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Protein Expression and Purification Nov 2018Lysostaphin, a bacteriolytic toxin from Staphylococcus simulans, is a Zn-dependent endopeptidase that cleaves pentaglycine cross-bridges found in peptidoglycan of...
Lysostaphin, a bacteriolytic toxin from Staphylococcus simulans, is a Zn-dependent endopeptidase that cleaves pentaglycine cross-bridges found in peptidoglycan of certain Staphylococci. Here, we have investigated a critical influence of Zn ions on lysostaphin-induced bioactivity. Initially, we succeeded in producing a large amount with high purity of the 28-kDa His-tagged mature lysostaphin via soluble expression in Escherichia coli and subsequent purification via immobilized-Ni affinity chromatography (IMAC). The purified monomeric bacteriocin exhibited concentration-dependent bioactivity against S. aureus and its methicillin-resistant strain through cell-wall hydrolysis rather than membrane perturbation. Following pre-incubation of the purified lysostaphin with exogenous Zn, a marked inhibition in staphylolytic activity was observed. When the pre-mixture was exposed to 1,10-phenanthroline (PNT, a Zn-chelator), the adverse effect of the exogenous Zn on bioactivity was greatly decreased. Conversely, lysostaphin pre-treated with excess PNT retained relatively high bioactivity, indicating ineffective chelation of PNT to detach the catalytic Zn from the active-site pocket. Structural analysis of the lysostaphin-catalytic domain together with amino acid sequence alignments of lysostaphin-like endopeptidases revealed a potential extraneous Zn-binding site found in close proximity to the Zn-coordinating active site. Overall our results provide more insights into an adverse influence of exogenous Zn ions on staphylolytic activity of the purified Zn-dependent endopeptidase lysostaphin, implicating the presence of an extraneous inhibitory metal-binding site.
Topics: Anti-Bacterial Agents; Biocatalysis; Cations, Divalent; Cell Wall; Chelating Agents; Coordination Complexes; Drug Resistance, Bacterial; Escherichia coli; Lysostaphin; Phenanthrolines; Recombinant Proteins; Staphylococcus; Staphylococcus aureus; Zinc
PubMed: 29944958
DOI: 10.1016/j.pep.2018.06.013 -
Journal of Dairy Science Jun 2018The purpose of this study was to investigate non-aureus Staphylococcus spp. intramammary infections (IMI) in periparturient heifers and determine the relationship of...
The purpose of this study was to investigate non-aureus Staphylococcus spp. intramammary infections (IMI) in periparturient heifers and determine the relationship of precalving body site isolation with precalving IMI and postcalving IMI using molecular speciation and strain-typing methods. Primiparous heifers were enrolled at approximately 14 d before expected calving date. Precalving mammary quarter secretions and body site swabbing samples (teat skin, inguinal skin, muzzle, and perineum) were collected. Postcalving, mammary quarter milk samples were collected for culture and somatic cell counting. Precalving body site samples were cultured, and up to 10 staphylococcal colonies were saved for characterization. Staphylococcal isolates were speciated using matrix-assisted laser/desorption ionization time-of-flight mass spectrometry or sequencing of rpoB or tuf. Pulsed-field gel electrophoresis was used to strain type a subset of isolates. Overall, Staphylococcus chromogenes, Staphylococcus agnetis, and Staphylococcus simulans were the most common species identified in precalving mammary secretions, whereas S. chromogenes, Staphylococcus xylosus, and S. agnetis were the most common species found in postcalving milk samples. The most common species identified from body site samples were S. chromogenes, S. xylosus, and Staphylococcus haemolyticus. Mammary quarters that had a precalving mammary secretion that was culture positive for S. agnetis, S. chromogenes, or Staphylococcus devriesei had increased odds of having an IMI with the same species postcalving. A S. chromogenes IMI postcalving was associated with higher somatic cell count when compared with postcalving culture-negative quarters. Among heifers identified with a non-aureus Staphylococcus spp. IMI either precalving or postcalving, heifers that had S. agnetis or S. chromogenes isolated from their teat skin had increased odds of having the same species found in their precalving mammary secretions, and heifers with S. chromogenes, S. simulans, and S. xylosus isolated from their teat skin precalving were at increased odds of having an IMI with the same species postcalving. Overall, 44% of all heifers with a S. chromogenes IMI around the time of parturition had the same strain isolated from a body site. Based on pulsed-field gel electrophoresis, a high level of strain diversity was found.
Topics: Animals; Cattle; Cell Count; Electrophoresis, Gel, Pulsed-Field; Female; Mammary Glands, Animal; Mastitis, Bovine; Milk; Staphylococcal Infections; Staphylococcus
PubMed: 29525303
DOI: 10.3168/jds.2017-13910 -
Veterinary Microbiology Apr 1994Ninety three staphylococci isolated from clinical specimens from cats were characterized and identified. Because the biochemical characteristics of Staphylococcus felis...
Ninety three staphylococci isolated from clinical specimens from cats were characterized and identified. Because the biochemical characteristics of Staphylococcus felis were very similar to those of Staphylococcus simulans, results were submitted to numerical analysis and DNA homology. Forty-two isolates (45%) were identified as S. felis, and 4 isolates (4%) as S. simulans. The other species identified, in order of their frequency were, 12 Staphylococcus aureus (13%), 9 Staphylococcus intermedius (10), 6 Staphylococcus sciuri (6), 6 Staphylococcus epidermidis (6), 2 Staphylococcus haemolyticus (2), 2 Staphylococcus xylosus (2), 1 Staphylococcus capitis (1), 1 Staphylococcus equorum (1), 1 Staphylococcus gallinarum (1) and 1 Staphylococcus lentus (1).
Topics: Animals; Cat Diseases; Cats; DNA, Bacterial; Nucleic Acid Hybridization; Staphylococcal Infections; Staphylococcus
PubMed: 8042273
DOI: 10.1016/0378-1135(94)90162-7 -
Veterinary Microbiology Nov 2010Nukacin 3299 (formerly designated simulancin 3299), produced by a Staphylococcus simulans strain involved in bovine mastitis in Brazil, is the first peptide bacteriocin...
Nukacin 3299 (formerly designated simulancin 3299), produced by a Staphylococcus simulans strain involved in bovine mastitis in Brazil, is the first peptide bacteriocin described in this staphylococcal species. With the intent to elucidate some aspects of its biology, nukacin 3299 was purified and characterized. The mass of the purified bacteriocin was shown to be 2957.3 Da, and the peptide N-terminal amino acids (KKKSGVI) were identified by Edman degradation. The nukacin 3299 structural gene, nukA, was detected by PCR and DNA sequencing, showing that this bacteriocin is identical to nukacin ISK-1, a 27-amino acid type-A (II) lantibiotic produced by Staphylococcus warneri ISK-1, isolated from a "nukadoko", in Japan. The genes involved in nukacin 3299 biosynthesis are located on plasmid pRJ97 (>27 kb). They have an organization similar to that of the nukacin ISK-1 gene cluster, excepted for the presence of an IS257/431 element (791 bp) present between the orf1 and nukA genes of the nukacin 3299 gene cluster. The presence of this insertion sequence is expected to affect the expression of orf1, whose function is presently unknown. Nukacin 3299 proved to be sensitive to proteolytic enzymes and relatively stable at different temperatures and between pH 3.0-9.0. Nukacin 3299 exhibited activity towards staphylococcal strains involved in bovine mastitis, showing a potential application on mastitis control, a disease with great economic impact.
Topics: Animals; Bacteriocins; Base Sequence; Cattle; Cloning, Molecular; Female; Genes, Bacterial; Mastitis, Bovine; Molecular Sequence Data; Multigene Family; Polymerase Chain Reaction; Sequence Alignment; Sequence Analysis, DNA; Staphylococcal Infections; Staphylococcus
PubMed: 20627619
DOI: 10.1016/j.vetmic.2010.04.032 -
Scientific Reports Jul 2017Herein we describe an association between activation of inflammatory pathways following transient hypoxia and the appearance of the multidrug resistant bacteria...
Herein we describe an association between activation of inflammatory pathways following transient hypoxia and the appearance of the multidrug resistant bacteria Staphylococcus simulans in the fetal brain. Reduction of maternal arterial oxygen tension by 50% over 30 min resulted in a subseiuent significant over-expression of genes associated with immune responses 24 h later in the fetal brain. The activated genes were consistent with stimulation by bacterial lipopolysaccharide; an influx of macrophages and appearance of live bacteria were found in these fetal brains. S. simulans was the predominant bacterial species in fetal brain after hypoxia, but was found in placenta of all animals. Strains of S. simulans from the placenta and fetal brain were equally highly resistant to multiple antibiotics including methicillin and had identical genome sequences. These results suggest that bacteria from the placenta invade the fetal brain after maternal hypoxia.
Topics: Animals; Brain; Drug Resistance, Multiple, Bacterial; Female; Fetal Hypoxia; Gene Expression Regulation, Developmental; Macrophages; Microglia; Placenta; Pregnancy; Sheep; Staphylococcus
PubMed: 28743956
DOI: 10.1038/s41598-017-06789-6 -
International Journal of Systematic and... May 2010Gram-positive, catalase-positive, coagulase-negative, non-motile, non-fermentative and novobiocin-susceptible cocci were isolated from a human brain abscess sample...
Gram-positive, catalase-positive, coagulase-negative, non-motile, non-fermentative and novobiocin-susceptible cocci were isolated from a human brain abscess sample (strain 5402776(T)). This novel strain was analysed by a polyphasic taxonomic approach. The respiratory quinones detected were MK-7 (93 %) and MK-6 (7 %) and the major fatty acids were C(15 : 0) iso (60.5 %), C(17 : 0) iso (8.96 %) C(15 : 0) anteiso (7.93 %) and C(19 : 0) iso (6.78 %). The peptidoglycan type was A3alpha l-Lys-Gly(2-3)-l-Ser-Gly. Based on cellular morphology and biochemical criteria, the new isolate was assigned to the genus Staphylococcus, although it did not correspond to any recognized species. The G+C content of the DNA was 36.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that the new isolate was most closely related to Staphylococcus piscifermentans, Staphylococcus condimenti, Staphylococcus carnosus subsp. carnosus, S. carnosus subsp. utilis and Staphylococcus simulans (97.7 %, 97.6 %, 97.6 %, 97.6 % and 96.5 % sequence similarity, respectively). Comparison of tuf, hsp60, rpoB, dnaJ and sodA gene sequences was also performed. In phylogenetic analysis inferred from tuf, dnaJ and rpoB gene sequence comparisons, strain 5402776(T) clustered with Staphylococcus pettenkoferi (93.7 %, 82.5 % and 89 % sequence similarity, respectively) and on phylogenetic analysis inferred from sodA gene sequence comparisons, it clustered with Staphylococcus chromogenes (82.8 %). On the basis of phenotypic and genotypic data, this isolate represents a novel species for which the name Staphylococcus massiliensis sp. nov. is proposed (type strain 5402776(T)=CCUG 55927(T)=CSUR P23(T)).
Topics: Bacterial Proteins; Bacterial Typing Techniques; Base Composition; Brain Abscess; DNA, Bacterial; DNA, Ribosomal; Fatty Acids; Genes, rRNA; Genotype; Humans; Male; Middle Aged; Molecular Sequence Data; Phenotype; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Species Specificity; Staphylococcal Infections; Staphylococcus
PubMed: 19666814
DOI: 10.1099/ijs.0.006486-0 -
Journal of Zoo and Wildlife Medicine :... Jun 2011Staphylococcus simulans, a coagulase-negative staphylococcal species, can cause several diseases in humans and animals. This report describes a case of dermatosis...
Staphylococcus simulans, a coagulase-negative staphylococcal species, can cause several diseases in humans and animals. This report describes a case of dermatosis characterized by broad, well-circumscribed hyperkeratosis and alopecia on the back of a household pygmy hedgehog (Erinaceous albiventris). Quills and exudates were studied by microscopy. The microscopic examination of the exudates collected from the lesion revealed several leukocytes and numerous gram-positive cocci. An aerobic bacterial culture revealed overgrowth of the same gram-positive bacteria. The bacterium was identified as S. simulans by polymerase chain reaction amplification and direct sequencing targeted to the 16S ribosomal RNA gene. This report is the first to show that S. simulans could be related to the dermatitis of hedgehogs.
Topics: Animals; Animals, Zoo; Dermatitis; Female; Hedgehogs; Phylogeny; Staphylococcal Skin Infections; Staphylococcus
PubMed: 22946405
DOI: 10.1638/2009-0140.1 -
Veterinary Microbiology Apr 1999This study was conducted to characterize Staphylococcus simulans isolated from cases of bovine mastitis. A total of 134 isolates of S. simulans selected from 80 quarters...
This study was conducted to characterize Staphylococcus simulans isolated from cases of bovine mastitis. A total of 134 isolates of S. simulans selected from 80 quarters from 61 cows or heifers in 37 different herds were characterized by EcoRI ribotyping. From 22 quarters two to seven consecutive isolates taken at weekly intervals were selected. Furthermore, three isolates from clinical infections in humans and two reference strains were included. A total of 16 different ribotypes were found, however, two types predominated. In most herds more than one type was found. From the 22 different quarters, where 76 paired or multiple isolates were at disposal, the same ribotype was constantly found in the same quarter. This study showed that S. simulans causing bovine mastitis could be divided into relatively large number of different types, but that two types predominated. More than one type could be found in the same herd and within different quarters of the same cow, but ribotyping confirmed that S. simulans could be the cause of persistent and stable infections.
Topics: Animals; Cattle; Cattle Diseases; Deoxyribonuclease EcoRI; Female; Mastitis, Bovine; Staphylococcal Infections; Staphylococcus
PubMed: 10227477
DOI: 10.1016/s0378-1135(99)00005-x -
Proceedings of the National Academy of... Mar 1987A 1.5-kilobase-pair fragment of DNA that contains the lysostaphin gene from Staphylococcus simulans and its flanking sequences has been cloned and completely sequenced....
A 1.5-kilobase-pair fragment of DNA that contains the lysostaphin gene from Staphylococcus simulans and its flanking sequences has been cloned and completely sequenced. The gene encodes a preproenzyme of Mr 42,000. The NH2-terminal sequence of the preproenzyme is composed of a signal peptide followed by seven tandem repeats of a 13-amino acid sequence. Conversion of prolysostaphin to the mature enzyme occurs extracellularly in cultures of S. simulans and involves removal of the NH2-terminal portion of the proenzyme that contains the tandem repeats. The high degree of homology of the repeats suggests that they have arisen by duplication of a 39-base-pair sequence of DNA. In S. simulans, the lysostaphin gene is present on a large beta-lactamase plasmid.
Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; Escherichia coli; Genes; Genes, Bacterial; Lysostaphin; Staphylococcus; Transcription, Genetic
PubMed: 3547405
DOI: 10.1073/pnas.84.5.1127 -
Protein Expression and Purification Aug 2001The Staphylococcus simulans gene encoding lysostaphin has been PCR amplified from pRG5 recombinant plasmid (ATCC 67076) and cloned into Escherichia coli expression...
The Staphylococcus simulans gene encoding lysostaphin has been PCR amplified from pRG5 recombinant plasmid (ATCC 67076) and cloned into Escherichia coli expression pTYB12 vector (IMPACT-CN System, New England BioLabs) which allows the overexpression of a target protein as a fusion to a self-cleavable affinity tag. The self-cleavage activity of the intein allows the release of the lysostaphin enzyme from the chitin-bound intein tag, resulting in a single-column purification of the target protein. This abundant overproduction allows purifying milligram amounts of the enzyme.
Topics: Affinity Labels; Amino Acid Sequence; Base Sequence; Chitin; Cloning, Molecular; Escherichia coli; Lysostaphin; Molecular Sequence Data; Plasmids; Recombinant Fusion Proteins; Staphylococcus
PubMed: 11483010
DOI: 10.1006/prep.2001.1454