PCR-based identification ofStaphylococcus epidermidistargetinggseAencoding the glutamic-acid-specific protease

2004 ◽  
Vol 50 (7) ◽  
pp. 493-498 ◽  
Author(s):  
Y Ikeda ◽  
Y Ohara-Nemoto ◽  
S Kimura ◽  
K Ishibashi ◽  
K Kikuchi
Keyword(s):  
Glutamic Acid ◽  
Staphylococcus Epidermidis ◽  
Single Step ◽  
Clinical Isolates ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Coagulase Negative Staphylococci ◽  
Gene Encoding ◽  
Staphylococcus Capitis ◽  
Specific Protease

The frequency of the gseA gene encoding a glutamic acid-specific serine protease, GluSE, of Staphylococcus epidermidis was investigated. DNA hybridization analysis demonstrated that gseA existed exclusively in S. epidermidis but not in other bacteria examined. A single step PCR assay with a set of designed primers yielded amplification of gseA from all 69 clinical isolates of S. epidermidis taken from patients and healthy adults, whereas production of GluSE was observed in 74% (51/69) of the isolates. Furthermore, none of the 46 clinical isolates of other species of coagulase-negative staphylococci and 45 clinical isolates of Staphylococcus aureus showed amplification, except a Staphylococcus capitis strain. However, this strain was positive for a S. epidermidis-specific DNA region and the DNA sequence of the 16S rRNA gene showed 99% identity with that of S. epidermidis. Therefore, these results indicated that the present PCR assay for gseA was ubiquitous and highly specific for detection of S. epidermidis.Key words: Staphylococcus epidermidis, glutamic acid-specific protease, PCR assay, molecular identification.

scholarly journals Molecular Analysis of Cross-Resistance to Capreomycin, Kanamycin, Amikacin, and Viomycin in Mycobacterium tuberculosis

2005 ◽  
Vol 49 (8) ◽  
pp. 3192-3197 ◽  
Author(s):  
Courtney E. Maus ◽  
Bonnie B. Plikaytis ◽  
Thomas M. Shinnick
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Molecular Basis ◽  
Multidrug Resistant ◽  
Single Step ◽  
Clinical Isolates ◽  
Cross Resistance ◽  
Resistance Pattern ◽  
Rrna Gene ◽  
Gene Encoding ◽  
Treatment Regimens

ABSTRACT Capreomycin, kanamycin, amikacin, and viomycin are drugs that are used to treat multidrug-resistant tuberculosis. Each inhibits translation, and cross-resistance to them is a concern during therapy. A recent study revealed that mutation of the tlyA gene, encoding a putative rRNA methyltransferase, confers capreomycin and viomycin resistance in Mycobacterium tuberculosis bacteria. Mutations in the 16S rRNA gene (rrs) have been associated with resistance to each of the drugs; however, reports of cross-resistance to the drugs have been variable. We investigated the role of rrs mutations in capreomycin resistance and examined the molecular basis of cross-resistance to the four drugs in M. tuberculosis laboratory-generated mutants and clinical isolates. Spontaneous mutants were generated to the drugs singularly and in combination by plating on medium containing one or two drugs. The frequencies of recovery of the mutants on single- and dual-drug plates were consistent with single-step mutations. The rrs genes of all mutants were sequenced, and the tlyA genes were sequenced for mutants selected on capreomycin, viomycin, or both; MICs of all four drugs were determined. Three rrs mutations (A1401G, C1402T, and G1484T) were found, and each was associated with a particular cross-resistance pattern. Similar mutations and cross-resistance patterns were found in drug-resistant clinical isolates. Overall, the data implicate rrs mutations as a molecular basis for resistance to each of the four drugs. Furthermore, the genotypic and phenotypic differences seen in the development of cross-resistance when M. tuberculosis bacteria were exposed to one or two drugs have implications for selection of treatment regimens.

scholarly journals sodA and gap Genes as Markers for The Identification of Staphylococcus capitis

2019 ◽  
Vol 11 (3) ◽  
pp. 314-9
Author(s):  
Rininta Firdaus ◽  
Hege Hartz Jartun ◽  
Miriam Khider
Keyword(s):  
Pcr Primers ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Conventional Pcr ◽  
Pcr Assay ◽  
Coagulase Negative Staphylococci ◽  
Accurate Identification ◽  
Bioinformatic Tools ◽  
Gap Genes ◽  
Staphylococcus Capitis

BACKGROUND: Rapid and accurate identification of Staphylococcus capitis is required to provide a better prognosis for endocarditis patients and tackle the emergence of multidrug resistant strains of the bacteria in hospitals. The current study was aimed to develop polymerase chain reaction (PCR) assay for specific identification of S. capitis using sodA and gap genes as markers.METHODS: Five sequences of sodA and sixteen sequences of gap registered in GeneBank were analysed using bioinformatic tools. PCR primers were designed based on the conserved and specific regions of sodA and gap. Four clinical isolates of S. capitis (named no. 56-59) and six reference strains of coagulase-negative staphylococci (CoNS) species including S. epidermidis ATCC 35984, S. epidermidis 48951I/09, S. lugdunensis 44987/09, S. sciuri 109645I/08, S. warneri 135612/09, S. hominis 114202/08 were used to validate the conventional PCR system.RESULTS: The current PCR system only amplified the DNA template of S. capitis. Current primers specifically targeted S. capitis as the agarose images only showed bands from S. capitis samples.CONCLUSION: The sodA and gap genes might serve as effective markers for identification of S. capitis using conventional PCR. The PCR assay in the current study was able to identify five clinical isolates of S. capitis accurately without mispriming, misamplification and misidentification. The PCR system was also able to discriminate other CoNS including S. epidermidis, S. lugdunensis, S. sciuri, S. warneri and S. hominis.KEYWORDS: Staphylococcus, S. capitis, sodA, gap, PCR

scholarly journals Development of a real-time PCR assay for the detection and identification of Staphylococcus capitis, Staphylococcus haemolyticus and Staphylococcus warneri

2007 ◽  
Vol 56 (10) ◽  
pp. 1346-1349 ◽  
Author(s):  
Tadayuki Iwase ◽  
Keiko Seki ◽  
Hitomi Shinji ◽  
Yoshimitsu Mizunoe ◽  
Shogo Masuda
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
High Specificity ◽  
Pcr Assay ◽  
Coagulase Negative Staphylococci ◽  
Staphylococcus Warneri ◽  
Staphylococcus Haemolyticus ◽  
Staphylococcus Capitis ◽  
Detection And Identification ◽  
Species Specific

Staphylococcus capitis, Staphylococcus haemolyticus and Staphylococcus warneri are coagulase-negative staphylococci. Each species has different characteristics, and a difference in pathology is also seen in compromised hosts. Therefore, the development of a species-specific simple detection method for the identification of these staphylococci is important. Here, a species-specific real-time PCR assay is reported that targets the superoxide dismutase A-encoding gene of these bacteria. Primers were designed with a base that was non-complementary with regard to the other bacteria. This base was at the 3′ end of the primer (3′ mismatch primer) and conferred high specificity. These primers were then evaluated using real-time PCR. They reacted only with the target bacterium. In addition, stable quantitative reactions were observed when experiments were performed using genomic DNA extracted from varying numbers of staphylococci cells (101–107 cells). These results indicate that this method is useful for the identification and quantitative analysis of S. capitis, S. haemolyticus and S. warneri.

scholarly journals Rapid differentiation of Staphylococcus aureus, Staphylococcus epidermidis and other coagulase-negative staphylococci and meticillin susceptibility testing directly from growth-positive blood cultures by multiplex real-time PCR

2010 ◽  
Vol 59 (12) ◽  
pp. 1456-1461 ◽  
Author(s):  
Leanne Jukes ◽  
Jane Mikhail ◽  
Naledi Bome-Mannathoko ◽  
Stephen J. Hadfield ◽  
Llinos G. Harris ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Real Time ◽  
Real Time Pcr ◽  
Staphylococcus Epidermidis ◽  
Susceptibility Testing ◽  
Rapid Test ◽  
Blood Cultures ◽  
Identification System ◽  
Coagulase Negative Staphylococci ◽  
Staphylococcus Capitis

This study evaluated a multiplex real-time PCR method specific for the mecA, femA-SA and femA-SE genes for rapid identification of Staphylococcus aureus, Staphylococcus epidermidis and non-S. epidermidis coagulase-negative staphylococci (CoNS), and meticillin susceptibility testing directly in positive blood cultures that grew Gram-positive cocci in clusters. A total of 100 positive blood cultures produced: 39 S. aureus [12 meticillin-resistant S. aureus (MRSA), 31 % of all the S. aureus]; 30 S. epidermidis (56.6 % of the CoNS), 8 Staphylococcus capitis (15.1 %), 3 Staphylococcus saprophyticus (5.7 %), 4 Staphylococcus hominis (7.5 %), 3 Staphylococcus haemolyticus (5.7 %), 2 Staphylococcus warneri (3.8 %), 1 Staphylococcus cohnii (1.9 %) and 2 unidentified Staphylococcus spp. (3.8 %); and 1 Micrococcus luteus in pure culture. Two blood cultures had no growth on subculture and five blood cultures grew mixed CoNS. For the 95 blood cultures with pure growth or no growth on subculture, there was very good agreement between real-time PCR and the BD Phoenix identification system for staphylococcal species categorization in S. aureus, S. epidermidis and non-S. epidermidis CoNS and meticillin-resistance determination (Cohen's unweighted kappa coefficient κ=0.882). All MRSA and meticillin-susceptible S. aureus were correctly identified by mecA amplification. PCR amplification of mecA was more sensitive for direct detection of meticillin-resistant CoNS in positive blood cultures than testing with the BD Phoenix system. There were no major errors when identifying staphylococcal isolates and their meticillin susceptibility within 2.5 h. Further studies are needed to evaluate the clinical benefit of using such a rapid test on the consumption of glycopeptide antibiotics and the alteration of empiric therapy in the situation of positive blood cultures growing staphylococci, and the respective clinical outcomes.

Characterization of the gene encoding the glutamic-acid-specific protease of Streptomyces griseus

1993 ◽  
Vol 71 (9-10) ◽  
pp. 454-461 ◽  
Author(s):  
Sachdev S. Sidhu ◽  
Gabriel B. Kalmar ◽  
Thor J. Borgford
Keyword(s):  
Glutamic Acid ◽  
Serine Protease ◽  
Streptomyces Griseus ◽  
Gene Encoding ◽  
Lysobacter Enzymogenes ◽  
Glutamic Acid Residue ◽  
Specific Protease ◽  
Mature Enzyme ◽  
Additional Segment ◽  
Pro Region

The complete gene sequence (sprE) for the glutamic-acid-specific serine protease (SGPE) of the gram-positive bacterium Streptomyces griseus is reported. The sprE gene encodes a 355 amino acid pre–pro–mature enzyme. The presence of a glutamic acid residue at the junction of the pro and mature segments of the protein suggests that the enzyme is self-processing. SGPE was found to have extensive homology with the S. griseus proteases A and B. However, there is an additional segment to the pro region of SGPE, lacking in proteases A and B, which has significant homology to the pro region of the α-lytic protease of the gram-negative bacterium Lysobacter enzymogenes. Expression of recombinant SGPE in Bacillus subtilis is also reported, and the enzyme is shown to be self-processing.Key words: serine protease, maturation, expression, propeptide.

scholarly journals In VitroActivities of Arylomycin Natural-Product Antibiotics againstStaphylococcus epidermidisand Other Coagulase-Negative Staphylococci

2010 ◽  
Vol 55 (3) ◽  
pp. 1130-1134 ◽  
Author(s):  
Peter A. Smith ◽  
Michael E. Powers ◽  
Tucker C. Roberts ◽  
Floyd E. Romesberg
Keyword(s):  
Natural Product ◽  
Staphylococcus Epidermidis ◽  
Clinical Isolates ◽  
Signal Peptidase ◽  
Type I ◽  
Potential Utility ◽  
Coagulase Negative Staphylococci ◽  
Inhibitor Binding ◽  
Important Species ◽  
Geographical Locations

ABSTRACTThe arylomycins are a class of natural-product antibiotics that act via the inhibition of type I signal peptidase (SPase), and we have found in diverse bacteria that their activity is limited by the presence of a resistance-conferring Pro residue in SPase that reduces inhibitor binding. We have also demonstrated thatStaphylococcus epidermidis, which lacks this Pro residue, is extremely susceptible to the arylomycins. Here, to further explore the potential utility of the arylomycins, we report an analysis of the activity of a synthetic arylomycin derivative, arylomycin C16, against clinical isolates ofS. epidermidisand other coagulase-negative staphylococci (CoNS) from distinct geographical locations. Against many important species of CoNS, includingS. epidermidis,S. haemolyticus,S. lugdunensis, andS. hominis, we find that arylomycin C16exhibits activity equal to or greater than that of vancomycin, the antibiotic most commonly used to treat CoNS infections. While the susceptibility was generally correlated with the absence of the previously identified Pro residue, several cases were identified where additional factors also appear to contribute.

scholarly journals Purification and characterization of a glutamic acid-specific protease from Staphylococcus epidermidis.

1998 ◽  
Vol 40 (5) ◽  
pp. 542-548 ◽  
Author(s):  
Minoru Sasaki ◽  
Yuko Ohara-Nemoto ◽  
Shihoko Tajika ◽  
Masahiko Kobayashi ◽  
Yoriko Ikeda ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Staphylococcus Epidermidis ◽  
Purification And Characterization ◽  
Specific Protease

Emergence of cfr-harbouring coagulase-negative staphylococci among patients receiving linezolid therapy in two hospitals in China

2013 ◽  
Vol 62 (6) ◽  
pp. 845-850 ◽  
Author(s):  
Xue-Jing Yang ◽  
Yan Chen ◽  
Qing Yang ◽  
Ting-Ting Qu ◽  
Li-Lin Liu ◽  
...  
Keyword(s):  
Nosocomial Infection ◽  
Staphylococcus Epidermidis ◽  
Control Strategies ◽  
Resistance Mechanism ◽  
Mechanisms Of Resistance ◽  
Pfge Analysis ◽  
Coagulase Negative Staphylococci ◽  
Staphylococcus Capitis ◽  
Linezolid Therapy ◽  
Staphylococcus Hominis

This study reports on the emergence of cfr-harbouring coagulase-negative staphylococci (CoNS) among patients who received linezolid therapy in two hospitals in Hangzhou, China. The mechanisms of resistance and transmission were analysed for these resistant isolates. Eight Staphylococcus capitis isolates, one Staphylococcus epidermidis isolate and one Staphylococcus hominis isolate, obtained from patients who had received linezolid therapy in two hospitals in Hangzhou, China, were confirmed as linezolid resistant, with MICs ranging from 8 to >256 mg l−1. The linezolid usage data of the ten patients before isolation of the linezolid-resistant CoNS were collected. PFGE analysis showed that the eight S. capitis isolates from the two hospitals belonged to the same clone. Nine of the linezolid-resistant CoNS isolates carried the cfr gene, which was located on plasmids of a similar size. A 5.3 kb fragment containing the cfr gene, revealing 99 % identity to the sequence of the cfr-harbouring plasmid pSS-01 reported previously, was determined by PCR mapping for all cfr-positive isolates, and the cfr gene was flanked by two copies of IS256-like elements. Thus, these results document the emergence of linezolid-resistant CoNS isolates carrying the cfr gene in Hangzhou, China. Effective nosocomial infection control strategies and the judicious use of antibiotics will be required to prevent further spread of this resistance mechanism.

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