-
International Journal of Molecular... Jul 2021The best-characterized members of the M23 family are glycyl-glycine hydrolases, such as lysostaphin (Lss) from or LytM from . Recently, enzymes with broad specificities...
The best-characterized members of the M23 family are glycyl-glycine hydrolases, such as lysostaphin (Lss) from or LytM from . Recently, enzymes with broad specificities were reported, such as EnpA from , that cleaves D,L peptide bond between the stem peptide and a cross-bridge. Previously, the activity of EnpA was demonstrated only on isolated peptidoglycan fragments. Herein we report conditions in which EnpA lyses bacterial cells live with very high efficiency demonstrating great bacteriolytic potential, though limited to a low ionic strength environment. We have solved the structure of the EnpA H109A inactive variant and analyzed it in the context of related peptidoglycan hydrolases structures to reveal the bases for the specificity determination. All M23 structures share a very conserved β-sheet core which constitutes the rigid bottom of the substrate-binding groove and active site, while variable loops create the walls of the deep and narrow binding cleft. A detailed analysis of the binding groove architecture, specificity of M23 enzymes and D,L peptidases demonstrates that the substrate groove, which is particularly deep and narrow, is accessible preferably for peptides composed of amino acids with short side chains or subsequent L and D-isomers. As a result, the bottom of the groove is involved in interactions with the main chain of the substrate while the side chains are protruding in one plane towards the groove opening. We concluded that the selectivity of the substrates is based on their conformations allowed only for polyglycine chains and alternating chirality of the amino acids.
Topics: Amino Acid Sequence; Bacterial Proteins; Catalytic Domain; Endopeptidases; Enterococcus faecalis; N-Acetylmuramoyl-L-alanine Amidase; Peptide Hydrolases; Peptidoglycan; Prophages; Protein Binding; Staphylococcus; Staphylococcus aureus; Substrate Specificity
PubMed: 34281200
DOI: 10.3390/ijms22137136 -
Journal of Clinical Microbiology Feb 1983Species of coagulase-negative staphylococci isolated from urine specimens submitted from both inpatients and outpatients to the clinical microbiology laboratory of a...
Species of coagulase-negative staphylococci isolated from urine specimens submitted from both inpatients and outpatients to the clinical microbiology laboratory of a teaching hospital were identified with a biotyping system, with species then correlated by clinical features and antimicrobial susceptibility. Of 145 isolates, 102 (70%) were Staphylococcus epidermidis, 24 (17%) were Staphylococcus saprophyticus, 7 (4.7%) were Staphylococcus haemolyticus, 4 (2.8%) were Staphylococcus hominis, 3 (2.1%) were Staphylococcus simulans, and 5 (3.4%) were other species. Features characterizing persons with bacteriuria with S. saprophyticus compared with bacteriuria with any other species included female sex (95% versus 52%), young age (median age, 22 years versus 61 years), ambulatory status (hospital outpatients, 86% versus 23%), and absence of indwelling catheters (4.5% versus 49%). All other coagulase-negative staphylococci were isolated in a setting suggesting nosocomial acquisition, were more frequently resistant to common antimicrobial agents (42% multiply resistant versus 4.2% of S. saprophyticus), and were not distinguished by clinical features. Novobiocin susceptibility, with a sensitivity of 100% and specificity of 96%, provided a simple and reliable test for differentiation of S. saprophyticus from other coagulase-negative staphylococci and should be routinely used for urinary tract specimens in the clinical laboratory.
Topics: Anti-Bacterial Agents; Coagulase; Humans; Microbial Sensitivity Tests; Staphylococcal Infections; Staphylococcus; Urinary Tract Infections
PubMed: 6833480
DOI: 10.1128/jcm.17.2.267-271.1983 -
Journal of Dairy Science Oct 2009Subclinical mastitis caused by intramammary infections (IMI) with coagulase-negative staphylococci (CNS) is common in dairy cows and may cause herd problems. Control of...
Subclinical mastitis caused by intramammary infections (IMI) with coagulase-negative staphylococci (CNS) is common in dairy cows and may cause herd problems. Control of CNS mastitis is complicated by the fact that CNS contain a large number of different species. The aim of the study was to investigate the epidemiology of different CNS species in dairy herds with problems caused by subclinical CNS mastitis. In 11 herds, udder quarter samples were taken twice 1 mo apart, and CNS isolates were identified to the species level by biochemical methods. The ability of different CNS species to induce a persistent infection, and their associations with milk production, cow milk somatic cell count, lactation number, and month of lactation in cows with subclinical mastitis were studied. Persistent IMI were common in quarters infected with Staphylococcus chromogenes, Staphylococcus epidermidis, and Staphylococcus simulans. The results did not indicate differences between these CNS species in their association with daily milk production, cow milk somatic cell count, and month of lactation in cows with subclinical mastitis. In cows with subclinical mastitis, S. epidermidis IMI were mainly found in multiparous cows, whereas S. chromogenes IMI were mainly found in primiparous cows.
Topics: Animals; Cattle; Coagulase; Female; Lactation; Mastitis, Bovine; Milk; Species Specificity; Staphylococcal Infections; Staphylococcus; Staphylococcus epidermidis
PubMed: 19762813
DOI: 10.3168/jds.2009-2184 -
AIMS Microbiology 2019The increasing spread of antibiotic-resistant microorganisms has led to the necessity of developing alternative antimicrobial treatments. The use of peptidoglycan...
The increasing spread of antibiotic-resistant microorganisms has led to the necessity of developing alternative antimicrobial treatments. The use of peptidoglycan hydrolases is a promising approach to combat bacterial infections. In our study, we constructed a 2 kb-triple-acting fusion gene () encoding the N-terminal amidase-5 domain of streptococcal LambdaSA2 prophage endolysin (D-glutamine-L-lysin endopeptidase), a mid-protein amidase-2 domain derived from the staphylococcal phage 2638A endolysin (N-acetylmuramoyl-L-alanine amidase) and the mature version (246 residues) of the Lysostaphin bacteriocin (glycyl-glycine endopeptidase) at the C-terminus. The gene was expressed in plants using the non-replicating (CPMV)-based vector pEAQ-HT and the replicating AltMV)-based pGD5TGB123-MCS-CP3 vector, and in using pET expression vectors pET26b+ and pET28a+. The resulting poor expression of this fusion protein in plants prompted the construction of a gene codon-optimized for expression in tobacco plants, resulting in an improved codon adaptation index (CAI) from 0.79 ( gene) to 0.93 ( gene). Incorporation of the nt gene into the pEAQ-HT vector, followed by transient expression in , led to accumulation of TFnt to an approximate level of 0.12 mg/g of fresh leaf weight. Antimicrobial activity of purified plant- and bacterial-produced TFnt proteins was assessed against two strains of Gram-positive 305 and Newman. The results showed that plant-produced TFnt protein was preferentially active against 305, showing 14% of growth inhibition, while the bacterial-produced TFnt revealed significant antimicrobial activity against both strains, showing 68 (IC 25 µg/ml) and 60% (IC 71 µg/ml) growth inhibition against 305 and Newman, respectively. Although the combination of codon optimization and transient expression using the non-replicating pEAQ-HT expression vector facilitated production of the TFnt protein in plants, the most functionally active antimicrobial protein was obtained using the prokaryotic expression system.
PubMed: 31384710
DOI: 10.3934/microbiol.2019.2.158 -
Journal of Dairy Science Apr 1999A 1-yr field investigation of clinical mastitis in heifers was carried out in 24 veterinary districts in Norway. Quarter lacteal secretions from cases that occurred...
A 1-yr field investigation of clinical mastitis in heifers was carried out in 24 veterinary districts in Norway. Quarter lacteal secretions from cases that occurred prepartum or within 14 d postpartum were examined bacteriologically. The study included 1040 heifers with clinical mastitis, and the total number of quarters that were clinically affected was 1361. The organisms that were most frequently isolated from samples from these quarters were Staphylococcus aureus (44.3%), Streptococcus dysgalactiae (18.2%), Staph. aureus together with Strep. dysgalactiae (1.2%), coagulase-negative staphylococci (12.8%), Arcanobacterium pyogenes (3.5%), A. pyogenes together with Strep. dysgalactiae (0.5%) or Staph. aureus (0.4%), and Escherichia coli (6.4%). Of the coagulase-negative staphylococci, Staphylococcus simulans (53.7%), Staphylococcus hyicus (14.8%), and Staphylococcus chromogenes (14.8%) were the most prevalent species. Except for a higher relative percentage of A. pyogenes in cases that occurred before parturition (8.2%) than in cases that occurred after parturition (2.7%), no significant differences were observed in the distribution of the various organisms among prepartum and postpartum cases. Regional variations were observed in the distribution of organisms. The proportions of Staph. aureus and A. pyogenes were highest, and the proportion of coagulase-negative staphylococci was lowest, in late autumn and early winter. The proportion of E. coli was highest in summer. In heifers in which mastitis was associated with increased rectal temperature or other systemic signs, the proportion of clinically affected quarters that were infected with Staph. aureus was larger than that in heifers without systemic reaction.
Topics: Actinomyces; Actinomycosis; Animals; Cattle; Escherichia coli; Escherichia coli Infections; Female; Mastitis, Bovine; Norway; Pregnancy; Staphylococcal Infections; Staphylococcus; Streptococcal Infections; Streptococcus
PubMed: 10212457
DOI: 10.3168/jds.S0022-0302(99)75288-4 -
Revista Da Sociedade Brasileira de... 2009Coagulase-negative staphylococci are frequently associated with nosocomial infections, and healthcare professionals can be reservoirs and spread them in hospitals and in...
Coagulase-negative staphylococci are frequently associated with nosocomial infections, and healthcare professionals can be reservoirs and spread them in hospitals and in the community. The aim of this study was to identify species of coagulase-negative staphylococci isolated from the saliva of nursing professionals, determine the resistance profile and detect the mecA gene. One hundred coagulase-negative staphylococci were selected: 41 were identified as Staphylococcus epidermidis, 25 as Staphylococcus saprophyticus, 18 as Staphylococcus haemolyticus, eight as Staphylococcus cohnii, four as Staphylococcus lugdunenses, three as Staphylococcus capitis and one as Staphylococcus simulans. Of these, 32% presented oxacillin resistance, 84.4% mupirocin resistance and 32% cefoxitin resistance, and all were vancomycin sensitive. Among the oxacillin-resistant coagulase-negative staphylococci, 93.7% developed in oxacillin agar (6microg/ml) and the mecA gene was detected in 75%. The results indicate that higher investments should be directed towards identifying coagulase-negative staphylococcus species in healthcare institutions and in the community.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Humans; Microbial Sensitivity Tests; Nursing Staff, Hospital; Oxacillin; Penicillin Resistance; Penicillin-Binding Proteins; Polymerase Chain Reaction; Saliva; Staphylococcus
PubMed: 19802475
DOI: 10.1590/s0037-86822009000400008 -
Journal of Bacteriology Jun 1998Many of the genes coding for extracellular toxins, enzymes, and cell surface proteins in Staphylococcus aureus are regulated by a 510-nucleotide (nt) RNA molecule,...
Many of the genes coding for extracellular toxins, enzymes, and cell surface proteins in Staphylococcus aureus are regulated by a 510-nucleotide (nt) RNA molecule, RNAIII. Transcription of genes encoding secreted toxins and enzymes, including hla (alpha-toxin), saeB (enterotoxin B), tst (toxic shock syndrome toxin 1), and ssp (serine protease), is stimulated, while transcription of genes encoding cell surface proteins, like spa (protein A) and fnb (fibronectin binding proteins), is repressed. Besides being a regulator, RNAIII is also an mRNA coding for staphylococcal delta-lysin. We have identified RNAIII homologs in three different coagulase-negative staphylococci (CoNS), i.e., Staphylococcus epidermidis, Staphylococcus simulans, and Staphylococcus warneri. RNAIII from these CoNS turned out to be very similar to that of S. aureus and contained open reading frames encoding delta-lysin homologs. Though a number of big insertions and/or deletions have occurred, mainly in the 5' half of the molecules, the sequences show a high degree of identity, especially in the first 50 and last 150 nt. The CoNS RNAIII had the ability to completely repress transcription of protein A in an RNAIII-deficient S. aureus mutant and the ability to stimulate transcription of the alpha-toxin and serine protease genes. However, the stimulatory effect was impaired compared to that of S. aureus RNAIII, suggesting that these regulatory functions are independent. By creating S. epidermidis-S. aureus RNAIII hybrids, we could also show that both the 5' and 3' halves of the RNAIII molecule are involved in the transcriptional regulation of alpha-toxin and serine protease mRNAs in S. aureus.
Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; Coagulase; DNA, Bacterial; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genetic Complementation Test; Hemolysin Proteins; Hybridization, Genetic; Molecular Sequence Data; RNA, Antisense; RNA, Bacterial; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Staphylococcus; Staphylococcus aureus; Staphylococcus epidermidis; Trans-Activators; Transcription Factors; Virulence
PubMed: 9620969
DOI: 10.1128/JB.180.12.3181-3186.1998 -
Applied and Environmental Microbiology Jun 2008Chitosan is a polysaccharide biopolymer that combines a unique set of versatile physicochemical and biological characteristics which allow for a wide range of...
Chitosan is a polysaccharide biopolymer that combines a unique set of versatile physicochemical and biological characteristics which allow for a wide range of applications. Although its antimicrobial activity is well documented, its mode of action has hitherto remained only vaguely defined. In this work we investigated the antimicrobial mode of action of chitosan using a combination of approaches, including in vitro assays, killing kinetics, cellular leakage measurements, membrane potential estimations, and electron microscopy, in addition to transcriptional response analysis. Chitosan, whose antimicrobial activity was influenced by several factors, exhibited a dose-dependent growth-inhibitory effect. A simultaneous permeabilization of the cell membrane to small cellular components, coupled to a significant membrane depolarization, was detected. A concomitant interference with cell wall biosynthesis was not observed. Chitosan treatment of Staphylococcus simulans 22 cells did not give rise to cell wall lysis; the cell membrane also remained intact. Analysis of transcriptional response data revealed that chitosan treatment leads to multiple changes in the expression profiles of Staphylococcus aureus SG511 genes involved in the regulation of stress and autolysis, as well as genes associated with energy metabolism. Finally, a possible mechanism for chitosan's activity is postulated. Although we contend that there might not be a single classical target that would explain chitosan's antimicrobial action, we speculate that binding of chitosan to teichoic acids, coupled with a potential extraction of membrane lipids (predominantly lipoteichoic acid) results in a sequence of events, ultimately leading to bacterial death.
Topics: Anti-Bacterial Agents; Cell Membrane; Cell Wall; Chitosan; Colony Count, Microbial; Gene Expression; Gene Expression Profiling; Membrane Potentials; Microbial Sensitivity Tests; Microbial Viability; Microscopy, Electron, Transmission; Permeability; Potassium; Staphylococcus
PubMed: 18456858
DOI: 10.1128/AEM.00453-08 -
Microbiology (Reading, England) Nov 1997Bovine mastitis is caused mainly by certain Staphylococcus and Streptococcus species. The sequences of the 16S-23S rRNA spacer regions were determined for the nine...
Bovine mastitis is caused mainly by certain Staphylococcus and Streptococcus species. The sequences of the 16S-23S rRNA spacer regions were determined for the nine species which cause mastitis: Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus hyicus, Staphylococcus simulans, Staphylococcus xylosus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis. Significant variation was found between the spacer sequences of different species with the lengths of the spacers varying from 240 to 461 bp. Between genera the spacers shared only short conserved regions (8-9 bp) and within genera the sequence identities varied from 53 to 85%. This variation made it possible to construct specific primer pairs for these species and genera. The specificities of these primers were tested with 25 bacterial species and 51 isolates from cattle with clinical mastitis. The DNA-based identification of the mastitis species was mostly successful.
Topics: Animals; Base Sequence; Cattle; DNA Primers; DNA, Bacterial; DNA, Ribosomal; Female; Genetic Variation; Mastitis, Bovine; Milk; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Ribosomal, 16S; RNA, Ribosomal, 23S; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Nucleic Acid; Species Specificity; Staphylococcus; Streptococcus
PubMed: 9387227
DOI: 10.1099/00221287-143-11-3491 -
Iranian Journal of Microbiology Apr 2023secretes an antimicrobial compound called lysostaphin, which has bactericidal properties. It destroys staphylococci through the hydrolysis of peptidoglycan in the cell...
BACKGROUND AND OBJECTIVES
secretes an antimicrobial compound called lysostaphin, which has bactericidal properties. It destroys staphylococci through the hydrolysis of peptidoglycan in the cell wall. Therefore, this unique property indicates the high ability of lysostaphin in the treatment of staphylococcal infections and is considered as an anti-staphylococcal agent.
MATERIALS AND METHODS
BL21 (DE3) competent cells were transformed with pET32a-lysostaphin clone and induced by isopropyl-β-D-thio-galactoside (IPTG). The recombinant protein was purified by affinity chromatography. Recombinant lysostaphin -A-based ointment was used for external wound healing in animal model. activity of ointment was evaluated by clinical evidences and cytological microscopic assessment.
RESULTS
Our results showed the recombinant protein was produced exactly. The results of checkerboard tests showed MIC, MBC and antibacterial activity test an acute reduction of cell viability during the use of lysostaphin, and SEM results approved the intense wrecking effects of lysostaphin in combination on bacterial cells. Macroscopic findings and microscopic data showed that the recombinant lysostaphin ointment was effective on excisional wound healing.
CONCLUSION
Our findings proved that the recombinant lysostaphin ointment was effective on wound healing due to infection.
PubMed: 37193239
DOI: 10.18502/ijm.v15i2.12476