scholarly journals Characterization of Phage Obtained from Methicillin -Resistant Staphylococcus aureus (MRSA)

2021 ◽  
Author(s):  
Özlem Ulusan Bağcı ◽  
Fikret Şahin ◽  
Mehmet Kıyan
Keyword(s):  
Staphylococcus Aureus ◽  
Phage Therapy ◽  
Relevant Literature ◽  
Open Reading Frames ◽  
Recombinant Phage ◽  
Protein Map ◽  
Genes Encoding ◽  
New Antibiotics ◽  
Lysogenic Phage

Objective: The emergence of Staphylococcus aureus strains resistant to all antimicrobials and failure to discover new antibiotics have led researchers to phage therapy, which lost popularity after the discovery of antibiotics. The development of recombinant technology introduced the idea of creating lysogenic recombinant phages that provide controlled bacterial death and this required small- sized phages that were easy to manipulate. Our aim is to identify small-sized lysogenic bacteriophages that can be used safely in therapy. Method: The gene and protein map of the phage was created by analysis of sequencing after extracting a phage from the MRSA strain that is known to contain a small phage. Results: The phage was classified in Caudovirales spp. as it contains genes encoding tail proteins, and in Podoviridae spp. due to its genomic size and arrangement. Conclusion: To date, there are only sixteen phages from Podoviridae family uploaded on NCBI, and the phage described in this study is the seventeenth one. Only 41.4% of the ORFs (Open Reading Frames) in the genome could be matched with proteins using the NCBI BLAST. Recent studies suggest that 50-75% of bacteriophage ORFs do not correspond to any organism in GenBank. For better understanding of bacteriophages and their utilization in phage therapy, it is essential to sequence greater number of phages, and to discover their genomes and corresponding proteins. Since the genes and proteins of a lysogenic phage that can be used safely in recombinant phage therapies have been identified in our study, it will contribute to the relevant literature.

scholarly journals Characterization of the Accessory Sec System of Staphylococcus aureus

2008 ◽  
Vol 190 (18) ◽  
pp. 6188-6196 ◽  
Author(s):  
Ian R. Siboo ◽  
Donald O. Chaffin ◽  
Craig E. Rubens ◽  
Paul M. Sullam
Keyword(s):  
Staphylococcus Aureus ◽  
Surface Expression ◽  
Complete Loss ◽  
Transport Pathway ◽  
Gram Positive Bacteria ◽  
Difference Gel Electrophoresis ◽  
Genes Encoding ◽  
Sec System ◽  
Specialized System

ABSTRACT The SraP adhesin of Staphylococcus aureus is a member of a highly conserved family of serine-rich surface glycoproteins of gram-positive bacteria. For streptococci, export of the SraP homologs requires a specialized transport pathway (the accessory Sec system). Compared to streptococci, however, SraP is predicted to differ in its signal peptide and glycosylation, which may affect its dependence on a specialized system for transport. In addition, two genes (asp4 and asp5) essential for export in Streptococcus gordonii are missing in S. aureus. Thus, the selectivity of the accessory Sec system in S. aureus may also differ compared to streptococci. To address these issues, the five genes encoding the putative accessory Sec system (secY2, secA2, and asp1-3) were disrupted individually in S. aureus ISP479C, and the resultant mutants were examined for SraP export. Disruption of secA2 resulted in the near complete loss of SraP surface expression. Similar results were seen with disruption of secY2 and asp1, asp2, or asp3. To assess whether the accessory Sec system transported other substrates, we compared secreted proteomes of ISP479C and a secA2 isogenic mutant, by two-dimensional fluorescence difference gel electrophoresis. Although two consistent differences in proteome content were noted between the strains, neither protein appeared to be a likely substrate for accessory Sec export. Thus, the accessory Sec system of S. aureus is required for the export of SraP, and it appears to be dedicated to the transport of this substrate exclusively.

scholarly journals The Concerted Action of Two B3-Like Prophage Genes Excludes Superinfecting Bacteriophages by Blocking DNA Entry into Pseudomonas aeruginosa

2020 ◽  
Vol 94 (15) ◽  
Author(s):  
Marco Antonio Carballo-Ontiveros ◽  
Adrián Cazares ◽  
Pablo Vinuesa ◽  
Luis Kameyama ◽  
Gabriel Guarneros
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Pathogenic Bacteria ◽  
Phage Therapy ◽  
Alternative Therapy ◽  
Multidrug Resistant ◽  
Open Reading Frames ◽  
Resistant Bacteria ◽  
Content Type ◽  
Secondary Infections ◽  
Genes Encoding

ABSTRACT In this study, we describe seven vegetative phage genomes homologous to the historic phage B3 that infect Pseudomonas aeruginosa. Like other phage groups, the B3-like group contains conserved (core) and variable (accessory) open reading frames (ORFs) grouped at fixed regions in their genomes; however, in either case, many ORFs remain without assigned functions. We constructed lysogens of the seven B3-like phages in strain Ps33 of P. aeruginosa, a novel clinical isolate, and assayed the exclusion phenotype against a variety of temperate and virulent superinfecting phages. In addition to the classic exclusion conferred by the phage immunity repressor, the phenotype observed in B3-like lysogens suggested the presence of other exclusion genes. We set out to identify the genes responsible for this exclusion phenotype. Phage Ps56 was chosen as the study subject since it excluded numerous temperate and virulent phages. Restriction of the Ps56 genome, cloning of several fragments, and resection of the fragments that retained the exclusion phenotype allowed us to identify two core ORFs, so far without any assigned function, as responsible for a type of exclusion. Neither gene expressed separately from plasmids showed activity, but the concurrent expression of both ORFs is needed for exclusion. Our data suggest that phage adsorption occurs but that phage genome translocation to the host’s cytoplasm is defective. To our knowledge, this is the first report on this type of exclusion mediated by a prophage in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is a Gram-negative bacterium frequently isolated from infected immunocompromised patients, and the strains are resistant to a broad spectrum of antibiotics. Recently, the use of phages has been proposed as an alternative therapy against multidrug-resistant bacteria. However, this approach may present various hurdles. This work addresses the problem that pathogenic bacteria may be lysogenized by phages carrying genes encoding resistance against secondary infections, such as those used in phage therapy. Discovering phage genes that exclude superinfecting phages not only assigns novel functions to orphan genes in databases but also provides insight into selection of the proper phages for use in phage therapy.

scholarly journals Postgenomic Scan of Metallo-β-Lactamase Homologues in Rhizobacteria: Identification and Characterization of BJP-1, a Subclass B3 Ortholog from Bradyrhizobium japonicum

2006 ◽  
Vol 50 (6) ◽  
pp. 1973-1981 ◽  
Author(s):  
Magdalena Stoczko ◽  
Jean-Marie Frère ◽  
Gian Maria Rossolini ◽  
Jean-Denis Docquier
Keyword(s):  
Bradyrhizobium Japonicum ◽  
Catalytic Efficiency ◽  
Open Reading Frames ◽  
Human Pathogens ◽  
Microbial Genomes ◽  
E Coli ◽  
Genes Encoding ◽  
And Function ◽  
Identification And Characterization

ABSTRACT The diffusion of metallo-β-lactamases (MBLs) among clinically important human pathogens represents a therapeutic issue of increasing importance. However, the origin of these resistance determinants is largely unknown, although an important number of proteins belonging to the MBL superfamily have been identified in microbial genomes. In this work, we analyzed the distribution and function of genes encoding MBL-like proteins in the class Rhizobiales. Among 12 released complete genomes of members of the class Rhizobiales, a total of 57 open reading frames (ORFs) were found to have the MBL conserved motif and identity scores with MBLs ranging from 8 to 40%. On the basis of the best identity scores with known MBLs, four ORFs were cloned into Escherichia coli for heterologous expression. Among their products, one (blr6230) encoded by the Bradyrhizobium japonicum USDA110 genome, named BJP-1, hydrolyzed β-lactams when expressed in E. coli. BJP-1 enzyme is most closely related to the CAU-1 enzyme from Caulobacter vibrioides (40% amino acid sequence identity), a member of subclass B3 MBLs. A kinetic analysis revealed that BJP-1 efficiently hydrolyzed most β-lactam substrates, except aztreonam, ticarcillin, and temocillin, with the highest catalytic efficiency measured with meropenem. Compared to other MBLs, BJP-1 was less sensitive to inactivation by chelating agents.

scholarly journals Characterization of the Genes for Two Protocatechuate 3,4-Dioxygenases from the 4-Sulfocatechol-Degrading Bacterium Agrobacterium radiobacter Strain S2

2000 ◽  
Vol 182 (21) ◽  
pp. 6123-6129 ◽  
Author(s):  
Matthias Contzen ◽  
Andreas Stolz
Keyword(s):  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Agrobacterium Radiobacter ◽  
Sequence Alignments ◽  
Degrading Bacterium ◽  
Regulator Protein ◽  
Putative Operon ◽  
Genes Encoding ◽  
Reading Frames

ABSTRACT The genes for two different protocatechuate 3,4-dioxygenases (P34Os) were cloned from the 4-sulfocatechol-degrading bacteriumAgrobacterium radiobacter strain S2 (DSMZ 5681). ThepcaH1G1 genes encoded a P34O (P34O-I) which oxidized protocatechuate but not 4-sulfocatechol. These genes were part of a protocatechuate-degradative operon which strongly resembled the isofunctional operon from the protocatechuate-degrading strainAgrobacterium tumefaciens A348 described previously by D. Parke (FEMS Microbiol. Lett. 146:3–12, 1997). The second P34O (P34O-II), encoded by the pcaH2G2 genes, was functionally expressed and shown to convert protocatechuate and 4-sulfocatechol. A comparison of the deduced amino acid sequences of PcaH-I and PcaH-II, and of PcaG-I and PcaG-II, with each other and with the corresponding sequences from the P34Os, from other bacterial genera suggested that the genes for the P34O-II were obtained by strain S2 by lateral gene transfer. The genes encoding the P34O-II were found in a putative operon together with two genes which, according to sequence alignments, encoded transport proteins. Further downstream from this putative operon, two open reading frames which code for a putative regulator protein of the IclR family and a putative 3-carboxymuconate cycloisomerase were identified.

scholarly journals Structural Characterization of ISCR8, ISCR22, and ISCR23, Subgroups of IS91-Like Insertion Elements

2010 ◽  
Vol 54 (10) ◽  
pp. 4321-4328 ◽  
Author(s):  
Kathleen M. Schleinitz ◽  
Tatiana Vallaeys ◽  
Sabine Kleinsteuber
Keyword(s):  
Open Reading Frames ◽  
Broad Host Range ◽  
Active Member ◽  
Opportunistic Pathogens ◽  
Candidate Sequence ◽  
Genes Encoding ◽  
Insertion Elements ◽  
Nitro Aromatics ◽  
Definition Of

ABSTRACT Analysis of ISCR8 (ISPps1) revealed that this group of insertion elements has to be subdivided into three subgroups: ISCR8, ISCR22, and ISCR23. The distinction of three subgroups is supported by phylogenetic analysis of the transposase open reading frames (ORFs). Comparison of over 20 complete and partial ISCR8/22/23 elements identified oriIS candidate sequences for all groups and a terIS candidate sequence for ISCR8. The oriIS sequences, their distance to the transposase ORFs, and the sequence of this intervening region are group specific, further supporting the definition of two new ISCR elements. ISCR8/22/23 have a very broad host range, including Gram-positive and Gram-negative bacteria, among which are several (opportunistic) pathogens. The IS often resides on plasmids or in the vicinity of other mobile elements and is mostly associated with genes for the degradation of halo- or nitro-aromatics. However, in one case ISCR8 was found in the neighborhood of an antibiotic resistance determinant in Klebsiella pneumoniae. ISCR8 resembles other IS91 family elements in mediating genetic rearrangements by homologous recombination between two copies. In Delftia acidovorans this led to the loss of the genes encoding dichlorprop cleavage. In conclusion, this study shows that ISCR8 could be a fully functional and active member of the IS91 family of insertion elements.

scholarly journals Genomic Characterization of a Novel Freshwater Cyanophage Reveals a New Lineage of Cyanopodovirus

2022 ◽  
Vol 12 ◽  
Author(s):  
Dong Zhang ◽  
Yiliang He ◽  
Karina Yew-Hoong Gin
Keyword(s):  
Open Reading Frames ◽  
Metabolic Genes ◽  
Genes Encoding ◽  
Genomic Characterization ◽  
Ecological Importance ◽  
Auxiliary Metabolic Genes ◽  
Reading Frames ◽  
Tropical Freshwater ◽  
Core Genes

Cyanobacteria are one of the dominant autotrophs in tropical freshwater communities, yet phages infecting them remain poorly characterized. Here we present the characterization of cyanophage S-SRP02, isolated from a tropical freshwater lake in Singapore, which infects Synechococcus sp. Strain SR-C1 isolated from the same lake. S-SRP02 represents a new evolutionary lineage of cyanophage. Out of 47 open reading frames (ORFs), only 20 ORFs share homology with genes encoding proteins of known function. There is lack of auxiliary metabolic genes which was commonly found as core genes in marine cyanopodoviruses. S-SRP02 also harbors unique structural genes highly divergent from other cultured phages. Phylogenetic analysis and viral proteomic tree further demonstrate the divergence of S-SRP02 from other sequenced phage isolates. Nonetheless, S-SRP02 shares synteny with phage genes of uncultured phages obtained from the Mediterranean Sea deep chlorophyll maximum fosmids, indicating the ecological importance of S-SRP02 and its related viruses. This is further supported by metagenomic mapping of environmental viral metagenomic reads onto the S-SRP02 genome.

scholarly journals Characterization of a CC1153 PVL-producing community-acquired methicillin-resistant Staphylococcus aureus from a dog bite wound

2017 ◽  
Vol 11 (06) ◽  
pp. 513-516 ◽  
Author(s):  
Edet Ekpenyong Udo ◽  
Samar Saed Boswihi ◽  
Tsonyu Ivanov Dimitrov ◽  
Bobby Noronha ◽  
Bindu Mathew ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Companion Animals ◽  
Dog Bite ◽  
Bite Wound ◽  
Methicillin Resistant ◽  
Wound Culture ◽  
Genes Encoding ◽  
Disease Hospital

The isolation of a rare community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) strain from an infected dog bite wound is here reported. A 27–year old man presented with a deep open wound on his right hand caused by his dog’s bite at the outpatient clinic of Infectious Disease Hospital (IDH), Kuwait. A wound swab was obtained for bacteriological culture and susceptibility testing. The wound culture yielded pure heavy growth of an MRSA isolate, designated IDH70, which was susceptible to vancomycin, teicoplanin, erythromycin, clindamycin, trimethoprim, fusidic acid and rifampicin. The patient was successfully treated with a combination of rifampicin and cotrimoxazole twice daily for 10 days. Molecular characterization revealed that IDH70 was positive for genes encoding Panton-Valentine leucocidin. IDH70 also carried the SCCmec V genetic element, belonged to coagulase type XIIIa, spa type t903, and was assigned to clonal complex 1153 and sequence type ST1153 (ST1153-V-t903). The report highlights the increasing burden of CA-MRSA in the community and the risk of its acquisition from bites of companion animals.

scholarly journals Interconnections between Sigma B, agr, and Proteolytic Activity in Staphylococcus aureus Biofilm Maturation

2009 ◽  
Vol 77 (4) ◽  
pp. 1623-1635 ◽  
Author(s):  
Katherine J. Lauderdale ◽  
Blaise R. Boles ◽  
Ambrose L. Cheung ◽  
Alexander R. Horswill
Keyword(s):  
Staphylococcus Aureus ◽  
Hydrolase Activity ◽  
Extracellular Proteases ◽  
Sigma B ◽  
Genes Encoding ◽  
Usa300 Strain ◽  
Sigma Factor B ◽  
Murein Hydrolase ◽  
Host Tissues

ABSTRACT Staphylococcus aureus is a proficient biofilm former on host tissues and medical implants. We mutagenized S. aureus strain SH1000 to identify loci essential for ica-independent mechanisms of biofilm maturation and identified multiple insertions in the rsbUVW-sigB operon. Following construction and characterization of a sigB deletion, we determined that the biofilm phenotype was due to a lack of sigma factor B (SigB) activity. The phenotype was conserved in a sigB mutant of USA300 strain LAC, a well-studied community-associated methicillin-resistant S. aureus isolate. We determined that agr RNAIII levels were elevated in the sigB mutants, and high levels of RNAIII expression are known to have antibiofilm effects. By introducing an agr mutation into the SH1000 or LAC sigB deletion strain, S. aureus regained biofilm capacity, indicating that the biofilm phenotype was agr dependent. Protease activity is linked to agr activity and ica-independent biofilm formation, and we observed that the protease inhibitors phenylmethylsulfonyl fluoride and α-macroglobulin could reverse the sigB biofilm defect. Similarly, inactivating genes encoding both the aureolysin and Spl extracellular proteases in the sigB mutant restored biofilm capacity. Due to the growing link between murein hydrolase activity and biofilm maturation, autolysin zymography was performed, which revealed an altered profile in the sigB mutant; again, the phenotype could be repaired through protease inactivation. These findings indicate that the lack of SigB activity results in increased RNAIII expression, thus elevating extracellular protease levels and altering the murein hydrolase activity profile. Altogether, our observations demonstrate that SigB is an essential regulator of S. aureus biofilm maturation.

scholarly journals Epidemiology and microbiological characterization of clinical isolates of Staphylococcus aureus in a single healthcare region of the UK, 2015

2016 ◽  
Vol 145 (2) ◽  
pp. 386-396 ◽  
Author(s):  
C. HORNER ◽  
L. UTSI ◽  
L. COOLE ◽  
M. DENTON
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistance ◽  
Current Data ◽  
Clinical Isolates ◽  
Dilution Method ◽  
Genes Encoding ◽  
Mupirocin Resistance ◽  
The Uk ◽  
Resistance Rates

SUMMARYWe investigated the epidemiology and characterization of isolates of Staphylococcus aureus within the Yorkshire and Humber (YH) region in the UK. In July 2015, each laboratory within YH (n = 14) was assigned two consecutive days during which all clinical isolates of S. aureus were collected. Isolates were tested for antibiotic susceptibilities and the presence of genes encoding methicillin resistance (mecA and mecC), Panton–Valentine leukocidin (PVL) (lukS-PV), and efflux-mediated chlorhexidine resistance (qacA); isolates were also characterized by spa-types. Minimum inhibitory concentrations (MICs) to chlorhexidine were determined by the broth dilution method. Of 520 isolates collected, 6·2% were methicillin-resistant S. aureus (MRSA, all mecA-positive) and mupirocin resistance was low [0·8%, 95% confidence interval (CI) 0·3–2·0] and only found in MRSA. Carriage of the qacA gene was identified in 1·7% (95% CI 0·8–3·3) of isolates and 3·5% (95% CI 2·2–5·4) had a chlorhexidine MIC of 4 mg/l. The PVL gene was infrequent (3·7%, 95% CI 2·4–5·6). Genotyping identified 234 spa-types that mapped to 22 clonal complexes. Comparison of these current data with previous work suggest that the widespread use of staphylococcal decolonization regimens over the past decade or more has not had an adverse impact on resistance rates, PVL carriage or the prevalence of specific S. aureus lineages.

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