The Isolation And Cloning of Chromosomal Dna Specific for a Clonal Population of Staphylococcus Aureus By Subtractive Hybridisation

AbstractObjective:To report an outbreak of methicillin-resistant Staphylococcus aureus (MRSA) in our burn unit and the steps we used to eradicate the organism.Design And Setting:Outbreak investigation in the burn unit of a 900-bed tertiary-care medical center.Outbreak:Between March and June 1993, MRSA was isolated from 10 patients in our burn unit. All isolates had identical antibiograms and chromosomal DNA patterns.Control Measures:Infection control personnel encouraged healthcare workers to wash their hands after each patient contact. The unit cohorted all infected or colonized patients, placed each affected patient in isolation, and, if possible, transferred the patient to another unit. Despite these measures, new cases occurred. Infection control personnel obtained nares cultures from 56 healthcare workers, 3 of whom carried the epidemic MRSA strain. One healthcare worker cared for six affected patients, and one cared for five patients. We treated the three healthcare workers with mupirocin. Subsequently, no additional patients became colonized or infected with the epidemic MRSA strain.Conclusions:The outbreak ended after we treated healthcare workers who carried the epidemic strain with mupirocin. This approach is not appropriate in all settings. However, we felt it was justified in this case because of a persistent problem after less intrusive measures.

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Species-Specific and Ubiquitous-DNA-Based Assays for Rapid Identification of Staphylococcus aureus

Journal of Clinical Microbiology ◽

10.1128/jcm.36.3.618-623.1998 ◽

1998 ◽

Vol 36 (3) ◽

pp. 618-623 ◽

Cited By ~ 215

Author(s):

Francis Martineau ◽

François J. Picard ◽

Paul H. Roy ◽

Marc Ouellette ◽

Michel G. Bergeron

Keyword(s):

Staphylococcus Aureus ◽

Genomic Library ◽

Antibiotic Resistance Genes ◽

Pcr Amplification ◽

Rapid Identification ◽

Clinical Microbiology Laboratory ◽

Chromosomal Dna ◽

Pcr Assay ◽

Serious Infections ◽

Species Specific

Staphylococcus aureus is the cause of serious infections in humans, including endocarditis, deep-seated abscesses, and bacteremia, which lead to toxic and septic shock syndromes. Rapid and direct identification of this bacterium specifically and ubiquitously directly from clinical specimens would be useful in improving the diagnosis of S. aureus infections in the clinical microbiology laboratory. A wide variety of kits based on biochemical characteristics efficiently identify S. aureus, but the rapidity and the accuracy of each of these methods combined with testing of clinically relevant antibiotic resistance genes need to be improved. On the basis of hybridization assays with randomly selected clones from an S. aureus genomic library, we have identified a chromosomal DNA fragment which is specific for S. aureus and which detected all 82 S. aureus isolates tested. This 442-bp fragment was sequenced and was used to design a set of PCR amplification primers. The PCR assay was also specific and ubiquitous for the identification from bacterial cultures of 195 clinical strains of S. aureus isolated from a variety of anatomical sites and obtained from hospitals throughout the world. The PCR assay that we have developed is simple and can be performed in about 1 h. This DNA-based test provides a novel diagnostic tool for the diagnosis of S. aureus infections.

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Peptide Deformylase in Staphylococcus aureus: Resistance to Inhibition Is Mediated by Mutations in the Formyltransferase Gene

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.7.1825-1831.2000 ◽

2000 ◽

Vol 44 (7) ◽

pp. 1825-1831 ◽

Cited By ~ 92

Author(s):

Peter S. Margolis ◽

Corinne J. Hackbarth ◽

Dennis C. Young ◽

Wen Wang ◽

Dawn Chen ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Genomic Library ◽

Specific Inhibitor ◽

Peptide Deformylase ◽

Cross Resistance ◽

Loss Of Function ◽

Wild Type ◽

Chromosomal Dna ◽

Resistant Mutants

ABSTRACT Peptide deformylase, a bacterial enzyme, represents a novel target for antibiotic discovery. Two deformylase homologs, defA and defB, were identified inStaphylococcus aureus. The defA homolog, located upstream of the transformylase gene, was identified by genomic analysis and was cloned from chromosomal DNA by PCR. A distinct homolog, defB, was cloned from an S. aureus genomic library by complementation of the arabinose-dependent phenotype of a P BAD -def Escherichia coli strain grown under arabinose-limiting conditions. Overexpression in E. coli of defB, but not defA, correlated to increased deformylase activity and decreased susceptibility to actinonin, a deformylase-specific inhibitor. ThedefB gene could not be disrupted in wild-type S. aureus, suggesting that this gene, which encodes a functional deformylase, is essential. In contrast, thedefA gene could be inactivated; the function of this gene is unknown. Actinonin-resistant mutants grew slowly in vitro and did not show cross-resistance to other classes of antibiotics. When compared to the parent, an actinonin-resistant strain produced an attenuated infection in a murine abscess model, indicating that this strain also has a growth disadvantage in vivo. Sequence analysis of the actinonin-resistant mutants revealed that each harbors a loss-of-function mutation in the fmt gene. Susceptibility to actinonin was restored when the wild-type fmt gene was introduced into these mutant strains. An S. aureusΔfmt strain was also resistant to actinonin, suggesting that a functional deformylase activity is not required in a strain that lacks formyltransferase activity. Accordingly, thedefB gene could be disrupted in an fmt mutant.

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Pulsed field gel electrophoresis of chromosomal DNA reveals a clonal population structure toBacillus thuringiensisthat relates in general to crystal protein gene content

FEMS Microbiology Letters ◽

10.1016/s0378-1097(03)00347-1 ◽

2003 ◽

Vol 223 (1) ◽

pp. 61-66 ◽

Cited By ~ 22

Author(s):

Adelaida M Gaviria Rivera ◽

Fergus G Priest

Keyword(s):

Population Structure ◽

Gel Electrophoresis ◽

Protein Gene ◽

Pulsed Field Gel Electrophoresis ◽

Crystal Protein ◽

Gene Content ◽

Chromosomal Dna ◽

Clonal Population ◽

Pulsed Field ◽

Clonal Population Structure

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Transposon Disruption of the Complex I NADH Oxidoreductase Gene (snoD) in Staphylococcus aureus Is Associated with Reduced Susceptibility to the Microbicidal Activity of Thrombin-Induced Platelet Microbicidal Protein 1

Journal of Bacteriology ◽

10.1128/jb.188.1.211-222.2006 ◽

2006 ◽

Vol 188 (1) ◽

pp. 211-222 ◽

Cited By ~ 38

Author(s):

Arnold S. Bayer ◽

Peter McNamara ◽

Michael R. Yeaman ◽

Natalie Lucindo ◽

Tiffanny Jones ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Complex I ◽

Enzyme Inhibitor ◽

Nadh:Ubiquinone Oxidoreductase ◽

Gene Products ◽

Chromosomal Dna ◽

In Vitro Susceptibility ◽

Ph Tolerance ◽

Platelet Microbicidal Protein

ABSTRACT The cationic molecule thrombin-induced platelet microbicidal protein 1 (tPMP-1) exerts potent activity against Staphylococcus aureus. We previously reported that a Tn551 S. aureus transposon mutant, ISP479R, and two bacteriophage back-transductants, TxA and TxB, exhibit reduced in vitro susceptibility to tPMP-1 (tPMP-1r) compared to the parental strain, ISP479C (V. Dhawan, M. R. Yeaman, A. L. Cheung, E. Kim, P. M. Sullam, and A. S. Bayer, Infect. Immun. 65:3293-3299, 1997). In the current study, the genetic basis for tPMP-1r in these mutants was identified. GenBank homology searches using sequence corresponding to chromosomal DNA flanking Tn551 mutant strains showed that the fourth gene in the staphylococcal mnh operon (mnhABCDEFG) was insertionally inactivated. This operon was previously reported to encode a Na+/H+ antiporter involved in pH tolerance and halotolerance. However, the capacity of ISP479R to grow at pH extremes and in high NaCl concentrations (1 to 3 M), coupled with its loss of transmembrane potential (ΔΨ) during postexponential growth, suggested that the mnh gene products are not functioning as a secondary (i.e., passive) Na+/H+ antiporter. Moreover, we identified protein homologies between mnhD and the nuo genes of Escherichia coli that encode components of a complex I NADH:ubiquinone oxidoreductase. Consistent with these data, exposures of tPMP-1-susceptible (tPMP-1s) parental strains (both clinical and laboratory derived) with either CCCP (a proton ionophore which collapses the proton motive force) or pieracidin A (a specific complex I enzyme inhibitor) significantly reduced tPMP-induced killing to levels seen in the tPMP-1r mutants. To reflect the energization of the gene products encoded by the mnh operon, we have renamed the locus sno (S. aureus nuo orthologue). These novel findings indicate that disruption of a complex I enzyme locus can confer reduced in vitro susceptibility to tPMP-1 in S. aureus.

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Evaluation of Interhospital Spread of Methicillin-Resistant Staphylococcus aureus in Sao Paulo, Brazil, Using Pulsed-Field Gel Electrophoresis of Chromosomal DNA

Infection Control and Hospital Epidemiology ◽

10.1086/646921 ◽

1994 ◽

Vol 15 (5) ◽

pp. 320-323 ◽

Cited By ~ 6

Author(s):

Helio S. Sader ◽

Antonio C. Pignatari ◽

Richard J. Hollis ◽

Ronald N. Jones

Keyword(s):

Staphylococcus Aureus ◽

Gel Electrophoresis ◽

Methicillin Resistant Staphylococcus Aureus ◽

Sao Paulo ◽

Pulsed Field Gel Electrophoresis ◽

São Paulo ◽

Chromosomal Dna ◽

Methicillin Resistant ◽

Pulsed Field

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Extra-Chromosomal DNA Sequencing Reveals Episomal Prophages Capable of Impacting Virulence Factor Expression in Staphylococcus aureus

Frontiers in Microbiology ◽

10.3389/fmicb.2018.01406 ◽

2018 ◽

Vol 9 ◽

Cited By ~ 7

Author(s):

Douglas R. Deutsch ◽

Bryan Utter ◽

Kathleen J. Verratti ◽

Heike Sichtig ◽

Luke J. Tallon ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Dna Sequencing ◽

Virulence Factor ◽

Chromosomal Dna ◽

Factor Expression

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Mutations in Topoisomerase IV and DNA Gyrase of Staphylococcus aureus: Novel Pleiotropic Effects on Quinolone and Coumarin Activity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.1.121 ◽

1998 ◽

Vol 42 (1) ◽

pp. 121-128 ◽

Cited By ~ 64

Author(s):

Bénédicte Fournier ◽

David C. Hooper

Keyword(s):

Staphylococcus Aureus ◽

Dna Gyrase ◽

Single Step ◽

Pleiotropic Effects ◽

Fluoroquinolone Resistance ◽

Wild Type ◽

Chromosomal Dna ◽

Topoisomerase Iv ◽

Gene Encoding ◽

Double Mutation

ABSTRACT Previous studies have shown that topoisomerase IV and DNA gyrase interact with quinolones and coumarins in different ways. The MICs of coumarins (novobiocin and coumermycin) for MT5, a Staphylococcus aureus nov mutant, are higher than those for wild-type strains. Sequencing the gyrB gene encoding one subunit of the DNA gyrase revealed the presence of a double mutation likely to be responsible for this resistance: at codon 102 (Ile to Ser) and at codon 144 (Arg to Ile). For single-step flqA mutant MT5224c9, previously selected on ciprofloxacin, the fluoroquinolone MIC was higher and the coumarin MIC was lower than those for its parent, MT5. Sequencing the grlB andgrlA genes of topoisomerase IV of MT5224c9 showed a single Asn-470-to-Asp mutation in GrlB. Genetic outcrosses by transformation with chromosomal DNA and introduction of plasmids carrying either the wild-type or the mutated grlB gene indicated that this mutation causes both increased MICs of fluoroquinolones and decreased MICs of coumarins and that the mutant grlBallele is codominant for both phenotypes with multicopy alleles. Integration of these plasmids into the chromosome confirmed the codominance of fluoroquinolone resistance, butgrlB + appeared dominant over grlB(Asp-470) for coumarin resistance. Finally, the gyrA(Leu-84) mutation previously described as silent for fluoroquinolone resistance increased the MIC of nalidixic acid, a nonfluorinated quinolone. Combining the grlA (Phe-80) and grlB(Asp-470) mutations with this gyrA mutation also had differing effects. The findings indicate that alterations in topoisomerases may have pleiotropic effects on different classes of inhibitors as well as on inhibitors within the same class. A full understanding of drug action and resistance at the molecular level must take into account both inhibitor structure-activity relationships and the effects of different classes of topoisomerase mutants.

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Dissemination of a pSCFS3-Likecfr-Carrying Plasmid in Staphylococcus aureus and Staphylococcus epidermidis Clinical Isolates Recovered from Hospitals in Ohio

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00071-13 ◽

2013 ◽

Vol 57 (7) ◽

pp. 2923-2928 ◽

Cited By ~ 29

Author(s):

Rodrigo E. Mendes ◽

Lalitagauri M. Deshpande ◽

Hector F. Bonilla ◽

Stefan Schwarz ◽

Michael D. Huband ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Staphylococcus Epidermidis ◽

Southern Blotting ◽

Surveillance Program ◽

Chromosomal Dna ◽

Content Type ◽

Medical Institutions ◽

Antimicrobial Surveillance ◽

Content Index ◽

Index Strain

ABSTRACTNineteen linezolid-resistantStaphylococcus epidermidisand twoStaphylococcus aureusisolates recovered from two medical institutions in northeast Ohio and anS. aureuscfrindex strain previously collected in the same facilities during the 2007 SENTRY Antimicrobial Surveillance Program were investigated for the genetic basis of oxazolidinone resistance and the location ofcfr. S. aureusisolates were typed by pulsed-field gel electrophoresis (PFGE),spatyping, and multilocus sequence typing (MLST). The location ofcfrwas determined by Southern blotting and hybridization. Plasmid sequencing was performed using the 454 Life Sciences (Roche) GS-FLX DNA platform. The twoS. aureusisolates showed unique PFGE patterns but were multilocus sequence type 5 (ST5) andspatype t002, whereas theS. aureusindex strain was ST239 and t037. Southern blot and hybridization experiments showed thatcfrwas plasmid located and that theS. epidermidisisolates, one of theS. aureusisolates, and theS. aureusindex strain shared an identicalcfr-carrying plasmid (39.3 kb). Sequencing results confirmed these findings. A 10-kb fragment containingcfrshowed the highest identity (99.9%) to a 9.5-kb fragment of plasmid pSCFS3 from a bovineStaphylococcus lentusisolate from Germany. In addition, these 39.3-kb plasmids from humanS. epidermidisandS. aureusexhibited BglII restriction profiles very similar to that observed for plasmid pSCFS3. Thecfr-carrying plasmid detected in the remainingS. aureusisolate (7.9 kb) was distinct and showed the highest identity to the chromosomalcfrintegrate found in the chromosomal DNA of aProteus vulgarisisolate from a pig in China.

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