Streptococcus pyogenes produces eight different bacteriocins , only one bacteriocin of Streptococcus agalactiae  and one of Streptococcus dysgalactiae subsp. equisimilis  has been described. Expression of class I bacteriocins is regulated by a two-component system, typically with autoinduction by the bacteriocin itself. In contrast, a separate quorum sensing system regulates expression of class II bacteriocins. Both identified class III bacteriocins are plasmid-encoded and regulation has not been elucidated.Beta-hemolytic streptococci cause a variety of infectious diseases associated with high morbidity and mortality. A key factor for successful infection is host colonization, which can be difficult in a multispecies environment. Secreting bacteriocins can be beneficial during this process. Bacteriocins are small, ribosomally produced, antimicrobial peptides produced by bacteria to inhibit the growth of other, typically closely related, bacteria. In this systematic review, bacteriocin production and regulation of beta-hemolytic streptococci was surveyed. While  Bacteriocins are small antimicrobial peptides or proteins of bacterial origin. They inhibit the growth of other, often closely related, bacteria, resulting in a colonization advantage of the producer . The mosblp) region in Streptococcus pneumoniae. The peptide pheromone BlpC is exported via the ATP-binding cassette transporter BlpAB and sensed by the histidine kinase BlpH, which phosphorylates the transcription activator BlpR [blp and bacteriocin-genes. Secretion of BlpC is induced by the competence-stimulating peptide CSP and couples bacteriocin production to competence [blp locus is highly variable and at least 16 bacteriocin encoding genes have been described in connection to the blp locus of S. pneumoniae [Streptococcus mutans, Streptococcus thermophilus, and Streptococcus salivarius [Regulation of bacteriocin expression is often dependent on cell density ,10, whictor BlpR . Phosphompetence . The blpeumoniae ,16. An alivarius ,18,19,20In regulators of bacteriocin production, cross-reactivity of the autoinducing peptide between different species can occur . For claStreptococcus pyogenes , Streptococcus agalactiae , and Streptococcus dysgalactiae subsp. equisimilis (SDSE). These species cause a variety of infectious diseases and are associated with high levels of morbidity and mortality. Colonization often precedes streptococcal infections and the production of bacteriocins provides a colonization advantage for the producing organism. Understanding the determinants of streptococcal colonization may thus provide the knowledge required for interfering with the development of streptococcal infections. Several bacteriocins of S. pyogenes have been described already. In contrast, only very few aspects are known about bacteriocin production in SDSE and S. agalactiae. An overview is given in Beta-hemolytic streptococci relevant for human infections mainly comprise S. pyogenes. Bacteriocin production of S. pyogenes was first described in 1971 with the identification of Streptococcin A-FF22 [scnM), transport (scnT), immunity (scnFEG), and regulation (scnKR) of the bacteriocin are present in the A-FF22 gene cluster. Two structural genes with scnA and scnA’ are also part of the scn gene cluster [scnA’ is however unclear since no actual peptide appears to be produced. Pore formation and the subsequent disruption of the membrane potential leads to the death of sensitive target bacteria [S. pyogenes strains [S. pyogenes background, a minor variant of A-FF22 exists, Streptococcin A-M49. It is encoded by two slightly different structural genes, scnA’ and scnA´´, while the mature bacteriocin A-M49 is identical to mature Streptococcin A-FF22 [ScnA´ has the same aa sequence as Streptococcin A-FF22, whereas scnA´´ differs in four aa of the leader peptide sequence. Streptococcin A-M49 is produced by 33% (7 of 21) of M-type 49 strains under anaerobic conditions and shows the same spectrum of antimicrobial activity as A-FF22 [scnA-like gene can furthermore be found in S. salivarius, S. dysgalactiae and S. macedonicus. Regulation of Streptococcin A-FF22 is dependent on a two-component system formed by the response regulator gene scnR and the histidine kinase gene scnK. An inactivation of one of these genes leads to a loss of Streptococcin A-FF22 production [Staphylococcus citreus, Micrococcus spp., Bacillus spp., Corynebacterium spp., and various streptococci, including S. pyogenes, Streptococcus. agalactiae, Streptococcus equi, and Streptococcus cremoris [So far, eight different bacteriocins have been identified and characterized for n A-FF22 . This 26 cluster . The rolbacteria . Further strains . In a din A-FF22 . ScnA´ hs A-FF22 . In addioduction . A-FF22 cremoris .S. pyogenes. It self-regulates its expression via QS [SrtA, the structural gene for Streptin, is part of a 10-gene operon [srtBC), regulation (srtRK), and transport and immunity . The genes srtRK appear to constitute a two-component system that is activated by Streptin [S. pyogenes strains. However, only 10 of these strains were able to produce active Streptin [srt locus showed mutations, but several strains had an intact srt locus without producing Streptin. Concerning its spectrum of antibacterial activity, the bacteriocin inhibits the growth of S. pyogenes strains and Micrococcus luteus.Another lantibiotic, called Streptin, has only been described for n via QS . Two fore operon . SimilarStreptin . The genStreptin . In someS. pyogenes is Salivaricin A. It is a type IIA lantibiotic that was first described in S. salivarius, where it can be found in approximately 10% of the strains [S. pyogenes, Salivaricin A seems to be exclusively expressed by M4-isolates, even though the majority of analyzed strains contain the sal locus [S. dysgalactiae and S. agalactiae [salA1-5) [salA is autoinduced and all genes necessary for quorum sensing  as well as for modification  and immunity  are part of one gene cluster [S. salivarius is active against S. pyogenes, M. luteus, S. pneumoniae and Corynebacterium spp. [S. pyogenes has not been investigated.A third lantibiotic expressed by  strains . In S. pal locus ,41. The  cluster ,29,30. A cluster . Even th cluster . Salivarium spp. . To our S. pyogenes, class II bacteriocins are regulated by an independent quorum sensing mechanism. In this context, only the streptococcal invasion locus (sil) has been described. The sil locus was first identified by Hidalgo-Grass and has been demonstrated to be important for the virulence of S. pyogenes [S. pneumoniae, which is linked to bacteriocin production [sil locus consists of a two-component system (SilAB), an autoinducing peptide (SilCR), and an ATP-binding cassette transporter system (SilDE) [In contrast to class I bacteriocins of pyogenes . Furtheroduction ,43. The  (SilDE) . The hisS. pyogenes isolates contain a sil system, only 9% seem to have a functional system, since the other strains carry relevant mutations of this locus [sil originally provided an advantage to S. pyogenes strains for survival in a polymicrobial environment, but degenerated during adaptation to the host [sil may be beneficial. Strains carrying an incomplete sil locus can still produce immunity proteins, which provide protection against bacteriocins of competitor strains. Essential in this context is an intact sensing system (SilAB) enabling a reaction to SilCR present in the environment.While 16% of clinical is locus . One posthe host . Howeversil region, two class II bacteriocins have been described. Armstrong et al. investigated the bacteriocin (spb) genes of S. pyogenes strain MGAS8232 [sil system does not seem to be functional. SpbM and spbN encode putative bacteriocins, which share homologies to blpMN of S. pneumoniae. Both peptides have a double-glycine-leader peptide, which is necessary for export of the mature peptide [S. pyogenes strains as well as for Streptococcus dysgalactiae, Streptococcus uberis, Micrococcus luteus, and Lactococcus lactis.Adjacent to the MGAS8232 . In this peptide . Further peptide . When tesil were described by Hertzog et al., Blp1 and Blp2 [S. pyogenes strain JS12, which, in contrast to other S. pyogenes strains, encodes a functional sil system. Hallmark traits of class II bacteriocins including a double-glycine-leader peptide and a GxxxG motif can be detected in the peptides Blp1.1 and Blp1.2 that are encoded in the blp1 region. These two peptides work as a two-component bacteriocin (Blp1). Another functional bacteriocin Blp2 is encoded in the blp2 region. Both of these bacteriocins are produced by the strain J12 and inhibit other S. pyogenes strains. Analyzing publicly available genomes of S. pyogenes strains harboring a sil system revealed that putative bacteriocin genes are commonly found adjacent to sil. Bioinformatic tools are available to efficiently identify and characterize these bacteriocins [A second and third class II bacteriocin encoded by a genetic locus adjacent to and Blp2 . The stueriocins .S. pyogenes strains [S. pyogenes strains but targets several other Gram-positive bacteria such as Micrococcus luteus, Lactococcus lactis, Listeria spp., Bacillus megaterium, and Staphylococcus simulans. Due to structural similarities of Streptococcin A-M57 to Dysgalacticin (see SDSE), a similar mode of action, including impaired glucose uptake and disturbed membrane potential, is proposed.A class III bacteriocin designated Streptococcin A-M57 is exclusively produced by M-type 57  strains ,36,48. Ssil locus was first described in S. pyogenes, it is much more prevalent in SDSE with 82% of the strains harboring this genetic region [S. pyogenes, a class II bacteriocin blpM gene cluster can be found adjacent to this locus. However, the functionality of these genes has not been investigated. Nevertheless, at a descriptive level, there are reports of SDSE showing inhibitory activity against other streptococci and Corynebacterium spp., but no link to known bacteriocins or bacteriocin genes has been established [While the c region . Like inablished ,51,52.S. pyogenes. It is a 21.5kDa large protein encoded on an indigenous plasmid [sec dependent pathway, as described for Zoocin A [One bacteriocin of SDSE has been characterized at a functional level. It is Dysgalacticin, which belongs to the class III bacteriocins and shows similarities in secondary structure to Streptococcin A-M57 of  plasmid . Of its Zoocin A .S. pyogenes, leading to a starvation of the bacterial cell [dysA, the gene dysI, encoding an immunity factor, is found [S. pyogenes strains.Dysgalacticin was shown to target the phosphoenolpyruvate-dependent glucose and mannose phosphotransferase system (PTS) of ial cell . Furtheris found . DysI isS. agalactiae isolates showed any inhibition of other bacteria. However, characteristics of a class III bacteriocin  and of a small, heat-resistant bacteriocin were described, but in the years following the initial observation, no further molecular genetic studies were reported on these findings.In 1983, the production of antimicrobial inhibitory substances was described for Group B streptococcal strains, isolated from humans and animals . All in S. agalactiae [S. agalactiae-type strain ATCC 13813 [agaBC), transport (agaT), immunity (agaI and agaFEG), regulation (agaRK), and leader peptidase (agaP) are present within the gene cluster. Agalacticin shows antimicrobial activity against several Gram-positive species . However, regulation of the expression of Agalacticin has not yet been investigated. The similarity to the Nisin gene cluster as well as the presence of lanRK, which encodes a putative two-component regulatory system, point in the direction of a quorum sensing system autoregulated by Agalacticin. Another bacteriocin described for S. agalactiae is Nisin P, which was identified in S. agalactiae strain DPC7040 [Just recently, a first detailed molecular description of a bacteriocin was provided for alactiae . AgalactCC 13813 . It show DPC7040 . Nisin PS. pyogenes a variety of bacteriocins have been described, only a few are reported for SDSE and S. agalactiae. The identified bacteriocins are mainly active against other Gram-positive bacteria and closely related streptococcal species, providing a colonization advantage to bacteriocin producers. Understanding the regulation of these peptides might help in elucidating the role they play in microbial communities and their importance for host colonization.Beta-hemolytic streptococcal species are part of polymicrobial environments that require competing with the commensal microbiota. Interestingly, while for


Diospyros kaki cv. Luotiantianshi is a rare germplasm of Diospyros Linn in the world. In this study, we generated the complete chloroplast (cp) genome of D. kaki cv. Luotiantianshi. The complete cp genome was 157,773 bp in length, containing a large single copy region (LSC) of 87,066 bp, a small single copy region (SSC) of 18,529 bp, and two inverted repeat (IR) regions of 26,089 bp. The new sequence has a total of 131 genes, including 86 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Further, phylogenetic analysis showed that the D. kaki cv. Luotiantianshi has a close relationship with Diospyros kaki. This study provides important information for future evolution, genetic and molecular biology studies of Diospyros. Diospyros kaki Thunb 1780 cv. Luotiantianshi, also is called Chinese PCNA (pollination-constant non-astringent) type persimmon (Diospyros kaki Thunb 1780), only distributes in Luotian and Macheng of Hubei province. Diospyros kaki cv. Luotiantianshi, which is morphologically and genetically different from Japanese pollination constant and non-astringent (JPCNA) or other persimmons by natural de-astringency model of fruits  technologies and a phylogenetic analysis of this species and its relatives was carried out.http://risfcaf.caf.ac.cn/, contact person: Bang-chu Gong, email: gongbc@126.com) under the voucher number: MBLS-LTTS84. The DNA libraries was constructed and sequenced on Illumina HiseqXten platform  for 150 bp paired-end sequencing by Biodata Biotechnologies Inc. . The cp genome was assembled using a de novo strategy according to the work of Xu et al.  using CTAB method  and larger than Diospyros oleifera Cheng 1935  , a small single-copy region , and two inverted repeats . The total GC content of new chloroplast genome was 37.4%, and while it was 43.1% of IRs, which was higher than LSC and SSC regions . The new sequence has 86 protein-coding genes, was one less than that in D. kaki (87), 37 tRNA genes, and 8 rRNA genes.The size of the complete chloroplast genome of D. kaki cv. Luotiantianshi and other 14 species in Diospyros. Meanwhile, two species from the Actinidia (Actinidia arguta Sieb. & Zucc 1864 and Actinidia chinensis Planch 1847) were used as outgroups. The sequences were aligned using MAFFT version 7 (Kazutaka and Standley D. kaki cv. Luotiantianshi has a close relationship with D. kaki under 100% bootstrap support values. The complete cp genome sequence of D. kaki cv. Luotiantianshi can be great benefit to further study on its population genetics and molecular breeding.A phylogenetic analysis was carried out using whole cp genome sequences of


Sporothrix schenckii is a worldwide-distributed thermally dimorphic fungus, which usually causes a subacute to chronic infection through traumatic implantation or inoculation of its infectious propagules. The fungus encompasses a group of phylogenetically closely related species, thus named the S. schenckii complex, of which S. schenckiisensu stricto and S. brasiliensis are main causative species of sporotrichosis. Owing to a multifaceted molecular dynamic, the S. schenckii complex can switch between the mycelium and the yeast form. This characteristic along with a varying cell wall composition account for significant species-specific differences in the host range, virulence, and susceptibility to antifungal drugs. While culture remains the gold standard to diagnose sporotrichosis, polymerase chain reaction (PCR) or matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry-based methods have become an essential for accurate species identification in many clinical laboratories. If directly applied on tissue samples, molecular methods are helpful to improve both sensitivity of and time to the etiological diagnosis of sporotrichosis. This mini-review aims to put together the old and new knowledge on the S. schenckii complex biology and identification, with particular emphasis on the laboratory diagnosis-related aspects of disease. Sporothrix schenckii (sensu lato) really consists of phylogenetically closely related species, which are embedded in the order Ophiostomatales, class Pyrenomycetes, division Ascomycota within the Fungi kingdom [S. schenckii complex encompasses four clinically relevant species  and two species  occasionally isolated from humans. Although Sporothrix schenckii (sensu lato) is regarded as a cosmopolitan fungus, most sporotrichosis cases occur in tropical and subtropical areas from Latin America, Africa, and Asia [S. schenckii sensu stricto and S. globosa are distributed throughout the world, S. brasiliensis has emerged in the Rio de Janerio state of and is restricted to Brazil [Originally known as the only causative agent of human and animal sporotrichosis , the the kingdom . Accordi kingdom ,4, the Sand Asia ,5. Whereo Brazil ,5. Aparto Brazil ,7,8.S. schenckii sensu stricto and S. globosa, a zoonotic transmission by which S. brasiliensis spread through deep scratches and bites from infected felines (cats) has been associated with more severe clinical presentations in humans [Sporotrichosis is a subacute or chronic implantation (formerly subcutaneous) mycosis that follows the traumatic inoculation of infectious propagules , released from the fungus in the environment, into the skin, mucosal, or osteoarticular body sites . Consistn humans .Sporothrix species identified was S. brasiliensis [S. schenckii sensu stricto about 3.8–4.9 million years ago, which suggests that a recent event of speciation has occurred in the genus Sporothrix.If not limited to skin (cutaneous-lymphatic or cutaneous-fixed sporotrichosis), sporotrichosis manifestations include cutaneous-disseminated, disseminated , and extracutaneous forms , which are prominently diagnosed in immunocompromised (human immunodeficiency virus (HIV)-AIDS, chronic alcoholism, diabetes, hematological cancer, or transplantation) patients . Primaryiliensis  that, aciliensis , divergeS. schenckii complex, to emphasize relevant aspects of biology and identification of human and animal sporotrichosis agents.In this review, we summarize recently or less recently published findings on the Sporothrix species cluster in a derived clade around S. schenckii sensu stricto, resulting in at least four “cryptic” species  within the S. schenckii complex [By means of molecular taxonomic markers as discussed below, it has become clear that the main pathogenic  complex . As alre complex ,17.S. schenckii complex as a whole but not that of the single species within the S. schenckii complex [Microscopic examination of potassium hydroxide-pretreated clinical samples obtained from skin lesions (pus and exudates), sputum, or synovial fluid from patients with sporotrichosis allows  to visualize yeast forms of 2–6 µm in diameter. Despite a slight Gram-staining positivity, yeast cells are usually observed close to giant multinucleated or polymorphonuclear cells . Otherwi complex .Sporothrix-specific antibody detection in the sera from Sporothrix-infected patients using agglutination and immunoenzymatic  assays [S. schenckii rabbit serum. The resulting antigen, thus named SsCBF (S. schenckii Con A-binding fraction), was specifically recognized by IgG antibodies present in patients’ clinical samples [S. schenckii strains showed that the antigen isolated from strain 1099-18 provided an accurate means for the serodiagnosis of sporotrichosis [S. schenckii complex [Immunological diagnoses of sporotrichosis rely on the ) assays . To deve samples . Furtherrichosis , serologrichosis , whereas complex .S. schenckii isolates do not belong to the same species. The authors described four S. schenckii (sensu stricto)-related species  through a combination of phenotypic and genetic features and proposed the partial calmodulin (CAL) gene as a molecular tool to differentiate  closely related species. However, Zhou et al. [CAL, translation elongation factor-1 (TEF1), and -3 (TEF3), to investigate the phylogeny of 99 clinical and 36 environmental strains belonging to the main pathogenic Sporothrix clade species . Multilocus sequence data were compared with amplified fragment length polymorphism (AFLP) genotyping. The four species of the clade were recognized, and the clade comprised nine subclusters. The combined tree based on CAL, TEF-1, and TEF-3 of 135 strains had a topology similar to that of the ITS tree. However, the authors noticed that the genes analyzed differed in PCR performance, whereas CAL showed a small intraspecific variability, which makes CAL an optimal diagnostic marker [CAL gene sequences identified specific PCR-based markers for clinically relevant members of the Sporothrix genus  and its relative Ophiostoma genus (O. stenoceras). The assay proved to be effective in identifying etiological agents of sporotrichosis, detecting Sporothrix DNA in tissues of infected animals, and using fungal conidia as a source of genomic DNA for PCR. Compared to PCR sequencing or other available molecular assays for S. schenckii complex identification [CAL gene sequences from the Sporothrix complex clinical clade isolates retrieved from GenBank . Sequence alignment using MEGA7 software  resulted in a tree showing that S. schenckii sensu stricto strains  clustered separately from S. brasiliensis , S. globosa , or S. luriei (ATCC 18616T) strains.In 2007, Marimon et al.  supporteu et al.  and Rodru et al.  showed tu et al.  used fouc marker . Accordic marker  using CAfication , the PCRfication  yielded CAL gene of clinically relevant Sporothrix species, such as S. globosa, S. schenckii sensu stricto, and S. brasiliensis. The diagnostic performance of the novel assay was evaluated in comparison with culture and species-specific PCR. Regarding clinical samples, the positive detection rates by culture, species-specific PCR, and the multiplex real-time PCR assay were 87.9% (29/33), 39.4% (13/33), and 93.9% (31/33), respectively. Regarding spiked samples, the positive detection rates were both 100% for S. schenckii sensu stricto and S. brasiliensis. Based on these findings, the authors claimed that the assay might transform into a new commercial kit for the diagnosis of infections caused by these clinically relevant Sporothrix species. Concomitantly, Hayashi et al. [Sporothrix-specific nested PCR assay for diagnosing cutaneous sporotrichosis from formalin-fixed and paraffin-embedded (FFPE) tissues. The authors found that all (100%) of 52 samples from patients with a positive culture result tested positive by PCR, whereas 58 (98.3) of 59 samples from patients in the control group tested negative by PCR. Thus, the assay showed an excellent sensitivity (100%) and specificity (98.7%). Finally, de Carvalho et al. [Sporothrix sequences, which resulted in six primer pair combinations to be tested in vitro. Three combinations provided the best diversity indices and lowest error rates. In general, the DNA fingerprint assay described in that study enabled the authors to assess the degree of intraspecific variability among pathogenic Sporothrix species, providing insights into their ecology and evolution [Recently, searching for faster and easier PCR-based methods culminated in the development of a multiplex real-time PCR assay by Zhang et al. , who desi et al.  exploredo et al.  used an volution .S. schenckii infections. In this context, Oliveira et al. [Sporothrix complex, which had previously been characterized by morphology . The authors adopted a two-step approach that has become a standard for studies dealing with the MALDI-TOF MS methods’ development for diagnostic applications. First, spectra from six isolates  were used to construct a reference database according to minimal adjustments in the manufacturer’s guidelines. Second, the database was challenged with spectra from remaining 64 isolates . Thus, the MALDI-TOF MS method distinguishing strains of S. brasiliensis, S. globosa, S. mexicana, S. schenckii, S. luriei, and S. pallida allowed the authors to identify all clinical and environmental isolates at the species level. This was in accordance with partial CAL gene sequence analyses performed on the same isolates [Despite still depending on fungal culture , matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry has become in many clinical microbiology laboratories a powerful and indispensable tool for the etiological diagnosis of filamentous fungal infections ,28, incla et al.  reportedisolates .S.schenckii complex was used to identify S. brasiliensis isolated from an immunocompetent patient’s subconjunctival infiltrative lesion. The fungus grown on SDA was identified as Sporothrix species by morphology , whereas its MALDI spectrum profile matched that of S. brasiliensis [Similarly, but more recently , MALDI-Tiliensis .Taken together, these findings suggest the potential of complementary (self-constructed) in-house databases that, to date, support the MALDI-TOF MS-based fungal identification at least in large clinical microbiology laboratories . FurtherS. schenckii sensu stricto (hereafter referred to as S. schenckii) and S. brasiliensis are two  fungal pathogens phenotypically similar to the human/animal pathogenic Onygenales  but phylogenetically closely related to plant associated Sordariomycetes . A comparative genomic analysis of the two Sporothrix species with the tree-pathogenic fungus G. clavigera showed S. schenckii and S. brasiliensis genomes with a G + C content being one of the highest within Ascomycota (62% versus 53.4% in G. clavigera) [Sporothrix-restricted genes, whereas transport or metabolism genes showed a higher conservation degree than those from other Ascomycota. Remarkably, comparing S. schenckii and S. brasiliensis with fungal species from other Sordariomycetes or dimorphic fungal pathogens  unveiled the expansion of some protein domains and the lack of (polysaccharide lyase) genes associated with plant biomass decay [Sporothrix lineage, which allowed S. schenckii or S. brasiliensis (close relatives of plant associated Sordariomycetes) to adapt to mammalian parasitism. In this context, dimorphism would be a congruent evolution case, reinforcing the role of this morphogenetic shift into fungal pathogenesis [Despite being evolutionarily divergent species, avigera) . Of 4788ss decay . Accordiss decay , these fogenesis .Sporothrix species [Sporothrix species are closely related to Ophiostoma species, one study using a gene family evolutionary analysis revealed that virulence, stress response, protease, cell wall composition, or transporter genes were expanded in Sporothrix species, supporting the genomic evolution of pathogenic Sporothrix species from Ophiostoma species [S. brasiliensis, S. schenckii, S. globosa, and S. luriei had significantly smaller genome sizes than other environmental and clinical Sporothrix species. This contraction mostly involved the genes associated with plant biomass decay such as CAZyme (Carbohydrate Active enZyme) or peptidase encoding genes, as well as the genes associated with loss of pathogenicity and reduced virulence [Sporothrix species from a saprobic life in plant materials to a pathogenic life in mammals but also an increase in their pathogenicity during the evolution [Sporothrix species [Very recently, comparative studies extended our knowledge on phylogenomics and/or genomic evolution, as well as on pathogenicity/virulence-associated genes, of pathogenic  species ,33. When species . Another species , despite species .Sporothrix species to generate two types of vegetative cells, i.e., those that are either hyphal or yeast in morphology, which correspond to the environmental (observable at 30 °C) and the host (observable at 37 °C) form, respectively. In the first form, hyaline, septate hyphal cells produce several morphologically different asexual conidia, some of which are hyaline, oval or elongate, and originate individually or in small clusters  from sympodial conidiogenous cells. Alternatively, conidia can be thick-walled, darkly pigmented, and usually produced individually. In the second form, yeast cells are round, oval, or lengthened (hence termed “cigar-shaped”) and divide by budding. While dimorphism is essential for virulence in the mammalian host [Sporothrix species may present different levels of pathogenicity, which in turn depend on the interaction of the fungus with the innate and adaptive immune response cells through which the mammalian host controls and/or eradicates fungal pathogens [Sporothrix species [S. schenckii and S. brasiliensis showed that the two Sporothrix species differed regarding the ability to elicit cytokine production by human peripheral blood mononuclear cells (PBMCs). Three S. schenckii morphologies elicited higher levels of pro-inflammatory cytokines than S. brasiliensis whereas S. brasiliensis yeast-like cells and germlings elicited a higher level of the anti-inflammatory cytokine IL-10, perhaps accounting for the apparently lower pathogenicity of S. schenckii compared to S. brasiliensis. Additionally, dectin-1 was a key receptor for the S. schenckii elicited cytokine production whereas was dispensable for the cytokine production elicited by S. brasiliensis germlings [As for other thermally dimorphic fungi , dimorphian host , differeathogens . Consist species , Martíne species  using coermlings .S. schenckii morphological (yeast-to-hypha) shift, Giosa et al. [S. schenckii yeast-to-hypha shift. Furthermore, a transcriptome sequencing analysis of 48 h induced yeast and mycelial S. schenckii stages allowed Zheng et al. [S. schenckii dimorphic transition.To elucidate the transcriptional networks involved in the a et al.  used RNAa et al. . Thus, ng et al.  to map tSporothrix cell wall components’ interaction with human PBMCs showed that S. schenckii sensu stricto and S. brasiliensis share a similar cell wall composition but display morphology-dependent changes. Interestingly, in S. schenckii sensu stricto, chitin  content was significantly different across the three morphologies  studied by the authors. Conversely, in S. brasiliensis, β-glucan  content was significantly lower in yeast-like cells compared to germlings [S. schenckii and S. brasiliensis, according to ultrastructural data by Lopes-Bezerra et al. [S. brasiliensis cell wall layers were found to be thicker than those of S. schenckii were, and this finding correlated with increased contents of chitin and rhamnose cell wall components [Sporothrix species are lacking the α-glucan component in their cell wall. Interestingly, in the same study [According to well-established fungal pathogenesis models, the cell wall is the place where pathogen-associated molecular patterns interact with pattern recognition receptors (PRRs) on innate immune cells. One of these PRRs is dectin-1. The study by Martínez-Álvarez et al.  which inermlings . In botha et al. , a bi-lamponents . Unlike me study , Lopes-BS. schenckii complex cell wall contains a glycoconjugate composed of rhamnose, mannose, glucuronic acid, and proteins, named peptidorhamnomannan (PMR). This cell wall component harbors Sporothrix-specific immunogenic epitopes [S. brasiliensis clinical isolates tested by Castro et al. [S. schenckii complex pathogenicity. While the PRM carbohydrate moiety has been extensively studied, only recently García-Carnero et al. [S. schenckii virulence. When Galleria mellonella larvae were inoculated with S. schenckii yeast-like cells pre-incubated with anti-rHsp60 or anti-rPap1 antibodies, larvae displayed increased survival rates compared to control groups [In addition to β-glucans and chitin, the epitopes ,40 and, o et al.  in a muro et al.  exploredl groups .Sporothrix species produce dihydroxynaphthalene-melanin (DHN-melanin) in conidia and yeast cells as well as L-dihydroxyphenylalanine (L-DOPA), which may enhance melanin production on both these structures and hyphae. To corroborate the role of melanin in the virulence of the S. schenckii complex, Almeida-Paes et al. [S. brasiliensis, S. schenckii sensu stricto, and S. globosa can produce a melanoid pigment, named pyomelanin, in the presence of tyrosine, in both mycelial and yeast stages. Thus, pyomelanin-producing fungal cells were more resistant to nitrogen-derived oxidants and to UV light. Using a murine model of disseminated sporotrichosis, Texeira et al. [S. schenckii yeast cells after they had been cultured on two media differing in their brain (and heart) infusion’s proportions. Such a difference allowed S. schenckii to modulate melanin expression on the surface of yeast cells and, consequently, virulence in mice. Interestingly, yeast cells grown in a brain-infusion poor medium supplemented with L-DOPA recovered their virulence, underlining rich brain-infusion medium’s phenolic compounds as a substrate for the melanin biosynthesis pathway.Similar to other pathogenic fungi, s et al.  showed ta et al.  comparedS. brasiliensis, extracellular vesicles (EVs), i.e., lipid bilayers exhibiting an array of biological properties, may aid the fungus to infect the host. In one study, Ikeda et al. [S. brasiliensis EVs to modulate dendritic cells (DCs) and to control the infection in vivo. The study showed that EVs induced an increase in the phagocytic index and fungal burden in DCs, whereas BALB/c mice inoculated with a high concentration of EVs before subcutaneous infection showed an increase in the fungal burden and lesion diameter at 21 days after infection. In another study [S. brasiliensis, S. schenckii, and S. globosa allowed to identify 247 proteins, of which 137 as differentially expressed. Most of differences between S. brasiliensis and S. schenckii or S. globosa regarded the amino acid metabolism and cell wall remodeling, along with downregulation of glycolytic enzymes, possibly explaining a more virulent behavior of S. brasiliensis compared to S. schenckii and S. globosa [S. brasiliensis is more resistant to different oxidants, such as H2O2 and menadione, than S. schenckii, perhaps because of mutations found in the S. brasiliensis Hog1 stress activated protein kinase. These mutations would allow S. brasiliensis to better adapt to the toxic reactive oxygen species generated by the host’s immune system.In a et al.  used S. er study , yeast p globosa . FinallyS. schenckii complex, Sánchez-Herrera et al. [S. schenckii biofilm contains mannose-rich glycoproteins, carbohydrates, lipids, and nucleic acids. However, S. schenckii biofilm’s ECM has greater carbohydrate and less protein amounts compared to those in the ECM from biofilms formed by Candida species , which are the most common pathogenic fungal species in clinical settings [In a very recent analysis of biofilm formation in the a et al.  tried tosettings .S. schenckii biofilm ECM may not only contribute to the biofilm structure integrity but also to the biofilm-associated antifungal resistance in S. schenckii infections [Sporothrix species have increasingly been reported to be resistant to amphotericin B (AMB), azoles, and echinocandins [S. schenckii sensu stricto, S. brasiliensis, S. globosa, and S. luriei), showed that AMB, 5FC, CSF had no antifungal activity against any Sporothrix species. Conversely, ITZ and PCZ were moderately effective against S. schenckii sensu stricto and S. brasiliensis. Interestingly, studying a panel of genetically diverse clinical Sporothrix isolates  revealed the presence of genotypes (haplotypes) within each (clade of) Sporothrix species that were linked to the antifungal-susceptibility profiles shown by the isolates from each species [Consistent with previous studies , a substfections . In thisocandins ,51,52. Tocandins , determi species .S. schenckii complex isolates to form biofilms in vitro as well as the growth kinetics, morphology, and antifungal susceptibility of these biofilms. The authors assessed the effects of exposure to antifungal drugs  at MIC against planktonic cells as well as at 10 × MIC and 50 × MIC against biofilm cells. When confirming a strong biofilm formation by all S. brasiliensis (n = 10), S. schenckii sensu stricto (n = 2), S. globosa (n = 2), and S. mexicana (n = 4) isolates, Brilhante et al. [S. schenckii complex. Only for some antifungal drugs, exposure of S. schenckii biofilms to planktonic MIC values allowed to observe anti-biofilm activity, which yet was more evident at 50 × MIC values [In 2018, Brilhante et al.  investige et al.  also fouC values .Sporothrix species. While melanin has been associated with lower AMB susceptibility and with protection against the effects of terbinafine (which may be used instead of AMB for sporotrichosis therapy), chromosomal polymorphism, amplifying antifungal resistance genes, may help Sporothrix species to overcome the selective pressure exerted by antifungal drugs [Sporothrix isolates. Additionally, in silico analysis performed by Matowane et al. [As recently reviewed , melaninal drugs . Howevere et al.  showed tSporothrix schenckii complex [Sporothrix isolates for antifungal susceptibility in vitro using the Clinical and Laboratory Standards Institute (CLSI) reference methods [S. globosa isolates to echinocandins , 5FC, azoles , and AMB. The authors found that ITZ was the most active antifungal drug against S. globosa, whereas the yeast phase of the same isolate was more susceptible than that of the mycelium phase, leading to consider the lower content of melanin present in the fungus’ yeast phase after repeated cultures as a possible explanation [According to recently established epidemiological cut-off values , non-wil complex . In this complex ,52 teste methods ,58. It i methods . However methods , a CLSI-lanation .Sporothrix species has represented [S. brasiliensis, a one-health approach aimed to optimize health outcomes, through collaborative efforts on the inter-relationships among humans, animals, plants, and the environment [The threat of emerging and re-emerging pathogenic resented  and so fresented  a continironment , should S. schenckii complex as well as the genome sequences of single species within the complex will permit to develop new identification or molecular typing tools that will improve the specificity of human or feline sporotrichosis diagnosis. Meanwhile, refinement of existing molecular laboratory methods is expected to embrace the possibility of targeting both virulence and antifungal resistance of Sporothrix species, two features thought to cause the selection and clonal expansion of resistant isolates during epidemics. From the therapeutic standpoint, emergence of ITZ-resistant S. schenckii complex isolates has prompted the research on new antifungal compounds such as essential oils. In one very recent study [Rosmarinus officinalis Linn.) proved to protect rats from the fungal spread to systemic organs, thus providing new options for treatment of sporotrichosis.From the diagnostic standpoint, elucidating the fine structure of the surface in both the morphological stages of nt study , the essIn summary, great future advances in this important fungal topic will make the prospect of significantly reducing the burden of sporotrichosis for human and animal health a not-so-distant reality for us.


There are several examples using SARS-CoV-2 genomes and Dengue virus serotype 1 E-genes sequence.The worldwide eruption of coronavirus disease 2019 (COVID-19) that began in Wuhan, China in late 2019 reached 10 million cases by late June 2020. In order to understand the epidemiological landscape of the COVID-19 pandemic, many studies have attempted to elucidate phylogenetic relationships between collected viral genome sequences using haplotype networks. However, currently available applications for network visualization are not suited to understand the COVID-19 epidemic spatiotemporally due to functional limitations that motivated us to develop Haplotype Explorer, an intuitive tool for visualizing and exploring haplotype networks. Haplotype Explorer enables to dissect epidemiological consequences via interactive node filters and provides the perspective on infectious disease dynamics depend on regions and time, such as introduction, outbreak, expansion, and containment. Here, we demonstrate the effectiveness of Haplotype Explorer by showing features and an example of visualization. The demo using severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes are available at  To control infectious diseases, it is important to quickly identify emerging infection clusters before they become critical issues. Many applications have been developed to assist researchers understanding the latest epidemiology. Indeed, the recent intensification of the coronavirus disease 2019 (COVID-19) pandemic, which began in late 2019 in Wuhan, China, has prompted development of new software to support investigations of this virus. For example, Nextstrain  nodes that can be dynamically filtered with metadata by complex search queries, (2) nodes can be indicated by real-time pie charts, which reflect sample size and content proportions at a given time span, and (3) creating interactive distribution files which require no external software installation. Hence, we endeavored to develop Haplotype Explorer, a specialized network viewer which assists onsite actions against emerging pathogens. Haplotype Explorer is a novel platform for network analysis displaying the network data from small scale to large scale and helping users to dissect the network by complex metadata filters. It also can export not only figure at a certain timepoint but continuous sections for constructing a movie to help people understanding the expansion of the pathogen at a glance.Haplotype Explorer is a JavaScript application executable in web browser, so it does not require uploading data to an external web server . This ale.g., removing low-quality sequences, such as those containing spaces, gaps, degenerated bases, and ambiguous collection dates  using seqkit   on June https://github.com/TKSjp/HaplotypeExplorer/. Supplemental Material available at figshare: https://doi.org/10.25387/g3.14349563.All DNA sequences used in this study were downloaded from the GISIAD. Source codes are available from the following URL: The primary feature of Haplotype Explorer is a vibrant and interactive visualization function utilizing D3.js . Users cThe epidemic context of the SARS-CoV-2 from January 1 to March 21 was visualized by Haplotype Explorer . We begaManuscript preparation: T.K.-S. M.K. Data analysis: T.K.-S., M.K., K.Y., and T.S. Data collection: K.I., M.H., and R.T.


Pseudomonas aeruginosa and Staphylococcus aureus. The bacterial factors that modulate this unique intracellular niche and its implications in contributing to resistance are discussed. We then briefly discuss some recent research that focused on the promises of boosting host immunity as a combination therapy with antimicrobials to eradicate these two particular pathogens. Finally, we summarize the importance of various strategies, including surveillance and vaccines, in mitigating the impacts of antimicrobial resistance in general.Globally, infectious diseases are one of the leading causes of death among people of all ages. The development of antimicrobials to treat infectious diseases has been one of the most significant advances in medical history. Alarmingly, antimicrobial resistance is a widespread phenomenon that will, without intervention, make currently treatable infections once again deadly. In an era of widespread antimicrobial resistance, there is a constant and pressing need to develop new antibacterial drugs. Unraveling the underlying resistance mechanisms is critical to fight this crisis. In this review, we summarize some emerging evidence of the non-canonical intracellular life cycle of two priority antimicrobial-resistant bacterial pathogens:  Infectious diseases remain one of the most pressing challenges faced by humanity today. The recently emerged severe acute respiratory syndrome coronavirus-2 (SARS–CoV-2), the causative agent of the ongoing pneumonia-like pandemic of coronavirus disease 2019 (COVID-19), has infected over 355 million and killed over 5.6 million people worldwide as of January 2022 ,5,6,7,8.Antimicrobial Resistance: Tackling a crisis for the health and wealth of nations”, which predicted that by 2050 there could be about ten million deaths per year due to AMR [The World Health Organization (WHO) declared AMR as one of the top 10 global public health threats facing humanity in 2019 ,14,15,16e to AMR . This ree to AMR . The deve to AMR , marked e to AMR . The broe to AMR . Mycobacterium, Salmonella, Chlamydia, Listeria, Shigella, Legionella, Brucella, and Rickettsia. Intracellular bacteria possess unique resistance strategies [Mycobacterium tuberculosis exemplifies this sophisticated strategy [Mycobacteria eventually establish latent infections in granulomas, clusters of infected macrophages, neutrophils, epithelioid cells, and foam cells that are encircled by the recruited T and B lymphocytes [In addition, a group of bacteria known as intracellular bacteria can gain entry to host cells, followed by bacterial manipulations of host cell biology to create a unique niche within the host cell environment as part of their infection cycles . Classicrategies . To trearategies , whereasrategies   patients [fections . Typicalatheters . P. aerupatients . P. aeruginosa to establish both acute and chronic infections. These virulence factors include but are not limited to phospholipase C, proteases, exotoxins, cytotoxins, flagella, pili, and secretion systems [P. aeruginosa utilizes five secretion systems: type 1, type 2, type 3, type 5, and type 6 [P. aeruginosa to deliver toxins directly into mammalian and bacterial target cells [P. aeruginosa’s intracellular life cycle as will be discussed below.Multiple virulence factors contribute to the ability of  systems . Proteind type 6 . The typet cells . Both thP. aeruginosa infections are difficult to treat and are becoming an increasing threat due to multiple antibiotic resistance mechanisms including intrinsic, acquired, and adaptive mechanisms. Intrinsic antimicrobial resistance features of P. aeruginosa include efflux pumps, low outer membrane permeability, and the synthesis of enzymes that are antibiotic inhibiting/inactivating [P. aeruginosa to produce biofilms plays a major role in infection and contributes to adaptive resistance; this form of resistance is particularly difficult to treat in infections [tivating . Acquiretivating . The abifections . P. aeruginosa that makes it difficult to treat is its striking strain-to-strain diversity. While the average genome size of P. aeruginosa ranges from 5.5 to 7 Mbp, the core genome consists of only 321 genes [P. aeruginosa genome contributes to its ability to evade antibiotics and cause a wide range of infections [P. aeruginosa infections.Another aspect of 21 genes ,42. The fections . While cfections ,44. TherP. aeruginosa is traditionally known to be an extracellular pathogen, there is increasing evidence that shows it can invade and survive within host cells. The ability to switch between being an extracellular and intracellular pathogen has been reported for other bacterial species and is a commonly used tactic to evade host defenses [E. coli can become an intracellular pathogen, allowing it to establish an intracellular reservoir in bladder epithelial cells, which makes eradication challenging [P. aeruginosa infections are already difficult to eradicate, the ability of P. aeruginosa to establish intracellular reservoirs poses additional complications for treatment. Therefore, understanding how P. aeruginosa invades, survives, and persists within host cells is crucial for eradication of infections. While defenses . For exallenging . Since PP. aeruginosa has been shown to aggregate in blebs or biofilm-like structures, alter host cytoskeletal elements, and be tolerant to antibiotics [P. aeruginosa has also been found intracellularly in multiple epithelial cell types including lung, corneal, and bladder, as well as phagocytic cells such as macrophages [P. aeruginosa to function as an intracellular pathogen are not well understood; however, the following sections will highlight some of the preliminary discoveries around potential intracellular invasion and survival mechanisms. Within host cells, ibiotics ,47,48,49rophages ,49,50,51P. aeruginosa possesses three type 6 secretion systems (T6SS) located on three different loci—they are named H1, H2, and H3, respectively [P. aeruginosa to deliver toxins to both prokaryotic and eukaryotic target cells [P. aeruginosa into eukaryotic cells; H2-T6SS mutants showed a 75% decrease in P. aeruginosa internalization into lung epithelial cells [P. aeruginosa prefers to enter injured and damaged cells from the basolateral surface and can subvert the cytoskeleton remodeling pathways to invade cells from the apical surface [P. aeruginosa binds to polarized epithelial cells, phosphatidylinositol 3-kinase (PI3K) is activated, resulting in the activation of protein kinase B/Akt (Akt) and the synthesis of phosphatidylinositol 1,4,5-triphosphate (PIP3) [P. aeruginosa’s ability to activate PI3K signaling and subsequently subvert cytoskeleton remodeling pathways was dependent on the H2-T6SS [clpV2) cocultured with lung epithelial cells resulted in decreased P. aeruginosa invasion and phosphorylation of Akt [ectively . The T6Set cells . The H2-al cells . The T6Sal cells . The H2-al cells . P. aeru surface . When P.e (PIP3) ,53. Sana H2-T6SS  , but when the stress dissipates, the wildtype phenotype is restored [As mentioned above, biofilm formation plays a critical role in lication . It has al cells . These bironment . Heterogironment . Heterogbiofilms . While trestored . Importarestored . The abirestored . Within restored . P. aeruginosa [P. aeruginosa cells treated with concentrations of ciprofloxacin 10 to 35 times the minimum inhibitory concentration resulted in a biphasic kill curve, where higher concentrations of the antibiotic resulted in increased numbers of tolerant cells [P. aeruginosa is developing persister cells in the intracellular environment [P. aeruginosa is developing persister cells in the intracellular environment. The idea that an intracellular pathogen could develop persister cells within the host is not unheard of: Salmonella typhimurium cells within macrophage vacuoles adopted a non-replicative state typical of persister cells, allowing it to achieve long term survival against host damage and antibiotics [A transient antibiotic tolerant phenotype has been observed in intracellular ruginosa ,48. Intrnt cells . This biironment . As wellironment . Garcia-ibiotics ,70. P. aeruginosa in the intracellular environment. However, information around specific mechanisms, such as how NF-kB signaling provides protection or how ExoS induces membrane blebbing, is still unknown. Even more questions arise around mechanisms underlying the development of intracellular biofilms and persister cells. What is clear is that P. aeruginosa possesses the ability to not only invade host cells but to persist within epithelial cells for extended periods of time, allowing for a potential reservoir of bacteria, contributing to the already difficult task of eradicating P. aeruginosa infection. There are multiple factors that allow for intracellular invasion and promote survival of Staphylococcus aureus is a Gram-positive commensal bacterial species that is well-recognized as a major human pathogen [S. aureus, with the human nose acting as the predominant reservoir [S. aureus is responsible for a wide spectrum of infections ranging from various soft tissue infections to invasive systemic infections [S. aureus is additionally recognized as a facultative intracellular pathogen: it is capable of entering host cells, replicating, and persisting in the host cells as part of its survival strategy [S. aureus avoid detection by professional phagocytes, evading host bactericidal defenses and detection by host pattern recognition receptors; it additionally offers concurrent protection from antibiotic therapies. Such factors impact patient recovery from staphylococcal infection while concomitantly increasing the severity of illness [S. aureus has been suggested to contribute to the often prolonged, chronic course and frequent relapse of severe infections, such as endocarditis and osteomyelitis [pathogen . Roughlyeservoir . S. aurefections . As of 2fections . Despitestrategy ,76,77. T illness . Intracemyelitis ,80,81.S. aureus invades a broad range of non-professional phagocytic cells (NPPCs) including epithelial/endothelial cells, osteoblasts, fibroblasts, and keratinocytes, amongst others [S. aureus is typically achieved by the well-documented zipper-type mechanism, “FnBP-Fn-α5β1 integrin-mediated uptake”, involving staphylococcal fibronectin-binding proteins A and B (FnBPA and FnBPB) [S. aureus binding to host cells, contributing to the endocytosis of the bacterium during cellular invasion. In the model described in S. aureus into keratinocytes at various stages of development [t others ,82. The d FnBPB) ,84. Varielopment . S. aureus virulence factors and host cell components that are involved in or mediate FnBP-independent cellular invasion by S. aureus. These mechanisms involve major staphylococcal proteins such as autolysin (Atl), α-hemolysin (HLA), clumping factor A (ClfA), clumping factor B (ClfB), iron-regulated surface determinant B (IsdB), lipoprotein-like lipoproteins (Lpls), serine-rich adhesin for platelets (SraP), and bacterial serine aspartate repeat-containing protein D (SdrD), whose variable interactions with host cell receptors are summarized in While the FnBP-Fn-α5β1 integrin pathway is historically recognized as the main internalization process, there are numerous “secondary” mechanisms between S. aureus virulence factors that have been shown to act as both primary and secondary entrance mechanisms are unequally distributed throughout S. aureus strains and between the core genome and accessory genomes of those strains [S. aureus strains [S. aureus strains, the authors selected a set of known virulence factors present in 64 different strains based on literature and database searches. They discovered that multiple virulence factors considered “core genome members” were not found across all 64 strains. Examples of this include protein A (encoded for by spa), which was found in 90% of strains, and α-hemolysin (encoded for by hla), found in 96% of strains [hla was detected in 90.3% of the 186 S. aureus isolates that were paneled [S. aureus [S. aureus is summarized in S. aureus strains’ genomes.Many of the  strains . Genome- strains . However strains  to inves strains . Similar strains  discover paneled . These v. aureus . A comprS. aureus can remain there for significant periods of time [S. aureus and the survival strategies that it employs to persist in the infected host cell depend on a wide variety of factors that aid in intracellular persistence, including staphylococcal isolate and corresponding virulence factors and differential susceptibility of host cells to virulence factors based upon host cell type and respective gene expression [Once endocytosed by host cells,  of time ,126,127. of time  note thapression .S. aureus must defend against the cell’s intracellular antibacterial measures. S. aureus has been shown not only to survive but even to replicate inside the harsh phagosomal environment. S. aureus variably alters the phagosomal acidification process and its eventual fusion with the lysosome, although such interaction is heavily dependent upon the staphylococcal strain and host cell type. The S. aureus-containing endosome acquires early endosomal markers such as the small GTPase Rab5 that transition to a Ras-related protein Rab7a and lysosome-associated membrane protein 1 (LAMP1)-associated phagosome that has an acidic intracellular environment [S. aureus within the cell, with some studies reporting different staphylococcal strategies used to cope with acidification. Acidification in macrophages promotes the expression of the accessory gene regulator (agr) system, an intracellular quorum sensing system of S. aureus crucial for the upregulation of numerous virulence factors [S. aureus will upregulate multiple peptide resistance factor (MprF) through its GraRS system to begin the process of replication within acidified phagolysosomes [S. aureus proliferation: the presence of inhibitors of phagosomal acidification was shown to significantly decrease the intracellular survival rates of S. aureus in THP-1 macrophages [S. aureus-containing cells [S. aureus disruption of beta-glucuronidase and cathepsin D activation [S. aureus at all [To remain in professional phagocytes, ironment ,130,131.ysosomes . Other sng cells , as the ng cells . This mativation . Still oS. aureus additionally recruits or produces multiple factors to deal with the host cell’s innate immune response to invasion: genetic factors, including the dlt operon, will encode for proteins responsible for the d-alanylation of teichoic acids in the S. aureus membrane. These S. aureus proteins provide a physical barrier against host cell defensins and antimicrobial peptides [S. aureus intracellular persistence by aiding in wildtype S. aureus transition to small colony variants (SCVs), which are adapted for long-term intracellular persistence [S. aureus persistence in human monocyte-derived macrophages, but their specific roles remain unclear [peptides . Multiplpeptides . Finallysistence . Other p unclear .S. aureus offers more than a place for replication; in some cases, macrophages may serve as vehicles for the dissemination of infection [S. aureus replicates within macrophages during a critical window of time where overexposure to infectious agents has resulted in infected macrophages briefly losing their ability to kill pathogens (termed as “exhausted” macrophages) [S. aureus emerges from these cells via host-cell lysis [S. aureus can be phagocytosed again by macrophages to produce a cyclical pattern of uptake and escape that creates a pool of S. aureus within macrophages [S. aureus within exhausted macrophages is hypothesized to cause the dissemination of bacteria throughout the host, potentially resulting in deeper and more aggressive infections [S. aureus to demonstrate higher levels of bacterial dissemination to the kidneys and brain when compared with infections with planktonic bacteria [S. aureus will replicate long-term in liver macrophages, and the eventual release of massive amounts of S. aureus from the lysis of dying cells will cause devastating bloodstream infections. Similarly, infected liver neutrophils have been shown to re-enter circulation after infection and disseminate the pathogen to secondary infection sites [Extended intracellular persistence of nfection . S. aureophages) . S. aurell lysis ,134,136.rophages ,134,136.bacteria . Surewaabacteria  found thon sites .S. aureus phagosomal escape and subsequent cytoplasmic replication is observed in many non-professional phagocytes such as epithelial cells [S. aureus phagosomal escape usually relies upon upregulation of the agr system [S. aureus mutants lacking agr will not translocate to the host cell cytoplasm [S. aureus strains [S. aureus translocation, PSMα production alone is not sufficient for the induction of escape: overexpression of PSMα in escape-deficient S. aureus strains did not result in bacterial translocation to the host cytoplasm [al cells ,140, endal cells ,129, andal cells ,141. S. r system , and S. ytoplasm . Agr act strains ,144. DesS. aureus escape may include the β-type PSMs, as proteomic analysis of S. aureus strains capable of phagosomal escape has found that these strains produce both PSMα and PSMβ proteins. Still others additionally upregulate the δ-toxin gene hld [S. aureus [Other contributing factors to gene hld  or expregene hld . The ina. aureus ,145. The. aureus ,146,147.. aureus ,148. Add. aureus .S. aureus has been shown to trigger an autophagic response in a wide variety of non-professional phagocytes (NPPCs) as well as dendritic cells, macrophages, and neutrophils [S. aureus has been shown to evade host autophagy, and S. aureus strains that undergo agr upregulation will block the process of autophagy within dendritic cells, leading to the cytotoxic accumulation of autophagosomes [S. aureus-containing phagosomes can mature into phagolysosomes without triggering the autophagy pathway [S. aureus [S. aureus, these phagosomes still continue to express selective xenophagy receptor p62 on these phagosomes, which is contrastingly associated with host protection [S. aureus, the retained expression of p62-positive phagosomes represents the host still struggling to eliminate S. aureus [Autophagy is a degradation procedure responsible for the elimination of unwanted cytoplasmic components and is utilized by the cell to degrade invading pathogens into presentable antigens for host defense ,150,151.trophils ,153. S. agosomes ,154. Oth pathway . However pathway . The LAP. aureus ,154,156.S. aureus in NPPC in multiple ways. However, it remains unclear whether S. aureus specifically manipulates autophagy to mitigate host cell defenses or increase its nutrient load or whether it uses autophagic machinery as a replicative niche for increased dissemination. S. aureus releases the α-hemolysin (Hla) toxin to trigger the autophagic pathway within NPPC [S. aureus. To combat this host-innate immune response, intracellular S. aureus develops mutations in the agr regulon  has also been observed in polymorphonuclear leukocytes, wherein S. aureus is found surviving in autophagosomes and replicating while simultaneously inhibiting apoptosis [S. aureus. In response, S. aureus activated a strong anti-autophagic response via upregulation of the MAPK14/p38α MAP kinase-mediated blockade of autophagy, which inhibits fusion with lysosomes to escape degradation [S. aureus did not require autophagosomes to further its development and actively inhibited autophagosomal maturation. A study suggesting yet another alternative benefit for autophagy noted that a significant number of autophagosomes did not contain S. aureus in infected HeLa cells [S. aureus significantly altered the host metabolic state, simultaneously activating autophagy. By doing so, it began to divert the nutrients generated by this pathway to meet its metabolic needs [S. aureus survival decreased, potentially due to loss of nutrients from alteration of host metabolism. While S. aureus likely exploits host cell autophagy, it remains unclear whether this is critical for nutrient uptake, self-defense, the creation of a replication niche, or a combination of all three [Host cell autophagic processes are manipulated by intracellular hin NPPC ,157,158.hin NPPC  through hin NPPC , which iy et al. ) that coy et al.  report tpoptosis . Neumannpoptosis  demonstrradation . S. aureLa cells . Insteadic needs ,164. It ll three .S. aureus has been identified as manipulating all known principal mechanisms of PCD, including apoptosis and pro-inflammatory pathways necroptosis or pyroptosis, in NPPCs [S. aureus modulation of host cell death. First, controlling the timing and mode of host cell death may help intracellular S. aureus maintain its replicative niche for an extended period of time [S. aureus with shelter from host immune responses and antibacterial therapies [in NPPCs ,168,169. of time ,167. Finherapies .S. aureus has factors that specifically inhibit apoptosis in NPPCs to delay host cell death and control bacterial exit from host cells [S. aureus utilizes EsxA, an effector protein secreted by the staphylococcal type VII secretion system, to inhibit apoptosis. This may potentially give the bacterium time to replicate and disseminate into the host tissues [S. aureus in keratinocytes, as shown by the activation of apoptosis caspases  [S. aureus strain caused significantly more caspase-dependent apoptosis in keratinocytes than a strain lacking PVL production.st cells . Korea est cells  suggeste tissues . Contras and -8) ,172. The and -8)  demonstrS. aureus additionally interacts with pyroptosis pathways in NPPCs. Pyroptosis induction has been shown to be a crucial step required for S. aureus penetration through keratinocyte barriers, with Soong et al. [S. aureus may activate pyroptosis of keratinocytes to enhance dissemination [g et al.  suggestimination .S. aureus produces a broad range of pro-PCD virulence factors that may facilitate infiltration of the bacteria into the cells and surrounding tissues, as reviewed previously [S. aureus may attempt to prolong NPPC life and avoid triggering these pathways to ensure safety from antibacterial treatment. For example, Kindi et al. [S. aureus entered and proliferated within keratinocytes without killing them or triggering any host cell death pathways. The majority of anti-staphylococcal antibiotics at 20-fold their standard minimal inhibitory concentration, including flucloxacillin, teicoplanin, clindamycin, and linezolid, did not kill internalized S. aureus during this time. The internalization of S. aureus by human skin keratinocytes and subsequent lack of host cell death pathway completion in response allowed S. aureus to evade killing by most anti-staphylococcal antibiotics in this study [eviously ,173, to i et al.  noted this study .S. aureus may trigger more than one of the above pathways at once: Steltzner et al. [S. aureus disturbs Ca2+ homeostasis and induces cytoplasmic Ca2+ overload in HeLa cells, which results in both apoptotic and mitochondrial permeability transition-driven necrotic cell death occurring simultaneously (or in quick succession). However, they suggest that the damage induced by strong perturbations of the cellular Ca2+ homeostasis and Ca2+ overload may not allow reliable detection of specific signaling pathways. With so many potential interactions between S. aureus and PCD pathways, it is difficult to target what the specific pathogenic goal of PCD may be; the damage resulting from this Ca2+ overload may be the reason for much of the conflicting information [r et al.  suggest ormation .S. aureus has also been shown to trigger pyroptosis, necroptosis, and other necrotic pathways in THP-1 macrophages, monocytes, and neutrophils, with minimal activation of apoptosis reported in phagosomes of these respective cells [S. aureus-infected macrophages showed characteristics of both apoptosis and necrosis [S. aureus-infected neutrophils have shown that the cells will undergo morphological changes consistent with apoptosis, before necroptotic RIPK-1-dependent lysis occurred [Intracellular ve cells ,177,178.ve cells  found thnecrosis . Other soccurred .S. aureus maintain human monocyte-derived macrophages for extended periods despite the appearance of early apoptotic features in host cells. S. aureus strongly upregulated the expression of B-cell lymphoma 2 (BCL2) and myeloid cell leukemia 1 (MCL1) genes, which stabilize mitochondrial membrane potential and delay apoptosis. The protective effects of S. aureus were confirmed by the finding that S. aureus-infected macrophages were more resistant to staurosporine-induced cell death than control cells [S. aureus infection in dendritic cells delayed host cell apoptosis, implicating staphylococcal protein EsxA as a critical mediating factor during this process [Bacterial control over professional phagocytic cell death is thought to support bacterial dissemination throughout the body as they travel within these mobile cells. Koziel et al.  suggesteol cells . Similar process .S. aureus has now been recognized as a facultative intracellular pathogen, with its ability to adeptly shift from an extracellular to an intracellular lifestyle playing a crucial role during recurrent and aggressive infections. S. aureus enters a wide range of host cell types and can replicate and survive within host cells, utilizing an incredible range of host cell pathways to enable its survival. As a result of this, effectively eliminating intracellular S. aureus remains a major challenge.S. aureus and P. aeruginosa, no vaccines are currently available, as discussed below. Additionally, both bacterial species discussed here are listed as species of concern by the WHO due to their AMR, and as discussed above, are both highly adaptable pathogens, common in the environment, and capable of forming biofilms and both have a multitude of virulence factors that can aid in infection, host evasion, and antibiotic resistance/tolerance. Consequently, treating these two pathogens is both challenging and situational, depending on the type or location of the infection and the antibiotic tolerance and resistance profile of the specific strains involved [For a treatment to successfully eradicate intracellular bacterial pathogens, the treatment must overcome several physical barriers due to the location of the pathogens. An obvious treatment strategy would be to enhance and manipulate the host’s immune response to the intracellular pathogen, with the most logical treatment choice being a vaccine. Vaccines are one of the most effective strategies for engaging and enhancing the host’s natural immune response to prevent and/or eradicate an infection by a pathogen. However, in the case of involved ,182.P. aeruginosa or S. aureus in various stages of clinical trials, as reviewed in [P. aeruginosa. The bispecific DMAb, MEDI3902, showed synergy with the antibiotics ciprofloxacin and tobramycin in mouse models of infections and even at subtherapeutic doses of MEDI3902 with either antibiotic, the combination treatments were effective in preventing death in mice [Combination therapies show promise for treating these two non-canonical intracellular pathogens, including therapies that combine traditional antimicrobials with treatments that harness the host’s immunity. While vaccines are an active immune-based therapy, the use of antibodies or antisera are common passive immune-based therapies. There are many antibody-based treatments that target various antigens from iewed in ,184,185.iewed in  designed in mice . Unfortu in mice .P. aeruginosa and S. aureus infections. An antibody–antibiotic conjugate (AAC) was constructed by Lehar et al. [S. aureus, and once they are phagocytized, the rifalogue is cleaved from the antibody by endoenzymes within the host cells, and the antibiotic effectively kills intracellular S. aureus, since rifalogue is effective against viable, replicating, and non-replicating bacteria. In vitro and in vivo testing showed this AAC was highly effective at reducing pathogen loads. Additionally, in a mouse infection model, the AAC was more effective at treating S. aureus bacteremia than a traditional treatment with vancomycin antibiotic [P. aeruginosa [P. aeruginosa inside macrophages. While demonstrating great in vitro efficacy, unfortunately the 26F8-cBuCit-G2637 AAC was not therapeutically effective in a mouse model of acute P. aeruginosa pneumonia, and the researchers suggest that one possible solution to this hindrance could be to use a MAb with stronger opsonic activity to improve the effectiveness in vivo [Other antibody-based therapies have shown potential against intracellular r et al.  from Gentibiotic . Recentlruginosa . Buildin in vivo .S. aureus infections. While each statin drug acted on the host and reduced invasion of S. aureus due to reductions in actin stress fiber reordering and fibronectin binding, only the co-treatment of ML141 and rifampicin decreased intracellular S. aureus compared with the rifampicin-alone treatments. Thus, the host-directed therapy of ML141 inhibited bacterial invasion and enhanced rifampicin-killing of S. aureus, as this lipophilic antibiotic is effective against intracellular and extracellular bacteria [P. aeruginosa and CRPA (carbapenem-resistant P. aeruginosa) infections in vitro and in vivo. The researchers found that MEM indirectly promoted ROS generation in neutrophils by increasing expression of the p67phox subunit of NADPH oxidase, which enhanced the bactericidal effect of neutrophils against P. aeruginosa. Further, MEM was found to be synergistic with AMK, and in vivo experiments showed that the combination therapy reduced the severities of bacteremia and inflammation and that the bacterial burden load was reduced in rats [While the above two examples with AAC-based therapies only passively utilize the host in the therapy against intracellular pathogens, there are some promising new studies that utilize repurposed drugs to directly enhance the host’s response to the pathogen in combination with traditional antibiotics. Evans et al.  investigbacteria . A diffebacteria , as they in rats .S. aureus and P. aeruginosa. Several recent encouraging studies have been published on host-directed monotherapies, including: using the kinase inhibitor Ibrutinib to treat intracellular S. aureus [2+-ATPases (SERCA) inhibitor thapsigargin to treat intracellular S. aureus [S. aureus, P. aeruginosa, and Streptococcus pyogenes [P. aeruginosa in a CF model [Host-directed therapies offer the benefit of targeting the host, which greatly reduces the risk of inducing drug resistance in the bacteria. However, the infection environment is complicated, and monotherapies could become ineffective against infections where the bacteria are highly adaptable and have the ability to survive intracellularly, such as what we have described here for the non-canonical intracellular pathogens . aureus ; using t. aureus ; treatinpyogenes ; and utiCF model . These hP. aeruginosa and S. aureus is critical for proper treatment and for reducing the spread of AMR. Additionally, global surveillance and prevention options are essential not only for controlling the infections caused by these two specific priority ESKAPE pathogens but also for controlling the broad spread of AMR in general.Recognizing the full life cycle and niche capabilities in infection caused by Surveillance lays the groundwork for the control of AMR spread. The WHO Global Antimicrobial Resistance Surveillance System (GLASS) AMR surveillance initiative was launched in 2015 to standardize global reporting of AMR data. The fourth GLASS report published in 2021  includedBordetella pertussis-caused whooping cough [Mycobacterium bovis that is still widely used in some regions [Mycobacterium tuberculosis, the causative agent of tuberculosis. Similarly, Salmonella Typhi strain Ty2 [Yersinia pestis strain EV76 [Vibrio [Shigella and enterotoxigenic Escherichia coli [Staphylococcus aureus [Pseudomonas aeruginosa [Acinetobacter baumannii, Klebsiella pneumoniae, and E. coli [Treponema pallidum, Chlamydia, and Neisseria gonorrhoeae) [Limiting the use of antimicrobial drugs is another key component of AMR control. Vaccines for both humans and animals are instrumental in preventing infections and reducing antimicrobial use, therefore limiting the development of resistance. The use of vaccines to fight against infectious diseases is the most significant advance in medical history. As reviewed previously , vaccineng cough , while tng cough . The att regions  to elicirain Ty2 ,210 and ain EV76  were use [Vibrio ) or are hia coli ,214). Othia coli . The subhia coli ,217. Dess aureus  and carbruginosa , Acineto E. coli . Moreoverrhoeae) ,222 are rrhoeae) , no licerrhoeae) ,225.Excessive use of antibiotics has accelerated the emergence of multidrug-resistant and extensively drug-resistant bacteria, against which even the most effective drugs are ineffective. The WHO has been sounding the alarm about antimicrobial resistance (AMR) as a severe threat to global health and the global economy ,9,226. EP. aeruginosa along with methicillin- and vancomycin-resistant S. aureus cause notoriously difficult-to-treat recalcitrant infections that are associated with the highest risk of mortality and increased health care costs [P. aeruginosa and S. aureus exploit the host intracellular environment actively by the use of multiple virulence factors to promote their intracellular life cycles, potentially contributing to their survival in the presence of antibiotics. This poses an additional challenge to eradicate infections caused by these bacteria, which are already highly resistant to antibiotics. Further research into the mechanisms driving these two bacterial species’ intracellular life cycles are desperately needed. Additionally, research in developing novel therapeutics and treatment strategies are urgently needed. In our review, we did not try to exhaustively cover all novel therapies, such as all combination therapies, phage therapies, etc. Instead, we focused on highlighting recent research on therapies that harness the host’s immune response, since it is more difficult for the bacteria to develop resistance to the treatment because it is host-directed. Given the complexity of these intracellular infections, we believe a multifaceted treatment approach will be required to avert catastrophes caused by the increasing number of AMR infections worldwide. Using the host’s immune response to assist in effectively eradicating the intracellular pathogen will be a key component in slowing the spread of AMR infections, and combining these treatments with traditional antimicrobials has the potential to accelerate patient care and recovery.Among the list of these AMR-associated bacteria, carbapenem-resistant re costs . Neither