Staphylococcus coagulans is among the three most frequent pathogens of canine pyoderma. Yet, studies on this species are scarce. Twenty-seven S. coagulans and one S. schleiferi, corresponding to all pyoderma-related isolations from these two species at two veterinary laboratories in Lisbon, Portugal, between 1999 and 2018 (Lab 1) or 2018 (Lab 2), were analyzed. Isolates were identified by the analysis of the nuc gene and urease production. Antibiotic susceptibility towards 27 antibiotics was evaluated by disk diffusion. Fourteen antibiotic resistance genes were screened by PCR. Isolates were typed by SmaI-PFGE. Two S. coagulans isolates  were methicillin-resistant  and four  displayed a multidrug-resistant (MDR) phenotype. We observed resistance to penicillin , fluoroquinolones , erythromycin and clindamycin , fusidic acid  and tetracycline . The blaZ and erm(B) genes were carried by 16 and 1 isolates resistant to penicillin and erythromycin/clindamycin, respectively. Only three S. coagulans carried plasmids. The single S. schleiferi isolate presented an MDR phenotype. SmaI-PFGE revealed a limited genetic diversity of S. coagulans, with a predominant lineage present from 2001 to 2018. This study describes the first MRSC causing canine infection in Portugal and reveals a high burden of antimicrobial resistance, with the emergence of MDR phenotypes within the main lineages. Staphylococcus pseudintermedius accounts for up to 90% of pyoderma-related staphylococcal infections, followed by Staphylococcus coagulans (previously known as Staphylococcus schleiferi subsp. coagulans) and Staphylococcus aureus , the remaining two resistant isolates could carry other determinants, such as additional erm genes like erm(43) or lnu(B), which are not usually linked to plasmids [S. coagulans and S. schleiferi collected from 2016 onwards, highlighting the recent emergence of resistance to this antibiotic. However, none carried the two resistance determinants screened, fusB/fusC, which are usually associated with plasmids or other mobile genetic elements. Resistance to this antibiotic could be linked to mutations occurring in the fusA gene, which are common in other staphylococcal species [tet genes screened was found in the single tetracycline-resistant S. coagulans detected. However, this isolate could carry the tet(O) determinant, as observed in the study by Chanchaithong and colleagues [In the last decades, a decrease has been observed in the susceptibility to penicillin, erythromycin, clindamycin and to a lesser extent gentamicin or other aminoglycosides and tetracycline in cumented ,41,42,43plasmids . Resista species . RegardiS. coagulans in Portugal in the last two decades, resistance phenotypes are still mainly restricted to a few antibiotic classes, namely beta-lactams, fluoroquinolones, macrolide-lincosamides and fusidic acid. Reports from other countries such as Thailand [S. coagulans. The low diversity of resistance genes encountered in our collection could be associated with the low abundance of plasmids.Despite the increase registered in antimicrobial resistance in Thailand  showed aS. coagulans is rather limited to a few studies with a limited number of isolates. These have used SmaI-PFGE to evaluate the clonality of this species, indicating a high clonality within the species and that S. coagulans and S. schleiferi often present indistinguishable PFGE patterns [S. coagulans collected in Portugal (Lisbon area) throughout the last two decades present a low genetic diversity , with a predominant lineage encompassing over half of the isolates collected through almost the entire time span of the collection. Nevertheless, some heterogeneity was found within this predominant lineage, with isolates varying in resistance phenotypes and resistance determinants.Our knowledge on the molecular epidemiology of patterns ,29,33,43WT values for six antibiotics  with no breakpoints available for S. coagulans. These antibiotics are not used or rarely recommended (mupirocin) for the treatment of skin infections in dogs [S. coagulans.We also analyzed the ZD distributions and corresponding CO in dogs ,2. Never in dogs . Thus, tS. coagulans may reflect the usage of antibiotics recommended for the treatment of canine pyoderma.The results gathered in this study are particularly worrisome when analyzed in conjunction with data on the classes of antibiotics most prescribed in small animal practice. Data from the European Medicines Agency (EMA) and a report by Oliveira and colleagues highlight that beta-lactams, fluoroquinolones, macrolides and tetracyclines represent the majority of antibiotics prescribed for treatment of skin infections in small animals ,58. Althn = 89) included all isolates previously identified as S. schleiferi or Staphylococcus spp. (non-S. pseudintermedius and non-S. aureus) causing skin infections in pets collected over 19 years (1999–2018) at a veterinary research laboratory providing diagnostic services for a veterinary teaching hospital and private veterinary clinics in the Lisbon area  and at a private veterinary diagnostic clinic during 2018   and included in the study as controls. All isolates were grown in tryptone soya broth or agar  at 37 °C, at 180 rpm (for broth cultures). Bacterial stocks were kept at −80 °C in TSB supplemented with 10% (v/v) glycerol.The initial collection studied  . This cous study . The typHindIII (Thermo Scientific™).Total DNA was isolated by the boiling method  and usedS. coagulans or S. schleiferi was confirmed by a species-specific nuc PCR approach described by Sasaki et al. [S. pseudintermedius DSM 21284T (DSMZ GmbH), S. aureus ATCC® 259323™ and S. epidermidis ATCC® 12228™  were included as negative controls. The differentiation between S. coagulans and S. schleiferi was carried out, for each isolate, by detection of urease activity after growth in Christensen urea agar at 35 °C for up to 48 h [S. schleiferi DSM 4807T and Escherichia coli ATCC® 2922™, and the urease-positive strains S. coagulans DSM 6628T and Klebsiella pneumoniae ATCC® BAA 1706™ were included as controls.The identification of the isolates as i et al. , after d to 48 h . ConversS. coagulans. Antibiotic disks were acquired from Thermo Scientific™ Oxoid™ or MAST Group (pradofloxacin) . Susceptibility profiles were interpreted according to CLSI VET01S-ED5 recommendations for enrofloxacin (5 μg), clindamycin (2 μg) and tetracycline (30 μg) [S. aureus ATCC®25923™ and S. aureus ATCC®29213™ and the type strains S. coagulans DSM 6628T and S. schleiferi DSM 4807T were used as quality controls. Multidrug resistance (MDR) was considered as not-susceptibility to, at least, one agent of, at least, three classes of antimicrobials [Antibiotic susceptibility was determined by agar disk diffusion on Mueller-Hinton agar plates (Thermo Scientific™ Oxoid™) according to CLSI recommendations for bacteria isolated from animals  or EUCAS (30 μg)  or accor (30 μg) ; or acco (30 μg) . Other a (30 μg) . The refcrobials .WT values were estimated using the Normalized Resistance Interpretation (NRI) method [WT corresponds to the smallest inhibition zone diameter presented by the putative WT population and is calculated at 2.5X the SD above the mean value and rounded up to the lowest absolute value [WT allows the distinction between putative WT populations  and non-wild-type populations  [WT estimated by the NRI method will include 99.4% of the WT population [http://www.bioscand.se/nri/ (accessed on 2 December 2020).The ZD-based CO) method ,61. Thiste value ,61. Thushanisms) . The COWblaZ and mecA was screened by PCR for all isolates, whereas the genes erm(A), erm(B), erm(C), msrA, mph(C), vga(A) and vga(C) for resistance to macrolides and/or lincosamides, genes tet(K), tet(L), tet(M) for resistance to tetracyclines and genes fusB and fusC for resistance to fusidic acid were only screened by PCR for isolates a resistant or intermediate phenotype. The presence of mutations associated with fluroquinolone resistance was screened by sequencing of the quinolone-resistance determining region (QRDR) of the genes grlA and gyrA. Amplification products were purified using the kit NZYGelpure  and sequenced. Sequences were analyzed using the programs SnapGene Viewer , translated to polypeptide sequnces, aligned in MEGA v. 7.0.26 and compared against the GrlA and GyrA sequences of the type strain S. coagulans DSM6628T (GenBank assembly accession no. GCA_002901995.1). All primers used for the detection of resistance genes or mutations are listed in Carriage of the beta-lactam resistance genes SmaI-PFGE typing. Briefly, agarose disks containing genomic DNA were prepared and restricted with SmaI, as previously described for S. aureus [S. aureus NCTC8325  and the PFG DNA Ladder (New England Biolabs). Macrorestriction profiles were analyzed with BioNumerics software version 7.6  using the unweighted pair group method using arithmetic averages and the Dice similarities coefficient. Band position tolerance and optimization were set at 1% and 0.5%, respectively. Isolates with a similarity coefficient ≥ 81% were considered as belonging to the same PFGE type, whereas isolates with a similarity coefficient ≥ 97% were considered as belonging to the same PFGE sub-type [All isolates in study were subjected to . aureus . Restric. aureus . In eachsub-type . The gensub-type .S. coagulans associated with canine pyoderma highlights a high burden of antimicrobial resistance in S. coagulans in Portugal during the last two decades, including the emergence of MRSC. This work also reveals a low genetic diversity and a low content of plasmids in the circulating S. coagulans. Yet, new lineages with relevant resistance phenotypes were identified. This scenario may have a high impact on the future management of canine pyoderma caused by S. coagulans.This study on


Camelus dromedarius) are widely distributed in Africa, the Middle East and northern India. In this study, we aimed to detect tick-borne pathogens through investigating prokaryotic and eukaryotic microorganisms in camel blood based on a metagenomic approach and then to characterize potentially pathogenic organisms using traditional molecular techniques. We showed that the bacteria circulating in the blood of camels is dominated by Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria. At the genus level, Sediminibacterium, Hydrotalea, Bradyrhizobium and Anaplasma were the most abundant taxa. Eukaryotic profile was dominated by Fungi, Charophyta and Apicomplexa. At the genus level, Theileria was detected in 10 out of 18 samples, while Sarcocystis, Hoplorhynchus and Stylocephalus were detected in one sample each. Our metagenomic approach was successful in the detection of several pathogens or potential pathogens including Anaplasma sp., Theileria ovis, Th. separata, Th. annulate, Th. mutans-like and uncharacterized Theileria sp. For further characterization, we provided the partial sequences of citrate synthase (gltA) and heat-shock protein (groEL) genes of Candidatus Anaplasma camelii. We also detected Trypanosoma evansi type A using polymerase chain reaction (PCR) targeting the internal transcribed spacer 1 (ITS1) region. This combined metagenomic and traditional approach will contribute to a better understanding of the epidemiology of pathogens including tick-borne bacteria and protozoa in animals.Dromedary camels ( Camelus dromedarius), the Bactrian camel (Camelus bactrianus) and the wild Bactrian camel (Camelus ferus) [Hyalomma anatolicum, Hy. excavatum, Hy. scupense, Hy. dromedarii, Hy. impeltatum, Hy. marginatum, Hy. rufipes, Hy. truncatum, Ornithodoros savignyi, Rhipicephalus praetextatus and Rh. turanicus [Rickettsia, Anaplasma, Ehrlichia, Coxiella, Babesia, Hepatozoon and Theileria [Camels are divided into three species, the Arabian or dromedary camel (s ferus) ,2. Arabis ferus) . There auranicus . Such tiheileria ,6,7,8,9,heileria .The metagenomic approach utilizing high-throughput sequencing techniques has contributed largely to the description of pathogenic and nonpathogenic microbes in many animal species. It is not limited to the simultaneous detection of known pathogens but also detection of novel potential pathogens . The mosThe study of blood parasites in mammals by conventional PCR or quantitative PCR can only detect genetically identified species . HoweverThe present study aims to describe both the prokaryotic and eukaryotic profiles in the blood of dromedary camels in Egypt. The results were further verified by conventional genetic assays amplifying genus or species-specific genes. Our data indicate the usefulness of metagenomic approach for the detection of pathogens, but at the same time, suggest the necessity to refine current methods to capture a full picture of the blood microbiome in camels. Pseudomonas, Escherichia-Shigella, Bacillus, Serratia, Novosphingobium, Curvibacter, Halomonas, Candidatus Lariskella and Coxiella (Sediminibacterium (mean = 13.8%), Hydrotalea (mean = 8.6%), Bradyrhizobium (mean = 7.9%), Anaplasma (mean = 6.4%) and Ralstonia (mean = 3.4%) were the highest abundant . The possible contaminants in the negative control composed of two main phyla, the Proteobacteria and Firmicutes, which contain nine genera: Coxiella . After rabundant . The gen samples . Other kTheileria, which was detected in 10 samples  of the camels were positive by PCR targeting 16S rDNA of Anaplasmataceae (EHR-PCR). Partial gene sequences of the 16S rDNA from the 29 positive individuals were 99.7% (two samples) and 100% (27 samples) identical to Candidatus Anaplasma camelii (MT510533) from a dromedary camel in Laisamis, Kenya. The maximum likelihood (ML) phylogenetic tree based on the 16S rDNA sequences showed that Anaplasma sp. detected from this study clustered with the previously published Candidatus A. camelii and Anaplasma platys .The partial sequences of the citrate synthase (gltA) gene obtained from 11 samples were 78.7% similar to A. platys (MH716422) from a tick in Shaanxi Province, China and and 77.4% to A. platys strain S3 (CP046391) from a dog in Saint Kitts and Nevis. The ML phylogenetic tree based on the gltA gene sequences showed the partial sequences from Anaplasma-positive samples formed an independent cluster separated from the other Anaplasma spp. . The partial sequences of the heat-shock protein (groEL) gene obtained from 11 Anaplasma-positive samples showed 99–100% identity to each other. These sequences showed 88.3% and 86.3% identity to A. platys (MH716435) found from a Rhipicephalus microplus tick from China and (CP046391) and a dog in Saint Kitts and Nevis, respectively. The obtained sequences were all clustered together in a separate clade from other Anaplasma spp. in the ML phylogenetic analysis .PCR targeting 18S rDNA of piroplasma species (RLB-PCR) yielded amplicons in 39% (39/100) of the camels. However, the sequencing analysis revealed that the amplified RLB-PCR products did not belong to piroplasma species and were classified as Cercomonas species.PCR targeting internal transcribed spacer 1 (ITS1) region of Trypanosoma species (ITS1-PCR) was positive for 19% (19/100) of the camels. All ITS1-PCR positive samples were also positive by PCR using the primers ILO7957 and ILO8091 (ILO-PCR) targeting the RoTat1.2 VSG region but negative by EVAB-PCR targeting a Tr. evansi type B-specific minicircle sequence, indicating that all Trypanosoma-positive samples were Tr. evansi type A.Theileria in our samples. We implemented a phylogenetic analysis of the genus Theileria using the obtained 18S rDNA sequences from NGS. Breifly, features belonging to the genus Theileria were exported and searched in BLAST to identify the species. The assigned taxonomic identities of these features by silva-132-99-nb classifier in QIIME2 were represented as follows: Th. ovis (n = 6), Th. separata (n = 2), Theileria sp. strain MSD (n = 2), Th. annulate (n = 1) and Theileria sp. (n = 1). Phylogenetic analysis showed that the detected Theileria spp. formed four clades clustered separately with Th. ovis, Th. separata, Th. annulate and Th. mutans. In addition, one Theileria sequence (LC592660) did not form a clade with Theileria sequences available in GenBank  ,26. ThisTheileria spp. using this NGS approach for the first time in camels. Moreover, it facilitated the detection of coinfections between several Theileria species . The number of Theileria reads obtained for these samples was 65 and 314, suggesting that they were not false positives. More investigations are required to explore the prevalence of the newly detected Theileria species in camels and ticks.Despite the detection of several eukaryotes using our NGS-based approach, we could not succeed in the complete elimination of camel-derived sequences. This caused the dominance of Metazoa (mean abundance of 72.9% before removing contaminants) in our samples, suggesting that increasing the depth of sequencing can obtain more eukaryotic members if they exist . However species . The resin Sudan ,32. Othe lepidum . These t lepidum ,34,35, sn camels  as well  in 2014 . Interesanalysis . AlthougEthiopia . In thisTh. annulata, Th. equi and Th. ovis in camel blood using conventional PCRs [Theileria-positive by the NGS-based approach. One possibility is the presence of other eukaryotes that can be amplified by the same primer set. In fact, most of the sequences from RLB-PCR products were classified to the genus Cercomonas by Sanger sequencing method. To overcome this cross-reactivity issue, RLB-PCR products can be subjected to NGS. This strategy was applied to detect several Theileria spp. in African buffalo from South Africa [Although previous studies reported the detection of nal PCRs ,38,39, oh Africa  and piroh Africa .Tr. evansi type A was one of the most common parasites detected in camel blood in our study. Our NGS-based approach was not successful in detecting Trypanosoma spp. in the examined samples that were positive by conventional PCR. This is possibly due to a sequence mismatch between UNonMet-PCR primers and Tr. evansi 18S rDNA. We found that the forward primer (18S-EUK581-F) has a single nucleotide mismatch with the 18S rDNA sequences of Tr. evansi in GenBank. In addition, the failure of detecting Tr. evansi by NGS might be due to low parasitic load in the samples. Moreover, the high abundance of host DNA fragments can also contribute to hindering the amplification of Tr. evansi. Therefore, further investigations and experimental studies are required to improve this approach.Tr. evansi infections detected in this study (19%) differed from what was previously published in Egypt [The prevalence of in Egypt ,42,43, wCandidatus A. camelii in camels from Saudi Arabia [Candidatus A. camelii. In fact, this recently described bacterium has also been reported from cattle (Bos taurus) and deer (Rusa timorensis) in Malaysia [Anaplasma were detected in camel blood in Egypt, however, species identification was not attempted [Candidatus A. camelii and A. platys makes it difficult to describe the evolutionary relationship and host range of both species. This study provided partial sequences of gltA and groEL genes for the first time from Candidatus A. camelii. The sequences obtained were 78.7% and 88.3% identical to the gltA and groEL partial gene sequences of A. platys, respectively, suggesting that both genes are useful markers to differentiate between the two closely related Anaplasma species. Although the prevalence of Candidatus A. camelii was relatively high, this study could not confirm whether this bacterium can pose a potential veterinary or zoonotic risk.There are several studies reporting i Arabia , Kenya [i Arabia , Nigeriai Arabia , Moroccoi Arabia  and Irani Arabia . Our stuMalaysia , suggestttempted . The higMetagenomics of the host-associated eukaryotic populations has been challenging primarily because most universal primers amplify the 18S rDNA of host animals rather than those of animal-associated eukaryotes . In the Tr. evansi type A, Candidatus A. camelii, Th. ovis, Th. separata, Th. annulata, Th. mutans and undescribed Theileria species. In addition, we provided gltA and groEL partial gene sequences of Candidatus A. camelii that can be used to differentiate this pathogen from A. platys. Our study results support the hybrid approach to characterize the microbe of interest in mammals using NGS and conventional PCR combined with Sanger sequencing as was found in humans [This study implemented a metagenomic investigation targeting the blood circulating prokaryotes and eukaryotes in camels, which could be used in other species of mammals. We revealed that the camels imported from Sudan were infected with a total of seven pathogens or potential pathogens, including n humans . This wiBlood samples were collected from the jugular vein of 100 camels from Abu Simbel near the Egypt–Sudan border. All camels were adult males imported from Sudan and kept in quarantine during the sampling process. The blood samples were collected on Na-EDTA tubes and DNA was extracted using innuPREP Blood DNA Mini Kit  following the manufacturer’s recommendations and stored at −20 °C until analyzed.A total of 18 camel blood samples were used for investigation by a metagenomic approach. Since all camels showed no clinical signs, we selected these samples randomly. We performed PCR targeting the V3-V4 region of the 16S rDNA of prokaryotes using Illumina barcoded primers  Illumina_16S_341F and Illumina_16S_805R as recommended by Illumina .The V4 region of the 18S rDNA of eukaryotes was amplified using the following nested PCR assays. The 1st PCR (UNonMet-PCR) reaction was performed to decrease the amplification of host DNA fragments in the blood samples using the primers 18S-EUK581-F and 18S-EUK1134-R . The resThe resulting fastq files were analyzed in QIIME2 (version 2019.10.0) . The forTrypanosoma-positive samples were further examined for Tr. evansi by ILO-PCR using the primers ILO7957 and ILO8091 [Anaplasma-positive samples were further characterized by amplifying a 650 bp and 1100 bp segments of gltA and groEL genes, respectively, as previously described [A total of 100 camel blood samples were tested by conventional PCRs targeting 18S rDNA (RLB-PCR), ITS1 region and 16S rDNA (EHR-PCR) of apicomplexan protozoa, trypanosomes and Anaplasmataceae, respectively.  ILO8091 . Anaplasescribed .PCR reactions consisted of a 25 μL mixture of 12.5 μL of 2× Gflex PCR Buffer, 0.5 μL of Tks Gflex DNA Polymerase (1.25 units/μL) , 0.5 μL of each primer, 1.0 μL of template DNA and molecular grade water . The PCRgltA-PCR and groEL-PCR were purified by ExoSAP-IT PCR Product Cleanup Reagent  and sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and an ABI Prism 3130xl genetic analyzer (Applied Biosystems).The positive PCR products that were obtained from RLB-PCR, EHR-PCR, Theileria were exported from QIIME2 (version 2019.10.0) [https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch) (accessed on 12 December 2020). The sequences were aligned using MAFFT v7.450 software [The obtained 18S rDNA features belonging to genus 19.10.0)  to Geneisoftware  and the software  and phylsoftware .gltA and LC592678-LC592688 for groEL.The sequences obtained in this study were submitted to the DDBJ under the accession numbers: LC592651-LC592666 for 18S rDNA, LC592622-LC592650 for 16S rDNA, LC592667-LC592677 for


Lactiplantibacillus plantarum is a species found in a wide range of foods and other commodities. It can be used as starter or adjunct culture in fermented foods. Herein the annotated high-quality draft genome (scaffolds) of six L. plantarum subsp. argentoratensis strains  isolated from various Greek wheat sourdoughs is presented. Raw sequence reads were quality checked, assembled into larger contiguous sequences and scaffolds were annotated. The total size of the genomes ranged from 3.13 Mb to 3.49 Mb and the GC content from 45.02% to 45.13%. The total number of coding and non-coding genes were between 3268 and 3723 . The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession numbers JAEQMR000000000, JAEQMQ000000000, JAEQMP000000000, JAEQMO000000000, JAEQMN000000000 and JAEQMM000000000. The version described in this paper is version JAEQMR010000000, JAEQMQ010000000, JAEQMP010000000, JAEQMO010000000, JAEQMN010000000 and JAEQMM010000000. Raw sequence reads have been submitted in the Sequence Read Archive (SRA) under the BioProject accession number PRJNA689714 . Therefore, analysis of the genome of the L. plantarum subsp. argentoratensis strains will provide insights regarding their genomic and functional features and their potential use as a starter and/or adjunct culture•Data could be of interest for third parties dealing with sourdough fermentations and/or other fermented foods, as well as with lactic acid bacteria as starters•Data available to scientific community for applying other bioinformatics approaches such as comparative genomics to investigate the genome evolution of this species and other technological characteristics•L. plantarum subsp. argentoratensis strain by providing high-quality whole-genome sequencesContributing to the limited number of available genomes of the 1L. plantarum subsp. argentoratensis strains, isolated from Greek wheat sourdoughs Lactobacillus (ca. 15%)  . Finallyca. 15%) ; Fig. 2.2L. plantarum subsp. argentoratensis strains were cultured in de Mann Rogosa and Sharpe (MRS) broth  and incubated overnight at 30°C. DNA was extracted from the microorganisms according to Syrokou et al. http://www.genomicepidemiology.org/). Contigs were organized into scaffolds using the Multi-Draft based Scaffolder (MeDuSa) v1.6 web server L. plantarum subsp. argentoratensis DSM 16365  and L. plantarum WCFS1  used as reference genomes. A re-implementation of the algorithm of CheckM tool https://www.ncbi.nlm.nih.gov/).Maria K. Syrokou: Conceptualization, Investigation, Writing – review & editing; Spiros Paramithiotis: Conceptualization, Investigation, Supervision, Writing – review & editing; Panagiotis N. Skandamis: Supervision, Writing – review & editing; Eleftherios H. Drosinos: Supervision, Writing – review & editing; Loulouda Bosnea: Supervision, Writing – review & editing; Marios Mataragas: Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Software, Supervision, Writing – original draft.The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article.


Pseudomonas aeruginosa infections. The mechanisms behind these new agents also need to be revealed at a molecular level. Materials and methods: The anti-biofilm effects of 10 plant-derived compounds on P. aeruginosa biofilms were investigated using minimum biofilm eradication concentration (MBEC) and virulence assays. The effects of ciprofloxacin and compound combinations on P. aeruginosa in mono and triple biofilms were compared. A metabolomic approach and qRT-PCR were applied to the biofilms treated with ciprofloxacin in combination with baicalein, esculin hydrate, curcumin, and cinnamaldehyde at sub-minimal biofilm inhibitory concentration (MBIC) concentrations to highlight the specific metabolic shifts between the biofilms and to determine the quorum sensing gene expressions, respectively. Results: The combinations of ciprofloxacin with curcumin, baicalein, esculetin, and cinnamaldehyde showed more reduced MBICs than ciprofloxacin alone. The quorum sensing genes were downregulated in the presence of curcumin and cinnamaldehyde, while upregulated in the presence of baicalein and esculin hydrate rather than for ciprofloxacin alone. The combinations exhibited different killing effects on P. aeruginosa in mono and triple biofilms without affecting its virulence. The findings of the decreased metabolite levels related to pyrimidine and lipopolysaccharide synthesis and to down-regulated alginate and lasI expressions strongly indicate the role of multifactorial mechanisms for curcumin-mediated P. aeruginosa growth inhibition. Conclusions: The use of curcumin, baicalein, esculetin, and cinnamaldehyde with ciprofloxacin will help fight against P. aeruginosa biofilms. To the best of our knowledge, this is the first study of its kind to define the effect of plant-based compounds as possible anti-biofilm agents with low MBICs for the treatment of P. aeruginosa biofilms through metabolomic pathways.Introduction: Alternative anti-biofilm agents are needed to combat  Pseudomonas aeruginosa, which is difficult to treat because of its resistance to many antibiotics and its ability to form a biofilm, is a significant pathogen that causes hospital-acquired infections [P. aeruginosa is frequently isolated together with Staphylococcus aureus and Enterococcus faecalis from the specimens of patients with biofilm-related infections [P. aeruginosa biofilm formation or display a microbicidal activity on the cells in preformed biofilms [P. aeruginosa biofilms, QS inhibitors are recommended as alternative anti-biofilm agents to antibiotics [P. aeruginosa biofilms has been studied. Although the inhibitory effects of some natural compounds on the P. aeruginosa QS system are known, their effects on P. aeruginosa cells in polymicrobial biofilm media are still under investigation [Biofilm-related infections have major importance because of their clinical challenges, such as persistence and treatment approach. fections . P. aerufections . There ibiofilms . Quorum biofilms . Becauseibiotics . QS inhiibiotics . Becausetigation ,8. InvesP. aeruginosa biofilm cells. The effect of sub-MBIC concentrations of curcumin, baicalein, cinnamaldehyde, and esculin hydrate, which show the best synergistic activity with ciprofloxacin, on the QS genes and virulence characteristics of P. aeruginosa was investigated. To observe the antimicrobial effects of these compounds and the ciprofloxacin combinations on polymicrobial biofilms at sub-MBIC concentrations, P. aeruginosa cells in mono- and triple-species biofilms were separately treated with them and the results were compared. A GC-MS based metabolomic approach was used to reveal the underlying mechanisms of the anti-biofilm and anti-QS activity of curcumin, baicalein, cinnamaldehyde, and esculin hydrate.The present study determined the minimal biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) of ciprofloxacin in combination with plant-derived compounds such as curcumin, azathioprine, resveratrol, catechin hydrate, baicalin hydrate, L-canavanine, 4-nitropyridine N-oxide, p-benzoquinone, esculin hydrate, and cinnamaldehyde against P. aeruginosa ATCC 47085 (PAO1), E. faecalis ATCC 47077, and S. aureus ATCC 35556 were used in the study. Fresh cultures were grown on brain heart infusion agar  at 37 °C for 24 h. The overnight cultures were prepared by subculturing bacterial strains in brain heart infusion broth  and incubated at 37 °C for 24 h. For all of the experiments, the final bacterial suspensions were diluted with BHI to 106 colony forming units per mL (cfu/mL).Curcumin, azathioprine, resveratrol, catechin hydrate, baicalin hydrate, L-canavanine, 4-nitropyridine N-oxide, p-benzoquinone, esculin hydrate, cinnamaldehyde, and ciprofloxacin were purchased from Sigma Chemical Co. . Stock solutions of ciprofloxacin and the compounds were prepared in dimethyl sulfoxide (pure DMSO), sterile water, or hydrochloric acid according to the manufacturer’s recommendations.P. aeruginosa ATCC 27853 was used as quality control for this test.The MICs of ciprofloxacin and the compounds were determined using the broth microdilution method according to Clinical Laboratory Standards Institute (CLSI) guidelines . P. aeruTM  was used to evaluate the anti-biofilm activity of the agents against P. aeruginosa PAO1 biofilms following the test protocol provided by the manufacturer. Briefly, 150 μL of P. aeruginosa suspension containing 106 cfu/mL was transferred to each test well and the MBEC assay plate lids with 96 pegs were placed in the microtiter plate wells to form 24 h mature biofilms at 37 °C. After washing, the peg lids were placed into the challenge plate wells, which contained a serial two-fold dilution of each agent alone and their combinations for 24 h of incubation. In columns 1 to 11, the tested concentration ranges of ciprofloxacin and the compounds were 1–0.001 mg/L and 8.2–0.008 mg/L, respectively. After the MBIC of ciprofloxacin was determined to be 0.128 mg/L, the test concentration ranges for use in combination with the compounds were determined as 0.128–0.00128 mg/L between columns 1 to 11. Following incubation, the sonication steps were performed to transfer the biofilms on the pegs to the recovery medium, followed by overnight incubation. The optical density of each well was measured at 550 nm. MBEC is defined as the lowest concentration of a compound that prevents the regrowth of bacteria from treated-biofilms and MBIC is the minimum concentration that inhibits bio-film formation. Sub-MBIC values corresponding to 1/2× MBIC of combinations of ciprofloxacin with compounds were used in the subsequent biofilm experiments, including the virulence, gene expressions, and metabolomic analyzes.MBEC AssayP. aeruginosa ATCC 47085 (PAO1) biofilms and triple-species biofilms formed by P. aeruginosa PAO1, E. faecalis ATCC 47077, and S. aureus ATCC 35556 were formed in 96-well microtiter plates as described by Kart et al. [E. faecalis and S. aureus in equal volume with P. aeruginosa was used.t et al. . For triP. aeruginosa in the mono- and triple-species biofilms, the plating was performed on BHIA and Cetrimide agar, respectively. After 24 h, the colonies were counted as cfu/mL. All of the experiments were carried out at least three times.Ciprofloxacin alone and the combination of ciprofloxacin with compounds at subMBIC (1/2× MBIC) concentrations were applied on the mature mono- and triple-species biofilms. For the quantification of P. aeruginosa, S. aureus, and E. faecalis strains containing ~106 CFU/mL in BHI were added alone (for P. aeruginosa) and in combination  onto the coverslips and were incubated at 37 °C for 24 h. Following the incubation time, the coverslips were washed twice with phosphate-buffered saline and fixed in a buffer containing 2% glutaraldehyde and 0.1 M cacodylate for 30 min, followed by rinsing three times for 10 min in a 0.2 M cacodylate buffer. After passing them through serial ethanol solutions, the samples were dried and then coated with gold-palladium [SEM analyses were performed to visualize mono- and triple-species biofilms . Brieflyalladium .P. aeruginosa biofilm cultures and incubated for 16 h at 37 °C. The zone diameter on the surfaces was measured to assess the swarming activity.Ciprofloxacin , compounds  and ciprofloxacin-compound combinations (1/2× MBIC) were added to nutrient agar (8 g/L) supplemented with 5% glucose. The plates were then inoculated with 2 μL of diluted P. aeruginosa were stabbed from the agar surface to the bottom of the plate with a sterile needle loop. The plates were then incubated at 37 °C for 48 h. After the agar was removed, the polystyrene surface of the plates was gently washed and stained with crystal violet . The twitching motility was assessed by measuring the diameter of the stained zone, indicating that bacteria were attached to the polystyrene surface.Luria–Bertani agar plates were prepared to contain ciprofloxacin , compounds , and ciprofloxacin–compound combinations (1/2× MBIC). The biofilm cultures of P. aeruginosa biofilm cultures was extracted with 1 mL of chloroform and then re-extracted into 1 mL of 0.2 N HCl to give a pink solution. The pyocyanin absorbance of this acidic solution was measured at 520 nm [Briefly, 1 mL of the treated (1/2× MBIC) and untreated t 520 nm . The resThe total RNAs in biofilm cells treated with sub-MBICs (0.0005 and 0.001 mg/L) of ciprofloxacin alone and ciprofloxacin (0.0005 and 0.001 mg/L) in combination with curcumin (0.004 mg/L), baicalein (0.004 mg/L), cinnamaldehyde (0.008 mg/L), and esculetin hydrate (0.008 mg/L) were obtained by RNA Isolation Kit (Roche Life Science) according to the manufacturer’s instructions. The measurements of the mRNA levels of the QS-related genes were carried out based on our previous reference study . The priMetabolites were extracted from the biofilm cells treated with ciprofloxacin (0.0005 and 0.001 mg/L) alone and ciprofloxacin (0.0005 and 0.001 mg/L) in combination with curcumin (0.004 mg/L), baicalein (0.004 mg/L), cinnamaldehyde (0.008 mg/L), and esculetin hydrate (0.008 mg/L). GC–MS-based measurement of the metabolites was carried out based on our previous reference study  Table S.p-value of <0.05 was considered significant.The colony counts were recorded for each treatment as Log10. All statistical calculations were performed on the log density values. The statistical significance of the data was determined by Student’s t-test for paired samples or by the non-parametric Wilcoxon signed-rank test. A P. aeruginosa are shown in P. aeruginosa biofilm cells were determined by the MBEC test. The compounds alone (8.2–0.004 mg/L) did not affect destroying cells in either biofilm. However, in the presence of the compounds, it was observed that the MBICs of ciprofloxacin were much lower compared with the MBIC of ciprofloxacin alone  and UMP metabolites  involved in pyrimidine synthesis were significantly reduced in cells treated with curcumin and cinnamaldehyde combinations compared with ciprofloxacin alone. Pyrimidine synthesis is a complex pathway that begins with the transformation of L-glutamate to uridine monophosphate (UMP) for RNA and DNA production [P. aeruginosa [Although it has been reported in the literature that some combinations tested in our study provide an antibiofilm effect, there are very few studies explaining these mechanisms . In the oduction ,28. Pyriruginosa . In phenruginosa .P. aeruginosa. In contrast to curcumin and cinnamaldehyde, the uracil and UMP metabolite levels and the upregulation of all genes increased in biofilm cells treated with combinations of ciprofloxacin with baicalein and esculin hydrate. These results show that one of the multifactorial mechanisms that can cause QS inhibition by curcumin and cinnamaldehyde is the uracil and UMP mediated pyrimidine mechanism.The down-regulated QS gene results obtained for curcumin and cinnamaldehyde, which provide a synergistic effect to ciprofloxacin in killing biofilm cells, and decreased uracil and UMP levels, support studies expressing the link between pyrimidine synthesis and QS inhibition in P. aeruginosa virulence by helping recover from the effects of antipseudomonal drugs and the host immune system [P. aeruginosa at the level of transcriptional regulation mediated by the PalgD activity [Alginate is a capsular polysaccharide that has a significant role in e system . Ahmar eactivity . SimilarP. aeruginosa [Lipopolysaccharide (LPS) is a complex glycolipid composed of lipid A, core o-saccharide and hypervariable long-chain o-polysaccharide and plays important roles in the integrity of the outer membrane permeability barrier of ruginosa . PhosphoP. aeruginosa [P. aeruginosa biofilm inhibition.Biogenic amines (BAs) regulate chromosomal and ribosomal organization; DNA, RNA, and protein synthesis in bacteria; and protect bacteria from osmotic stress. They are also known to regulate the swarming action and biofilm formation of ruginosa ,34. UnliP. aeruginosa biofilm inhibition. Curcumin, cinnamaldehyde, baicalein, and esculin reduced the inhibitory concentration of ciprofloxacin against biofilm cells by up to 64-fold. Using these compounds together with ciprofloxacin will help achieve successful results in the fight against ciprofloxacin-resistant P. aeruginosa biofilm infections.In conclusion, our multifactorial results, such as uracil and UMP-mediated pyrimidine synthesis, LPS biosynthesis, alginate production, and QS systems, indicate the reasons for curcumin and cinnamaldehyde-mediated


Helicobacter pylori (H. pylori) eradication treatment, antibiotic resistance to clarithromycin has increased considerably, resulting in eradication rates of less than 80%. We investigated the efficacy of tailored therapy after a clarithromycin resistance test compared with empirical therapy for H. pylori eradication. The cost-effectiveness of H. pylori eradication success was evaluated according to the average medical cost per patient. A total of 364 patients were enrolled in the study. The first-line H. pylori eradication rate was significantly higher in patients who received tailored therapy than in those who received empirical therapy. The total medical costs for the tailored and empirical groups were 46,374 Won and 53,528 Won. The total treatment period for each ultimately successful eradication in the tailored group was 79.8 ± 2.8 days, which is shorter than that of the empirical group (99.2 ± 7.4 days). The rate of eradication-related adverse events for the tailored group and empirical group was 12.9% and 14.8%, respectively. Tailored therapy could be a useful option to achieve a higher successful eradication rate, shorter treatment periods, and lower medical costs than empirical therapy in the era of increasing antibiotic resistance.Recently in Korea, where triple therapy is accepted as the first-line  Helicobacter pylori (H. pylori) is associated with peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric cancer [H. pylori infection [c cancer ,2,3, andnfection . Howevernfection ,6, and snfection ,8,9. Thenfection ,11, but nfection .H. pylori infection in Taiwan. Dual resistance to both clarithromycin and levofloxacin was found in 73.2%. This study highlighted ways to decide the optimum H. pylori eradication strategy according to the results of antibacterial susceptibility analysis [H. pylori eradication. Bismuth-based and bismuth-free therapies are equally effective for first-line H. pylori eradication [Kuo et al. recently reported on patients with refractory analysis . In Italdication . Therefodication .H. pylori resistance is extremely complex. The H. pylori virulence factors are involved in the induction of inflammatory responses, and control and regulate those responses, maintaining chronic inflammation [H. pylori exhibit an expanded complex of mechanisms that alter host cellular responses and signaling pathways. H. pylori elicit numerous adaptive mechanisms that enable effective bacterial adherence, colonization, and cellular alterations that provide the induction of further premalignant changes in the gastric microenvironment [An understanding of the mechanism of ammation . The H. ironment .H. pylori.In the Maastricht V/Florence guidelines, bismuth-containing quadruple therapy or concomitant therapy is recommended in areas where the clarithromycin resistance rate is higher than 15% . RecentlH. pylori infection. However, the cost-effectiveness of this test has not yet been definitively identified. The DPO-based multiplex PCR is more expensive than the Giemsa stain and rapid urease tests. It is difficult to perform this test routinely in clinical practice before the eradication of H. pylori. Therefore, we aimed to evaluate the eradication rate, adverse drug events, and cost-effectiveness of empirical and tailored therapies for the treatment of H. pylori infection.Tailored therapy based on the DPO-PCR test could be a useful regimen to increase the eradication rate of H. pylori was confirmed by rapid urease test, histology such as Giemsa stain, urea breath test (UBT), or DPO-PCR test; (2) patients receiving empirical therapy or tailored therapy based on the DPO-PCR results. Subjects (n = 71) were excluded in the following conditions: (1) patients aged >80 years (n = 13); (2) history of gastrectomy (n = 18); (3) severe systemic illness, such as severe cardiopulmonary dysfunction, liver cirrhosis, or renal failure (n = 17); (4) history of any allergic reaction to antibiotics (n = 8); (5) loss to follow-up (n = 15).This retrospective study was performed at two university hospitals in South Korea  from January 2017 to December 2019. We included 435 subjects who met the following criteria: (1) the presence of H. pylori eradication treatment  were enrolled in this study. Data for January 2017 and January 2018 were collected through chart review in Ewha Womans University Mokdong Hospital, and data for February 2018 and December 2019 were collected using data extracted from the Clinical Data Warehouse of the Ewha Womans University Seoul Hospital. This study was approved by the Institutional Review Board .A total of 364 patients who received H. pylori was regarded as positive when at least one positive result was obtained in UBT , rapid urease test , or histologic assessment (Giemsa staining) conducted using gastric biopsy specimens from the antrum and greater curvature of the body. In the tailored therapy group, DNA was extracted from frozen gastric biopsy specimens to detect clarithromycin-resistant H. pylori mutants. DPO-based multiplex PCR  was performed. Point mutations were identified by PCR amplification of a portion of the 23S ribosomal RNA gene. The amplified DNA products were visualized on a UV transilluminator after electrophoresis on a 2% agarose gel. The amplified DNA products were determined to have point mutations.Infection with H. pylori eradication treatment was stopped due to adverse drug effects. UBT, rapid urease test, or Giemsa staining of gastric biopsy specimens were performed to confirm successful H. pylori eradication.There were a total of three regimens for empirical therapy, which depended on the doctor’s preference: (1) triple therapy  for 14 days; (2) sequential therapy  for 10 days; (3) bismuth-containing quadruple therapy  for 14 days. In the tailored therapy group, patients received the eradication regimen based on the results of the DPO-PCR test. Six weeks after the end of the treatment, compliance with therapy and side effects were assessed through personal interviews. An adverse event was defined as an unscheduled early visit to the outpatient clinic with symptoms during eradication or when the The total medical cost per patient was assessed as the sum of the diagnostic and regimen costs. The cost of the DPO-PCR test was 38,350 Won, while those of the Giemsa stain and rapid urease test were 11,427 Won and 10,504 Won, respectively. The cost of 14-day triple therapy was calculated to be 34,300 Won. The cost of 10 day sequential therapy was 41,800 Won and that of the 14-day quadruple therapy was 25,746 Won.t-tests or Mann–Whitney U tests for continuous variables and Pearson’s chi-squared tests or Fisher’s exact tests for categorical variables. Categorical variables are presented as numbers and proportions. All statistical analyses were 2-sided, and results were considered statistically significant at p < 0.05. The Statistical Package for the Social Science (SPSS) software  was used for statistical analysis.Continuous variables were presented as mean ± standard deviation, and categorical variables were presented as the number of subjects and percentage. Group comparisons were performed using independent samples H. pylori eradication treatment. After excluding 71 patients, 364 patients were finally enrolled in the study. Empirical therapy was given to 155 patients, and 209 patients received tailored therapy. There were older and more current smokers in the tailored therapy group. The most common cause of need for H. pylori eradication was H. pylori associated gastritis in both groups  received 7-day triple therapy as the first-line H. pylori eradication regimen. As for the eradication rate according to regimen, the 14-day triple therapy was 81.5%, bismuth-containing quadruple therapy was 88.2%, and sequential therapy was 82.0% in the empirical therapy group. In the tailored therapy group, the eradication rate for 7-day triple therapy was 89.2%, bismuth-containing quadruple therapy was 91.7%, and sequential therapy was 80.0%. The eradication rate was significantly higher in those who received tailored therapy than in those who received empirical therapy   received sequential therapy as the first-line = 0.023) .p < 0.001). The cost of the diagnostic method was higher in the tailored therapy group . The cost of the eradication regimen was higher in the empirical group . The total treatment duration for each ultimately successful eradication in the tailored therapy group was 79.8 ± 2.8 days, which is significantly shorter than that of the empirical group’s 99.2 ± 7.4 days (p = 0.013). . p = 0.028). The most common adverse event was nausea or vomiting in both groups (26.1% vs. 29.7%)  in the empirical therapy group who did not want the next eradication treatment, but none of the patients in the tailored therapy group denied further treatment.Adverse drug events were more common in the empirical therapy group than in the tailored therapy group  . Five paH. pylori was significantly higher in those who received tailored therapy as their first-line treatment in comparison to those who received empirical therapy. The H. pylori eradication regimen is more cost-effective in tailored therapy than empirical therapy. The total treatment periods for ultimately successful eradication were shorter in the tailored therapy group, and fewer eradication-related adverse drug events were observed compared to the empirical therapy group.In this study, the eradication rate of H. pylori eradication rate was significantly higher with tailored therapy than with empirical therapy. Recently, as the clarithromycin resistance rate of H. pylori has increased, the eradication rate of the existing triple therapy for seven days has decreased. To overcome this situation, prolonged treatment periods, various regimens such as quadruple therapy with bismuth, sequential and concurrent therapy, or tailored therapy may be used [H. pylori eradication regimen requires a large amount of medicine, including two antibiotics to be taken. In particular, if you are already taking other drugs due to a comorbid disease, the H. pylori eradication treatment might a great burden. Moreover, antibiotic resistance is the leading cause of failure of the H. pylori eradication treatment, and we should try to reduce antibiotic resistance. Empirical quadruple therapy with bismuth as the first-line therapy raises public health concerns regarding increasing resistance to the constituent antibiotics. Finally, in tailored therapy, the risk of misuse of the antibiotics is lower due to reduced H. pylori eradication retreatment.The first-line  be used ,23,24,25H. pylori eradication regimen was more cost-effective in the tailored therapy than in the empirical therapy in this study. Different studies that have evaluated H. pylori tailored therapy have achieved contradictory results. Liou et al. in Taiwan found that 6920 USD would be required to additionally cure one patient using the genotype resistance guide therapy, compared to empirical therapy, which is clearly not a cost-effective option [H. pylori antibiotic resistance varies among different geographic areas, the cost-effectiveness may vary according to the cost of care in a given country, and therefore the same conclusion may not be applicable to other healthcare systems [The e option . Chang ee option . Howevere option . Gweon ee option . Since H systems .H. pylori infections is emerging in microbiological point. The changes of gut microbiota are greatly implicated in the pathogenicity of H. pylori Infections. The antimicrobial peptides (AMPs) have great importance in the innate immune reactions to H. pylori and participate in conservative co-evolution with an intricate microbiome. During H. pylori infections, AMP expression is able to eradicate the bacteria, thereby preventing H. pylori infections in the gastrointestinal tract [H. pylori infection [A novel view of al tract . The β-Dnfection .H. pylori infection, COX-2 is modulated by DNA methylation. The DNA methylation changes at the COX-2 promoter are associated with transcriptional activation and precede histone modifications in gastric cells exposed to H. pylori [H. pylori infection. The gastric cancer is regulated by methylation mechanism rather than genetic linkage, and H. pylori leads DNA methylation [In chronic inflammation induced by . pylori . Woo et hylation . TherefoH. pylori treatment [H. pylori eradication may be superior in quality, with fewer adverse events, compared to empirical therapy in Korea, where clarithromycin resistance is high.The prevalence of 23S rRNA point mutations was 28.7% in the study population. A total of 139 patients (66.5%) who received 7-day triple therapy were included in the tailored therapy group. This regimen might decrease drug-related adverse events and result in shorter treatment duration and minimal antibiotic overuse. Better compliance and fewer adverse effects were observed in patients in the tailored therapy group, similar to those reported in a previous study ,36. ChoiH. pylori infection were not enrolled in this study, and the total medical cost would be underestimated. Second, as this study was conducted retrospectively, there were limitations in obtaining detailed medical information that could have an influence on eradication failure or diagnosis of infection. Third, this study enrolled patients from only two medical centers, and a majority of them resided in Seoul. This study may have been subject to a selection bias. Fourth, DPO-based multiplex PCR could determine only the presence of clarithromycin resistance, and we did not check for resistance to other antibiotics such as A2115G, G2141A, A2142T, and T2182C.There are several limitations in this study. First, patients with a negative In conclusion, this study showed that tailored therapy could be a useful option to achieve a higher successful eradication rate, shorter treatment periods, and lower medical costs than empirical therapy in the era of increasing antibiotic resistance.


Candida yeast are the most common cause of opportunistic fungal infections resistant to certain antifungal drugs. Therefore, it is necessary to detect more effective antifungal agents that would be successful in overcoming such infections. Among them are some herbal products and their active constituents.The purpose of this review is to summarize the current state of knowledge onherbal products and their active constituents havingantifungal activity against drug-resistant Candida sp. used alone and in combination with antifungal drugs.The possible mechanisms of their action on drug-resistant Candida sp. including (1) inhibition of budding yeast transformation into hyphae; (2) inhibition of biofilm formation; (3) inhibition of cell wall or cytoplasmic membrane biosynthesis; (4) ROS production; and (5) over-expression of membrane transporters will be also described.Clinical isolates of  Candida yeastare considered to be one of the highly resistant fungi to most commercially known antifungal drugs [Candida is an opportunistic pathogen that can cause local and systemic infections in predisposed individuals, commonly affecting immunologically compromised patients and those undergoing prolonged antifungal drug treatment [C. albicans, C. tropicalis, C. krusei, C. parasilopsis, and C. glabrata [Candida sp. belongazoles , polyenes , echinocandins , and allylamine (terbinafine) [C. albicans toless sensitive species such as C. glabrata and C. krusei [C. glabrata is naturally about 8-fold more resistant to fluconazole than C. albicans and easily develops further fluconazole resistance in prolonged therapy with this drug [Candida sp. acting alone or synergistically with the antifungal drug [Candida sp. used alone and in combination with antifungal drugswassummarized based onseveral electronic databases and hand-searched references. Moreover, the mechanism of such herbal products’action will be also described.In recent years, multidrug-resistant pathogens have become a serious health problem worldwide. Improper and extensive usage of antibiotics results in selective pressure supporting the rise of antibiotic-resistant microbes. At present, clinical isolates of al drugs . Candidareatment . The mosglabrata . To maininafine) ,4. Due tinafine) . Moreove. krusei . C. glabhis drug ,8. Therehis drug ,12,13,14gal drug ,16. Thisgal drug . Moreovegal drug . In thisCandida sp.’, ‘herbal products in combination with antifungal drugagainst drug-resistant Candida sp.’,‘herbal products against fluconazole-resistant Candida sp.’, ‘active constituents from herbs against drug-resistant Candida sp.’, ‘active constituents in combination with antifungal drug against drug-resistant Candida sp.’and ‘active constituents from herbs against fluconazole-resistant Candida sp.’. References from reviews presentingherbal products and their active constituents against drug-resistant Candida sp. were searched for additional articles and case reports. A manual search was also conducted based on citations in the published literature.The Scopus and Google Scholar databases were searched for articles published from 2011 to the present. Search terms included ‘herbal products against drug-resistant Candida sp.without determining their drug-resistance. In addition, publications in languages other than English were excluded.Selection criteria excluded articles that examined antifungal activity of herbal products and their active constituentsagainst Overall, 20,044 articles were found in the databases. Of these, 13,634 articles were excluded at the title level, among them were alsoduplicates and unrelated articles. Furthermore, 6410 articles were excluded asnot meeting the inclusion criteria. Finally, 47 articles were usedfor the review .Candida sp. [Herbs have been used in traditional herbal medicine for many years. Herbal products may be also promising drugs against drug-resistant dida sp. ,10,11.Lupinus various seeds [Echinophoraplatyloba extract [Andrographispaniculata and Achyranthesaspera [Hibiscus sabdariffa extract [C. abicans, while Coriandrumsativum, Menthapiperita, and Punicagranatum extracts showed superior antifungal activity against fluconazole-resistant C. glabrata [C. albicans growth [Azadiractaindica, Allium sativum, Cordiadichotoma, Ocimum sanctum, Syzygiumcumini, and Trigonellafoenumgrecum were effective against all multidrug-resistant  Candida isolates  [Candida isolates was the ethanolic extract of Allium sativum.The aqueous extracts of us seeds , Echinop extract , hydroalesaspera , as well extract  stronglyglabrata . Thechers growth . The ethlabrata) . Among tCandida strains is scacre. In some studies, it was shownthat Ocoteaglomerata extract [Thymusbroussonetii and Thymus maroccanus essential oils [Thymus vulgaris essential oil [Citrusaurantium essential oil [Lavandulaangustifolia essential oils [Ocoteaglomerata extract [Thymus maroccanus and Thymus broussonetii essential oils [Citrusaurantium essential oil [C. albicans, C. glabrata, C. tropicalis,and C. krusei. However, it is not known whether these Candida strains weredrug-resistant or not. Extending the research on the interaction of herbs in combination with conventional antifungal drugs against drug-resistant Candida strains would enrich the scientific literature with new data.Unfortunately, data presenting theinteractions of herbs used in combination with conventional antifungal drugs against drug-resistant  extract , Thymusbial oils , Thymus tial oil , Citrusatial oil  in combiial oils , Ocoteag extract  in combiial oils , and Cittial oil  in combiCandida sp.as well as significantly enhance the anti-candidal activity of the drug in treating drug-resistant Candida sp.In recent years, an increasing number of studies was performed to discover new bioactive compounds of plant origin which may possibly control multidrug-resistant human pathogens . MoreoveCandida sp.  [Candida isolates [Candida sp. [Pseudolarixkaempferi Gordon, possesses inhibitory activity against fluconazole-resistant and fluconazole-susceptible strains of C. tropicalis [Theberberineshowed antifungal activity against fluconazole-resistant  krusei) .Thecurcuisolates . Among adida sp. . Pseudolopicalis .Candida strains. The gypenosides, the main active components of Gynostemmapentaphyllum in combination with fluconazole, revealed a synergistic antifungal activity against fluconazole-resistant C. albicans [C. tropicalis is were PAB in combination with fluconazole [C. albicans [C. albicans. The trans-resveratrol and cis-resveratrol enhanced the azoles  susceptibility on fluconazole-resistant Candida isolates [C. albicans and Streptococcusmutans mixed biofilms [C. auris and C. albicans [C. auris biofilms [C. albicans and C. tropicalis [It was also observedthat some active constituents isolated from herbs in combination with conventional antifungal drug showed synergistic antifungal activity against drug-resistant albicans . Highly conazole . Eucalypalbicans . Alsogeralbicans  and magnalbicans  enhancedisolates .The combbiofilms . The mosalbicans . The intbiofilms . Synergiopicalis .Candida sp. is onlypartially known [Candida sp. have developed many mechanismsby which they become resistant to the antifungal drugaction.In several antifungal-resistant Candida clinical isolates, the over-expression of membranes transporters , Candida multidrug resistance— CaMDR1), altered ergosterol biosynthesis via mutation and/or over-expression of ergosterol pathway genes ERG3  double bond into episterol, a precursor in ergosterol biosynthesis), ERG6 (encodingdelta(24)-sterol C-methyltransferase, which converts zymosterol to fecosterol by methylating C-24 in the ergosterol biosynthetic pathway), and ERG11  as wellas altered sterol import were observed [Candida clinical isolates resistant to some antifungal drugs.The mechanism of antifungal drug action against ly known . It was ly known ,44. UnfoCandida sp.are based on (1) inhibition of budding yeast transformation into hyphae; (2) inhibition of biofilm formation; (3) inhibition of cell wall or cytoplasmic membrane biosynthesis; (4) reactive oxygen species (ROS) production; and (5) over-expression of membrane transporters [Candida sp.Whereas herbal products/active constituentsare used in combination with antifungal drug; in the first step, they decrease the drug resistance of yeast through the suppression of CDR1 and multidrug resistant 1 (MDR1) gene expression.Then, they elevate the intracellular concentration of antifungal drugs and in turnincrease the effectiveness of those drugs against resistant Candida strains.The mechanism of action of herbal products and their active constituents may be multi-directional . The possporters ,46. MoreC. albicans grows as budding yeast which can transform into hyphae in response to various environmental or biological stimuli [C. albicans. Hyphae can attach to host cells, damage host tissue, and escape from host immune defenses [Pelargonium capitatum and Cymbopogon martini essential oils can inhibit the major virulence factor of C. albicans as the germ tube formation [Gynostemmapentaphyllum in combination with fluconazole [Pseudolarixkaempferi Gordon in combination with fluconazole [C. albicans and C. tropicalis biofilm. The (+)-Lyoniresinol-3α-O-β-d-glucopyranoside induced the accumulation of intracellular trehalose on C. albicans as a stress response to the drug, and it disrupted the dimorphic transition that forms hyphae [C. albicans cells with both apigetrin and its aglyconeapigeninlowers the number of cells growing in the hyphal form. Berberine hydrochloride (BBH) and fluconazole combination inhibited yeast adhesion, morphological hyphae transformation, and biofilm formation by downregulating the hyphal-specific genes ALS3 (agglutinin-like protein 3), HWP1 , and ECE1 (extent of cell elongation protein 1) [YVC1) and vacuolar calcium pump 1 gene (PMC1) are key targets for BBH and fluconazole combination, which increase cytoplasmic Ca2+ in resistant isolates, which might be critical for reversing biofilm-positive fluconazole-resistant C. albicans through yeast apoptosis induced by intracellular or mitochondrial high Ca2+ levels. stimuli . Yeast cdefenses . Moreoveormation . Thegypeconazole  and PAB,conazole  significs hyphae . Cinnamas hyphae . The sels hyphae . Treatmeotein 1) . MoreoveCandia sp. infections associated with biofilms. An alternative to antifungal drugs used in the treatment of candidiasis can be herbal products and their active constituents with anti-biofilm potency. The Ononisspinosa effectively inhibited biofilms formed by Candida strains through inhibition of ergosterolsynthesis and leakage of cellular components [Rubusidaeus, Vacciniummyrtillus, Vacciniummacrocarpon, Malpighiapunicifolia) extracts showed a lack of antifungal activity but a significant anti-adhesion and anti-biofilm potency on C. albicans and C. glabrata,especiallythecranberry extract [Hibiscus sabdariffa extract was found to be significantly effective against fluconazole-resistant C. albicans isolated from patients but also to be a substitute for eradicating pre-formed biofilm and inhibiting the growth of C. albicans [Thymus kotschanus essential oil exhibited anti-biofilm activity by a significant decrease of als gene expression, which leads to a decrease in the synthesis of ALS3—an important protein for fungal adhesion and biofilm formation [Boesenbergia rotunda inhibitsthe biofilm formation of C. albicans, especially during the biofilm development stage, by reducing the cell surface hydrophobicity and suppressing the ALS3 mRNA expression [B. rotunda had a stronger effect on ALS3 mRNA expression  and significantly decreased the ACT1 (actin 1) mRNA level (pinocembrin).The filamentous form decreased with pinocembrin rather than with pinostrobin and inhibitedthe stage of C. albicans biofilm development. The 6-shogaol extracted from ginger exhibited anti-biofilm activity by inhibiting biofilm formation and eradicating the preformed biofilms of C. auris [Gynostemmapentaphyllum, in combination with fluconazole inhibit early biofilm formation, suppress drug efflux, and inhibit yeast–hyphalconversion [C. glabrata, C. guilliermondii, and C. parapsilosis [C. albicans. Moreover, the combination of berberine and amphotericin B against C. albicans/S. aureus dual-species biofilms revealed that hyphalfilamentation of C. albicans and co-adhesion between C. albicans/S. aureus were considerably impaired by the treatment [The formation of biofilms makes treatment difficult and contributes to high rates of morbidity and mortality, thus representing one of the main virulence factors that contribute to the pathogenesis of candidiasis ,54,55. Tmponents . Somered extract . The Hibalbicans . Thymus ormation . In addipression . FurtherC. auris . The gypnversion . The antpsilosis . The isopsilosis . It was psilosis  and berbpsilosis  inhibitereatment .Ononisspinosa [Coriarianepalensis essential oil [C. albicans with apigenin and rutin led to lower expression levels of ergosterol biosynthesis enzyme (ERG11), while apigenin and isoquercitrin up-regulated the expression of ERG11, since their application can lead to reduced susceptibility to azole antifungals [C. albicans, C. glabrata, and C. tropicalis. The cinnamaldehyde fungicidal mechanism of action is likely related to ergosterolcomplexation through binding to enzymes involved in the formation of the cytoplasmic membrane in yeast cells [Coriandrumsativum essential oil [Candida sp., probably by apoptosis. The MCh-AMP1, a natural peptide from Matricariachamomilla L. flowers caused C. albicans cell death via increasing the cell membrane permeability by induced potassium leakage from the yeast cells [C. albicans and C. tropicalis [Candida activity through cell wall remodeling induction was observed after sodium houttuyfonate, berberine, palmatine, jatrorrhizine, cinnamaldehyde, and their combinations [The numerous herbal products and their active constituents target the biosynthesis of ergosterol, which is a unique cell membrane component, present only in fungi. The methanolic extract of sspinosa  and Coritial oil  effectivifungals . Thekaloifungals , and β-cifungals  decreasest cells . The funtial oil , protobetial oil , and bertial oil  is a resst cells . Pseudolst cells . The (R)opicalis . Moreoveinations .Ocoteaglomerata did not reveal effects on ergosterol biosynthesis; however, it led to an increase in intracellular ROS levels, decreased cell viability, and consequently, cell death [C. albicans, causing the leakage of intracellular trehalose, the entrance of extracellular impermeable substance, and the decrease of ergosterol content, all of these contributed to the death of C. albicans cells [C. glabrata, C. guilliermondii, and C. parapsilosis [Matricariachamomilla flowers, induced ROS production and caused cell death via increasing cell membrane permeability induced by potassium leakage from the C. albicans cells [2+/calcineurinpathway [C. albicans Metacaspase-1) in C. albicans.Theberberine serves as a potent ROS-inducing agent, disrupting the antioxidant system, especially in fluconazole-resistant C. albicans [C. albicans exhibited efficient antioxidant response at lower concentrations but could not sufficiently alleviate berberine-induced oxidative stress occurring at concentrations greater than 250 μg/mL.The inhibition of cell wall or cytoplasmic membrane biosynthesis, cell wall remodeling, and disruption in the integrity of cell membrane leading to theleakage of cellular components outside the cell is one of the main mechanisms of action of herbal products and their constituents, but it is not the only one. For example, methanol extract of ll death . Thekalons cells . The purpsilosis . The MChns cells . Thecarvnpathway . Fu et anpathway  reportedalbicans . InteresCandida sp.correlates with the over-expression of the CDR1 and CDR2 genes encoding transporters of the ATP-binding cassette(ABC) family and the CaMDR1gene coding a major facilitator superfamily (MFS) transporters [Candida sp. [C. albicans is the failure of cells to accumulate the drug due to increased expression of the efflux proteins encoded by the CDR1, CDR2, and MDR1 genes [CDR1 and MDR1 genes and thereby inhibit the activity of these pumps, whichincrease the intracellular concentration of antifungal drugs , thereby increasing the effectiveness of these drugs onthe resistant Candida strain. Keereedach et al. [Melaleucacajuputi in combination with fluconazole inhibited the growth of fluconazole-resistant C. albicans clinical isolates by significant reduction of the MDR1 gene expression level. The β-lapachone isolated from the lapacho tree reverted fluconazole resistance of C. albicans strains over-expressing transporters CaCdr2p  and CaMdr1p  by inhibiting these proteins activities [CDR1 gene in C. auris [CDR1 downregulation, whereas fluconazole alone induced high CDR1 transcription in resistant C. albicans strains [C. albicans-resistant isolates. Eucalyptal D revealed the upregulation of CDR1 and CDR2 genes [C. albicans. Furthermore, geraniol [CDR1 and CDR2 expression as well as competitiveinhibition offluconazole efflux from fluconazole-resistant C. albicans.Literature data showed that a reduced level of intracellular antifungal drug accumulation in resistant sporters ,45,46. Bdida sp. . The mosh et al.  notedthativities . The 6-sC. auris . The antC. auris .Apigenin strains . BBH as R2 genes . Eucalypgeraniol  and magngeraniol  were fouCandida sp. Moreover, the use of herbal products and their active constituents with antifungal drugs combination is likely to reduce the minimum effective dose of the drugs, thus minimizing their toxic side effects and the treatment cost. Unfortunately, little is known about the bioavailability of herbs and their active constituents. Most of the presented data arebased on in vitroexperiments carried out on drug-resistant Candida sp. isolated from skin, vagina, blood, pus swab, sputum, urine, gastric aspirate, or clinical isolates from a culture collection. Despite the fact that herbal compounds showed better antimicrobial activity against drug-resistant Candida strains when used in synergy with an antifungal drug, there are no preclinical and clinical in vivo studies confirming that these combinations can inhibit diseases caused by Candida yeasts. Therefore, more advancedin vivostudies are needed to fully evaluate this strategy of Candida treatment. However, the advancement in the techniques ofseparation, purification, and identification of bioactive compounds may enable obtaining new compoundsof plant origin which will be used as drugs for the treatmentofdrug-resistant Candida strains in the near future.This review suggests that the usage of herbs may be considered as important support for conventional antifungal therapy. Herbal products and their active constituents are reported to be potentially active against a wide variety of fungi including drug-resistant


Nosema ceranae primarily infects honey bee gut epithelial cells, leading to impaired memory, suppressed host immune responses and colony collapse under certain circumstances. As the genome of N. ceranae is challenging to assembly due to very high genetic diversity and repetitive region, the genome was re-sequenced using long reads. We present a robust 8.8 Mbp genome assembly of 2,280 protein coding genes, including a high number of genes involved in transporting nutrients and energy, as well as drug resistance when compared with sister species Nosema apis. We also describe the loss of the critical protein Dicer in approximately half of the microsporidian species, giving new insights into the availability of RNA interference pathway in this group. Our results provided new insights into the pathogenesis of N. ceranae and a blueprint for treatment strategies that target this parasite without harming honey bees. The unique infectious apparatus polar filament and transportation pathway members can help to identify treatments to control this parasite.Microsporidia comprise a phylum of single cell, intracellular parasites and represent the earliest diverging branch in the fungal kingdom. The microsporidian parasite  As the earliest branch from the fungal kingdom, microsporidia comprise a large and widespread group of obligate intracellular animal parasites . In humaNosema ceranae is a microsporidian parasite which infects honey bee mid-gut epithelial cells  with assembled genomes were retrieved from NCBI and MicrosporidianDB1,2   . In ordeeviously . The seqeviously . Additioh Muscle . These ah Muscle . The speN. ceranae were used to query this library. Through blast with an E-value cutoff of ≤1e−5, the best aligned sequence was used for further analysis. To identify PTPs in N. ceranae, about 10 kb sequences of upstream and downstream of PTPs in E. cuniculi and N. bombycis were obtained, then candidate sequences were retrieved by BLAST . Synteny blocks were identified manually from BLAST coordinates.In order to further characterize the PTP genes and SWP genes, protein and CDS sequences encoded SWP were downloaded from NCBI and used as a library. The genome sequences of E. cuniculi and T. hominis  and two yeast species (Schizosaccharomyces pombe and Saccharomyces cerevisiae). The sequences of fungal ABC transporters were obtained from previous studies, which were used as seed sequences to query the downloaded protein sequence sets using BLAST with cutoff P < 0.05 . By aligning the RNA-seq reads back to the assembly, all the predicted protein-coding genes were expressed, suggesting these protein-coding genes were functional. Out of 600 conserved BUSCO groups, 541 complete, 6 fragmented and 53 missing BUSCOs were identified, indicating the assembly is nearly complete  and the cysteine desulphurase complex Nfs1–Isd11, respectively. The electrons are delivered by ferredoxin (Yah1). Then, the Fe–S cluster pass from Isu1 and Isu2 to target apoproteins with the support from a Hsp70 (Ssq1) protein, co-chaperone Jac1, and the monothiol glutaredoxin Grx5.Microsporidia are highly obligate intracellular parasites of animals with extremely compact genomes and decreased cellular and biochemical reactions . The micN. ceranae genome for Isu1  were relatively close and clustered. However, significant intra-microsporidian divergence was observed for Isu1 and Nfs1. Interestingly, all three phylogenetic trees suggest that the microsporidian sequences are branched relatively early from other species, indicating microsporidia may contain specific features of the Fe-S cluster assembly, supported by the Siddal and Whiting method  cluster proteins, multidrug resistance, and antigen processing  were present in genomes of 21 microsporidian species, and among these ABC subfamilies, members of ABC-G proteins were the most abundant, followed by ABC-B subfamily . ABC-A, ocessing . Genomicocessing . In our ocessing . The numary tree . AlternaN. apis, E. bieneusi, E. canceri, and V. culicis to 12 copies in E. aedis (N. ceranae (four ABC-G) showed higher copy number of ABC-G than that of its most closely related species N. apis (one ABC-G). However, the other three ABC-G orthologs were clustered into a single group  and N. bombycis (one ATP/ADP carrier). N. ceranae Nn.00g020520 and Nn.00g004970 are, respectively, homologous to N. apis EQB60298.1 and EQB60147.1, while N. ceranae Nn.00g004860 is homologous to N. bombycis EOB13854.1  IPL-LD-65Y cell-based system of N. ceranae infection  and polar tube. After germination, the polar tube protein PTP1 can interact with lectin receptors on the host cell surface , 2004. Pstinalis . Remarkastinalis . Similarstinalis . Accordistinalis . In the stinalis . Proteinstinalis . In N. bin coats . The homst cells . NcHSWP1st cells . The lowN. ceranae destroys the gut integrity of honey bees, leading to impaired flying and memory abilities, which can lead to the loss of colonies. We anticipate that the updated genome resource and comparative analyses provided here lead to novel methods to control this parasite without harming honey bees. The provided genome also reveals numerous evolutionary features compared with other microsporidian parasites, which may help to clarify the evolution of virulence and co-evolution with hosts.https://www.ncbi.nlm.nih.gov/, SRR8536193.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Apis mellifera), which is neither an endangered nor a protected species.The apiaries for bee sample collection are the property of the USDA-ARS Bee Research Laboratory, Beltsville, MD, United States. No specific permits are required for the described studies. Studies involved the European honey bee (QH and JE designed the investigation. QH assembled the genome. WL performed Fe-S phylogenetic analysis. RG performed ABC and ATP/ADP carriers’ phylogenetic analysis. JX, XD, and ZM performed SW and Polar tube protein phylogenetic analysis. QH, ZW, WL, RG, JX, XD, ZM, YC, and JE 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.


Leishmania species, threatening millions of people in approximately 100 endemic countries. The emergence of antimony-resistant Leishmania strains have brought difficulties to the treatment and elimination of leishmaniasis. This study performed genome-wide resequencing and phylogenetic analysis of five isolates from the Leishmania donovani complex, focusing on finding mutations related to antimony resistance and virulence of the newly isolated Leishmania strain L_HCZ in 2016. By combining whole-genome sequencing and whole-genome phylogenetic analysis, Leishmania isolates L_801, L_9044 and L_Liu were identified as Leishmania donovani, and L_HCZ as Leishmania infantum. By discovering genome-wide single-nucleotide polymorphisms and structural variations, we identified mutations of drug resistance-related genes in the antimony-resistant Leishmania isolate L_HCZ. The new Leishmania isolate L_HCZ has strong virulence and strong drug resistance, which should be taken seriously by the relevant health departments and scientific researchers.Leishmaniasis, a neglected tropical disease, is caused by infection with the Leishmania strains have brought difficulties to the treatment and elimination of leishmaniasis. This study performed genome sequencing, phylogenetic analysis and mutation analysis of five Leishmania clinical isolates, especially the Leishmania strain L_HCZ isolated in 2016, which shows strong virulence and antimony resistance. By phylogenetic analysis, four isolates  were identified as Leishmania donovani, the isolate L_HCZ was identified as Leishmania infantum and the isolate L_DD8 as a standard strain of L.donovani. Genome-wide mutation analysis was applied to identify mutations related to the drug resistance and virulence of the newly isolated L_HCZ. Compared with the other four Leishmania isolates, L_HCZ had the most mutations in genes associated with antimony resistance, including the ABC transporter, ascorbate-dependent peroxidase, gamma–glutamylcysteine synthetase, glucose-6-phosphate 1-dehydrogenase, ATP-binding cassette protein subfamily A and multi-drug resistance protein-like genes. Among the genes associated with virulence, L_HCZ had the most mutations in cysteine peptidase A, cysteine peptidase B, cysteine peptidase C, heat-shock protein 70, gp63, acid phosphatase, kinesin k39, kinesin, phosphoglycan beta 1, amastin-like surface protein and amastin-like proteins. The mutations in L_HCZ might possibly contribute to its antimony resistance and strong virulence in clinical patients. Whole-genome resequencing has exhibited broad application prospects and may be put into clinical use in the future for parasite identifying and epidemiological investigations.Leishmaniasis is a neglected tropical disease threatening millions of people worldwide. The emergence of antimony-resistant  Leishmania parasites, which are transmitted by insects known as sandflies. The main clinical symptoms of VL are long-term irregular fever, anemia, weight loss and hepatosplenomegaly [Leishmania spp. that causes VL is mainly derived from the Leishmania donovani complex (including Leishmania donovani and Leishmania infantum) [Leishmania drugs are available, and their effectiveness is severely limited by toxicity, cost and drug resistance [Leishmania strains have appeared in clinics, especially with resistance to antimony. Understanding the Leishmania genomes, their natural variation and genetic relationships is essential to support and strengthen public health monitoring and intervention strategies.Visceral leishmaniasis (VL) is a neglected zoonotic parasitic disease and is caused by nomegaly . Failurenfantum) . At presnfantum) . Only a sistance . Some reLeishmania is a protist belonging to the family trypanosomidae [Leishmania species are known, and at least 20 species are pathogenic for human beings [Leishmania was initially based on eco-biological criteria such as vector, geographical distribution, tropism, antigenic properties and clinical manifestations [Leishmania began to be based on combined molecular data. The currently accepted classification of Leishmania species is divided into two major phylogenetic lineages: Euleishmania and Paraleishmania [Euleishmania section is composed of at least four subgenera: Leishmania, Viannia, Sauroleishmania and Mundinia [Leishmania has four main species complexes: L. donovani, L. major, L. mexicana and L. tropica [Leishmania species will represent a keystone for biological and epidemiological research programs and can help guide clinical treatment and prevention [Leishmania at the specific or subspecific level. The advent of molecular sequence data provided a large extent of additional information for phylogenetic analyses [cytB) and the heat-shock protein 70 gene (hsp70) were used for the phylogenetic analysis of Leishmania, but some evolutionary relationships are still not well resolved.osomidae ,6. Curren beings . The clastations . With thishmania . The EulMundinia ,10,11. T tropica ,11. A reevention . Howeveranalyses . MoleculLeishmania varied among different subspecies, L. amazonensis genome is 29 Mb in size, while L. infantum, L. major and L. braziliensis genomes are 32 Mb in size [L. amazonensis and L. mexicana, 35 in L. brasiliensis and 36 in L. major, L. donovani and L. infantum [Leishmania genome has more than 8300 coding genes [L. infantum, L. major and L. braziliensis are conserved, and only a few species-specific genes were found [Leishmania clinical isolates were collected, especially the newly isolated strain MHOM/CN/2016/SCHCZ (L_HCZ) [Leishmania isolates and looked for mutations in the genome that are potentially related to antimony resistance and virulence, especially in the L_HCZ isolate.The genome size of  in size ,12,13. Tinfantum . The Leing genes . More thre found ,17. Withre found . In recere found . Moreovere found . In this (L_HCZ) . The L_HLeishmania clinical isolates were used in this study , Chinese Center for Disease Control and Prevention . L_HCZ is a new Leishmania strain isolated from the bone marrow of a patient with kala-azar by our experimental team.Five is study . All fivLeishmania isolates were preserved in Novy–MacNeal–Nicolle (NNN) medium. The parasites, preserved in NNN medium, were transferred to M199 medium  supplemented with 10% fetal bovine serum  and antibiotics  at pH 7.4 and 28 °C for extended culture. After cultivating the parasites for 5–7 days to reach the logarithmic growth phase, the medium was added logarithmically. The morphology of Leishmania promastigotes was observed by Wright’s staining. When the total amount of M199 medium reached 80 mL or more, the parasites were collected by centrifuge at 800× g for 10 min to extract genomic DNA using TIANamp Genomic DNA Kit . The concentration of DNA was detected by Qubit Fluorometer, and the integrity of DNA was detected by agarose gel electrophoresis. The qualified DNA was sent to The Beijing Genomics Institute for sequencing.The promastigotes of the five Leishmania donovani BPK282A1 was used. The genome contains sequences of 36 chromosomes and is 32,444,968 bp in length in total. The GC content of the genome is 59.40%, and the gap rate of the genome is 3.676%. The N50 length of the genome is 1.024 MB and the N90 of the genome is 558.17 KB.The reference genome (GCF_000227135.1) of the strain http://annovar.openbioinformatics.org/en/latest/, accessed on 9 August 2020). CNVnator [https://github.com/abyzovlab/CNVnator, accessed on 9 August 2020) to detect copy number variation (CNV) and BreakDancer [https://github.com/genome/breakdancer, accessed on 9 August 2020) to detect structural variation (SV).The main steps of GATK best practices for variation detection were as follows: clean reads obtained by sequencing were compared with the reference genome using BWA software . Picard CNVnator   with a quality threshold of 20, a length threshold of 50 bp and a front trimming of 5 bp in both ends. The trimmed reads were subjected to assembly by Shovill  with default parameters. Contigs with low coverage of less than 3 and lengths less than 1000 bp were filtered. FastANI [https://github.com/ParBLiSS/FastANI, accessed on 21 October 2021) was used to calculate pair-wise genomic similarities between all 33 genomes and a similarity matrix was generated. MultiDendrograms [https://github.com/sergio-gomez/MultiDendrograms, accessed on 21 October 2021) was used to construct the phylogeny of the strains based on the similarity matrix with the UPGMA algorithm.We downloaded 28 genome sequences of rom NCBI . These cby fastp   and core regions with a length ≥ 1000 bp for each were determined by Samtools [http://samtools.github.io, accessed on 21 October 2021) and Bedtools [https://bedtools.readthedocs.io, accessed on 21 October 2021). The sequences from the core regions were extracted by Maf parase [https://github.com/CshlSiepelLab/phast, accessed on 21 October 2021) from the MAF file generated by minimap2. The sequences from each core region were aligned using Mafft [https://mafft.cbrc.jp/alignment/software/, accessed on 21 October 2021) and then trimmed by Trimal [http://trimal.cgenomics.org, accessed on 21 October 2021). The alignments of all the core regions were concatenated into a single core-genome alignment, which was then used for phylogenetic analysis by iqtree [http://www.iqtree.org/, accessed on 21 October 2021) using the TVM + F + G4 model as determined automatically and by 1000 bootstrap replicates.The complete or draft genomes of the 33 strains were mapped to the genome of the strain Friedlin (GCA_000002725.2) by Minimap2  , followed by isolates L_801, L_DD8 and L_DD8, and the isolate L_HCZ had the lowest map rate (90.01%). The genomic coverages based on 1×, 4×, 10× depths for the samples were all greater than 90%, suggesting that the five isolates had high similarity with the reference genome .Leishmania isolates were found , insertion (about 3%), inversion (about 10%), intrachromosomal translocation (about 32%) and interchromosomal translocation (about 20%) were basically the same B. It is Leishmania strains were downloaded from NCBI and analyzed together with the whole-genome sequences of the five Leishmania clinical isolates. The pairwise comparison matrix of the whole genomic nucleic acids of the 33 Leishmania strains is shown in Leishmania strains were divided into four groups: L. donovani complex, L. major complex, L. tropica complex and L. mexcicana complex. The results of the phylogenetic tree show that L_801, L_DD8 and three L. donovani strains were clustered into a small branch, proving that they were closely related. The relationship between L_DD8 and L_801 also showed a high correlation on the matrix diagram. L_DD8 is the internationally common standard strain of L. donovani. Therefore, it could be determined that L_801 is L. donovani. L_9044 and L_Liu were clustered together in the tree, suggesting that their evolutionary relationship is close, but they were slightly distant from the rest of the L. donovani strains. L_9044 and L_Liu were temporarily incorporated into the Leishmania donovani complex. Due to the close evolutionary relationship between L_801 and L_DD8, the SNPs, InDels and CNV data of L_801 and L_DD8 were basically consistent and could be explained. Similarly, the consistent SNPs, InDels and CNV results of L_9044 and L_Liu may also be caused by their close evolutionary relationship. It should be noted that L_HCZ was clustered with two canine L. infantum strains and one human L. infantum strain in the tree. Consequently, the L_HCZ isolate could be identified as L. infantum. However, because of the close genetic relationship between L. donovani and L. infantum [L. donovani and L. infantum.The whole-genome sequences of 28 infantum , the genCompared with the reference genome, L_DD8 had 883 mutations in exons, 773 mutations were aneuploid, and the proportion of aneuploidy was 87.54%. L_801 had 1022 mutations in exons, 918 mutations were aneuploid, and the proportion of aneuploidy was 89.82%. L_9044 had 37,796 mutations in exons, 1648 mutations were aneuploid, and the proportion of aneuploidy was 4.36%. L_Liu had 38,078 mutations in exons, 2054 mutations were aneuploid, and the proportion of aneuploidy was 5.39%. L_HCZ had 49,352 mutations in exons, 1322 mutations were aneuploid, and the proportion of aneuploidy was 2.68%. We found that although L_HCZ, L_Liu and L_9044 had more exon mutations, most of them were euploid. However, although L_801 and L_DD8 had fewer exon mutations, most of them were aneuploid .Leishmania isolates is shown in The number of mutations in nine antimony resistance-related genes of five Leishmania isolates is shown in The number of mutations in nine virulence genes of five Leishmania: kinesin [Leishmania: ATP-binding cassette protein subfamily A [Leishmania is shown in We counted genes with more than 50 mutations in the L_HCZ isolate and finally obtained 18 genes. Genes involved in cell signaling and various biological functions: protein kinase , ubiquit kinesin , calpain kinesin , phospho kinesin , amastin kinesin  and amas kinesin . A gene  kinesin . A gene  kinesin . Genes afamily A , folate/family A  and protfamily A . UnknownLeishmania genomes [Leishmania isolates, L_HCZ had the largest number of SNPs and deletions, and had the largest number of variations in upstream, downstream, the exonic region of ncRNA and the 5′ UTR region. Furthermore, in coding regions, the number of SNV, insertion, deletion, synonymous, nonsynonymous, frameshift and non-frameshift mutations was also the highest in L_HCZ. It is interesting that L_HCZ had the lowest number of CNV and SV, especially the number of CNV duplication. The CNV duplication number of L_DD8 and L_801 was almost twice that of L_HCZ. Gene duplication would represent an important means of innovation by expanding the genomic information, and normal copies of the gene would compensate deleterious mutations, whereas successful mutations could be expanded [Recently, the development of whole-genome sequencing and computational analysis has improved the resolution and accuracy of in-depth exploration and comparison of  genomes . Althougexpanded . In trypexpanded ,56. TherLeishmania isolates. In the phylogenetic tree, L_DD8 and L_801 were clustered together, while the data of the two isolates were similar in the SNP/InDel results. The same was true for the phylogenetic and SNP/InDel results of L_9044 and L_Liu. Genome-wide SNP variation provided new evolutionary insights into the ongoing diversification of the phenotypically variable set of Leishmania strains [L. donovani and had a close genetic relationship with L_DD8. L_Liu was isolated from a patient with cutaneous leishmaniasis in Karamay, Xinjiang, China [L. infantum [L. donovani [L. donovani. Moreover, L_HCZ was grouped with three L. infantum isolates in the phylogenetic tree, which basically confirmed L_HCZ as L. infantum. Whole-genome resequencing is economical, fast and convenient. It can be used for the identification of Leishmania isolates and searches for drug resistance targets. It has broad application prospects and may be put into clinical use with phylogenetic analysis in the future for identifying pathogens and epidemiological investigations [The results of the phylogenetic tree corresponded to the SNP/InDel results of five  strains . Based og, China . An earlinfantum . Howeverinfantum . In the donovani ,60. Therigations ,61.Leishmania is to regulate the expression of drug target, drug transporter or other drug resistance determinants by changing gene dose through copy number variations  [Leishmania isolates, the number of mutations in virulence genes and drug resistance-related genes of L_HCZ was larger. Except for L_HCZ, the other four Leishmania isolates were isolated from the 1980s and 1990s. As a newly isolated Leishmania strain, the mutations in virulence genes and drug resistance-related genes of L_HCZ might be caused by the variation accumulation during its evolution [L. infantum (L_HCZ) had more mutations in virulence genes than those of L. donovani , which might be one of the reasons why L. infantum is more virulent than L. donovani. In this study, we investigated the mutation characteristics of five Leishmania clinical isolates based on the results of whole-genome resequencing, and analyzed the phylogenetic relationship of these isolates by constructing a phylogenetic tree with the published whole-genome sequences of 28 Leishmania isolates. Genome-wide phylogenetic analysis would be helpful to improve the molecular identification of Leishmania species, and genome-wide mutation analysis revealed possible mechanisms of pathogenicity and drug resistance in different Leishmania isolates. The datasets of the five Leishmania clinical isolates will provide a molecular basis for future research on the pathogenic mechanism, drug resistance mechanism and vaccine development of Leishmania. In our previous study, we focused on verifying the virulence and antimony resistance of the Leishmania clinical isolate (L_HCZ) at molecular levels and proved that L_HCZ has high-level pathogenicity and evident antimony resistance in vitro, using an integrative approach of genome sequencing, proteomic profiling and phenotypic analysis to find the target spot of virulence and antimony resistance of L_HCZ [Leishmania isolates may become targets for vaccines or drugs, as well as the molecular markers for screening of Leishmania drug-resistant strains and different virulence strains [SNPs in drug targets or transporters can cause drug resistance without changing gene expression, so single-nucleotide mutations constitute a strategy to avoid drug pressure . Single-eletion) ,63. In tvolution . The mutof L_HCZ . Accordiof L_HCZ . Similar strains .Leishmania donovani complex, focusing on finding mutations related to antimony resistance and virulence of the newly isolated Leishmania strain L_HCZ in 2016. By combining whole-genome sequencing and whole-genome phylogenetic analysis, Leishmania isolates L_801, L_9044 and L_Liu were identified as L. donovani, and L_HCZ was L. infantum. By discovering genome-wide single-nucleotide polymorphisms and structural variations, we identified mutations of drug resistance-related genes in the antimony-resistant Leishmania isolate L_HCZ. The new Leishmania isolate L_HCZ has strong virulence and strong drug resistance, which should be taken seriously by relevant health departments and scientific researchers.This study performed genome-wide resequencing and phylogenetic analysis of five isolates from the