Diversity and Standard Nomenclature of Staphylococcus aureus Hyaluronate Lyases HysA and HysB

ABSTRACT Bacterial hyaluronate lyases (Hys) are enzymes that degrade hyaluronic acid in their host and are known to contribute to the pathogenesis of several illnesses. The first two identified Hys genes in Staphylococcus aureus were registered as hysA1 and hysA2. However, their annotations have been mistakenly reversed in some registered assembly data, and different abbreviations (hysA and hysB) in some reports complicates comparative analysis of Hys proteins. We investigated the hys loci of S. aureus genome sequences registered in public databases, analyzed the homology, and defined hysA as hys located in the core genome surrounded by a lactose metabolic operon and a ribosomal protein cluster present in almost all strains and hysB as that located on the genomic island νSaβ of the accessory genome. Homology analysis of the amino acid sequences of HysA and HysB revealed that they are conserved among clonal complex (CC) groups with a few exceptions. Thus, we propose a new nomenclature for S. aureus Hys subtypes: HysACC*** for HysA and HysBCC*** for HysB, with the asterisks representing the clonal complex number of the S. aureus strain producing the Hys subtype. The application of this proposed nomenclature will facilitate the intuitive, straightforward, and unambiguous designation of Hys subtypes and contribute to enhancing comparative studies in this regard. IMPORTANCE Numerous whole-genome sequence data for Staphylococcus aureus harboring two hyaluronate lyase (Hys) genes have been registered. However, the assigned gene names for hysA1 and hysA2 are incorrect in some assembled data, and in some cases, the genes are annotated differently as hysA and hysB. This creates confusion with respect to the nomenclature of Hys subtypes and complicates analysis involving Hys. In this study, we compared the homology of Hys subtypes and observed that to some extent, their amino acid sequences are conserved in each clonal complex group. Hys has been implicated as an important virulence factor, but relative sequence heterogeneity among S. aureus clones raises the question of whether Hys activities are different among these clones. Our proposed Hys nomenclature will facilitate comparison of the virulence of Hys, as well as discussions of the subject

Therapeutic Potential of an Endolysin Derived from Kayvirus S25-3 for Staphylococcal Impetigo

Impetigo is a contagious skin infection predominantly caused by Staphylococcus aureus. Decontamination of S. aureus from the skin is becoming more difficult because of the emergence of antibiotic-resistant strains. Bacteriophage endolysins are less likely to invoke resistance and can eliminate the target bacteria without disturbance of the normal microflora. In this study, we investigated the therapeutic potential of a recombinant endolysin derived from kayvirus S25-3 against staphylococcal impetigo in an experimental setting. First, the recombinant S25-3 endolysin required an incubation period of over 15 minutes to exhibit efficient bactericidal effects against S. aureus. Second, topical application of the recombinant S25-3 endolysin decreased the number of intraepidermal staphylococci and the size of pustules in an experimental mouse model of impetigo. Third, treatment with the recombinant S25-3 endolysin increased the diversity of the skin microbiota in the same mice. Finally, we revealed the genus-specific bacteriolytic effect of recombinant S25-3 endolysin against staphylococci, particularly S. aureus, among human skin commensal bacteria. Therefore, topical treatment with recombinant S25-3 endolysin can be a promising disease management procedure for staphylococcal impetigo by efficient bacteriolysis of S. aureus while improving the cutaneous bacterial microflora

Emergence of Staphylococcus aureus Carrying Multiple Drug Resistance Genes on a Plasmid Encoding Exfoliative Toxin B

Job file for the creation/design of stained glass from either the Charles J. Connick Studio (1912-1945) or the Charles J. Connick Associates studio (1945-1986). The job file contains a job number, location information, date of completion, size, contact information, price, and a description of the project. This particular job file contains information on a job located at: Albany, New York. Saint James Church

Cellular Entry of Clostridium perfringens Iota-Toxin and Clostridium botulinum C2 Toxin

Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin are composed of two non-linked proteins, one being the enzymatic component and the other being the binding/translocation component. These latter components recognize specific receptors and oligomerize in plasma membrane lipid-rafts, mediating the uptake of the enzymatic component into the cytosol. Enzymatic components induce actin cytoskeleton disorganization through the ADP-ribosylation of actin and are responsible for cell rounding and death. This review focuses upon the recent advances in cellular internalization of clostridial binary toxins

Plasmid map of pBACi, CRISPRi plasmid for <i>S</i>. <i>aureus</i>.

<p>Plasmid map of pBACi, CRISPRi plasmid for <i>S</i>. <i>aureus</i>.</p

First genomic characterization of blavim-1 and mcr-9-coharbouring enterobacter hormaechei isolated from food of animal origin

We describe here the complete genome sequence of an Enterobacter hormaechei ST279 coharbouring blaVIM-1 and mcr-9 recovered from uncooked beef patty in June 2017, Egypt. The tested isolate was resistant to carbapenem but susceptible to colistin (minimum inhibitory concentration (MIC), 0.5 μg/mL). The antimicrobial susceptibility profile and conjugation experiments were performed. The entire genome was sequenced by the Illumina MiniSeq and Oxford Nanopore methods. The blaVIM-1 and mcr-9 genes are carried on the same IncHI2/pMLST1 plasmid, pMS37a (Size of 270.9 kb). The mcr-9 gene was located within the physical boundaries demarcated by two insertion elements IS903 (upstream) and IS1 (downstream) but did not possess the downstream regulatory genes (qseC/qseB) which regulate the expression of mcr- 9. Therefore, the mcr-9 might be silently disseminated among carbapenem-resistant Enterobacterales. In addition to blaVIM-1 and mcr-9, plasmid pMS37a harbored various antibiotic resistance genes including aac(6’)-Il, ΔaadA22, aac(6’)-Ib-cr, sul1, dfrA1 and tetA. To the best of our knowledge, this is the first report of a blaVIM-1 and mcr-9-coharbouring E. hormaechei isolate of food origin worldwide. The identification of a multidrug-resistant VIM-1 and mcr-9 positive Enterobacter hormaechei isolate from food is worrisome as retail meat and meat products could serve as a vehicle for these MDR bacteria, which could be transferred between animals and humans through the food chain. It further highlights that Enterobacterales co-producing MCR and carbapenemases being found in the food chain indeed correspond to a One- Health issue, highlighting the need for serious steps to prevent their further dissemination

Tailor-made gene silencing of <i>Staphylococcus aureus</i> clinical isolates by CRISPR interference

<div><p>Preparing the genetically modified organisms have required much time and labor, making it the rate-limiting step but CRISPR/Cas9 technology appearance has changed this difficulty. Although reports on CRISPR/Cas9 technology such as genome editing and CRISPR interference (CRISPRi) in eukaryotes increased, those in prokaryotes especially in Staphylococci were limited. Thus, its potential in the bacteriology remains unexplored. This is attributed to ecological difference between eukaryotes and prokaryotes. Here, we constructed a novel CRISPRi plasmid vector, pBACi for <i>Staphylococcus aureus</i>. The transformation efficiency of <i>S</i>. <i>aureus</i> was ~10⁴ CFU/μg DNA using a vector extracted from <i>dcm</i> negative, which encoded one of DNA modification genes, <i>E</i>. <i>coli</i>. Further, pBACi was introduced into various clinical isolates including that not accepting the conventional temperature-sensitive vector. <i>dcas9</i> in the vector was expressed throughout the growth phases of <i>S</i>. <i>aureus</i> and this vector decreased various gene mRNA expressions based on the crRNA targeting sequences and altered the knockdown strains’ phenotypes. The targeted genes included various virulence and antibiotic resistant genes. Bioinformatics suggest this vector can be introduced into wide range of low-GC Gram-positive bacteria. Because this new CRISPR/Cas9-based vector can easily prepare knockdown strains, we believe the novel vector will facilitate the characterization of the function of genes from <i>S</i>. <i>aureus</i> and other Gram-positive bacteria.</p></div

Evaluation of the novel CRISPRi plasmid for <i>S</i>. <i>aureus</i>.

<p>2A. Transformation efficiencies of <i>S</i>. <i>aureus</i>. The transformation efficiencies of pBACi and pKFT are shown. Both plasmids were extracted from DH5α (<i>dcm</i>+) and BL21(DE3) (<i>dcm</i>-). Electroporation of all strains was performed at least three times. When no transformants were obtained until three trials, seven more electroporations (total n = 10) were conducted. *: Strains which could accept pKFT extracted from RN4220 (Transformation efficiency is 16.8~1118.2CFU/µg DNA). §: Strains which could not accept pKFT extracted from RN4220. 2B. Confirmation of correct transformation. The MW2 transformants were subjected to PCR. 1–3: Transformants using pBACi extracted from BL21(DE3). 4–6: Transformants using pBACi extracted from RN4220. 7: pBACi DNA. 8: No DNA (only purified water). M: 100bp marker. 2C. Growth curve of MW2/pBACi. Temporal measurement of OD₆₆₀ are shown. Three independent cultures were performed. After inoculation of 1/100 volume o/n pre-culture media into fresh media (OD₆₆₀: ~0.1), the temporal samplings (3 h: early-mid log phase, 6 h: late log phase, and 12 h: stationary phase) were performed. Vertical line: OD₆₆₀. Horizontal line: time (hours, h). Average and standard error (SE) are shown. 2D. Relative expression of <i>dcas9</i>. The temporal expression of <i>dcas9</i> in MW2/pBACi is shown. Three genes were used as reference genes. Gyrase subunit B (<i>gyrB</i>): white, glyceraldehyde 3-phosphate dehydrogenase (<i>gapDH</i>): gray and <i>femB</i>: black. Three independent samples of three cultures and three qPCR assays were conducted (n = 9/sample). The samplings were performed at three time points as described above. Average and SE are shown.</p

Repression of various virulence and antibiotic resistant factors in MW2 using CRISPRi.

<p>Knockdown strain names were as follows. C: MW2/pBACi (Vector control), 1. MW2/pYS69 (<i>icaA</i> silenced knockdown strain 1), 2: MW2/pYS70 (<i>icaA</i> silenced knockdown strain 2), 3: MW2/pYS106 (<i>sec</i> silenced knockdown strain 1), 4: MW2/pYS107 (<i>sec</i> silenced knockdown strain 2), 5: MW2/pYS108 (<i>sec</i> silenced knockdown strain 3), 6: MW2/pYS109 (<i>coa</i> silenced knockdown strain 1) 7: MW2/pYS110 (<i>coa</i> silenced knockdown strain 2), 8: MW2/pYS111 (<i>coa</i> silenced knockdown strain 3), 9: MW2/pYS112 (<i>blaZ</i> silenced knockdown strain 1), and 10: MW2/pYS113 (<i>blaZ</i> silenced knockdown strain 2). All phenotype assays were repeated at least three times (qPCR and ELISA: three independent cultures and three independent measurements. Other assays: three independent assays). Average and SE of each assay are shown in graphs. Statistics: Student’s <i>t</i>-test. 4A. crRNA binding regions. 100bp upstream sequences of four genes in MW2 and nucleotide sequences corresponding to spacer sequences designed in this study. Underline: spacer sequences, red boxes: PAM sequence (NGG), start codon: start codon of gene, capital letters: putative -35b, -10b and ribosome binding sites of genes. 4B. Biofilm formation on plastic surfaces. After 24 h culture, the amount of biofilm on surface was assayed. G-: without additional glucose, G+: with additional glucose. 4C. OD₅₉₀ value of extracts from G+ wells as shown in Fig 4B. 4D. Repression of <i>icaA</i> mRNA. 4E. SDS-PAGE of supernatants. Culture media were subjected to electrophoresis. Arrow head: Protein corresponding to SEC (SEC from MW2: 28kDa). M: molecular weight marker (kDa), 4F: Sandwich ELISA of supernatants. SEC production was quantified. 4G. Repression of <i>sec</i> mRNA. 4H. Coagulase test. White arrow indicates a fibrin clot. 4I. Repression of <i>coa</i> mRNA. 4J. Measurement of β-lactamase activity. OD₄₉₀ of nitrocephin degraded product was measured. The value of control was compared with silencing vectors (%, vertical line). 4K. Repression of <i>blaZ</i> mRNA.</p

Staphylococcal protein A (Spa) silenced using CRISPRi.

<p>No significant growth inhibition was observed between the control and the silenced knockdown strains. pBACi (C): Vector control, pYS103 [103]: <i>spa</i> silenced knockdown vector 1, pYS104 [104]: <i>spa</i> silenced knockdown vector 2, pYS105 [105]: <i>spa</i> silenced knockdown vector 3. 3A. The upstream sequence of <i>spa</i> in MW2 and nucleotide sequences corresponding to spacer sequences. The partial sequence between <i>spa</i> (MW0084) and <i>sarS</i> (MW0085) in MW2 (Accession number: BA000033) is shown (101,008 bp-101,207 bp in MW2). Underline: spacer sequence [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185987#pone.0185987.ref001" target="_blank">1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185987#pone.0185987.ref003" target="_blank">3</a>], red boxes: PAM sequence (NGG), start codon: start codon of <i>spa</i> (ttg), capital letters: putative -35b, -10b and SD sequence (ribosome binding site) of <i>spa</i> in MW2. 3B. Western blotting to detect Spa in vector controls and silenced knockdown strains. Seven strains were analyzed. Three independent cultures from a single colony were performed and representative data are shown. Brackets indicate Protein A. Due to posttranslational processing of the cell surface-exposed ProteinA, some strains show double bands. “Plasmid No.” indicates serial pYS number plasmid used in this study (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185987#pone.0185987.s001" target="_blank">S1 File</a>). M: molecular marker. 75, 50 and 37 mean 75kDa, 50kDa and 37kDa bands, respectively. C: pBACi control vector (No crRNA coding region for <i>spa</i> silencing), pYS103-105: silencing CRISPRi vector containing the nucleotide sequence 1–3 corresponding to Fig 3A. Arrow heads: Spa. Two bands were found. This might result from processed/non-processed bands [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185987#pone.0185987.ref052" target="_blank">52</a>]. 3C. Repression of <i>spa</i> mRNA in MW2 using CRISPRi. Relative <i>spa</i> gene expression (/<i>gyrB</i>) is shown. MW2/pBACi, MW2/pYS103, MW2/pYS104 and MW2/pYS105 were independently cultured three times. Three independent qPCR were performed (n = 9/strain). The average and SE are shown. Statistics: Student’s <i>t</i>-test.</p