Detection of methicillin-resistant Staphylococcus aureus in clinical specimens from cystic fibrosis patients by use of chromogenic selective agar

We evaluated the use of a chromogenic selective medium (MRSA ID) as a useful tool for the detection of methicillin-resistant Staphylococcus aureus (MRSA) in cystic fibrosis (CF) patient samples. Fifty-four MRSA isolates were detected by MRSA ID, while only 24/54 (44%) (odds ratio [OR], 2.79; 95% confidence interval [CI], 1.63 to 4.76) were detected by conventional methods. A chromogenic selective medium for MRSA detection may improve its surveillance in CF patients.     

Detection of Staphylococcus aureus in milk by PCR

The aim of this study was to compare the detection of Staphylococcus aureus (S. aureus) in raw milk samples by polymerase chain reaction (PCR) with detection by cultural methods. A total of 100 milk samples were collected from individual cattle in industrial dairy herds in Urmia, Iran. DNA was extracted from all samples directly. PCR amplifications were performed with a pair of primers specific for the coa gene of S. aureus. In order to isolate S. aureus by cultural methods, bacteriological examinations were done on all samples. Of the 100 milk samples tested, 14% were positive on the basis of the cultural results while 21% of the samples were positive using amplification of the coa gene. There were no culture positive samples that were negative using PCR. PCR amplification of the coa gene could be used as a powerful tool for the identification of S. aureus in milk samples.     

Pseudodiffuse-type growth of a Staphylococcus aureus strain in serum-soft agar.

Strain MC31 of Staphylococcus aureus showed diffuse-type growth in regular serum-soft agar (SSA) (pH 7.2), but in alkaline SSA (pH 8.4) the type of growth was compact. The organisms were of extra low virulence in the mouse and were unencapsulated. Based on three properties, the growth type of the S. aureus strains in SSA was designated as pseudodiffuse type.     

Accurate detection of oxacillin-resistant Staphylococcus lugdunensis by use of agar dilution

Background/purposeStaphylococcus lugdunensis is a Gram-positive coagulase-negative bacterium and is recognized as a critical pathogenic species recently. Here, we aimed to evaluate the cefoxitin disk diffusion (CDD), oxacillin agar dilution (OAD), and mecA PCR for detecting oxacillin-resistant S. lugdunensis (ORSL) isolates.MethodsMultilocus sequence typing (MLST) analysis was performed to determine the clonality of 117 S. lugdunensis isolates isolated between May 2009 and Jul 2014. CDD, OAD, and mecA PCR were used to identify oxacillin-resistant S. lugdunensis (ORSL).ResultsMLST results showed that the most common sequence type (ST) of our S. lugdunensis isolates was ST6 (35.9%) followed by ST3 (28.2%), ST27 (17.9%), and ST4 (6.8%). CDD and OAD showed that 39 and 43 isolates were ORSL, respectively. 4 ST3 CDD-susceptible S. lugdunensis (OSSL) isolates had MIC values ≥ 4 for oxacillin. mecA PCR results showed that 43 OAD-resistant S. lugdunensis and 3 OAD-susceptible ST27 S. lugdunensis had the mecA gene. Therefore, OAD was used as the gold standard to evaluate the performance of CDD and mecA PCR for identifying ORSL. The overall sensitivity, specificity, and accuracy of CCD for ORSL detection was 90.7%, 100%, and 96.8%, respectively. The sensitivity, specificity, and accuracy of mecA PCR for identifying ORSL was 100%, 95.9%, and 97.44%, respectively.ConclusionOur results indicate that OAD shows higher accuracy for ORSL detection compared with CDD and mecA PCR.     

Nature of the genetic determinant controlling encapsulation in Staphylococcus aureus Smith.

Two strains of Staphylococcus aureus, Smith and M, were studied for the elimination of encapsulation. For S. aureus M, encapsulation was stable. For S. aureus Smith, spontaneous loss of encapsulation was 1.3% and increased markedly in medium containing surface-active agents. In the presence of sodium dodecyl sulfate, unencapsulated cells had a considerable selective advantage. Attempts to demonstrate covalently closed circular plasmid deoxyribonucleic acid were unsuccessful. In cultures of unencapsulated cells, encapsulated cells were observed occasionally. These data argue against a plasmid location for the determinants controlling encapsulation in this organism in spite of a high spontaneous loss of this character.     

The expression of capsule in serum-soft agar by Staphylococcus aureus isolated from human clinical sources

Staphylococcus aureus isolates from human clinical sources were incubated for various times in modified 110 medium and tested for production of capsule by the serum-soft agar technique. Ten (5.7%) of 175 isolates were encapsulated after incubation for 24 h. A more detailed examination of 77 isolates showed that incubation period affected the production of capsule. After 2 h, 31% were encapsulated, but after 6 h and 24 h this decreased to 17% and 4% respectively. Rapid passage in vitro induced the expression of capsule in four of 50 unencapsulated strains. Only three of 20 encapsulated strains could be typed with standard antisera.     

Mueller–Hinton agar is superior to PDM blood agar for detection of methicillin-resistant Staphylococcus aureus

The aim of this study was to compare the expression of oxacillin resistance in methicillin-resistant Staphylococcus aureus (MRSA) on Paper Disc Method agar supplemented with 5% defibrinated blood (PDM blood agar) and Mueller–Hinton agar supplemented with 2% NaCl (MH NaCl agar) using different susceptibility tests. Fifty mecA- containing isolates of S. aureus, exhibiting 46 different pulsed-field gel electrophoresis patterns, were comparatively tested using the E test, the single disk diffusion test, and the multipoint inoculation technique, under various culture conditions. The E test incubated at 35 °C for 24 h (breakpoint of resistance ≥2.0 mg/L) detected 94% of the isolates on MH NaCl agar, compared with 28% for PDM blood agar ( P  < 0.05). The disk diffusion test (breakpoint ≤ 10 mm in diameter) under these incubation conditions detected resistance in 100% of the isolates on MH NaCl agar and in 80% of the isolates on PDM blood agar ( P  < 0.05). The multipoint technique (breakpoint ≥1 mg/L), applied at 35 °C for 24 h, detected 100% on MH NaCl agar and 46% on PDM blood agar ( P  < 0.05). Irrespective of the method of susceptibility testing evaluated, MH NaCl agar was superior to PDM blood agar for the detection of oxacillin resistance in mecA -containing S. aureus .     

Detection of methicillin-resistant Staphylococcus aureus by polymerase chain reaction]

The low-affinity penicillin-binding protein (PBP 2') is associated with methicillin-resistance of Staphylococcus aureus and its structural gene (mecA) not present in methicillin-susceptible S. aureus could be detected by the polymerase chain reaction (PCR) method, in which a 533 bp region of mecA was amplified and detected by agarose gel electrophoresis. Survey for the mecA gene in 210 clinical isolates of S. aureus revealed that, while there was a gross correlation between the presence of the mecA gene and the resistance level to beta-lactams, three strains of mecA (+) tested showed beta-lactam susceptibility similar to those of mecA (-) strains. These three strains did not produce a detectable amount of PBP 2' constitutively nor inducibly, which was the cause of their high susceptibility to beta-lactams. One of them yielded a typical methicillin-resistant variant at a low frequency with a concomitant recovery of PBP 2' production when the bacterial cells of high density were spread onto an agar plate containing 10 micrograms/ml of oxacillin. These findings suggested that typical methicillin resistant S. aureus occurred during chemotherapy with beta-lactam antibiotics even when resistant strains could not be detected by the susceptibility test and thus all mecA (+) strains including those with high susceptibility should be precisely detected.     

Serum albumin as an auxiliary factor enhancing compact-colony formation by Staphylococcus aureus in serum-soft agar.

Serum albumin was identified as an auxiliary factor enhancing the substance active in compact-colony formation by strains of S. aureus when cultivated in serum-soft agar.     

Development and evaluation of a chromogenic agar medium for methicillin-resistant Staphylococcus aureus

We describe here the development and evaluation of MRSA ID, a new chromogenic agar medium for the specific isolation and identification of methicillin-resistant Staphylococcus aureus (MRSA). We used S. aureus ID (bioMérieux, La Balme Les Grottes, France) and supplemented it with various antimicrobials, including cefoxitin, ciprofloxacin, oxacillin, and methicillin. Cefoxitin proved to be superior to the other antimicrobials for the selection of MRSA from other strains of S. aureus. MRSA ID (consisting of S. aureus ID supplemented with 4 mg of cefoxitin/liter) was evaluated by the use of 747 swabs from various clinical sites. All specimens were also cultured on CHROMagar MRSA and oxacillin resistance screening agar base (ORSAB) and in selective mannitol broth (SMB). A total of 85 MRSA strains were isolated by a combination of all methods. After 22 to 24 h of incubation, 80% of the MRSA strains were isolated as green colonies on MRSA ID, compared with 59 and 62% of the strains that were isolated as colored colonies on CHROMagar MRSA and ORSAB, respectively. After 48 h of incubation, 89, 72, and 78% of the MRSA strains were isolated on MRSA ID, CHROMagar MRSA, and ORSAB, respectively. Sixty-five percent of the strains were isolated by growth in SMB. The specificities of MRSA ID, CHROMagar MRSA, ORSAB, and SMB were 99.5, 99.3, 97.9, and 92.8%, respectively, after 22 to 24 h of incubation. We conclude that MRSA ID is a sensitive and specific medium for the isolation and identification of MRSA.     

Phenotypic detection of methicillin resistance in Staphylococcus aureus by disk diffusion testing and Etest on Mueller-Hinton agar

Cefoxitin is increasingly recommended for detection of methicillin resistance in Staphylococcus aureus (MRSA) when using disk diffusion testing. In this study, 95 mecA-negative S. aureus isolates and a highly genetically diverse collection of mecA-positive S. aureus types (n=50) were used to investigate the influence of technical factors such as disk potency, incubation time, and temperature on Mueller-Hinton agar. The use of cefoxitin MIC testing by Etest for the same purpose was investigated under similar conditions. For disk diffusion, the accuracy was high at both 35 degrees C and 36 degrees C using overnight incubation, while incubation at 30 degrees C or 37 degrees C was associated with slightly lower accuracy. Increasing incubation times from 18 to 24 h did not improve accuracy at either temperature. Cefoxitin Etest MICs for mecA-positive strains were 6 mg/liter or higher, while cefoxitin Etest MICs for mecA-negative strains were 4 mg/liter, corresponding to S>or=22 mm and Ror=17 mm and R相似文献   

Evaluation of S. aureus ID, a new chromogenic agar medium for detection of Staphylococcus aureus

S. aureus ID (bioMérieux, La Balme Les Grottes, France) is a new chromogenic agar medium designed to enable the isolation of staphylococci and the specific identification of Staphylococcus aureus. S. aureus produces green colonies on this medium due to production of α-glucosidase. To evaluate this medium, a total of 350 wound swabs were cultured onto S. aureus ID, CHROMagar Staph. aureus, and conventional media routinely used in our laboratory. After 18 to 20 h of incubation, 96.8% of strains formed green colonies on S. aureus ID compared with 91.1% of strains forming mauve colonies on CHROMagar Staph. aureus. A total of 94.3% of strains were recovered within 18 to 20 h with conventional media. The sensitivity was increased after 48 h of incubation to 98.7, 96.2, and 95.6% with S. aureus ID, CHROMagar Staph. aureus, and conventional media, respectively. A total of 97.4% of green colonies on S. aureus ID were confirmed as S. aureus compared with 94.4% of mauve colonies on CHROMagar Staph. aureus. We conclude that S. aureus ID is a highly sensitive and specific medium for the isolation and identification of S. aureus from wound swabs.     

Detection of low-level oxacillin resistance in mecA-positive Staphylococcus aureus

Detection of low-level oxacillin-resistant Staphylococcus aureus is a problem that needs special attention, particularly in relation to methicillin-resistant S. aureus (MRSA) strains in the community that belong to clonal lineage ST80. This study compared different phenotypic methods for the detection of 74 low-level oxacillin-resistant S. aureus strains (oxacillin MIC or=2 mg/L) and 117 methicillin-susceptible S. aureus strains. Determination of microbroth dilution MICs for oxacillin was wholly unsatisfactory, and gave a limited specificity for cefoxitin. The sensitivity of disk-diffusion performed according to CLSI recommendations was 92% with an oxacillin 1-microg disk, and 96% with a cefoxitin 30-microg disk; use of a 10-microg cefoxitin disk and a semi-confluent inoculum (breakpoint for resistance <18 mm zone diameter) gave a sensitivity of 97%. When disk-diffusion was performed on IsoSensitest agar with a zone diameter breakpoint for resistance of <22 mm (as recommended by the Swedish Reference Group for Antibiotics), the sensitivity was 95%.     

Improved Methods for Detection of Methicillin-Resistant Staphylococcus aureus

 In order to assess the performance of two detection methods, a set of 93 recent clinical isolates of Staphylococcus aureus, including a large number of strains that demonstrated low-level methicillin-resistance were evaluated using the MRSA-Screen (Denka Seiken, Japan), a commercial latex agglutination test to detect penicillin-binding protein 2′ (PBP2′), and a polymerase chain reaction assay using the LightCycler Instrument (Roche Diagnostics, Switzerland). The results show that the latex agglutination test is highly sensitive if performed after induction by cefoxitin. Inconclusive results can be rapidly confirmed on the same day by real-time polymerase chain reaction used to detect mecA and femA genes.     

Detection of Staphylococcus aureus and Staphylococcus epidermidis in Clinical Samples by 16S rRNA-Directed In Situ Hybridization

Staphylococcus epidermidis and Staphylococcus aureus are the most common causes of medical device-associated infections, including septicemic loosenings of orthopedic implants. Frequently, the microbiological diagnosis of these infections remains ambiguous, since at least some staphylococci have the capacity to reduce their growth rate considerably. These strains exhibit a small-colony phenotype, and often they are not detectable by conventional microbiological techniques. Moreover, clinical isolates of S. aureus and S. epidermidis adhere to polymer and metal surfaces by the generation of thick, multilayered biofilms consisting of bacteria and extracellular polysaccharides. This study reports improved detection and identification of S. aureus and S. epidermidis by an in situ hybridization method with fluorescence-labeled oligonucleotide probes specific for staphylococcal 16S rRNA. The technique has proven to be suitable for the in situ detection of staphylococci, which is illustrated by the identification of S. epidermidis in a connective tissue sample obtained from a patient with septicemic loosening of a hip arthroplasty. We also show that this technique allows the detection of intracellularly persisting bacteria, including small-colony variants of S. aureus, and the differentiation of S. epidermidis from other clinically relevant staphylococci even when they are embedded in biofilms. These results suggest that the 16S rRNA in situ hybridization technique could represent a powerful diagnostic tool for the detection and differentiation of many other fastidious microorganisms.