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Journal of Bacteriology Aug 1982As a further development of previous investigations showing that different staphylococcal species display different bacteriolytic activity patterns (lyogroups), the... (Comparative Study)
Comparative Study
As a further development of previous investigations showing that different staphylococcal species display different bacteriolytic activity patterns (lyogroups), the bacteriolytic enzymes excreted by three different Staphylococcus species, Staphylococcus aureus (lyogroup I), S. simulans (lyogroup II), and S. saprophyticus (lyogroup IV); have been purified and characterized. A representative strain from each species was grown in a preselected medium made of fully dialyzable products. Culture supernatants were collected in the appropriate growth phase. Two different affinity adsorbents were used for enzyme purification. One was obtained by coupling lysozyme-digested pure peptidoglycan from Micrococcus luteus to cyanogen bromide-activated Sepharose 4B. The second affinity adsorbent used was chitin. The S. aureus bacteriolytic enzyme bound to the solubilized peptidoglycan but not to chitin, whereas the opposite was true for the S. simulans enzyme. The bacteriolytic enzyme from S. saprophyticus did not bind to either the Sepharose 4B-peptidoglycan resin or to chitin, and its purification was achieved by two ion-exchange chromatography steps combined with gel filtration. All three enzymes were purified to apparent homogeneity. Their subsequent characterization indicated that all acted as endo-beta-N-acetylglucosaminidases. However, the three glucosaminidases differed significantly in their kinetics of activity and bacteriolytic spectrum against heat-killed cells of a variety of microorganisms. Very different values also resulted from molecular weight determinations: 80,000 for the S. aureus enzyme, 45,000 for the S. simulans enzyme, and 31,000 for the S. saprophyticus enzyme. Other important differences were observed in their stability, optimal pH and ionic strength for their activity, and their responses to temperature and divalent cations. These results confirmed the previous proposal that different staphylococcal species excrete different lytic enzymes.
Topics: Acetylglucosaminidase; Bacteriolysis; Hexosaminidases; Hydrogen-Ion Concentration; Kinetics; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Molecular Weight; Staphylococcus; Staphylococcus aureus; Temperature
PubMed: 6807958
DOI: 10.1128/jb.151.2.636-647.1982 -
Jundishapur Journal of Microbiology May 2014Staphylococcus aureus is one of the most common causes of nosocomial infections and its resistance to antibiotics is a global concern. Lysostaphin is an antimicrobial...
BACKGROUND
Staphylococcus aureus is one of the most common causes of nosocomial infections and its resistance to antibiotics is a global concern. Lysostaphin is an antimicrobial agent belonging to a major class of antimicrobial peptides and proteins known as the bacteriocins. It exhibits a high degree of anti-staphylococcal bacteriolytic activity.
OBJECTIVES
In this study, high level of recombinant mature lysostaphin in Escherichia coli was produced by using pET32a expression vector.
MATERIALS AND METHODS
The S. simulans gene encoding lysostaphin was extracted, amplified by polymerase chain reaction (PCR), and sub-cloned in prokaryotic expression vector pET32a. E. coli BL21 (DE3) plysS were transformed with pET32a-lys and gene expression was induced by IPTG. The expressed protein was purified by affinity-chromatography using (Ni-NTA) resin.
RESULTS
PCR and sequencing results confirmed the successful cloning of the target gene into the vector. The expression of protein was induced by IPTG and high concentration of the recombinant protein was obtained via the purification process by affinity-chromatography.
CONCLUSIONS
Our data showed that the recombinant mature lysostaphin protein produced by pET32a vector in E. coli system was very efficient.
PubMed: 25147708
DOI: 10.5812/jjm.10009 -
The Journal of Hospital Infection Dec 1996
Topics: Aged; False Positive Reactions; Humans; Methicillin Resistance; Nursing Homes; Staphylococcal Infections; Staphylococcus; United Kingdom
PubMed: 8971622
DOI: 10.1016/s0195-6701(96)90113-9 -
Biochimie Sep 2001Staphylococcus simulans strain secretes a non-induced lipase in the culture medium. Staphylococcus simulans lipase (SSL), purified to homogeneity, is a tetrameric... (Comparative Study)
Comparative Study
Staphylococcus simulans strain secretes a non-induced lipase in the culture medium. Staphylococcus simulans lipase (SSL), purified to homogeneity, is a tetrameric protein (160 kDa) corresponding to the association of four lipase molecules. The 30 N-terminal amino acid residues were sequenced. This sequence is identical to the one of Staphylococcus aureus PS54 lipase (SAL PS54) and exhibits a high degree of homology with Staphylococcus aureus NCTC8530 lipase (SAL NCTC8530), Staphylococcus hyicus lipase (SHL) and Staphylococcus epidermis RP62A lipase (SEL RP62A) sequences. But the cloning and sequencing of the part of the gene encoding the mature lipase show some differences from SAL PS54 sequence, which suggest that it is a new sequence. The lipase activity was maximal at pH 8.5 and 37 degrees C. SSL is able to hydrolyze triacylglycerols without chain length specificity. A specific activity of about 1000 U/mg was measured on tributyrin or triolein as substrate at 37 degrees C and at pH 8.5 in the presence of 3 mM CaCl(2). In contrast to other staphylococcal lipases previously characterized, Ca(2+) is not required to express the activity of SSL. SSL was found to be stable between pH 4 and pH 9. The enzyme is inactivated after a few minutes when incubated at 60 degrees C. Using tripropionin as substrate, SSL does not present the interfacial activation phenomenon. In contrast to many lipases, SSL is able to hydrolyze its substrate in the presence of bile salts or amphiphilic proteins.
Topics: Amino Acid Sequence; Base Sequence; Bile Acids and Salts; Enzyme Stability; Hydrogen-Ion Concentration; Kinetics; Lipase; Molecular Sequence Data; Sequence Homology, Amino Acid; Staphylococcus; Staphylococcus aureus; Staphylococcus epidermidis; Substrate Specificity; Temperature; Transformation, Bacterial
PubMed: 11698108
DOI: 10.1016/s0300-9084(01)01327-x -
Canadian Journal of Microbiology Dec 2010High-resolution melting analysis (HRMA) is a fast (post-PCR) high-throughput method to scan for sequence variations in a target gene. The aim of this study was to test...
High-resolution melting analysis (HRMA) is a fast (post-PCR) high-throughput method to scan for sequence variations in a target gene. The aim of this study was to test the potential of HRMA to distinguish particular bacterial species of the Staphylococcus genus even when using a broad-range PCR within the 16S rRNA gene where sequence differences are minimal. Genomic DNA samples isolated from 12 reference staphylococcal strains (Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus sciuri, Staphylococcus simulans, Staphylococcus warneri, and Staphylococcus xylosus) were subjected to a real-time PCR amplification of the 16S rRNA gene in the presence of fluorescent dye EvaGreen™, followed by HRMA. Melting profiles were used as molecular fingerprints for bacterial species differentiation. HRMA of S. saprophyticus and S. xylosus resulted in undistinguishable profiles because of their identical sequences in the analyzed 16S rRNA region. The remaining reference strains were fully differentiated either directly or via high-resolution plots obtained by heteroduplex formation between coamplified PCR products of the tested staphylococcal strain and phylogenetically unrelated strain.
Topics: Base Sequence; DNA Fingerprinting; DNA, Bacterial; DNA, Ribosomal; Heteroduplex Analysis; Molecular Sequence Data; RNA, Ribosomal, 16S; Sensitivity and Specificity; Sequence Alignment; Sequence Analysis, DNA; Species Specificity; Staphylococcus; Transition Temperature
PubMed: 21164574
DOI: 10.1139/W10-091 -
Veterinary Journal (London, England :... Jan 2015Since phenotypic methods to identify coagulase negative staphylococci (CNS) from the milk of ruminants often yield unreliable results, methods for molecular... (Review)
Review
Since phenotypic methods to identify coagulase negative staphylococci (CNS) from the milk of ruminants often yield unreliable results, methods for molecular identification based on gene sequencing or fingerprinting techniques have been developed. In addition to culture-based detection of isolates, culture-independent methods may be of interest. On the basis of molecular studies, the five CNS species commonly causing intramammary infections (IMI) are Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus simulans and Staphylococcus xylosus. Current knowledge suggests that S. chromogenes is a bovine-adapted species, with most cases of IMI due to this bacterium being opportunistic. S. haemolyticus also appears to be an opportunistic pathogen, but this bacterium occupies a variety of habitats, the importance of which as a source of IMI remains to be elucidated. S. xylosus appears to be a versatile species, but little is known of its epidemiology. S. epidermidis is considered to be a human-adapted species and most cases of IMI appear to arise from human sources, but the organism is capable of residing in other habitats. S. simulans typically causes contagious IMI, but opportunistic cases also occur and the ecology of this bacterium requires further study. Further studies of the ecology and epidemiology of CNS as a cause of IMI in cattle are required, along with careful attention to classification of these bacteria and the diseases they cause.
Topics: Animals; Bacteriological Techniques; Mastitis; Ruminants; Staphylococcal Infections; Staphylococcus
PubMed: 25467994
DOI: 10.1016/j.tvjl.2014.11.001 -
Antimicrobial Agents and Chemotherapy Sep 1990The presence of an additional penicillin-binding protein (PBP) was demonstrated in methicillin-resistant strains of Staphylococcus epidermidis, S. haemolyticus, S.... (Comparative Study)
Comparative Study
Presence of an additional penicillin-binding protein in methicillin-resistant Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, and Staphylococcus simulans with a low affinity for methicillin, cephalothin, and cefamandole.
The presence of an additional penicillin-binding protein (PBP) was demonstrated in methicillin-resistant strains of Staphylococcus epidermidis, S. haemolyticus, S. hominis, and S. simulans. In these four species, the apparent molecular mass of this protein was analogous to that of PBP 2' of methicillin-resistant S. aureus SR 1550-9. It exhibited a low affinity for methicillin, cephalothin, and cefamandole; and its synthesis was methicillin inducible. Peptide mapping of this PBP from the four species yielded identical results that were analogous to those obtained with S. aureus SR 1550-9. These results suggest that this protein is similar to, if not the same as, PBP 2' of S. aureus and that it is involved in methicillin resistance in the four species studied.
Topics: Bacterial Proteins; Carrier Proteins; Cefamandole; Cephalothin; Hexosyltransferases; Methicillin; Methicillin Resistance; Muramoylpentapeptide Carboxypeptidase; Penicillin-Binding Proteins; Peptide Mapping; Peptidyl Transferases; Phenotype; Staphylococcus; Staphylococcus epidermidis; beta-Lactamases
PubMed: 2285281
DOI: 10.1128/AAC.34.9.1691 -
Journal of Biomolecular Structure &... Dec 2023Cold-adapted and organic solvent tolerant lipases have significant potential in a wide range of synthetic reactions in industry. But there are no sufficient studies on...
Cold-adapted and organic solvent tolerant lipases have significant potential in a wide range of synthetic reactions in industry. But there are no sufficient studies on how these enzymes interacts with their substrates. Herein, the predicted structure and function of the lipase (SCL) are studied. Given the high amino acid sequence homology with the lipase (SSL), 3D structure models of closed and open forms of the lipase were built using the structure of SSL as template. The models suggested the presence of a main lid and a second lid that may act with the former as a double door to control the access to the active site. The SCL models also allowed us to identify key residues involved in binding substrates, calcium or zinc ions. By following this model and utilizing molecular dynamics (MD) simulations, the stability of the lipase at low temperatures could be explained in the presence and in the absence of calcium and zinc. Due to its thermolability, the SCL is extremely valuable for different biotechnological applications in a wide variety of industries from molecular biology to detergency to food and beverage preparation.Communicated by Ramaswamy H. Sarma.
Topics: Calcium; Staphylococcus capitis; Molecular Dynamics Simulation; Lipase; Zinc; Ions
PubMed: 36546696
DOI: 10.1080/07391102.2022.2159528 -
Central European Journal of Public... Jun 2022This work aimed to determine the representation and resistance of bacteria belonging to the genus Staphylococcus and Enterococcus on inanimate surfaces of two selected...
OBJECTIVES
This work aimed to determine the representation and resistance of bacteria belonging to the genus Staphylococcus and Enterococcus on inanimate surfaces of two selected workplaces of the University Hospital of L. Pasteur in Košice (UHLP) and to investigate their importance in the hospital environment. The men's ward of the Department of Internal Medicine (DIM) and the Department of Anaesthesiology and Intensive Care (DAIC) were chosen.
METHODS
Using sterile sampling kits, a total of 182 swabs were collected from the inanimate surfaces of both UHLP workplaces. The swabs were then transported to a microbiological laboratory and inoculated onto sterile culture media (blood agar containing 5% ram erythrocytes). After culturing (24-48 hours, in a thermostat at constant temperature 37 °C), bacterial colonies were identified by mass spectrometry on a MALDI TOF MS. Bacteria belonging to the genera Staphylococcus and Enterococcus were subsequently separated from the spectrum of identified bacteria. Nosocomial significant strains of staphylococci (Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus aureus) and all isolated enterococci were subjected to susceptibility testing for selected antibiotics using the disk diffusion method - E-tests.
RESULTS
Several members of the genus Staphylococcus were identified from the inanimate surfaces of both workplaces. These were mainly coagulase-negative strains - Staphylococcus epidermidis (45), Staphylococcus capitis (34), Staphylococcus haemolyticus (20), Staphylococcus hominis (45), Staphylococcus pasteuri (2), Staphylococcus sroph (1), Staphylococcus simulans (3), and Staphylococcus warneri (4). Staphylococcus aureus strains were also identified (2). Nosocomial significant isolates were tested for susceptibility to the antibiotics cefoxitin (FOX) and oxacillin (OXA). Two members of the genus Enterococcus - Enterococcus faecium (7) and Enterococcus faecalis (8) were isolated. All strains were subject to vancomycin susceptibility testing using the disk method.
Topics: Animals; Anti-Bacterial Agents; Bacteria; Cross Infection; Enterococcus; Hospitals; Humans; Male; Microbial Sensitivity Tests; Sheep; Staphylococcal Infections; Staphylococcus
PubMed: 35841227
DOI: 10.21101/cejph.a7241 -
Infection and Immunity May 1990An encapsulated strain of Staphylococcus simulans was observed to be more resistant to phagocytosis by human granulocytes than was a nonencapsulated strain. Phagocytosis...
An encapsulated strain of Staphylococcus simulans was observed to be more resistant to phagocytosis by human granulocytes than was a nonencapsulated strain. Phagocytosis of the encapsulated strain was enhanced by antisera to S. simulans, but opsonic activity of antisera was removed by absorption with S. simulans capsular material. The encapsulated strain of S. simulans was also more invasive than the nonencapsulated S. simulans in vivo. More encapsulated than nonencapsulated S. simulans were found in heart blood when equal numbers of organisms were injected intraperitoneally into mice. Invasion of the bloodstreams of mice by encapsulated S. simulans was prevented by passive immunization (rabbit antiserum). Thus, the capsule of S. simulans inhibited phagocytosis in vitro and contributed to virulence in vivo.
Topics: Animals; Antibodies, Bacterial; Antigen-Antibody Complex; Blood Bactericidal Activity; Female; Humans; In Vitro Techniques; Mice; Neutrophils; Peritoneal Cavity; Phagocytosis; Staphylococcus
PubMed: 2323819
DOI: 10.1128/iai.58.5.1350-1354.1990