Detection of vancomycin resistant Staphylococcus aureus: a comparative study of three different phenotypic screening methods

The objective of this study was to investigate screening methodologies, to detect Staphylococcus aureus strains with decreased susceptibility to vancomycin. Three methods were used to screen 160 Staphylococcus aureus clinical isolates along with ATCC quality control strains. Subsequently, MIC of all these 160 strains were determined by NCCLS methodology. The MIC of all the 160 clinical isolates was < or = 4 microg/mL and were classified as vancomycin susceptible by NCCLS criteria but 23 strains were positive by Hiramatshu method, two grew on MHA (5 microg/mL vancomycin) while CDC method correctly identified no vancomycin intermediate S.aureus (VISA) or vancomycin resistant S.aureus (VRSA) strains with reference to there MIC. CDC method was found to be the most appropriate screening methodology for detection of VISA or VRSA for diagnostic laboratories.     

Effect of sub‐lethal doses of vancomycin and oxacillin on biofilm formation by vancomycin intermediate resistant Staphylococcus aureus

Biofilms are means of protection to bacteria against antibiotics and antibodies. Catheters and others tube devices used by patients are prone to accumulation of thick layers of biofilms as hiding place for etiologic agents, resulting in substantial morbidity and mortality. Methicillin‐resistant Staphylococcus aureus (MRSA) is a major cause of hospital‐acquired infections. Vancomycin remains the only treatment of choice for MRSA infections. In the present study a vancomycin resistant S. aureus (VRSA) (Labeled as CP2) was isolated from the blood of a post‐operative cardiac patient. It harbors a plasmid which carry vanA gene and exhibited low‐level vancomycin resistance (MIC 16μg/ml), high level of oxacillin/methicillin resistance (MIC 500 μg/ml) and was sensitive to teicoplanin. CP2 also found to carry icaA gene on its chromosome. This strain exhibited resistance to triton‐X100 induced autolysis under sub‐inhibitory concentration of vancomycin and produced some extracellular matrix material that surrounding the cells. These characteristic features have warranted us to study the biofilm formation by CP2 on biomedical indwellings in presence of vancomycin and oxacillin. Our findings suggest that sub‐lethal dose of vancomycin induced the biofilm formation by CP2 on nylon and silicon indwellings whereas oxacillin facilitated the biofilm formation on glass surfaces exclusively. This implicates that not only the antibiotics but also the indwelling material influences biofilm formation. Therefore, these implants serve as potential surfaces for bacterial adhesion that lead to biofilm formation, thus provide hiding places for pathogens from the actions of antimicrobials. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Detection of intrinsically resistant (heteroresistant) Staphylococcus aureus with the Sceptor and AutoMicrobic systems.

Modified procedures for the Sceptor Gram-Positive MIC Panel and the Vitek AutoMicrobic System GPS-M Card were evaluated for their ability to detect methicillin-resistant (heteroresistant) Staphylococcus aureus. A total of 398 clinical isolates (including 222 methicillin-resistant S. aureus) obtained from 10 hospitals were tested. Both systems had 2% NaCl in the oxacillin wells. Sceptor MIC panels were inoculated with an organism suspension prepared from an 18- to 24-h blood agar plate and were inoculated for a full 24 h at 35 degrees C before MICs were read. All methicillin-resistant S. aureus isolates were detected as resistant to oxacillin at greater than or equal to 8 micrograms/ml by the Sceptor method and at greater than 2 micrograms/ml by the Vitek method. All 176 oxacillin-susceptible, methicillin-susceptible S. aureus isolates were correctly distinguished from methicillin-resistant S. aureus isolates by Sceptor. However, with the Vitek system 29 methicillin-susceptible S. aureus isolates tested as falsely resistant to oxacillin and four isolates tested as falsely resistant to vancomycin. The modified testing procedure with the Sceptor system can be used reliably for accurate susceptibility testing of methicillin-resistant and methicillin-susceptible S. aureus. The Vitek GPS-M card does not accurately discriminate between methicillin-resistant and methicillin-susceptible S. aureus with an oxacillin breakpoint of greater than 2 micrograms/ml.     

In vitro selection of resistance to vancomycin in bloodstream isolates ofStaphylococcus haemolyticus andStaphylococcus epidermidis

The purpose of the study was to determine whether vancomycin-resistant strains ofStaphylococcus haemolyticus could be selected regardless of the initial MIC of vancomycin. Twenty-one bloodstream isolates ofStaphylococcus haemolyticus were studied by broth and agar selection methods. The broth method selected strains for which MICs of vancomycin ranged from 4 to 32 µg/ml and MBCs from 16 to>128 µg/ml. The agar method selected strains for which MICs ranged from 8 to 32 µg/ml and MBCs from 8 to>128 µg/ml. For comparison, seven strains ofStaphylococcus epidermidis were evaluated by the agar selection method. Final MICs of vancomycin ranged from 8 to 16 µg/ml; MBCs ranged from 16 to 64 µg/ml. Clearly, in vitro exposure to vancomycin can select strains ofStaphylococcus haemolyticus andStaphylococcus epidermidis for which MIC values are beyond the susceptible breakpoint.     

Detection of methicillin-resistant Staphylococcus epidermidis.

To determine whether methods suggested for detecting methicillin-resistant Staphylococcus aureus apply equally to methicillin-resistant Staphylococcus epidermidis, 135 S. epidermidis isolates were tested by the Vitek AMS gram-positive susceptibility card (Vitek Systems, Inc., Hazelwood, Mo.) and by modifications of agar screen, disk diffusion, and microdilution methods. Modifications included 24- versus 48-h incubation, unsupplemented versus 2% NaCl-supplemented broth, and standard versus direct inoculum. At 24 h, the highest number of resistant strains, 59, was detected by oxacillin (1 microgram) disk diffusion. At 48 h, three additional strains were judged resistant. With one exception, results for oxacillin disk diffusion and agar screen were equivalent at 24 and 48 h. Vitek detected 50 resistant strains. Significantly fewer resistant strains were detected at 24 h by methicillin disk diffusion (5 micrograms) and methicillin microdilution with 2% NaCl. For oxacillin microdilution, neither 2% NaCl supplementation nor the method of inoculum preparation significantly affected the results. Oxacillin microdilution with cation- rather than non-cation-supplemented broth detected significantly fewer (n = 33) resistant strains at 24 h; 51 were resistant at 48 h. To detect methicillin-resistant S. epidermidis, a direct inoculum with either 24-h oxacillin disk diffusion and reincubation of intermediate strains for an additional 24 h or 24-h oxacillin agar screen and reincubation of strains with no growth for a total of 48 h is recommended.     

Detection of slime production by means of an optimised Congo red agar plate test based on a colourimetric scale in Staphylococcus epidermidis clinical isolates genotyped for ica locus

This investigation was conduced on a collection of 113 S. epidermidis strains isolated from biomaterial-associated infections. All strains were examined both for the presence of icaA and icaD genes responsible for slime synthesis by a PCR method and for the in vitro slime production ability by the Congo red agar (CRA) plate test. In the present study, the original CRA test was optimised adopting a six-colour reference scale for a fine classification of colonies colours. The six-colour tones of the scale were as follows: very black (vb), black (b), almost black (ab), which were considered as positive results, and bordeaux (brd), red (r), and very red (vr), interpreted as negative. 57.5% of all the strains were found to be icaA icaD-positive as well as slime-forming onto CRA, exhibiting the following colonies colours: vb (35.4%); b (15.9%); ab (6.2%). The percentage of icaA icaD-negative strains was 42.5% and all of them were negative onto CRA: brd (19.5%), r (14.2%), vr (8.8%). The comparison of colour classification with the information on ica genes confirmed the validity of the scale adopted, providing support to the criteria used for a correct interpretation of the colonies colour during the execution of the CRA test. Overall these results indicate a fine consistency between these two experimental methods and a good reliability of CRA plate test, especially when this is supported by a colourimetric scale.     

Comparison of inoculation methods for testing enterococci by using vancomycin screening agar.

One hundred four recent clinical isolates of Enterococcus species were screened for vancomycin resistance by using inocula of 10(5) or 10(6) CFU dispensed by pipet and by use of a cotton swab dipped in a 0.5 McFarland standard organism suspension applied to the surface of brain heart infusion agar containing 6 micrograms of vancomycin per ml. The three inoculation methods were equivalent in the detection of nonsusceptible isolates. The use of swab inoculation was convenient and less costly than the use of micropipets.     

Use of trehalose-mannitol-phosphatase agar to differentiate Staphylococcus epidermidis and Staphylococcus saprophyticus from other coagulase-negative staphylococci.

Using a plate medium containing trehalose, mannitol, and phenolphthalein diphosphate (TMPA), we differentiated significant clinical isolates of Staphylococcus epidermidis by their lack of acid production in 18 h from other coagulase-negative staphylococci, with our results having a sensitivity (R. S. Galen and S. R. Gambino, Beyond Normality: The Predictive Value and Efficiency of Medical Diagnoses) of 100%, a specificity of 89.9%, and a positive predictive value of 94.8%. With a Taxo A bacitracin disk, which differentiates Staphylococcus species from Micrococcus species, no zone of inhibition was seen for 96% of all staphylococcal strains, with 5 of 26 strains of Staphylococcus saprophyticus exhibiting zone diameters up to 10 mm. By using resistance to a 5-microgram novobiocin disk, we differentiated S. saprophyticus, with our results having a sensitivity of 100%, a specificity of 97.1%, and a positive predictive value of 83.9% on TMPA. These two species represented 77.8% of coagulase-negative staphylococci isolated. Reference strains fo Staphylococcus and Micrococcus species were differentiated by TMPA. The cost of TMPA was compared with that of another method. TMPA was found to offer an inexpensive, sensitive method for rapidly differentiating coagulase-negative Staphylococcus isolates.     

Epidemiologic application of a typing method for Staphylococcus epidermidis strains by the serum-soft agar technic

Using the serum-soft agar technic of Staphylococcus epidermidis typing, an epidemiologic study of pollution with S. epidermidis in the tuberculosis and pediatric wards of a hospital was conducted. Specimen samples were taken from 334 locations, including beds, bedspreads, pillows, doors and window knobs, chairs, tables, and incubators of premature infants. These were cultured on Staphylococcus 110 medium and the strains identified as S. epidermidis were obtained. Of the strains from both patients' rooms and the nurses' station in the tuberculosis ward a considerable number were of serotype 53, suggesting an interrelationship of this organism and these locations. In the rooms of newborns and premature infants in the pediatric ward, 55.5% of the strains of S. epidermidis isolated were of the serotype 53/408, indicating a high degree of pollution of these environments with these serotype strains.     

Targeted delivery of vancomycin to Staphylococcus epidermidis biofilms using a fibrinogen-derived peptide

This study reports on the use of a fibrinogen-derived peptide for the specific targeting and delivery of vancomycin to Staphylococcus epidermidis biofilms. One method by which S. epidermidis initially adheres to biomaterials uses the plasma protein fibrinogen as an intermediary, where the S. epidermidis surface protein SdrG binds to a short amino acid sequence near the amino terminus of the Bβ chain of fibrinogen. We mimicked this binding interaction and demonstrated the use of a synthetic fibrinogen-based β6-20 peptide to target and deliver vancomycin to S. epidermidis in vitro. The β6-20 peptide was synthesized and labeled with a Nanogold probe, and its targeting capabilities were examined through the use of scanning electron microscopy. The Nanogold component was then replaced by vancomycin, utilizing a flexible, variable length poly(ethylene glycol) linker between the peptide and antibiotic to create the targeted vancomycin products, β6-20-PEG(x) -VAN. Initial binding to surface adherent S. epidermidis was increased in a concentration-dependent manner relative to vancomycin for all equivalent concentrations ≥4 μg/mL, with targeted vancomycin content up to 22.9 times that of vancomycin alone. Retention of the targeted antibiotics was measured after an additional 24-h incubation period, revealing levels 1.3 times that of vancomycin. The results demonstrate the improved targeting and retention of vancomycin within a biofilm due to the incorporation of a specific targeting motif.     

Detection of encapsulation in Staphylococcus aureus by use of antiserum agar

We examined an antiserum agar method to study its reliability in screening Staphylococcus aureus strains for capsule production. The encapsulated S. aureus Smith diffuse strain was compared with its nonencapsulated variant, Smith compact, in CCY medium containing 0.5% NaCl and 5.0% Smith diffuse rabbit antiserum. A halo was visible surrounding colonies of the Smith diffuse strain but not the Smith compact strain. On this same medium, the protein A-producing Cowan I strain possessed a halo that was visible on photographs. Single high-salt medium is known to inhibit protein A production, halo formation by the strains was also compared in 7.5% NaCl medium. The halo surrounding the Cowan I strain was not present when the salt content of the medium was increased. In contrast, the halo surrounding the Smith diffuse strain persisted in the 7.5% NaCl medium. By use of this medium, the antiserum agar technique may be valuable for the identification of encapsulated staphylococci without appreciable interference from protein A.     

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.     

Endemic heteroresistant glycopeptide intermediate Staphylcoccus aureus (hGISA) comprising unrelated clonal types and not associated with vancomycin therapy

The detection of methicillin-resistant S. aureus (SA) (MRSA) refractory to glycopeptides is a serious clinical issue. The prevalence of hetero-resistant GISA (hGISA) strains at H. Maréchal Joffre, France is reported.858 non-repeat SA were isolated during 1999. 367 (43%) of these, from 257 patients, were MRSA (mean incidence 11.9/1000 admissions). All MSRA detected during 1999 were screened for vancomycin (VAN) resistance (BHI+4 mg/l VAN). Isolates recovered were retested using Etest strips (2 McFarland inoculum on BHI) and population analysis profile/area under the curve (PAP-AUC) analysis with hGISA SA Mu3 as a comparator. 58 selected strains were screened for teicoplanin resistance(TEI) using SFM recommended screen (2 McFarland inoculum on MH+5 mg/L TEI) and MIC (0.5 MF inoculum swabbed on MH agar) methods. 188 (51.3%) grew on VAN screen agar (6.1/1000 admissions). 58 strains (7.6%) possessed Etest VAN MIC > 8 mg/l all others being VAN < 8 mg/l. Of these 58 isolates, 10 were stably heterogeneously resistant to both VAN and teicoplanin (MIC > 8 mg/l). PAP-AUC showed 12 strains to have PAP-AUC ratios > 0.95 but < 1.5 (ie. hGISA, not GISA). All 7 isolates defined as hGISA by both Etest and PAP-AUC comprised 1 PFGE clone (< 3 bands difference).Additionally 2 distinct PFGE types were detected among the other 5 hGISA identified PAP-AUC. The 12 hGISAs, were derived from 12 patients with severe underlying disease. None were on glycopeptide therapy prior to hGISA isolation.This is the first report of endemic hGISA, comprising 3 clonal types. The isolation of hVISA seems not to be associated with patient-specific glycopeptide therapies.     

Staphylococcus heterogeneously resistant to vancomycin in China and antimicrobial activities of imipenem and vancomycin in combination against it

Of 115 methicillin-resistant Staphylococcus strains collected from sputum specimens, 34 strains reduced susceptibility to vancomycin, 9 of which emerged as heterogeneous vancomycin-resistant strains (hetero-VRS), with various degrees of vancomycin resistance at a frequency of 10(-6) or higher. Seventy-six percent (19 of 25) of non-hetero-VRS and 100% (9 of 9) of hetero-VRS were susceptible to synergistic treatment with vancomycin and imipenem. Clinical clearance between 9 hetero-VRS and 25 non-hetero-VRS had an obvious statistical significance (P = 0.001). The hetero-VRS may play an important role in vancomycin therapy failure.     

Surgical biomaterials and differential colonization by Staphylococcus epidermidis

The data presented in this communication demonstrate preferential colonization of certain biomaterials by Staphylococcus epidermidis. Using a laminar flow biomaterial colonization chamber and surgical-grade biomaterials (stainless steel, aluminium ceramic, methyl methacrylate and high-density polyethylene), the pattern of colonization was quantitated using plate count techniques and electron microscopy. Under comparable conditions, methyl methacrylate was colonized by S. epidermidis in greater numbers than the other biomaterials. Increased bacterial colonization and slime production on methyl methacrylate was time-dependent and 15 times higher than on stainless steel and aluminium and four times higher than on high-density polyethylene. The data reveal that certain biomaterials may promote infection by favouring colonization by potential pathogens. This variable should be explored extensively in an in vivo setting because of its implication in clinical infections.     

In vitro activity of antimicrobial agents against oxacillin resistant staphylococci with special reference to Staphylococcus haemolyticus

One hundred and sixty seven isolates of staphylococci isolated from the inpatients of a tertiary care referral hospital in South India were speciated and activity of oxacillin, glycopeptides, linezolid and quinupristin/dalfopristin against these isolates was tested by broth microdilution method. Of the 114 coagulase negative staphylococci (CoNS), 49.1 % were S. haemolyticus, isolated predominantly from urine (64.6%), while the rest belonged to 11 other species. More than half the isolates of S. aureus (52.8%) and 68.4% of the CoNS were oxacillin resistant. All the strains were uniformly susceptible to vancomycin, linezolid and quinupristin/dalfopristin; but 25.6% isolates of S. haemolyticus showed reduced susceptibility to teicoplanin (MIC: 8-16 mg/L). Our study demonstrates the high prevalence of oxacillin resistance among hospital isolates of S. aureus and CoNS in India. Vancomycin, along with the newer agents like linezolid and quinupristin/dalfopristin remains the drug of choice for treating multi drug resistant staphylococcal infections.     

Double triplex real-time PCR assay for simultaneous detection of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus haemolyticus and determination of their methicillin resistance directly from positive blood culture bottles

We developed and validated here a double triplex real-time PCR assay to simultaneously detect and identify Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus and their methicillin resistance in a single reaction directly from Gram-positive cocci-in-clusters (GPCs)-positive blood culture bottles. From August 15, 2009 through February 15, 2010, 238 GPC-positive samples were collected and identified by conventional methods as 11 methicillin-resistant S. aureus (MRSA), 28 methicillin-susceptible S. aureus (MSSA), 176 MR coagulase-negative staphylococci (MRCoNS), 21 MSCoNS and two Enterococcus faecalis. The double triplex real-time PCR assay was targeted and detected tuf, nuc and mecA genes in the first tube and atlE, gap and mvaA genes in the second tube which could be run simultaneously. The detection limit of the assay was found at 10³ CFU/ml for the atleE gene, 10⁴ CFU/ml for the mva gene and 10⁵ CFU/ml for gap, nuc, mecA and tuf genes based on seeding experiments. All Staphylococcus species except two S. epidermidis were correctly identified by the assay. The double triplex real-time PCR assay quickly and accurately detects S. aureus, S. epidermidis, S. hominis and S. haemolyticus and their methicillin resistance in a single reaction directly from positive blood culture bottles within 83 min.     

Hemagglutination and adherence to plastic by Staphylococcus epidermidis.

Staphylococcus epidermidis is an important nosocomial pathogen responsible for intravenous catheter-related bacteremia and infections of other prosthetic medical devices. We found that the ability of S. epidermidis to hemagglutinate erythrocytes correlated with the adherence of bacteria to plastic and to intravenous catheters. S. epidermidis isolates responsible for prosthetic-valve endocarditis (n = 61) and isolates from intravenous catheters (n = 59) were significantly more likely to cause hemagglutination than isolates from the skin of preoperative cardiac surgery patients (n = 19) (P = 0.027). S. epidermidis isolates (n = 23) recovered from the skin of patients 7 to 10 days after cardiac surgery were significantly more likely to exhibit hemagglutination than the preoperative isolates (P = 0.015). By a quantitative adherence assay, we also observed that the hemagglutination titer and number of species of erythrocytes agglutinated correlated directly with adherence to polystyrene (P less than 0.001). In addition, hemagglutinating isolates were significantly more likely to be recovered in high number from intravenous catheters when semiquantitative catheter culture techniques were used (P less than 0.001). We speculate that hemagglutinin(s) either plays a direct role in adherence to polymers and thus prosthetic-device infection or serves as an easily demonstrable marker for adherence-prone isolates.