginosa, B. subtilis, and E. coli [C. rotundus extracts revealed an excellent antibacterial activity against Klebsiella pneumoniae strains, and the observed inhibitory effects seemed to be associated with factors such as mechanical damage, oxidative damage, and genetic toxicity [The antimycobacterial activity of rominent . In addiactamase . Another E. coli . For suctoxicity .C. rotundus extracts has also been assessed; the mechanism of action also elucidated the influence on genetic expression. For example, human cervical cancer (HeLa) cell lines exposed to different doses of C. rotundus extracts revealed morphological modifications and changes in the degree of chromatin condensation. Microarray analysis also showed that the extract led to the upregulation of 449 genes and downregulation of 484 genes, classified into different interaction pathways, with gene expression induction being associated with apoptosis and cell cycle arrest [C. rotundus ethanol extracts were used to evaluate its effects on triple-negative breast cancer cells (TNBC)  protein overexpression). As main findings, the authors stated that the chemical components present in the extracts inhibited the TNBC cell proliferation, which might be related to cell cycle arrest at the G0/G1 phase, thus inducing apoptosis by promoting Bcl-2-associated X protein (Bax) expression and inhibiting B cell lymphoma (Bcl) expression. The n-hexane extract from C. rotundus rhizomes also exhibited an anticancer activity on Michigan Cancer Foundation-7 (MCF-7) breast cancer cell lines, by inducing apoptosis and halting them in G0-G1 stages of the cell cycle [The anticancer activity of e arrest . The maill cycle .Cyperus sp. roots and tubers were also studied in adenocarcinoma gastric (AGS) and human gastric cancer cell lines. As main achievements, the authors stated that benzoquinones exerted their toxic effect by activating stress in the endoplasmic reticulum, increasing expression of C/EBP homologous protein (CHOP) (mRNA and protein levels), intracellular ROS, changes in calcium dynamics, and caspase-4 activation. The proteasome inhibition caused by hydroxyl cyperaquinone (causing cell death) was first described by the inositol-requiring enzyme 1α- (IRE1α-) independent/(PKR-like ER kinase) PERK-dependent pathway in stomach cancer cells [C. conglomeratus extracts. The cytotoxic effect was assessed in MCF-7 breast cancer cells and normal fibroblasts using 3--2,5-diphenyl tetrazolium bromide (MTT), and a selective cytotoxicity against MCF-7 was stated, while in fibroblasts, no toxic effect was reported. Furthermore, the apoptotic effects were confirmed using annexin V-fluorescein isothiocyanate-propidium iodide (FITC-PI) and real-time PCR for apoptotic genes [Cyperus spp. are shown in The cytotoxic effects of benzoquinones isolated from er cells . Recentlic genes . VariousC. rotundus rhizome methanol extracts were analyzed as an alternative to hormone replacement therapy (HRT). Its estrogenic activity was evaluated in MCF-7 cell lines through its competitive binding to estrogen receptor- (ER-) α and ER-β. These isolated compounds revealed to be useful as an alternative to HTR [C. rotundus, Citrus unshiu, and Poria cocos, has been used in traditional Korean medicine for dysuria. In a study, a significant reduction in body weight and a decrease in weight of epididymal and visceral white adipose tissue were found following oral administration of TBS to male mice (C57BL/6N) for 11 weeks. TBS enhanced the expression of AMP-activated protein kinase (AMPK) and inhibited the expression of transcription factors, such as CCAAT-enhancer-binding proteins (C/EBPs), sterol regulatory element-binding transcription factor 1 (SREBP1), and peroxisome proliferator-activated receptors (PPARγ) in the liver and white adipose tissue of the epididymis [Six sesquiterpenes isolated from e to HTR . Tongbi-ididymis .Cyperus eragrostis seed extracts have also demonstrated vasodilatory properties and ability to inhibit the mammalian arginase enzyme in both an ex vivo experiment on rat aortic rings and an in vitro assay with purified bovine liver arginase [in vivo study in rats, mice, and chicks and in vitro study using isolated tissues of the jejunum and ileum of rabbits and rats, the antispasmodic, antidiarrheal, and antiemetic effects of Cyperus niveus Retz. were assessed. The presence of flavonoids, phenols, alkaloids, tannins, saponins, and glycosides in the extracts was described to be responsible for the significant inhibition of diarrhea in rats and for the marked decrease in the intestinal motility of mice [arginase . Through of mice .Cyperus articulatus ethanolic extracts revealed to prevent pentylenetetrazol-induced seizures (PTZ) and to increase the gamma aminobutyric acid (GABA) levels in mice with PTZ [in vivo and in vitro) were experiments performed with C. rotundus to evaluate its effect on gastrointestinal, bronchial, and vascular disorders, as well as pain, emesis, pyrexia, and bacterial infections. A study revealed that the crude extract from C. rotundus has remarkable spasmolytic, bronchodilator, and vasodilatory effects, possibly through blockade of calcium channels [Cyperus alternifolius L. rhizomes and aerial structures in fasted rats with orally administered indomethacin (30 mg/kg). The extracts led to a significant reduction in the number of ulcers and TNF-α content in the stomach. Histopathological examination revealed an improvement in damaged mucosa, with the effect generated by tubers being more effective than that of the control ranitidine [with PTZ . Other pchannels . A recennitidine .Cyperus spp. have been used over the years in folk medicine around the world to prevent and even treat different medical affections. C. rotundus is the most widely used and exploited species from this genus, and the metabolites present in this species are currently well elucidated [C. rotundus with other medicinal plants taken as capsules of dehydrated plants or in the form of decoction were found effective against overweight, obesity, Alzheimer's disease (AD), depression, and rheumatoid arthritis. However, no clinical trials on C. rotundus used alone could be found.As previously referred , Cyperusucidated , 129, soucidated . For exaC. rotundus tubers, Embelia ribes Burm.f. fruits and Plumbago zeylanica L. roots traditionally used for the management of overweight and obesity, was investigated by Salunke et al. [Emblica officinalis L., Terminalia bellirica (Gaertn.) Roxb., and Terminalia chebula Retz.) , Trimad, and placebo (dextrin) for the management of obesity and overweight, over 90 days. Significant differences were stated in visceral and subcutaneous fat, as well as other benefits, like positive bowel regulation and a decrease in fatigue in patients who received triphala [Berberis aristata, C. rotundus, Cedrus deodara, Emblica officinalis, Terminalia chebula, and Terminalia bellirica, following HPLC analysis revealed the presence of berberine (1.27%), quercetin (0.01%), and gallic acid (3.09%). The randomized study included 93 participants, and 48 of them received 3 g of the herbal treatment for 6 months. The study concluded that the formulation had a positive effect on blood glucose levels, on glycosylated hemoglobin, and on the lipid profile of the patients without showing any adverse effects [C. rotundus [Vitex negundo, C. rotundus, Nyctanthes arbor-tristis, Smilax glabra, Delphinium denudatum, and Withania somnifera, was combined with Maha yogaraj Guggulu Vaiswanar churna and Simhanada Guggulu. No information on its safety or chemical composition was provided in the article, despite the fact that patients (39%) who receive the preparation twice a day before meals for 1 year exhibited a good response to the treatment.An Ayurvedic polyherbal formulation, called Trimad, composed of e et al.  in 20 ovtriphala . Similar effects . Finallyrotundus  was founC. rotundus against neurocognitive disorders has also been reported. The combination of C. rotundus with Crocus sativus and Astragalus membranaceus honey was assessed for its ability to treat a major neurocognitive disorder. A double-blind clinical trial on 60 patients previously diagnosed was performed. The intervention group took two daily capsules of 500 mg each one of the combinations for three months. The results indicated that the combination could be useful to improve the cognitive and depression score. However, the extract used was not characterized, and the preparation method was not indicated [C. rotundus, Zingiber officinale, Acorus calamus, Piper nigrum, and Boswellia carterii has also been tested against AD. In the study, 24 patients with mild to moderate AD took 500 mg of the capsules 3 times daily for 12 weeks. Results showed that the herbal mixture might have an improvement in the memory of patients. However, despite the safety of the administered mixture which was tested, the preparation method was not detailed [Codonopsis pilosula, Atractylodes, Angelica sinensis, membranous milkvetch root, Polygala tenuifolia, and Arillus longan; 20 g of Poria cocos and magnesium; 30 g of calcined Os Draconis, calcined oyster shell, light wheat, and concha haliotidis; and 10 g of elecampane, fresh ginger, Chinese date, colla corii asini, mint, Albizia julibrissin, C. rotundus, jasmine, and Coptis chinensis as well as spina date seed of 40 g. The mixture was decocted, and patients received 400 ml of extract for a month. The control group was treated with sertraline. Results concluded that the mixture exerted a curative effect and improved the patients' quality of life by alleviating depression symptoms, although no significant differences were reported in blood pressure.The possible use of ndicated . The tradetailed . In traddetailed . So, Zhudetailed  tested tCyperus spp., but the species investigated were not specified in a large amount of cases. These are the cases of mixtures made with Cyperus for sexual dysfunction [Cyperus spp., which have been widely described via a plethora of in vitro and in vivo tests.Some other clinical trials have been performed using the extracts of function , as antifunction  agents afunction . AnotherCyperus plants have been widely used in folk medicine for multiple affections [Cyperus plants as a popular remedy among various ethnic groups, researchers have increasingly explored their therapeutic potential [Due to its richness in chemical constituents, fections , 142. Thotential . HoweverC. rotundus extracts have been reported by several investigators [Cyperus extracts as safe [C. rotundus extracts were studied in vivo for toxicity and biochemical activities in mice and rats. The lethal dose LD50 of C. rotundus root extract, when administered intraperitoneally, was 90 g/kg [50 > 0.5 mg/kg [50 of 681.0 mg/kg [50 of Cyperus essential oils was 5000 mg/kg in rats [C. rotundus ethanolic extract at 5000 mg/kg did not produce signs of toxicity, behavioral changes, mortality, or differences on gross appearance of internal organs in the animals. In subacute toxicity, all rats received a repeated oral dose of 1000 mg/kg of the ethanolic extract for 14 days. The parallel group received the ethanolic extract in the same period but was kept for further 14 days. Application did not produce any effects or reversibility of toxic effects. Thus, it was concluded that the extract did not cause changes in terms of general behaviors, mortality, weight gain, and hematological and clinical blood chemistry parameters in comparison to the control group [Cyperus extracts. C. rotundus methanolic extracts in mice at doses of 250 and 500 mg/kg body weight showed no toxic effects [C. rotundus tubers' hexane extracts for 60 days in rats stimulated a significant reduction in weight gain but without toxic effects. Raut and Gaikwad [C. rotundus crude extract at different concentrations and oral doses. Jebasingh et al. [C. rotundus extract and found no mortality or morbidity up to 2000 mg/kg body weight in rats. Toxicity studies also revealed no changes in food, water consumption, and body weight of animals with an increase in white blood cell count and hemoglobin level as well as improvement in kidney and liver function. Krisanapun et al. [C. rotundus water extracts in rats and reported the single oral LD50 > 5 g/kg body weight. The C. rotundus extract used at three doses, 10, 100, and 1000 mg/kg, did not exhibit any sign of toxicity. However, it was observed that at 1000 mg/kg, motor activity was slightly decreased. The effects of C. rotundus extract were also assessed on different biochemical parameters . Liver enzymes were found normal, and a nonsignificant increase in serum bilirubin, gamma-glutamyl transferase (GGT), and serum glutamic-pyruvic transaminase (SGPT) was recorded. Also, hematological studies did not show any significant toxic changes, and histopathological examination confirmed that the tested extract was safe and nontoxic [Cyperus spp. for biotechnological purposes, namely, regarding its use as a functional food additive. For example, Carvalho Barros and coworkers [C. esculentus) oil emulsion, to reduce total fat and saturated fatty acids in the studied samples. As main findings, the authors stated that total replacement of beef fat using tigernut oil emulsions in beef burger resulted in a well-accepted and healthier meat product with reduced total and saturated fat contents, as well as increased unsaturated fatty acids [Cyperus sp. agroindustrial and biotechnological potentialities.Toxicological data from tigators . Most of as safe , 109, wi 90 g/kg , 146. Et.5 mg/kg . Aqua-et.0 mg/kg . In turn in rats . Other sol group . Another effects . On the  effects  document Gaikwad  also obsh et al.  performen et al.  carried nontoxic . Finallyoworkers  evaluatety acids . HoweverCyperus sp. is popularly employed as a potent ethnomedicine owing to its plethora of pharmacological attributes, viz., antioxidant, anti-inflammatory, neuroprotective, antidepressive, antiarthritis, antiobesity, antimicrobial, anticancer, vasodilator, spasmolytic, bronchodilator, and estrogenic properties. This wide array of biological activities is closely linked to the presence of phytochemicals such as α-cyperone, α-corymbolol, α-pinene, caryophyllene oxide, cyperotundone, germacrene D, mustakone, and zierone. However, its wide-ranging use in folk medicine and expansive pharmacological properties are not corroborated with incontrovertible evidences employing animal models, where despite the bioactive phytochemicals of Cyperus spp., they have been well-deciphered. Comprehensive investigations on the pharmacological efficacies of isolated compounds are still inadequate. Moreover, structure-activity analyses on the obtained phytoconstituents have also uncovered the perception of the underlying molecular insights of action of its active extracts and/or phytochemicals. On the other side and although toxicological data have indicated the use of Cyperus spp. as safe and effective, conclusive studies encompassing its clinical, toxicological, and safety features are still sparse. In addition and given that reported clinical trials lack herbal mixtures' characterization containing Cyperus spp., further studies are needed to explore the precise active constituents. Moreover, the subchronic toxicity as well as their interactions with commonly used conventional drugs to assure a safe and long-term consumption by human subjects also needs further attention of the researchers. Lastly and not least interesting to underline is that, considering the extensive pharmacological potential of Cyperus spp., more in-depth research is needed to attain a greater clarity of its mechanism of action.Across the diverse traditional systems of medicine,


Drosophila and LGN in vertebrates) has been established in bilaterian models, but its function remains unclear. We used a phylogenetic approach to test whether the interaction is obligate in animals, and in particular whether Pins/LGN/GPSM2 evolved in multicellular organisms as a Dlg-binding protein. We show that Dlg diverged in C. elegans and the syncytial sponge Opsacas minuta and propose that this divergence may correspond with differences in spindle orientation requirements between these organisms and the canonical pathways described in bilaterians. We also demonstrate that Pins/LGN/GPSM2 is present in basal animals, but the established Dlg-interaction site cannot be found in either Placozoa or Porifera. Our results suggest that the interaction between Pins/LGN/GPSM2 and Dlg appeared in Cnidaria, and we therefore speculate that it may have evolved to promote accurate division orientation in the nervous system. This work reveals the evolutionary history of the Pins/LGN/GPSM2-Dlg interaction and suggests new possibilities for its importance in spindle orientation during epithelial and neural tissue development.The orientation of the mitotic spindle determines the direction of cell division, and therefore contributes to tissue shape and cell fate. Interaction between the multifunctional scaffolding protein Discs large (Dlg) and the canonical spindle orienting factor GPSM2 (called Pins in  Summary: Discs large and GPSM2 interact to drive cell division orientation. In this article the authors investigate the evolutionary history of this interaction and speculate on its importance to development. Discs large (Dlg) is a multifunctional scaffolding protein . Like otDrosophila, nuclear mitotic apparatus in vertebrates), GPSM2 , and the microtubule motor protein dynein  in the unstructured linker between the TPR and GoLoco domains  mutants are viable and demonstrate only a few defects: morphological changes in the mushroom bodies of the brain  in neuroblasts but not in epithelia. In bilaterian models, Insc orients asymmetrical neuroblast division in the apical–basal axis by apically localizing Pins/LGN/GPSM2 . Unlike While the importance of the interaction between Dlg and Pins/LGN/GPSM2 has been demonstrated in bilaterian models, whether these proteins and their interactions are conserved in more ancient animals remains unknown. If this interaction is conserved across all animal phyla, then the interaction between Pins/LGN/GPSM2 and Dlg may be essential to multicellularity. Alternatively, the interaction could have evolved as tissue types and architectures diversified within the animal kingdom. Therefore, determining when the Dlg and Pins/LGN/GPSM2 interaction arose may reveal the possible functions of the interaction in spindle orientation.Homo sapiens, M. musculus, Danio rerio, and Drosophila  with animals .The Dlg GUK domain is found in animals and the single-celled eukaryotes choanoflagellates and Filasterea . Our anaosophila , 2012. Iosophila C. We als animals C. Togeth animals . We alsoO. minuta, a member of Hexactinellida. Of the seven amino acids that interact with the Pins/LGN/GPSM2 linker, only three are conserved in O. minuta while five are conserved in Amphimedon queenslandica (Demospongiae) and all seven are conserved in Oscarella lobularis (Homoscleromorpha). Furthermore, the functional proline residue is conserved in A. queenslandica and O. lobularis but is replaced by a leucine in O. minuta .There are four classes of sponge within the Porifera phylum: Calcarea, Demospongia Hexactinellida, and Homoscleromorpha. Hexactinellida cell types are mainly syncytial, while Calcarea, Demospongia, and Homoscleromorpha contain entirely mononuclear cells. We found that the Dlg protein sequence has diverged in . minuta C .Before investigating conservation of the Dlg-interacting region in Pins/LGN/GPSM2, we first asked to what extent sequence conservation should be expected. To address this question, we examined the Dlg-interacting region in Khc-73, a well-established binding partner for Dlg, with particular attention paid to sequences in animals outside of the Bilaterian clade A. Khc73 czarzaki  and locaosophila . HoweverA. queenslandica. This sequence lacked an apparent linker region and GoLoco domains. Manual analysis of the Amphimedon queenslandica Annotation Release 102 genomic sequence revealed an additional annotated sequence with homology to the GoLoco domains  located 3769 nucleotides past the C-terminus of XP_019856151.1. These sequences were combined to generate a candidate full length Pins/LGN/GPSM2 sequence .We next sought to identify Pins/LGN/GPSM2 orthologs in basal animals. BLAST searching against Ctenophora and Porifera genomes recovered no hits for Pins/LGN/GPSM2 in ctenophores and only one hit, a Pins/LGN/GPSM2-like protein  in the sponge A. queenslandica to all other organisms included in this study. Based on this analysis, missing exons are likely to be located past the N-terminus of XP_019856151.1, as all organisms except A. queenslandica contain an additional 20-80 amino acids at the N-terminus, corresponding to the first TPR domain in H. sapiens Pins/LGN/GPSM2. It is also possible that an exon is missing past the C-terminus of XP_003389018.1, although other organisms in this study only contain an additional 5-20 amino acids at the C-terminus, and these do not correspond with any functional domains.We considered the possibility that exons past the N-terminus of XP_019856151.1 or past the C-terminus of XP_003389018.1 were missed in the manual reconstruction. We therefore performed global sequence alignment comparing the Pins/LGN/GPSM2 sequence in Hydra vulgaris XM_004209432.1) used in the earlier alignment is no longer annotated as part of the H. vulgaris genome and has been removed from the NBCI GenBank  . The conserved serine residue is predicted to be a phosphorylation site with >99% confidence . The efficiency (Kcat/Km) of human Aurora A phosphorylation of synthetic peptides is reduced by 85% when the B position is occupied by an arginine versus a leucine (The conserved phosphorylation target sequence in cnidarians does not match the bilaterian target. All identified cnidarian linker sequences contain either a charged or polar residue (R/K/N) at the B position  Pins/LGN/GPSM2 contains two predicted serine and one predicted threonine phosphorylation site between amino acids 381-420  (ig. S3A) . While aA. queenslandica (Poriferan) GPMS2 contains two predicted serine phosphorylation sites between amino acids 328-359, presenting the same challenge . Given similarity in length and sequence to other Pins/LGN/GPSM2 homologs, we expect that the entire length of the disordered linker region is included in our reconstruction . Only one of these [NW_003546593.1 ] contained phosphorylation sites , but the sequence was not similar to bilaterian or cnidarian linker sequences. Our analysis suggests that Dlg and Pins/LGN/GPSM2 do not interact in Placozoa or Porifera.truction B. Neverttruction . We idenDlg is necessary for spindle orientation across many bilaterian models. Here we provide evidence that the interaction between Dlg and Khc73 is present throughout the animal kingdom but that the interaction between Dlg and Pins/LGN/GPSM2 evolved in Cnidaria. While it remains unclear whether or not Dlg functions as a positioning cue or to assist in microtubule capture in bilaterian spindle orientation, our results suggest that neither function is required for multicellularity.O. minuta, a hexactinellid, showed an unusually low average percent identity. The same trend was observed in the global Dlg sequence. Given that the GB subdomain participates in many interactions important for spindle orientation, a likely explanation for this divergence is that spindle orientation is not required for the development of the trabecular syncytium. Therefore, it would not appear necessary to orient the plane of division in cells with only nuclear, and not cytoplasmic, divisions. Furthermore, how the spindle orientation complex, consisting of many cortical proteins, could interact with nuclei towards the center of the cytoplasmic mass is unclear.Hexactinellida is a class of sponges classified by the presence of the trabecular syncytium, a continuous multinucleated tissue that is the main component of the organism . This clC. elegans Pins/LGN/GPSM2 orthologs, GPR1/2, demonstrate low sequence conservation with Pins/LGN/GPSM2 proteins despite maintaining its role in spindle orientation  compared to other bilaterians. GPR1/2 and LIN-5 (Mud/NuMA) act downstream of cell polarity factors in C. elegans embryos (Drosophila Pins (Pins/LGN/GPSM2), Mud (Mud/NuMA), and polarity factors are mutually dependent on one another , which is implicated in cell polarity and spindle orientation (While site prediction software (NetPhos) predicts phosphorylation in the cnidarian linker with >99% confidence, the consensus sequence that we identified in these animals is markedly distinct from that in bilaterians. First, although human Aurora-A is unable to efficiently phosphorylate synthetic peptides with a charged residue at the B position, it is possible that Aurora-A can phosphorylate Pins/LGN/GPSM2 entation .Drosophila wing disc epithelium, spindle orientation is not disrupted by 1P20dlg mutants, which can still regulate apical-basal polarity factors but cannot interact with Pins due to a truncation in the GUK domain. Wing disc epithelium spindle orientation is also preserved in 62pins mutants, which are null  cells, which do not endogenously express Insc  database and PSI-BLAST Iteration 1  program H. sapiens Pins/LGN/GPSM2  and Dlg (NP_001353143.1) as the query sequence, we performed a BLAST search against common bilaterian model organisms . Since spindle orientation factors have not been studied in basal animals, the entire Cnidarian (taxid:6073), Placazoan (taxid:10226), Poriferan (taxid:6040) and Ctenophoran (taxid: 10197) phyla were searched for homologous sequences. With the exception of C. elegans, Pins/LGN/GPSM2 sequences were identified in the bilaterian models as well as N. vectensis, A. tenebrosa, E. diaphana, and T. adhaerens each with >30% identity to H. sapiens Pins/LGN/GPSM2 and an E-value <10−10. Similarly, Dlg sequences were identified in the bilaterian models, N. vectensis, A. tenebrosa, E. diaphana, T. adhaerens, O. lobularis, and O. minuta each with >30% identity to H. sapiens Dlg and an E-value of<10−10 . These results indicate that the identified sequences share significant similarity with human Pins/LGN/GPSM2 (LGN) and therefore are homologs of human Pins/LGN/GPSM2  and therefore this organism was not included in the Dlg alignment.Using GN/GPSM2 . While PH. sapiens Khc73 (KIF13B) as the query sequence (Q9NQT8), we identified potential orthologs in all organisms except for S. purpuratus and T. adhaerens. Reciprocal BLAST searching confirmed that identified sequences are the best hit Khc73 homologs in M. musculus, X. laevis, and D. rerio. However, the reciprocal BLAST for all other identified sequences indicated the best hit as KIF13A, a kinesin-motor protein that is closely related to Khc73. To confirm whether or not identified sequences are orthologs to KIF13A or Khc73, we created and analyzed the conservation domain map. While Khc73 contains an N-terminal Cap-Gly domain, KIF13A does not. Therefore, conservation at this region can be used to distinguish between the two proteins. The conservation domain map revealed a highly conserved region at the N-terminus of identified sequences, which we confirmed to be conserved at the H. sapiens Cap-Gly domain by analyzing the corresponding amino acid sequences. However, the identified sequence for C. elegans was truncated at the N-terminus and did not contain the Cap-Gly region, indicating it either is missing an exon at the N-terminus or is not the ortholog of Khc73. Therefore, C. elegans, along with T. adhaerens and S. purpuratus, were excluded from our analysis.Using Alignments were created using COBALT   and wereA. queenslandica exons were translated into amino acid sequences using EMBOSS EMBL-EBI Transeq  algorithm to predict the structure of identified Pins/LGN/GPSM2 and Dlg sequences . IdentifPredicted Pins/LGN/GPSM2 structures have conserved secondary structures at previously defined domains. Each TPR consisted of a helix-turn-helix motif while each GoLoco domain contains an alpha helix. These results provide further evidence that the sequences presented in this study are orthologous. However, due to high angstrom error estimates within unstructured regions of these proteins, including the Pins/LGN/GPSM2 linker region, these structures could not be used to predict interactions between Pins/LGN/GPSM2 and Dlg.


Bloodstream infections (BSI) are associated with inpatient morbidity in the United States. We sought to characterize the epidemiology of common bacterial BSIs in individuals receiving care within the US Military Health System (MHS), which actively prospectively captures clinical and microbiological data from both retired and active-duty US Uniformed Service members and their beneficiaries.We performed a retrospective cohort study analyzing MHS patients with blood cultures positive for all bacterial pathogens, between January 2010 and December 2019. Microbiological data captured by the Navy and Marine Corpse Public Health Center, excluding cultures isolating contaminants, were retrospectively collated with clinical and demographic data from the MHS Data Repository. Acinetobacter spp. represented 17,206 episodes of BSI from 14,531 individuals. The cohort was predominantly male (59.4%) and ≥65 years old (48.7%). Most individuals were retired  or active duty  service members and their dependents . Median Updated Charlson Comorbidity Index Score was 2. Chronic pulmonary disease was the most frequent comorbid condition. Hospital admission was associated with 13,733 (79.8%) BSI episodes, including 5,870 admissions to the ICU. Overall, inpatient mortality was 8.3%. E. coli  was isolated with the highest frequency, followed by S. aureus . Further, 9.5% of E. coli and 36.9% of S. aureus isolates were resistant to ceftriaxone and oxacillin, respectively. Beta-hemolytic streptococci represented the highest percentage (6.3%) of recurrent BSI episodes occurring at least 14 days post-initial BSI. Males or Native American race were most commonly infected with S. aureus. E. coli BSI was most common in all other demographic categories. The most frequent nine bacterial pathogens, as well as Frequency of Bacterial Blood Stream Infections in the US Military Health SystemThe most frequent nine bacterial pathogens, as well as Acinetobacter spp. in the US Military Health System.S. aureus. Further assessments are underway into BSI-related risk factors for occurrence, antimicrobial resistance and mortality, after controlling for comorbidities and disease severity.We assessed the epidemiologic features of all individuals with BSI receiving care in the MHS over a 10-year period. We noted demographic differences in the occurrence of microbiological causes of BSI including All Authors: No reported disclosures


Enterococcus faecalis and Staphylococcus aureus) were more susceptible to tea extracts than Gram-negative bacteria . Green tea extracts inhibited bacterial pathogens much more effectively in all four varieties of tea with the minimum inhibitory concentration (MIC) values at 20 mg/mL, 10 mg/mL, 35 mg/mL, and 16 mg/mL for E. faecalis, S. aureus, E. coli, and S. typhimurium, respectively. Catechins should be considered as the main antibiotic components of the four tea extracts. Total catechins were extracted from green tea and evaluated their antibacterial activity. Additional studies showed that the catechins damaged the cell membrane and increased cell membrane permeability, leading to changes in the relative electrical conductivity and the release of certain components into the cytoplasm. Tea extracts, especially green tea extracts, should be considered as safe antibacterial food additives.Tea is a popular beverage known for its unique taste and vast health benefits. The main components in tea change greatly during different processing methods, which makes teas capable of having different biological activities. We compared the antibacterial activity of four varieties of tea, including green, oolong, black, and Fuzhuan tea. All tea extracts showed antibacterial activity and Gram-positive bacteria ( Camellia sinensis (L.) O. Kuntze), have been reported to display good inhibitory effects against pathogenic bacteria and may support the development of antimicrobial supplements [Increased foodborne illness caused by foodborne spoilage and pathogenic bacteria has been a major food safety challenge and caused widespread concern . Pathogeplements ,6.Tea is one of the most widely consumed beverages worldwide and is considered to exert various pharmacological effects, such as hypoglycemic, anti-obesity, anti-carcinogenic, antioxidant, anti-arteriosclerotic, and antibacterial activities ,8,9. MorBacillus cereus, Campylobacter jejuni, Escherichia coli, Clostridi-um perfringens, Helicobacter pylori, Legionella pneumophila, Staphylococcus aureus, Salmonella typhimurium, Shigella flexneri, Pseudomonas aeruginosa, Vibrio cholerea, etc. [S. aureus and penicillinase-producing S. aureus by inhibiting the biosynthesis of β-lactams [Tea flush extract and extracts of various tea products have shown a wide range of an-timicrobial activities against ea, etc. ,21. A coea, etc. ,23,24. T-lactams ,26. The -lactams . Catechi-lactams . Kombuch-lactams . Tea tre-lactams . HoweverE. faecalis ATCC29212, S. aureus ATCC25923, E. coli ATCC25922, and S. typhimurium ATCC29213 were obtained from the College of Food Science and Engineering, Northwest Agriculture and Forestry University and maintained in slants of nutrient agar (NA) at 4 °C. All microorganisms were cultured in nutrient broth (NB) at 37 °C for 24 h. NA and NB were procured from Beijing Land Bridge Technology Company Limited . Catechin standards, gallic acid, and caffeine were purchased from Shanghai Yuanye Biotechnology . High-performance liquid chromatography (HPLC) grade methanol, acetonitrile, and trifluoroacetic acid were purchased from Thermo Fisher Scientific . Bradford protein assay kits were purchased from the Nanjing Jiancheng Bioengineering Institute . All other reagents and solvents were purchased from the China National Pharmaceutical Group Corporation  and were of analytical grade. Purified water (18.2 MΩ) was prepared using a Millipore Mill-Q Ultrapure Water System .Bacteria stains including Camellia sinensis cv. Fuding-dabaicha) were harvested from the tea station at Northwest Agriculture and Forestry University, Yangling, China. The same fresh green shoots were washed thoroughly with water and manufactured into green tea, oolong tea, black tea, and Fuzhuan tea by different manufacturing processes (Fresh tea leaves (rocesses .w/v = 1:20) for 7 min. The solutions were collected and concentrated by a RE-52AA vacuum rotary evaporator , and then freeze-dried .Tea extracts were prepared according to the described methods . BrieflyTotal catechins (TCs) were isolated from green tea as described previously . The teaThe total polyphenol content was determined according to the Folin-Ciocalteu method . The ami7 cfu/mL) was spread on the surface of agar plates. A sterile filter paper disc (6 mm) immersed in test solution was placed on the plate followed by incubation at 37 °C for 24 h. Tea extracts were used at the concentrations ranging from 20 mg/mL to 80 mg/mL and TCs were used at concentrations ranging from 1 mg/mL to 10 mg/mL. After incubation, the diameter of the inhibition zone (DIZ) was observed and measured in millimetres (mm), and the experiments were performed in triplicate. Sterile water was used as a control. The experiments were performed in triplicate with triplicate samples.The antibacterial activities were evaluated by the disc diffusion method in agar plates according to Goni et al. . Each st7 cfu/mL) was inoculated into a test tube containing 5 mL of NB followed by the addition of serially diluted aqueous test solutions at different concentrations. All tubes were incubated at 37 °C for 24 h. The percent inhibition was calculated by measuring the absorbance at 600 nm with a UV-Visible Spectrophotometer . The MIC values were determined as lowest concentration at which no growth or turbidity was seen. Negative control contained non-inoculated medium and positive control contained inoculated without extract samples.The lowest concentration of tea extract to inhibit bacterial growth was determined by the broth dilution method according to a previous method . In brieg for 10 min. The cells were washed and re-suspended in 5% glucose until the electric conductivities of the cells were stable. TCs (at the MIC concentration) were added into 5% glucose and measured electrical conductivity with a conductivity meter .The membrane permeability of the bacterial cells was evaluated by measuring the change in electrical conductivity according to the method of Kong et al. . The bacg for 10 min. The cells were washed and re-suspended in 0.1 M phosphate buffer solution  followed by the addition of TCs (at the MIC concentration). After treatment, the suspensions were collected using a centrifuge at 6000× g for 5 min. The absorption at 260 nm was measured to determine the change in nucleic acid content by using a UV-visible spectrophotometer . The suspension was also collected to determine the concentration of solute sugars according to the anthrone-sulfuric acid method [The membrane integrity of the bacterial cells was evaluated by measuring the release of cell constituents, including nucleic acids, solute sugars, and proteins, into the cell suspension . Bacterid method  and the p < 0.05.All analyses were performed in triplicate using triplicate samples, and the results are expressed as the mean ± standard deviation. Significance differences for multiple comparisons were determined using one-way ANOVA with SPSS  at E. faecalis and S. aureus) and Gram-negative (E. coli and S. typhimurium) bacteria were evaluated. As shown in The antibacterial activity of different tea extracts on Gram-positive  than Gram-negative bacteria. S. aureus was the most susceptible bacterium, and E. coli was the most resistant bacterium to all tea extracts. The destructive response to the tea extracts of the Gram-positive bacteria compared to the Gram-negative bacteria might be due to the structural differences in the cell walls of the bacteria. The cell walls of Gram-positive bacteria is mainly composed of peptidoglycan and teichoic acid, which are permeable to solute sugars, amino acids, and most ions [Comparing the inhibitory activities of the tea extracts against Gram-negative and Gram-positive bacteria, it was found that tea extracts had stronger antimicrobial activity against Gram-positive bacteria , S. aureus (200 μg/mL), E. coli (580 μg/mL), and Yersinia enterocolitica (620 μg/mL). Generally, Gram-positive bacteria were more susceptible to EGCG than Gram-negative bacteria.HPLC detection and quantitative analysis, as shown in . aureus ,27. Parv. aureus  observedu et al.  measuredE. coli and S. aureus were determined.The antimicrobial mechanism of polyphenols is believed to be associated with precipitation of bacterial cell membrane proteins by the reaction of polyphenols ,48. ManyE. coli and S. aureus similarly increased after treatment with TCs at their MICs . The growth trends rapidly increased in response to TCs during the first 2 h, and then the increases slowed down. However, overall, S. aureus showed higher electrical conductivity after TC treatment than E. coli, indicating that S. aureus was more susceptible to catechins increasing the permeability of their cell membrane, causing more cellular leakage.The cell membrane, as a protective barrier for bacteria, will be destroyed by strong antimicrobial agents, leading to internal electrolyte leakage. Changes in relative electrical conductivity were examined as a reflection of the permeability of bacterial cell membranes. As shown in E. coli and S. aureus, respectively). The release of intracellular nucleic acids, as an index of cell lysis, can be revealed by changes in optical density at 260 nm [E. coli and S. aureus treatment with TCs, respectively  and Gram-negative bacteria (E. coli and S. typhimurium). Catechins should be considered as the main antibacterial active ingredient in tea extract, and their antibacterial activity were confirmed by test DIZ and MIC values against the test bacteria. The membrane permeability and integrity of E. coli and S. aureus changed after treatment with catechins. The results suggest that the antibacterial activity of tea extracts is due to the interaction of catechins with the bacterial cell membrane.This study compared the antibacterial activity of four kinds of tea extracts  which were manufactured from the same fresh green shoots with different manufacturing processes. Green tea contained the highest the catechins and showed the best antibacterial activity against Gram-positive bacteria (


Haemophilus cause a wide range of diseases in humans. Recently, H. influenzae was classified by the WHO as priority pathogen due to the wide spread of ampicillin resistant strains. However, other Haemophilus spp. are often misclassified as H. influenzae. Therefore, we established an accurate and rapid whole genome sequencing (WGS) based classification and serotyping algorithm and combined it with the detection of resistance genes.Bacteria belonging to the genus Haemophilus (sub)species (training dataset). The algorithm was evaluated on 1329 public short read datasets  and used to reclassify 262 clinical Haemophilus spp. isolates from 250 patients (German cohort). In addition, the presence of antibiotic resistance genes within the German dataset was evaluated with SRST2 and correlated with results of traditional phenotyping assays.A gene presence/absence-based classification algorithm was developed, which employs the open-source gene-detection tool SRST2 and a new classification database comprising 36 genes, including capsule loci for serotyping. These genes were identified using a comparative genome analysis of 215 strains belonging to ten human-related Haemophilus species including, but not limited to, H. influenzae, H. haemolyticus, and H. parainfluenzae. It can also identify putative haemin-independent H. haemolyticus strains and determine the serotype of typeable Haemophilus strains. The algorithm performed excellently in the evaluation dataset (99.6% concordance with reported species classification and 99.5% with reported serotype) and revealed several misclassifications. Additionally, 83 out of 262 (31.7%) suspected H. influenzae strains from the German cohort were in fact H. haemolyticus strains, some of which associated with mouth abscesses and lower respiratory tract infections.The newly developed algorithm can differentiate between clinically relevant blaTEM-1D, and tetracycline resistance, associated with tetB, correlated well with available phenotypic data.Resistance genes were detected in 16 out of 262 datasets from the German cohort. Prediction of ampicillin resistance, associated with Haemophilus spp. and can easily be coupled with other public genotyping and antimicrobial resistance databases. Our data also point towards a possible pathogenic role of H. haemolyticus strains, which needs to be further investigated.Our new classification database and algorithm have the potential to improve diagnosis and surveillance of The online version contains supplementary material available at 10.1186/s13073-022-01017-x. Haemophilus are small, pleomorphic gammaproteobacteria that are characterized by their dependency on the blood factors haemin (X-factor) and/or nicotinamide adenine dinucleotide (V-factor) for growth [Haemophilus includes 10 (sub)species that exclusively or predominantly colonize and infect humans [H. influenzae are commonly found as commensal in the human nasopharynx but can also be pathogenic, especially for susceptible populations such as premature infants, elderly adults, and people with compromised immune systems [H. influenzae serotype b (Hib) strains were one of the major causes of bacterial meningitis in children worldwide until the introduction of an effective conjugate vaccine [H. influenzae (NTHi) strains have become the most common cause of H. influenzae related invasive disease, exacerbations of chronic obstructive pulmonary disease and localized infections such as otitis media, pneumonia, sinusitis, bronchitis, and conjunctivitis [H. influenzae strains led to the WHO classification of a priority pathogen for research and development of new antibiotics [H. haemolyticus and H. parainfluenzae have been considered as harmless respiratory tract commensals; however, evidence is accumulating that also strains of these species should be considered as opportunistic pathogens with the potential of causing a wide range of infections [H. para(phro)haemolyticus, H. pittmaniae, and H. sputorum have been linked to different diseases but are less frequently detected [Haemophilus (sub)species include H. (haemolyticus) quentini strains, a urogenital pathogen, H. (influenzae) aegyptius strains causing conjunctivitis and Brazilian purpuric fever [H. ducreyi strains, a causative agent of genital chancre and chronic skin ulceration [H. paraphrohaemolyticus, H. aegyptius, and H. quentini, which are closely related to H. parahaemolyticus, H. influenzae, and H. haemolyticus, respectively, as separate species is still under debate [Members of the genus r growth , 2. The t humans . Bacteri systems , 5. In p vaccine , 7. Sincctivitis , 8, 9. Ffections , 12. Alsdetected , 14. Othic fever , 15–17, ceration . The clar debate , 20.Haemophilus spp. strains and related disease types underlines the need for exact taxonomic differentiation of clinical Haemophilus strains. Current gold-standard methods, i.e., bacterial culture, PCR, and mass spectrometry, are challenged by similarities of genetic and phenotypic characteristics among Haemophilus spp. This is especially problematic for H. haemolyticus, which is often misclassified as H. influenzae or H. parainfluenzae [Haemophilus spp. that are currently mainly considered as benign commensals. Over the past decades, whole genome sequencing (WGS) has become widely adopted for the analysis of pathogens due to its unprecedented discriminatory power and increased cost-efficiency. However, currently available WGS-based classification tools are not tailored towards the identification of  human-related Haemophilus species, do not provide subspecies/serotyping information, have large memory requirements, and/or are time-consuming.The diversity in pathogenicity of fluenzae , 21–24. Haemophilus spp. direct from short read sequencing data. We evaluated the performance of this algorithm using both in silico generated and real WGS datasets downloaded from public repositories. Lastly, we used this algorithm in combination with in silico resistance prediction to resolve misclassifications and determine the horizontally acquired resistome of clinical isolates from a German cohort.To address this problem, we established a curated classification database and algorithm for the rapid identification of clinical human-related The number of datasets used for each species in the training, evaluation, and German cohort dataset are summarized in Additional file Haemophilus spp. reported to date [H. influenzae (n=68) and all H. haemolyticus genomes  that were available on September 22, 2020 were downloaded from the National Center for Biotechnology Information (NCBI)/RefSeq Assembly database [H. parainfluenzae (n=41), Haemophilus ducreyi (n=17), Haemophilus aegyptius (n=5), Haemophilus parahaemolyticus (n=8), Haemophilus paraphrohaemolyticus (n=4), Haemophilus pittmaniae (n=3), Haemophilus quentini (n=3), and Haemophilus sputorum (n=5) were also downloaded from RefSeq. Reads for these 215 assemblies were simulated using dwgsim [The training dataset consists of whole genome data from all human-related  to date , 25. Alldatabase   (n=1323) [H. parainfluenzae datasets from bioproject PRJEB37651 (n=51) and all available Ion Torrent datasets (one fastQ file per strain) from Haemophilus spp. that were available on July 27, 2021, were downloaded from SRA (n=100 H. influenzae datasets). All datasets with estimated mean coverage depth below 15 (using the median genome length reported at the NCBI website [n=19). Transformants created in the lab  and strains with the same sample name (n=38) were also removed. Finally, read datasets that corresponded to an assembly already included in the training dataset (and thus had the same biosample number) (n=48) or for which we could not retrieve an assembly  were also excluded. Unfortunately, all available H. sputorum and H. parahaemolyticus datasets were filtered out using these selection criteria, leaving no datasets left to analyze for these two species. The only three available datasets of Haemophilus pittmaniae could also not be used due to low quality . In total, 1329 Haemophilus datasets remained and constituted the “evaluation dataset”   that were available on November 12, 2020 and annotated as (n=1323) . In addi website ) were reBetween April 2008 and April 2013, different specimen types were collected from 250 in- and outpatients of the University Hospital Schleswig-Holstein, Medical Clinic Borstel and Wismar. These included blood, eye smears, wound smears, and tracheostomal samples, as well as upper respiratory tract  and lower respiratory tract specimens  , oleandomycin discs were used to suppress bacteria other than Haemophilus spp. strains. Single colonies  suggestive of Haemophilus were picked for processing  and subsequent freezing. In total, 262 Haemophilus isolates were obtained and identified at time of diagnosis (2008–2013) as H. influenzae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) . We also reanalyzed several strains that were still viable by current MALDI-TOF equipment (Bruker Biotyper® sirius System with Flex Control 3.4 software). Measuring and classification were performed using MBT compass software 4.1 equipped with MBT IVD library . All isolates were recultivated for 24h on chocolate agar plates before extraction of genomic DNA using Qiagen Genomic-tip 100/G. DNA was sequenced in 2013 with the Illumina Miseq benchtop sequencer using 251 bp paired-end reads and Nextera XT library preparation kit according to the manufacturer’s instructions . All datasets had a minimum mean coverage depth of at least 50-fold. Quality control of the WGS data and primary taxonomic analysis was performed using fastQC [H. influenzae). BBL Cefinase™ discs  were used for the detection of β-lactamase activity.Chocolate agar plates were inoculated and incubated at 35–37°C and 5% COg fastQC  and krakg fastQC  with a sg fastQC )  from the training dataset were re-annotated by prokka and used as input for the pangenome calculation tool Roary v.3.13.0 [The v.3.13.0 . Defaultv.3.13.0 . The optv.3.13.0  is also Core gene alignments were performed with Roary v.3.13.0  (defaulthttps://github.com/ngs-fzb/HaemoSeq) [H. influenzae serotype f strain KR494 were used [H. influenzae serotype c strain to take into account the sequence divergence between the two big serotype subclades described previously [H. influenzae strains, the six concatenated sequences  included in the seq_typing repository [H. sputorum and H. parainfluenzae [The new taxonomic classification database that was developed in this study comprises 74 alleles for 36 marker genes, including genes for serotyping, and is freely available as a SRST2-compatible multi-fasta file on Github  . Detailsere used  as well eviously . For regpository  were usefluenzae  were extfluenzae , this waThe public resistance database ARGannot_r3  was downloaded from the SRST2 repository . This daA reference database with MLST alleles and a file which defines the sequence type profiles as a combination of alleles were downloaded from pubMLST on July 25, 2020, using the getmlst.py script included in the SRST2 github repository .Haemophilus strains and was optimized to allow for accurate species/serotype identification. In-house developed bash scripts were used to apply SRST2 v0.2.0 [https://github.com/ngs-fzb/HaemoSeq) [The presence of specific genes in the genomes of bacterial strains was evaluated directly from raw reads using the mapping-based gene detection tool SRST2 v0.2.0 . Referen2 v0.2.0  on multi2 v0.2.0 . All scraemoSeq) . The scrhttps://github.com/ngs-fzb/HaemoSeq [H. influenzae strains and does not evaluate region II genes from other Haemophilus spp. [In silico serotyping was performed in two ways: (i) starting from raw sequencing reads using SRST2 and the decision algorithm developed in this study (see previous section)  and  publicly available H. haemolyticus genome assemblies and all (n=68) publicly available fully closed H. influenzae genomes  of these ten genes from a H. haemolyticus marker genes were identified with SRST2 among all the H. haemolyticus strains including phylogenetically related strains previously termed H. quentini, and no hits were detected among H. influenzae strains , H. parahaemolyticus (hem*-HpH), H. sputorum (hem*-HS), H. pittmaniae (hem*-HP), and H. haemolyticus (hem*-HH) genome to our classification database  exclusively to the reference alleles from the species the strain belonged to by employing conservative gene coverage and nucleotide identity thresholds (sequence divergence <15% and sequence coverage breadth ≥90%)  for strains from both species  were also found in all H. paraphrohaemolyticus strains  and H. parahaemolyticus , we added a β-galactosidase reference gene (lacZ) from a H. paraphrohaemolyticus strain to our classification database , region II (determines the serotype), and region III (hcsA/B) of three Haemophilus spp. to our classification database  and with the assembly-based in silico predictions from Hicap (limited to H. influenzae strains)  are required to classify a strain as H. pittmaniae, H. sputorum, H. parainfluenzae, or H. para(phro)haemolyticus. H. paraphrohaemolyticus is distinguished from H. parahaemolyticus by the presence of a lacZ gene. When all capsule loci are detected, the serotype is determined based on the presence of specific region II alleles. Absence of at least one capsule gene is translated into a capsule-deficient strain and absence of all capsule genes in H. influenzae strains defines non-typeable H. influenzae (NTHi). Finally, presence of all nine haemin biosynthesis genes in the genome of a H. haemolyticus strain is indicative of a haemin-independent phenotype related to H. haemolyticus subsp. intermedius. The detection of (unexpected) marker genes from different Haemophilus species could further point towards mixed cultures or a recombination event, but our classification approach cannot rule out mixed populations of two strains from the same Haemophilus sp. or contamination with non-Haemophilus spp. strains.Based on the analysis of the training dataset, we concluded the design of the taxonomic classification database, finally comprising 36 marker genes Fig. . Next, wn=215), our new script produced classification and serotyping results in less than 15 min using 30 CPU threads (on average 1.7 min per read dataset).Starting from the simulated read datasets of the training dataset . Predicted species classifications were in agreement with the SRA reported taxonomy for 1324/1329 (99.6%) datasets. Five reported H. influenzae strains were reclassified by our classification algorithm as H. parainfluenzae (n=4) and H. haemolyticus subsp. intermedius (n=1)  were in agreement with assembly-based Hicap predictions  with multiple serotypes and capsule-deficient strains (n=11) predicted by our algorithm were not identified as such by Hicap.Capsule loci were found in 534/1055  strains , 54, corHaemophilus isolates were collected from 250 patients in three Northern German hospitals between 2008 and 2013 and were at that time classified by MALDI-TOF MS as H. influenzae  datasets were unambiguously classified by our decision algorithm as H. influenzae, two of them belonging to serotype f  contained all genes involved in the biosynthesis of haemin and were therefore classified as H. haemolyticus subsp. intermedius strains by our algorithm  H. haemolyticus strains were still misclassified as H. influenzae with current MALDI-TOF MS equipment/software. The X-factor/haemin-independent phenotype was confirmed for 24 out of 25 of the H. haemolyticus subsp. intermedius strains while the haemin-dependent phenotype was confirmed for the other 19 H. haemolyticus strains  presumptive Haemophilus spp. was found by our classification algorithm, pointing towards a mixed population or laboratory contamination  patients with mouth abscesses as no other pathogen was detected  lower respiratory tract isolates were H. influenzae, 5 H. haemolyticus, and 2 putative mixed isolates , and all tetB (n=3) positive isolates were confirmed to be phenotypically resistant to ampicillin (but not ceftriaxone) and tetracycline, respectively. One H. haemolyticus strain was predicted ampicillin resistant based on the presence of the blaZ gene, but this was not confirmed by pDST . In addition, one H. influenzae strain with a blaTEM-1D gene, which codes for a β-lactamase that is typically inhibited by sulbactam [Finally, we also determined the exogeneously acquired resistome using the same gene detection tool, combined with a public antibiotic resistance database. One or more resistance genes were found in 16 out of 262 datasets, including 9 tes Fig. . All ninins Fig. . Retrospulbactam , was pheHaemophilus spp. has proven difficult with current diagnostics. Here, we present a taxonomic marker database and WGS-based classification/serotyping algorithm for human-related Haemophilus spp., which can easily be combined with the detection of acquired resistance genes.Correct species identification and antibiotic resistance prediction are crucial for proper diagnosis and antimicrobial stewardship. However, differentiation between some Haemophilus sp. marker genes as well as genes that are involved in important metabolic pathways (haemin biosynthesis) and virulence mechanisms (capsule loci). The classification algorithm is based on the detection of these marker genes directly from short reads (fastQ files) using SRST2 and allows to differentiate between H. influenzae, H. haemolyticus, H. parahaemolyticus, H. paraphrohaemolyticus, H. parainfluenzae, H. sputorum, H. ducreyi, and H. pittmaniae. H. quentini and H. aegyptius are not recognized as separate species but classified as H. haemolyticus and H. influenzae, respectively.The newly developed lightweight taxonomic database (only 110 kilobytes) comprises 36 genes, including newly identified Haemophilus spp. strains. We considered H. influenzae strains as NTHi when none of the capsule loci were detected. When at least one capsule loci was found, but not all, we labeled the strains as “capsule-deficient.” The genetics of these mutants have mainly been investigated for H. influenzae serotype b [bexA has been identified as the cause for the capsule loss. Consequences of other deletions would need further experimental investigation. Moreover, the in silico detection of capsule genes does not guarantee that a capsule is formed as gene expression can be affected by many other factors such as the presence of specific mutations/indels, the action of regulatory proteins, or the microenvironment [Haemophilus strains, but it is not a warranty for a fully functioning haemin biosynthesis pathway.The inclusion of capsule loci in our classification database allows to predict the serotype of rotype b . In thesironment , 58. Simn=1329). Based on our results, we propose that some presumed H. influenzae strains available at SRA should be reclassified as H. parainfluenzae and H. haemolyticus, respectively. However, more sequencing data for less prevalent Haemophilus spp. such as H. para(phro)haemolyticus, H. sputorum, H. ducreyi, and H. pittmaniae are needed to further validate the robustness of our classification algorithm.Our classification method showed high concordance with the reported serotype (99.5%) and species classification (99.6%) for the evaluation dataset . In this cohort, our algorithm reclassified almost one third of the H. influenzae strains as H. haemolyticus, also pointing towards a role of H. haemolyticus as human pathogen. Few strains could not be classified by our decision algorithm due to an aberrant marker pattern. These strains remind of previously described “fuzzy” species [H. influenzae strains. The high H. haemolyticus misclassification rate could be explained by an incomplete MALDI-TOF mass spectrum database that was used at the time of diagnosis [H. haemolyticus strains in our strain dataset is in clear contrast to findings of a similar study [H. haemolyticus strains were found among clinical isolates obtained from patients in Germany. Another interesting finding was the presence of H. haemolyticus in aspirates of mouth abscesses. H. influenzae has been isolated from peritonsillar abscesses before, but H. haemolyticus has not been linked with this condition to the best of our knowledge [H. haemolyticus strains in this study were isolated from lower respiratory tract (LRT) specimens from patients with suspected or proven pneumonia could point towards another unexpected pathogenic role of H. haemolyticus in LRT infections. However, as LRT samples can also be contaminated with the bacterial flora of the upper respiratory tract [H. haemolyticus.Next to publicly available data, we also analyzed a new dataset of 262  species , 60 and iagnosis , 62. Nevar study , in whicnowledge . The facry tract  and humary tract , 66, 67,Haemophilus species are currently scarce. To determine the horizontally acquired resistome, we used the same gene detection tool (SRST2) that was used to determine the presence of our classification markers, in combination with a public resistance database [blaTEM-1D gene, and tetracycline resistance, associated with the presence of the tetB gene, could be confirmed in vitro by routine pDST. The identification of Haemophilus spp. strains with predicted resistance to multiple antibiotics (including β-lactam antibiotics) highlight the need for close monitoring of resistance evolution and spread of resistant strains in the future. The detection of resistance genes is, however, limited to rule-in antimicrobial resistance, and the absence of resistance genes in a specimen does not imply antimicrobial susceptibility. A follow-up study is envisaged which complements the gene detection approach with a mutation database and comprehensive pDSTs to determine sensitivity and specificity of a molecular antimicrobial resistance prediction for clinical Haemophilus spp. strains.WGS-based resistance prediction is increasingly being used to monitor antimicrobial resistance, which has become a major global public health threat, as well as for personalized therapy , 69. Howdatabase , 70. In Haemophilus spp. as well as for in silico serotyping and prediction of haemin independency (ii), it is very fast and can be run on a basic laptop due to the lightweight classification database and low memory requirements, (iii) it outputs easy interpretable text files with gene presence/absence results and classification tags as well as iTol annotation files for visualization of the results on phylogenetic trees, and (iv) it can easily be combined with MLST typing and the detection of acquired resistance genes. On the other hand, the algorithm cannot detect species belonging to genera other than Haemophilus and only works well with short read data.Overall, our WGS-based classification algorithm offers several advantages compared to other identification tools/techniques: (i) it can be used for species classification of human-related Haemophilus spp. in clinical microbiology.At this stage, we believe that WGS-based classification and especially resistance prediction should be regarded as a tool that complements other routine diagnostic methods. An additional algorithm that detects chromosomal resistance-related mutations as well as more phenotype-genotype studies and larger sample sizes will be necessary to further evaluate the use of WGS as a diagnostic method for Haemophilus spp. direct from raw short read WGS data. In addition, the algorithm can also determine the serotype of typeable Haemophilus strains and identify putative haemin-independent H. haemolyticus strains. Together with drug resistance prediction, this opens the door for improved diagnosis, surveillance, and treatment of Haemophilus spp. infections.We developed a lightweight gene database and fast assembly-free classification algorithm that is customized to identify clinically relevant Additional file 1: Table S1. Summary of the number of short read datasets used for each species in the training set, evaluation set, and German cohort.Additional file 2: Table S2. Metadata and classification results for all samples from the training and evaluation set.Additional file 3: Table S3. Metadata and classification results for all samples from German cohort.Additional file 4: Table S4. Summary of all genes that were evaluated as taxonomic markers.Additional file 5: Table S5. Summary of all H. influenzae and H. haemolyticus marker candidates that were identified using a panGWAS analysis.Additional file 6: Figure S1. Genomic arrangement of selected marker genes.Additional file 7: Figure S2. Phylogenetic tree comprising training set strains and misclassified strains from SRA.Additional file 8: Table S6. SRST2 output for short read datasets from the evaluation set using the newly developed classification database.Additional file 9: Table S7. SRST2 output for Illumina sequencing datasets from the German cohort using a public MLST database.Additional file 10: Table S8. phenotypic drug susceptibility testing (pDST) results for isolates from the German cohort.