| 1. |
Møretrø T,
Hermansen L,
Holck AL,
Sidhu MS,
Rudi K,
Langsrud S,
( 2003 ) Biofilm formation and the presence of the intercellular adhesion locus ica among staphylococci from food and food processing environments. PMID : 12957956 : DOI : 10.1128/aem.69.9.5648-5655.2003 PMC : PMC194930 Abstract >>
In clinical staphylococci, the presence of the ica genes and biofilm formation are considered important for virulence. Biofilm formation may also be of importance for survival and virulence in food-related staphylococci. In the present work, staphylococci from the food industry were found to differ greatly in their abilities to form biofilms on polystyrene. A total of 7 and 21 of 144 food-related strains were found to be strong and weak biofilm formers, respectively. Glucose and sodium chloride stimulated biofilm formation. The biofilm-forming strains belonged to nine different coagulase-negative species of Staphylococcus. The icaA gene of the intercellular adhesion locus was detected by Southern blotting and hybridization in 38 of 67 food-related strains tested. The presence of icaA was positively correlated with strong biofilm formation. The icaA gene was partly sequenced for 22 food-related strains from nine different species of Staphylococcus, and their icaA genes were found to have DNA similarities to previously sequenced icaA genes of 69 to 100%. Northern blot analysis indicated that the expression of the ica genes was higher in strong biofilm formers than that seen with strains not forming biofilms. Biofilm formation on polystyrene was positively correlated with biofilm formation on stainless steel and with resistance to quaternary ammonium compounds, a group of disinfectants.
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2. |
Morikawa K,
Inose Y,
Okamura H,
Maruyama A,
Hayashi H,
Takeyasu K,
Ohta T,
( 2003 ) A new staphylococcal sigma factor in the conserved gene cassette: functional significance and implication for the evolutionary processes. PMID : 12875655 : Abstract >>
Staphylococcus aureus is a major human pathogen and causes a serious hospital infection due to the acquired multidrug resistance. Unlike the well-studied bacteria such as Escherichia coli and Bacillus subtilis, which have seven and 18 sigma factors, respectively, only two sigma factors have been known for S. aureus. We searched for possible sigma factor genes by examining the S. aureus genome with a special attention to the gene arrangement around the sigma factor genes of a close relative, B. subtilis. A new sigma factor gene was identified in Staphylococcus. The gene constituted a conserved gene cluster with other genes including translation- and transcription-related genes. Phylogenetic analysis and comparison of the gene sequences among species indicated that the staphylococcal sigma factor originated from a common ancestor of B. subtilis SigH. An over-expression of this sigma factor in S. aureus resulted in a drastic induction of the expression of the com operons that encode proteins required for the natural genetic competence. We demonstrated that the newly identified staphylococcal sigma factor participated in a regulatory network of transcription that controlled the genetic competence genes. In our phylogenetic tree, the factor was classified as a single group with a common function.
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3. |
Poyart C,
Quesne G,
Boumaila C,
Trieu-Cuot P,
( 2001 ) Rapid and accurate species-level identification of coagulase-negative staphylococci by using the sodA gene as a target. PMID : 11724835 : DOI : 10.1128/JCM.39.12.4296-4301.2001 PMC : PMC88539 Abstract >>
Simple PCR and sequencing assays that utilize a single pair of degenerate primers were used to characterize a 429-bp-long DNA fragment internal (sodA(int)) to the sodA gene encoding the manganese-dependent superoxide dismutase in 40 coagulase-negative staphylococcal (CNS) type strains. The topology of the phylogenetic tree obtained was in general agreement with that which was inferred from an analysis of their 16S rRNA or hsp60 gene sequences. Sequence analysis revealed that the staphylococcal sodA genes exhibit a higher divergence than does the corresponding 16S ribosomal DNA. These results confirm that the sodA gene constitutes a highly discriminative target sequence for differentiating closely related bacterial species. Clinical isolates that could not be identified at the species level by phenotypical tests were identified by use of this database. These results demonstrate the usefulness of this method for rapid and accurate species identification of CNS isolates, although it does not allow discrimination of subspecies. The sodA sequence polymorphisms observed with staphylococcal species offer good opportunities for the development of assays based on DNA chip technologies.
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4. |
Drancourt M,
Raoult D,
( 2002 ) rpoB gene sequence-based identification of Staphylococcus species. PMID : 11923353 : DOI : 10.1128/jcm.40.4.1333-1338.2002 PMC : PMC140360 Abstract >>
The complete sequence of rpoB, the gene encoding the beta subunit of RNA polymerase was determined for Staphylococcus saccharolyticus, Staphylococcus lugdunensis, S taphylococcus caprae, and Staphylococcus intermedius and partial sequences were obtained for an additional 27 Staphylococcus species. The complete rpoB sequences varied in length from 3,452 to 3,845 bp and had a 36.8 to 39.2% GC content. The partial sequences had 71.6 to 93.6% interspecies homology and exhibited a 0.08 to 0.8% intraspecific divergence. With a few exceptions, the phylogenetic relationships inferred from the partial rpoB sequences were in agreement with those previously derived from DNA-DNA hybridization studies and analyses of 16S ribosomal DNA gene sequences and partial HSP60 gene sequences. The staphylococcal rpoB sequence database we established enabled us to develop a molecular method for identifying Staphylococcus isolates by PCR followed by direct sequencing of the 751-bp amplicon. In blind tests, this method correctly identified 10 Staphylococcus isolates, and no positive results were obtained with 10 non-Staphylococcus gram-positive and gram-negative bacterial isolates. We propose partial sequencing of the rpoB gene as a new tool for the accurate identification of Staphylococcus isolates.
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5. |
Martineau F,
Picard FJ,
Ke D,
Paradis S,
Roy PH,
Ouellette M,
Bergeron MG,
( 2001 ) Development of a PCR assay for identification of staphylococci at genus and species levels. PMID : 11427566 : DOI : 10.1128/JCM.39.7.2541-2547.2001 PMC : PMC88182 Abstract >>
We have developed a PCR-based assay which allows the detection of staphylococci at the genus level by targeting the tuf gene, which encodes the elongation factor Tu. Degenerate PCR primers derived from consensus regions of several tuf genes were used to amplify a target region of 884 bp from 11 representative staphylococcal species. Subsequently, the entire nucleotide sequence of these amplicons was determined. The analysis of a multiple alignment of these sequences revealed regions conserved among staphylococci but distinct from those of other gram-positive bacteria genetically related to staphylococci. PCR primers complementary to these regions could amplify specifically and efficiently a DNA fragment of 370 bp for all of 27 different staphylococcal species tested. There was no amplification with genomic DNA prepared from 53 nonstaphylococcal species tested to verify the specificity of the assay (20 gram positive and 33 gram negative). Furthermore, this assay amplified efficiently all 27 American Type Culture Collection (ATCC) staphylococcal reference strains as well as 307 clinical isolates of staphylococci from the Qu?bec City region. Analysis of the multiple sequence alignment for the 884-bp fragment for the 11 staphylococcal species as well as comparison of the sequences for the 370-bp amplicon from five unrelated ATCC and clinical strains for each of the species S. aureus, S. epidermidis, S. haemolyticus, S. hominis, and S. saprophyticus demonstrated sufficient interspecies polymorphism to generate genus- and species-specific capture probes. This sequence information allowed the development of Staphylococcus-specific and species-specific (targeting S. aureus, S. epidermidis, S. haemolyticus, S. hominis, or S. saprophyticus) capture probes hybridizing to the 370-bp amplicon. In conclusion, this PCR assay is suitable for detection of staphylococci at both genus and species levels.
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6. |
Av-Gay Y,
Dovichi NJ,
Chow AW,
Bay SJ,
Su SC,
Reynolds RP,
( 1999 ) Species identification and phylogenetic relationships based on partial HSP60 gene sequences within the genus Staphylococcus. PMID : 10425778 : DOI : 10.1099/00207713-49-3-1181 Abstract >>
The phylogenetic relationships among 36 validly described species or subspecies within the genus Staphylococcus were investigated by cloning and sequencing their 60 kDa heat-shock protein (HSP60) genes using a set of universal degenerate HSP60 PCR primers. The cloned partial HSP60 DNA sequences from nine Staphylococcus aureus strains were highly conserved (97-100% DNA sequence similarity; mean 98%), indicating that the HSP60 gene of multiple isolates within the same species have little microheterogeneity. At the subspecies level, DNA sequence similarity among members of S. aureus, Staphylococcus schleiferi, Staphylococcus cohnii and Staphylococcus capitis ranged from 91 to 98%. At the interspecies level, sequence similarity among 23 distinct species of staphylococci ranged from 74 to 93% (mean 82%). By comparison, the highest sequence similarity of Bacillus subtilis and Escherichia coli with members within the genus Staphylococcus was only 70 and 59%, respectively. Importantly, phylogenetic analysis based on the neighbour-joining distance method revealed remarkable concordance between the tree derived from partial HSP60 gene sequences and that based on genomic DNA-DNA hybridization, while 16S rRNA gene sequences correlated less well. The results demonstrate that DNA sequences from the highly conserved and ubiquitous HSP60 gene offer a convenient and accurate tool for species-specific identification and phylogenetic analysis of staphylococci.
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7. |
Fitzgibbon JE,
Nahvi MD,
John JF,
( 1999 ) Topoisomerase sequences of coagulase-negative staphylococcal isolates resistant to ciprofloxacin or trovafloxacin. PMID : 10390214 : PMC : PMC89335 Abstract >>
Coagulase-negative staphylococcal isolates (n = 188) were screened for susceptibility to oxacillin, ciprofloxacin, and trovafloxacin, a new fluoroquinolone. At an oxacillin concentration of >/=4 microg/ml, 43% were methicillin resistant; of these, 70% were ciprofloxacin resistant (MIC, >/=4 microg/ml). Of the methicillin-resistant, ciprofloxacin-resistant isolates, 46% were susceptible to /=8 microg/ml) and increased trovafloxacin MICs (0.25 to 2 microg/ml) could be conferred by the combined presence of single mutations in each gyrA and grlA gene. Trovafloxacin MICs of >/=8 microg/ml also occurred, but these required an additional mutation in grlA.
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8. |
Layer F,
Ghebremedhin B,
König W,
König B,
( 2007 ) Differentiation of Staphylococcus spp. by terminal-restriction fragment length polymorphism analysis of glyceraldehyde-3-phosphate dehydrogenase-encoding gene. PMID : 17681623 : DOI : 10.1016/j.mimet.2007.06.015 Abstract >>
Classical phenotypic and biochemical testing do not lead to correct identification of the distinct Staphylococcus species. Therefore, the aim of our study was to develop a method for the reliable and accurate determination of distinct Staphylococcus species. In the present study, the 931-934-bp partial sequences of the glyceraldehyde-3-phosphate dehydrogenase-encoding (gap) gene of 28 validly described Staphylococcus species were amplified and sequenced. By using the respective sequence information we performed a terminal-restriction fragment length polymorphism (T-RFLP) analysis. For T-RFLP the partial gap gene was amplified with double-fluorescently labelled primers and digested with the restriction enzymes DdeI, BspHI and TaqI. Distinctive T-RFLP patterns were rendered by the use of capillary electrophoresis with laser-induced fluorescence detection. This molecular method allowed us to identify all 28 Staphylococcus species with high specificity. This was validated by analysis of 34 Staphylococcus epidermidis and 28 Staphylococcus haemolyticus isolates. These results demonstrate the feasibility and applicability of the T-RFLP method based on the partial gap gene sequences for rapid and accurate species identification.
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9. |
Layer F,
Ghebremedhin B,
Moder KA,
König W,
König B,
( 2006 ) Comparative study using various methods for identification of Staphylococcus species in clinical specimens. PMID : 16891498 : DOI : 10.1128/JCM.00226-06 PMC : PMC1594629 Abstract >>
Coagulase-negative staphylococci (CNS) play a predominant role in nosocomial infections. Rapid, reliable identification of these organisms is essential for accurate diagnosis and prompt effective treatment of these infections. Quite recently, the VITEK 2 g-positive (gram-positive [GP]) identification card (bioM?rieux) has been redesigned for greater accuracy in the identification of gram-positive cocci. We compared the BD Phoenix (Becton Dickinson) and VITEK 2 (bioM?rieux) automated microbiology systems, using their respective update version cards, and the API ID32 STAPH test. The glyceraldehyde-3-phosphate dehydrogenase (gap) gene-based T-RFLP (terminal restriction fragment length polymorphism) method was used for verifying the results. In total, 86 clinical isolates of CNS and 27 reference strains were analyzed. The results show that for identification of CNS, the automated identification methods using the newest VITEK 2 and BD Phoenix identification cards are comparable. However, API ID32 STAPH revealed more correct results compared to both automated microbiology systems. Despite the increased performance of the phenotypic automated identification systems compared to the former versions, molecular methods, e.g., the gap-based T-RFLP method, still show superior accuracy in identifying Staphylococcus species other than Staphylococcus aureus.
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10. |
Sakinc T,
Kleine B,
Gatermann SG,
( 2006 ) SdrI, a serine-aspartate repeat protein identified in Staphylococcus saprophyticus strain 7108, is a collagen-binding protein. PMID : 16861649 : DOI : 10.1128/IAI.01885-05 PMC : PMC1539602 Abstract >>
A gene encoding a serine-aspartate repeat protein of Staphylococcus saprophyticus, an important cause of urinary tract infections in young women, has been cloned and sequenced. In contrast to other SD repeat proteins, SdrI carries 21 additional N-terminal repeats with a consensus sequence of (P/A)ATKE(K/E)A(A/V)(T/I)(A/T/S)EE and has the longest SD(AD)(1-5) repetitive region (854 amino acids) described so far. This highly repetitive sequence contains only the amino acids serine, asparagine, and a distinctly greater amount of alanine (37%) than all other known SD repeat proteins (2.3 to 4.4%). In addition, it is a collagen-binding protein of S. saprophyticus and the second example in this organism of a surface protein carrying the LPXTG motif. We constructed an isogenic sdrI knockout mutant that showed decreased binding to immobilized collagen compared with wild-type S. saprophyticus strain 7108. Binding could be reconstituted by complementation. Collagen binding is specifically caused by SdrI, and the recently described UafA protein, the only LPXTG-containing protein in the genome sequence of the type strain, is not involved in this trait. Our experiments suggest that, as in other staphylococci, the presence of different LPXTG-anchored cell wall proteins is common in S. saprophyticus and support the notion that the presence of matrix-binding surface proteins is common in staphylococci.
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11. |
Hauschild T,
Lüthje P,
Schwarz S,
( 2006 ) Characterization of a novel type of MLSB resistance plasmid from Staphylococcus saprophyticus carrying a constitutively expressed erm(C) gene. PMID : 16487670 : DOI : 10.1016/j.vetmic.2006.01.007 Abstract >>
An erm(C)-carrying plasmid of unusual size and restriction map, designated pSES22, was identified in a Staphylococcus saprophyticus strain and sequenced completely. Constitutive expression of the erm(C) gene from pSES22 is based on a novel 22-bp tandem duplication in the erm(C) translational attenuator. Comparative analysis of the deduced Erm(C) amino acid sequence revealed that Erm(C) from pSES22 - together with an Erm(C) methylase from S. hyicus - represented a separate branch in the homology tree of Erm(C) methylases. Structural comparisons showed that plasmid pSES22 differed distinctly from all other completely sequenced erm(C)-carrying resistance plasmids. However, pSES22 was similar to several members of a diverse group of small plasmids, all of which carried closely related plasmid backbones consisting of the genes repU and pre/mob, but differed in their resistance genes.
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12. |
Sakinc T,
Woznowski M,
Ebsen M,
Gatermann SG,
( 2005 ) The surface-associated protein of Staphylococcus saprophyticus is a lipase. PMID : 16177313 : DOI : 10.1128/IAI.73.10.6419-6428.2005 PMC : PMC1230896 Abstract >>
Staphylococcus saprophyticus surface-associated protein (Ssp) was the first surface protein described for this organism. Ssp-positive strains display a fuzzy layer of surface-associated material in electron micrographs, whereas Ssp-negative strains appear to be smooth. The physiologic function of Ssp, however, has remained elusive. To clone the associated gene, we determined the N-terminal sequence, as well as an internal amino acid sequence, of the purified protein. We derived two degenerate primers from these peptide sequences, which we used to identify the ssp gene from genomic DNA of S. saprophyticus 7108. The gene was cloned by PCR techniques and was found to be homologous to genes encoding staphylococcal lipases. In keeping with this finding, strains 7108 and 9325, which are Ssp positive, showed lipase activity on tributyrylglycerol agar plates, whereas the Ssp-negative strain CCM883 did not. Association of enzyme activity with the cloned DNA was proven by introducing the gene into Staphylococcus carnosus TM300. When wild-type strain 7108 and an isogenic mutant were analyzed by transmission electron microscopy, strain 7108 exhibited the fuzzy surface layer, whereas the mutant appeared to be smooth. Lipase activity and the surface appendages could be restored by reintroduction of the cloned gene into the mutant. Experiments using immobilized collagen type I did not provide evidence for the involvement of Ssp in adherence to this matrix protein. Our experiments thus provided evidence that Ssp is a surface-associated lipase of S. saprophyticus.
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13. |
Kuroda M,
Yamashita A,
Hirakawa H,
Kumano M,
Morikawa K,
Higashide M,
Maruyama A,
Inose Y,
Matoba K,
Toh H,
Kuhara S,
Hattori M,
Ohta T,
( 2005 ) Whole genome sequence of Staphylococcus saprophyticus reveals the pathogenesis of uncomplicated urinary tract infection. PMID : 16135568 : DOI : 10.1073/pnas.0502950102 PMC : PMC1201578 DOI : 10.1073/pnas.0502950102 PMC : PMC1201578 Abstract >>
Staphylococcus saprophyticus is a uropathogenic Staphylococcus frequently isolated from young female outpatients presenting with uncomplicated urinary tract infections. We sequenced the whole genome of S. saprophyticus type strain ATCC 15305, which harbors a circular chromosome of 2,516,575 bp with 2,446 ORFs and two plasmids. Comparative genomic analyses with the strains of two other species, Staphylococcus aureus and Staphylococcus epidermidis, as well as experimental data, revealed the following characteristics of the S. saprophyticus genome. S. saprophyticus does not possess any virulence factors found in S. aureus, such as coagulase, enterotoxins, exoenzymes, and extracellular matrix-binding proteins, although it does have a remarkable paralog expansion of transport systems related to highly variable ion contents in the urinary environment. A further unique feature is that only a single ORF is predictable as a cell wall-anchored protein, and it shows positive hemagglutination and adherence to human bladder cell associated with initial colonization in the urinary tract. It also shows significantly high urease activity in S. saprophyticus. The uropathogenicity of S. saprophyticus can be attributed to its genome that is needed for its survival in the human urinary tract by means of novel cell wall-anchored adhesin and redundant uro-adaptive transport systems, together with urease.
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14. |
Frank KL,
Hanssen AD,
Patel R,
( 2004 ) icaA is not a useful diagnostic marker for prosthetic joint infection. PMID : 15472359 : DOI : 10.1128/JCM.42.10.4846-4849.2004 PMC : PMC522308 Abstract >>
A collection of 99 staphylococcal isolates associated with prosthetic joint infection and 23 coagulase-negative staphylococci isolated from noninfected arthroplasty-associated specimens were screened in order to determine whether the presence of icaA could be used to distinguish between pathogens and nonpathogens. All Staphylococcus aureus prosthetic joint infection isolates (n = 55) were icaA positive. A total of 46% (20 out of 44) of coagulase-negative staphylococcal prosthetic joint infection isolates were icaA positive, and 30% (7 out of 23) of arthroplasty-associated non-prosthetic joint infection-associated coagulase-negative staphylococcal isolates were icaA positive (P = 0.23). Certain coagulase-negative Staphylococcus species appeared more likely to be isolated as either arthroplasty-associated non-prosthetic joint infection-associated isolates (e.g., Staphylococcus warneri and Staphylococcus hominis) or pathogens (e.g., Staphylococcus lugdunensis). The presence of icaA in a coagulase-negative staphylococcal isolate associated with an arthroplasty is not a useful diagnostic indicator of pathogenicity.
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15. |
Sakinc T,
Kulczak P,
Henne K,
Gatermann SG,
( 2004 ) Cloning of an agr homologue of Staphylococcus saprophyticus. PMID : 15268951 : DOI : 10.1016/j.femsle.2004.06.030 Abstract >>
An agr homologue of Staphylococcus saprophyticus was identified, cloned and sequenced. The gene locus shows homologies to other staphylococcal agr systems, especially to those of S. epidermidis and S. lugdunensis. A putative RNAIII was identified and found to be differentially expressed during the growth phases. In contrast to the RNAIII molecules of S. epidermidis and S. aureus it does not contain an open reading frame that codes for a protein with homologies to the delta-toxin. Using PCR, the agr was found to be present in clinical isolates of S. saprophyticus.
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16. |
Gueneau de Novoa P,
Williams KP,
( 2004 ) The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts. PMID : 14681369 : DOI : 10.1093/nar/gkh102 PMC : PMC308836 DOI : 10.1093/nar/gkh102 PMC : PMC308836 Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
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17. |
Gueneau de Novoa P,
Williams KP,
( 2004 ) The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts. PMID : 14681369 : DOI : 10.1093/nar/gkh102 PMC : PMC308836 DOI : 10.1093/nar/gkh102 PMC : PMC308836 Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
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18. |
Capurro A,
Artursson K,
Waller KP,
Bengtsson B,
Ericsson-Unnerstad H,
Aspán A,
( 2009 ) Comparison of a commercialized phenotyping system, antimicrobial susceptibility testing, and tuf gene sequence-based genotyping for species-level identification of coagulase-negative staphylococci isolated from cases of bovine mastitis. PMID : 18930604 : DOI : 10.1016/j.vetmic.2008.08.028 Abstract >>
In order to evaluate the usefulness of some phenotypic and genotypic methods for species identification of coagulase-negative staphylococci (CNS), isolates were obtained from bovine cases of clinical and sub-clinical mastitis from different geographical areas in Sweden. By using the Staph-Zym test, antimicrobial susceptibility testing, and sequencing of part of the CNS tuf gene and, when needed, part of the 16S rRNA gene we characterized 82 clinical isolates and 24 reference strains of 18 different species of staphylococci. The genotypic methods identified nine different species of CNS among the 82 milk isolates. A comparison with results obtained by tuf gene sequencing showed that Staph-Zym correctly identified CNS reference strains to species level more often than bovine milk CNS isolates (83% and 61%, respectively). In addition, tests supplementary to the Staph-Zym were frequently needed in both groups of isolates (50% of reference strains and 33% of milk isolates) to obtain an identification of the strain. It is notable that Staph-Zym judged two isolates as CNS, although they belonged to other species, could not give a species name in 11% of the bovine CNS isolates, and gave 28% of the isolates an incorrect species name. The present study indicates that the studied phenotypic methods are unreliable for identification of CNS from bovine intra-mammary infections.
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19. |
Rossi P,
Aramini JM,
Xiao R,
Chen CX,
Nwosu C,
Owens LA,
Maglaqui M,
Nair R,
Fischer M,
Acton TB,
Honig B,
Rost B,
Montelione GT,
( 2009 ) Structural elucidation of the Cys-His-Glu-Asn proteolytic relay in the secreted CHAP domain enzyme from the human pathogen Staphylococcus saprophyticus. PMID : 18951393 : DOI : 10.1002/prot.22267 PMC : PMC2735724 Abstract >>
N/A
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20. |
Strauss C,
Hu Y,
Coates A,
Perreten V,
( 2017 ) A Novel erm(44) Gene Variant from a Human Staphylococcus saprophyticus Isolate Confers Resistance to Macrolides and Lincosamides but Not Streptogramins. PMID : 27799208 : DOI : 10.1128/AAC.01655-16 PMC : PMC5192153 Abstract >>
A novel erm(44) gene variant, erm(44)v, has been identified by whole-genome sequencing in a Staphylococcus saprophyticus isolate from the skin of a healthy person. It has the particularity to confer resistance to macrolides and lincosamides but not to streptogramin B when expressed in S. aureus The erm(44)v gene resides on a 19,400-bp genomic island which contains phage-associated proteins and is integrated into the chromosome of S. saprophyticus.
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21. |
Ma?yszko I,
Schwarz S,
Hauschild T,
( 2014 ) Detection of a new mecC allotype, mecC2, in methicillin-resistant Staphylococcus saprophyticus. PMID : 24569631 : DOI : 10.1093/jac/dku043 Abstract >>
N/A
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22. |
Marty E,
Buchs J,
Eugster-Meier E,
Lacroix C,
Meile L,
( 2012 ) Identification of staphylococci and dominant lactic acid bacteria in spontaneously fermented Swiss meat products using PCR-RFLP. PMID : 22202869 : DOI : 10.1016/j.fm.2011.09.011 Abstract >>
Pathogenic, spoilage, and technologically important microorganisms were monitored in 21 spontaneously fermented Swiss meat products manufactured with meat from wildlife or animals grown in natural habitat. Thereby, PCR-restriction fragment length polymorphism (RFLP) on rpoB and 16S rRNA gene sequences provided a powerful tool for fast and accurate identification of the main microbial population. Lactobacillus sakei and Lactobacillus curvatus dominated in fermented meat products followed by Staphylococcus species, which constituted 88.2% of all Gram-positive, catalase-positive cocci (GCC(+)) with cell counts varying from 2.6 to 7.0 log cfu/g during maturation. Staphylococcus equorum was prevalent in frequency and cell counts during maturation (18.0%; 5.0-7.3 log cfu/g) and in the end products (28.4%; 1.8-6.2 log cfu/g) implicating a new presumptive starter species for meat fermentation. Nine out of 14 end products indicated safety risks to consumers due to the high incidence of Staphylococcus saprophyticus or Staphylococcus epidermidis combined with cell counts of 7.4 and 4.9 log cfu/g, respectively. This fact was supported by the detection of Staphylococcus aureus and Enterobacteriaceae in ready-to-eat products strongly exceeding the tolerable limit of 2 log cfu/g. Spontaneously fermented meat products produced from wildlife or animals grown in natural habitats not only gave rise to hygienic and safety concerns but also provided new presumptive starter strains.
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23. |
Wang Y,
Zhang W,
Wang J,
Wu C,
Shen Z,
Fu X,
Yan Y,
Zhang Q,
Schwarz S,
Shen J,
( 2012 ) Distribution of the multidrug resistance gene cfr in Staphylococcus species isolates from swine farms in China. PMID : 22183168 : DOI : 10.1128/AAC.05827-11 PMC : PMC3294914 Abstract >>
A total of 149 porcine Staphylococcus isolates with florfenicol MICs of ? 16 �gg/ml were screened for the presence of the multiresistance gene cfr, its location on plasmids, and its genetic environment. In total, 125 isolates carried either cfr (16 isolates), fexA (92 isolates), or both genes (17 isolates). The 33 cfr-carrying staphylococci, which included isolates of the species Staphylococcus cohnii, S. arlettae, and S. saprophyticus in which the cfr gene has not been described before, exhibited a wide variety of SmaI pulsed-field gel electrophoresis patterns. In 18 cases, the cfr gene was located on plasmids. Four different types of cfr-carrying plasmids--pSS-01 (n = 2; 40 kb), pSS-02 (n = 3; 35.4 kb), pSS-03 (n = 10; 7.1 kb), and pBS-01 (n = 3; 16.4 kb)--were differentiated on the basis of their sizes, restriction patterns, and additional resistance genes. Sequence analysis revealed that in plasmid pSS-01, the cfr gene was flanked in the upstream part by a complete aacA-aphD-carrying Tn4001-like transposon and in the downstream part by a complete fexA-carrying transposon Tn558. In plasmid pSS-02, an insertion sequence IS21-558 and the cfr gene were integrated into transposon Tn558 and thereby truncated the tnpA and tnpB genes. The smallest cfr-carrying plasmid pSS-03 carried the macrolide-lincosamide-streptogramin B resistance gene erm(C). Plasmid pBS-01, previously described in Bacillus spp., harbored a Tn917-like transposon, including the macrolide-lincosamide-streptogramin B resistance gene erm(B) in the cfr downstream region. Plasmids, which in part carry additional resistance genes, seem to play an important role in the dissemination of the gene cfr among porcine staphylococci.
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24. |
Zong Z,
Peng C,
Lü X,
( 2011 ) Diversity of SCCmec elements in methicillin-resistant coagulase-negative staphylococci clinical isolates. PMID : 21637845 : DOI : 10.1371/journal.pone.0020191 PMC : PMC3102682 Abstract >>
Methicillin-resistant coagulase-negative staphylococci (MR-CoNS) are opportunistic pathogens and serve as a large reservoir of staphylococcal cassette chromosome mec (SCCmec). Characterization of SCCmec in MR-CoNS can generate useful information on the mobilization and evolution of this element. Non-repetitive MR-CoNS clinical isolates (n = 84; 39 S. epidermidis, 19 S. haemolyticus, 9 S. hominis, 6 S. capitis, 4 S. warneri, 2 S. cohnii, 2 S. saprophyticus, 1 S. kloosii, 1 S. simulans and 1 S. massiliensis) were collected. All isolates could grow on plates with 4 mg/L cefoxitin and all had mecA as detected by PCR. Strain typing using RAPD and ERIC-PCR revealed that almost all isolates were of different strains. SCCmec typing was performed using multiplex PCR published previously. For isolates in which SCCmec could not be typed, the mec complex classes were determined by additional PCR and the ccr genes were amplified with published or newly-designed primers and then sequenced. SCCmec types were assigned for 63 isolates by multiplex PCR and were assigned for 14 other isolates by PCR targeting mec and ccr. Among 77 isolates with determined SCCmec types, 54 had a single type, including type III (n = 19), IV (n = 14), V (n = 10), II (n = 2), I (n = 1), VIII (n = 1) and five unnamed types (n = 7), while 23 isolates had two types, III+V (n = 12), II+V (n = 8), II+IV (n = 2) or IV+V (n = 1). The five unnamed types were assigned UT1 (class A mec, ccrA1/ccrB4), UT2 (class C1 mec, ccrA4/ccrB4), UT3 (class A mec, ccrA5/ccrB3), UT4 (class C2 mec, ccrA2/ccrB2 plus ccrC1) and UT5 (class A mec, ccrA1/ccrB1 plus ccrC1). SCCmec types III, IV and V were prevalent in MR-CoNS and many isolates could harbor more than one type. Several new types of SCCmec were identified, highlighting the great genetic diversity and the need of developing classification schemes for SCCmec in MR-CoNS.
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25. |
( 1996 ) HSP60 gene sequences as universal targets for microbial species identification: studies with coagulase-negative staphylococci. PMID : 8815090 : PMC : PMC228899 Abstract >>
A set of universal degenerate primers which amplified, by PCR, a 600-bp oligomer encoding a portion of the 60-kDa heat shock protein (HSP60) of both Staphylococcus aureus and Staphylococcus epidermidis were developed. However, when used as a DNA probe, the 600-bp PCR product generated from S. epidermidis failed to cross-hybridize under high-stringency conditions with the genomic DNA of S. aureus and vice versa. To investigate whether species-specific sequences might exist within the highly conserved HSP60 genes among different staphylococci, digoxigenin-labelled HSP60 probes generated by the degenerate HSP60 primers were prepared from the six most commonly isolated Staphylococcus species (S. aureus 8325-4, S. epidermidis 9759, S. haemolyticus ATCC 29970, S. schleiferi ATCC 43808, S. saprophyticus KL122, and S. lugdunensis CRSN 850412). These probes were used for dot blot hybridization with genomic DNA of 58 reference and clinical isolates of Staphylococcus and non-Staphylococcus species. These six Staphylococcus species HSP60 probes correctly identified the entire set of staphylococcal isolates. The species specificity of these HSP60 probes was further demonstrated by dot blot hybridization with PCR-amplified DNA from mixed cultures of different Staphylococcus species and by the partial DNA sequences of these probes. In addition, sequence homology searches of the NCBI BLAST databases with these partial HSP60 DNA sequences yielded the highest matching scores for both S. epidermidis and S. aureus with the corresponding species-specified probes. Finally, the HSP60 degenerate primers were shown to amplify an anticipated 600-bp PCR product from all 29 Staphylococcus species and from all but 2 of 30 other microbial species, including various gram-positive and gram-negative bacteria, mycobacteria, and fungi. These preliminary data suggest the presence of species-specific sequence variation within the highly conserved HSP60 genes of staphylococci. Further work is required to determine whether these degenerate HSP60 primers may be exploited for species-specific microbic identification and phylogenetic investigation of staphylococci and perhaps other microorganisms in general.
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26. |
( 1994 ) Urease from Staphylococcus saprophyticus: purification, characterization and comparison to Staphylococcus xylosus urease. PMID : 8042901 : Abstract >>
Urease from Staphylococcus saprophyticus was purified more than 800-fold by liquid chromatography reaching homogeneity, as shown by isoelectric focussing, at a maximum specific activity of 1979 U/mg. The molecular weight of the native enzyme was 420,000; it consisted of subunits with molecular weights of 72,400 (alpha), 20,400 (beta), 13,900 (gamma) in an estimated (alpha beta gamma)4 stoichiometry. In native gradient polyacrylamide gel electrophoresis urease exhibited a multiple activity band pattern with molecular weights ranging from 420,000 to 100,000. In the native enzyme, 4.09 (+/- 0.25) atoms of nickel per molecule were detected. The N-terminal amino acids of the urease subunits were identical to those from Staphylococcus xylosus, and amino acid analysis revealed high similarities in both enzymes; no cysteine was detected after acid hydrolysis of vinylpyridinylated urease. Electron micrographs of negatively stained urease specimens from both staphylococci showed identical size and structure.
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27. |
Gricajeva A,
Bikut? I,
Kal?dien? L,
( 2019 ) Atypical organic-solvent tolerant bacterial hormone sensitive lipase-like homologue EstAG1 from Staphylococcus saprophyticus AG1: Synthesis and characterization. PMID : 30797006 : DOI : 10.1016/j.ijbiomac.2019.02.110 Abstract >>
Biocatalysts exerting activity against ester bonds have a broad range of applications in modern biotechnology. Some of the most industrially relevant enzymes of this type are lipolytic and their market is predicted to uphold leadership up till 2024. In this study, a novel bacterial hormone-sensitive lipase-like (bHSL) family homologue, designated EstAG1, was discovered by mining gDNA of bacteria isolated from fat contaminated soil in Lithuania. Putative lipolytic enzyme was cloned, overexpressed in E. coli, purified and characterized determining its biochemical properties. While the true physiological role of the discovered leaderless, ~36 kDa enzyme is unknown, metal-activated EstAG1 possessed optima at 45-47.5 �XC, pH 7.5-8, with a generally intermediate activity profile between esterases and lipases. Furthermore, EstAG1 was hyperactivated by ethanol, dioxane and DMSO, implicating that it could be industrially applicable enzyme for the synthesis of valuable products such as biodiesel, flavor esters, etc. Sequence analysis and structure modeling revealed that the highest sequence homology of EstAG1 with the closest structurally and functionally described protein makes up only 26%. It was also revealed that EstAG1 has some differences in the bHSL family-characteristic conserved sequence motives. Therefore, EstAG1 presents interest both in terms of biotechnological applications and basic research.
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28. |
Ras G,
Bailly X,
Chacornac JP,
Zuliani V,
Derkx P,
Seibert TM,
Talon R,
Leroy S,
( 2018 ) Contribution of nitric oxide synthase from coagulase-negative staphylococci to the development of red myoglobin derivatives. PMID : 29150355 : DOI : 10.1016/j.ijfoodmicro.2017.11.005 Abstract >>
As part of the microbial community of meat or as starter cultures, coagulase-negative staphylococci (CNS) serve several essential technological purposes in meat products, such as color development through the reduction of nitrate to nitrite. As the safety of nitrite as an additive has been questioned, we explored the potential of CNS to develop red myoglobin derivatives such as oxymyoglobin and nitrosomyoglobin. Nitrosoheme was extracted to evaluate NO production. This production could be due to a nitric oxide synthase (NOS) activity. In all CNS strains, a nos gene was identified. The NOS sequences deduced were highly conserved within CNS. A phylogenetic tree based on the NOS sequences revealed that the strains within species were clustered. Ninety-one percent of the strains, whatever the species, were able to form red myoglobin derivatives in aerobic conditions, but a high variability was observed between strains within species. However, NO production was low as nitrosomyoglobin represented 8% to 16% of the red pigments according to the species. Formation of oxymyoglobin, especially under aerobic conditions, was substantial, but varied greatly within species. The mechanism involved in the formation of oxymyoglobin could rely on staphylococcal reductases and remains to be explored.
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29. |
( 1998 ) The Staphylococcus qacH gene product: a new member of the SMR family encoding multidrug resistance. PMID : 9631545 : DOI : 10.1111/j.1574-6968.1998.tb13025.x Abstract >>
The prevalence of disinfectant-resistant food-related microorganisms is of concern to the food industry. The Staphylococcus saprophyticus strain ST2H6 isolated from a poultry processing plant contained a 2.4-kb plasmid (p2H6) harbouring qacH, which encodes resistance to disinfectants based on quaternary ammonium compounds. The complete p2H6 nucleotide sequence revealed an open reading frame encoding a putative protein of 107 amino acid residues with strong similarity to members of the small multidrug resistance protein family. QacH also conferred high-level ethidium bromide resistance and low-level proflavine resistance and thus differed phenotypically from the similar proteins Smr and QacG. Fluorimetry indicated that the high-level ethidium bromide resistance was due to improved efflux energised by the proton motive force. Site-directed mutagenesis substituting the Asp-24 residue with Glu-24 had no effect on resistance characteristics. An additional open reading frame on p2H6 encoded a putative protein with similarity to rolling circle replication proteins.
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30. |
( 1998 ) Cloning of aas, a gene encoding a Staphylococcus saprophyticus surface protein with adhesive and autolytic properties. PMID : 9723925 : DOI : 10.1046/j.1365-2958.1998.00983.x Abstract >>
A gene encoding a novel cell wall-associated protein of Staphylococcus saprophyticus that binds fibronectin and to sheep erythrocytes has been cloned and sequenced. The 4392 bp open reading frame codes for an amino acid sequence that is quite similar to the Atl, an autolysin, of Staphylococcus aureus and to the AtlE of S. epidermidis. The two regions of most pronounced homology code for an N-acetyl-muramyl-L-alanine amidase and for an endo-beta-N-acetyl-D-glucosaminidase. The cloned protein lysed cells of S. saprophyticus and Micrococcus luteus exogenously. Subcloning localized the enzymatic activities to the regions of high homology and demonstrated that the interposed sequence is responsible for the adhesive activities. Two allelic replacement mutants were constructed that lacked autolytic activity and adhesive properties. The N-terminal portion of the protein contains seven highly conserved, contiguous repeats with no similarity to published sequences. It lacks the motifs typical of Gram-positive surface proteins and shows a different overall organization. This autolysin/adhesin of S. saprophyticus (Aas) appears to represent a new class of staphylococcal adhesins.
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