Serological Typing of Staphylococcus epidermidis Strains by the Serum-Soft Agar Technique

Antisera to five different diffuse forms of Staphylococcus epidermidis were prepared in rabbits as a mean of serological differentiation of S. epidermidis strains. These antisera converted the growth types of the homologous strains from diffuse to compact form in serum-soft agar but did not convert the heterologous strains. The converting activities of the antisera were absorbed out with homologous organisms and not by heterologous strains, indicating the specific serological properties of these strains. Further, the converting activity was absorbable with cell surface substance extracted by sonic oscillation and was not absorbed with cells from which this surface substance had been removed. With these factor sera and the serum-soft agar technique, S. epidermidis strains were differentiated, and it was found that 102 of 116 strains or 87.9% were typable. Within the 102 typable strains, 49 and 53 typed as monovalent and polyvalent, respectively. No correlation between source and strain types of S. epidermidis was found.     

Isolation of an Additional Capsular-Type Strain of Staphylococcus aureus by the Serum-Soft Agar Technique

An additional capsular-type strain of Staphylococcus aureus was isolated by the serum-soft agar technique and designated as capsule type D.     

Comparison of Capsular Types of Staphylococcus aureus Strains

Staphylococcal capsular types 1 to 4 as determined by the "specific capsular reaction" belonged to type A capsule as determined by the serum-soft agar technique.     

Compact-Type Growth of Staphylococcus epidermidis Strains in Serum-Soft Agar

Of 120 Staphylococcus epidermidis strains tested, 25.8% were of the compact type in serum-soft agar. Compact variants showed higher production of metabolites than counterpart diffuse variants.     

Role of Protein A in the Serum-Soft Agar Technique

Formation of compact colonies of Staphylococcus aureus in serum-soft agar is mainly a result of a reaction between protein A and the Fc-part of immunoglobulin G and not a clumping factor-fibrinogen reaction.     

Detection of Capsular Antigen Production in Unencapsulated Strains of Staphylococcus aureus

Of 91 compact-type strains of Staphylococcus aureus in regular serum soft agar (SSA), 82 converted diffuse-type growth in serum soft agar (pH adjusted to 6.0). With the addition of four different rabbit anticapsular sera (anti-type A, B, C, and D sera) in low pH (6.0) SSA, 21 strains of S. aureus showed compact-type colonial morphologies. Eleven, one, and one strains of S. aureus reacted singly with rabbit anticapsular sera types A, B, and C, respectively, and no strain reacted with rabbit anticapsular type D. Eight of the e strains reacted with both rabbit anticapsular sera types A and B. When the ability to absorb the converting activities of the antisera (changes of colonial morphologies of anticapsular sera in SSA) was quantitatively tested, 7- to 27-fold of these organisms were capable of absorbing the activities compared with the Smith diffuse organisms. These results suggest that even unencapsulated S. aureus strains are capable of producing capsular substance, although the capability is quantitatively different from strain to strain.     

Novel Screening Agar for Detection of Vancomycin-Nonsusceptible Staphylococcus aureus

In January 2006, the Clinical and Laboratory Standards Institute (CLSI) updated breakpoints for vancomycin susceptibility testing for Staphylococcus aureus such that an MIC greater than 2 mg/liter was considered to represent nonsusceptibility to vancomycin. Previously, an MIC of 4 mg/liter had been considered to represent susceptibility. Additionally, in 2009, the CLSI altered the guidelines for staphylococci such that disk diffusion was no longer an acceptable means for testing vancomycin susceptibility in these organisms. To accommodate the change in breakpoints and methodological updates, we designed a medium consisting of brain heart infusion agar with 3 mg/liter vancomycin (BHI-V3) to screen for isolates of S. aureus with reduced susceptibility to vancomycin. We challenged this medium using a previously characterized collection of 100 isolates of S. aureus, including 55 vancomycin-susceptible isolates and 45 isolates representing vancomycin-intermediate strains of S. aureus (VISA) (with vancomycin MICs of 4 mg/liter or greater). All of the VISA isolates grew on the agar, for 100% sensitivity. Nineteen vancomycin-susceptible isolates also grew on the agar, for 65% specificity. We then incorporated BHI-V3 into clinical practice. In the first 60 days postimplementation, we identified 17 potential VISA isolates out of 421 S. aureus isolates tested. Thirteen out of the 17 were confirmed to represent VISA isolates. In light of the excellent sensitivity of this medium, we recommend that clinical laboratories incorporate BHI-V3 to screen for vancomycin-nonsusceptible isolates of S. aureus.At the site of this study, 60 to 70% of infections in which Staphylococcus aureus is the etiologic agent are caused by methicillin-resistant S. aureus (MRSA). For these infections, vancomycin is considered the mainstay of antimicrobial therapy. The ability to accurately identify isolates of S. aureus with reduced susceptibility to vancomyin is paramount for a successful clinical outcome.Until recently, strains of S. aureus with reduced susceptibility to vancomycin could be detected using a screening medium consisting of brain heart infusion agar (BHI) supplemented with 6 mg/liter vancomycin (BHI-V6). The medium is inoculated with 10 μl of a 0.5 McFarland standard suspension of the organism and incubated at 35 to 37°C for 24 h. Strains with reduced susceptibility to vancomycin demonstrate growth of one or more colonies after 24 h of incubation (1, 2, 8, 9). However, as of January 2006, the Clinical and Laboratory Standard Institute (CLSI) breakpoints for vancomycin susceptibility for S. aureus were updated such that strains with an MIC greater than 2 mg/liter were considered to be nonsusceptible to vancomycin, where those with an MIC of 4 mg/liter had previously been considered to be vancomycin susceptible (1-4). As a vancomycin MIC of 4 to 8 mg/liter is considered to represent intermediate susceptibility, the use of an agar medium such as BHI-V6 as a means to screen for vancomycin-intermediate strains of S. aureus (VISA) is not adequate for this purpose, as those strains having a vancomycin MIC greater than 2 but less than 6 mg/liter are not detected by this method. To accommodate the change in breakpoints, we designed a screening medium consisting of BHI with 3 mg/liter vancomycin (BHI-V3). BHI-V3 was manufactured for our laboratory by Remel (Lenexa, KS). A noninhibitory pink dye, amaranth, was added to the agar to assist in rapid visual differentiation of it from other screening agars in the laboratory, such as the 6 mg/liter vancomycin screening agar (yellow) and/or the oxacillin screening agar (clear). The agar is inoculated using the same method as for the BHI-V6 medium: 10 μl of a 0.5 McFarland standard suspension of organism is added to the agar and incubated for 24 h at 35 to 37°C. Several isolates can be placed onto one plate. For quality control purposes, Enterococcus faecalis ATCC 29212 was used as a positive growth control. This organism is the negative growth control for the 6 mg/liter vancomycin screening agar and has an MIC of 4 mg/liter by Etest in our laboratory. S. aureus ATCC 29213 was used as a negative growth control and has an MIC of 2 mg/liter by Etest in our laboratory. These organisms were chosen as quality control isolates because they bracket the desired concentration range of the agar. Any growth on BHI-V3 was interpreted as positive after confirmation that the isolate was S. aureus. Any isolate that grew on BHI-V3 was tested directly from this medium for vancomycin MIC confirmation, or, if there was not sufficient growth to do so, the isolate was subcultured to another 3 mg/liter BHI-V3 medium prior to performance of susceptibility testing to maintain vancomycin selective pressure.In the initial evaluation, we challenged the BHI V3 with eight clinical isolates of E. casseliflavus and E. gallinarum, which intrinsically have a vancomycin-intermediate phenotype. All of these isolates grew on the screening agar and had a vancomycin MIC of 6 or 8 mg/liter, as determined by Etest. Then, the ability of BHI-V3 medium to detect isolates of S. aureus with reduced vancomycin susceptibility was verified using a previously characterized collection of S. aureus isolates with MICs to vancomycin ranging from 0.5 to 8 mg/liter (7). The vancomycin MIC for these strains was determined by the Centers for Disease Control and Prevention (CDC) using CLSI reference broth microdilution (7). The MIC that was used for the purpose of deciding whether these isolates exhibited full or intermediate susceptibility to vancomycin was based on the Difco broth microdilution result. The collection included 55 vancomycin-susceptible isolates (i.e., with a vancomycin MIC of 2 mg/liter or less) and 45 vancomycin-intermediate isolates (VISA) with a vancomycin MIC of 4 or 8 mg/liter. For the purpose of this analysis, the MIC results of the strain collection were coded and not revealed until the testing was complete.All of the VISA isolates grew on the BHI-V3 agar, for 100% sensitivity for this screening method (Table ​(Table1).1). This is a significant improvement in sensitivity over what has been reported for several other methods for the detection of VISA isolates. For example, Swenson and colleagues have previously reported that BHI-V6 agar failed to detect 33% (12 of 36) of VISA isolates with an MIC of 4 mg/liter (7). Other methods that have shown poor sensitivity for the detection of VISA include the use of Kirby-Bauer disk diffusion, the Trek Sensititre broth microdilution panel, the Vitek Legacy, and the Vitek2 GP61 and GP67 antimicrobial susceptibility cards (7, 10). The Etest has previously been demonstrated to be sensitive for detection of VISA, but it did categorize one of 45 VISA isolates as vancomycin susceptible in a recent study (7).

TABLE 1.

Performance of BHI-V3 with previously characterized strains of S. aureus

Evaluation of Mannitol Salt Agar for Detection of Oxacillin Resistance in Staphylococcus aureus by Disk Diffusion and Agar Screening

Mannitol salt agar was evaluated for detection of oxacillin resistance in 136 Staphylococcus aureus isolates. All mecA-positive isolates (n = 54) were correctly categorized as oxacillin resistant by the disk diffusion test (1-μg disk; zone diameter, <16 mm); the specificity was 97.6%. Agar screening (2 μg of oxacillin per ml) revealed a sensitivity of 98.1% and a specificity of 95.1%.     

Capsular antibodies induce type-specific phagocytosis of capsulated Staphylococcus aureus by human polymorphonuclear leukocytes.

Capsular types 5 and 8, which account for about 70% of Staphylococcus aureus strains isolated from the blood of patients, resisted in vitro phagocytosis by human polymorphonuclear leukocytes (PMN). Antisera and monoclonal antibody to type 5 and 8 capsular polysaccharides (CPS) induced type-specific in vitro phagocytosis of capsulated organisms by PMN. Antibodies directed against the O-acetyl moiety of the type 8 CPS were more effective in inducing phagocytosis of type 8 organisms by PMN. Either type-specific antiserum or monoclonal antibody reactive with the native O-acetylated type 8 CPS was most effective in inducing in vitro phagocytosis of type 8 organisms by PMN. These results provide further evidence that CPS of S. aureus are associated with host immunity to this organism.     

Different Serum Requirements for Opsonization of Two Serologically Identical Strains of Pseudomonas Aeruginosa

Two strains of serotype 2 Pseudomonas aeruginosa and one species of E. coli were compared regarding their opsonic requirements using human sera and polymorphonuclear leukocytes. None of the three organisms tested activated exclusively one complement pathway. Both strains of Pseudomonas had an absolute requirement for antibody for opsonization, but differed in complement requirements. The studies described herein showed significant differences in the mechanisn of opsonization of the two strains of Pseudomonas.     

Different Serum Requirements for Opsonization of Two Serologically Identical Strains of Pseudomonas Aeruginosa

Two strains of serotype 2 Pseudomonas aeruginosa and one species of E. coli were compared regarding their opsonic requirements using human sera and polymorphonuclear leukocytes. None of the three organisms tested activated exclusively one complement pathway. Both strains of Pseudomonas had an absolute requirement for antibody for opsonization, but differed in complement requirements. The studies described herein showed significant differences in the mechanisn of opsonization of the two strains of Pseudomonas.     

Methicillin-Resistant Staphylococcus aureus Clonal Types in the Czech Republic

Molecular surveillance studies have documented the extensive spread of methicillin-resistant Staphylococcus aureus (MRSA) clones. Studies carried out by Centro de Epidemiologia Molecular-Network for Tracking Gram-Positive Pathogenic Bacteria (CEM/NET) led to the identification of two international multidrug-resistant strains, which were designated as the Iberian and Brazilian MRSA clones and which were defined by multiple genomic typing methods; these included ClaI restriction digests hybridized with mecA- and Tn554-specific DNA probes and pulsed-field gel electrophoresis (PFGE). The genotypic characteristics of these clones are distinct: the Iberian clone is defined as mecA type I, Tn554 type E (or its variants), and PFGE pattern A (I:E:A), whereas the Brazilian clone is defined as mecA type XI (or its variants), Tn554 type B, and PFGE pattern B (XI:B:B). In this study, we characterized 59 single-patient isolates of MRSA collected during 1996 and 1997 at seven hospitals located in Prague and five other cities in the Czech Republic by using the methodologies mentioned above and by using ribotyping of EcoRI and HindIII digests hybridized with a 16S-23S DNA probe. The Brazilian MRSA clone (XI:B:B) was the major clone (80%) spread in two hospitals located in Prague and one located in Brno; the Iberian MRSA clone (I:E:A or its variant I:DD:A), although less representative (12%), was detected in two hospitals, one in Prague and the other in Plzen. Almost all the strains belonging to clone XI:B:B (45 of 47) corresponded to a unique ribotype, E1H1, whereas most strains of the I:E:A and I:DD:A clonal types (6 of 7) corresponded to ribotype E2H2.     

Rapid Detection of Epidemic Strains of Methicillin-Resistant Staphylococcus aureus

Fifty methicillin-resistant Staphylococcus aureus (MRSA) initial isolates obtained from patients hospitalized in the orthopedic clinic of the Frankfurt University Hospital and 150 methicillin-sensitive Staphylococcus aureus (MSSA) isolates were investigated in this study to determine whether the Slidex Staph-Kit is capable of differentiating between MRSA and MSSA owing to its unique performance characteristics. The Slidex Staph-Kit is a combined latex hemagglutination test designed to detect clumping factor, protein A, and a specific surface immunogen for S. aureus. Clumping factor-positive strains cause erythrocytes sensitized with fibrinogen to hemagglutinate, thereby resulting in visible red clumps. S. aureus strains deficient in clumping factor agglutinate latex particles sensitized with specific antibodies against surface proteins of S. aureus, thereby resulting in visible white clumps. Our results demonstrate that white clumping has a 99% specificity as well as a 98% positive predictive value for MRSA. Clumping factor-negative MRSA, which have been reported to occur in several countries, are epidemic in the Frankfurt area and account for 80% of all MRSA initial isolates in the orthopedic clinic of the Frankfurt University Hospital. Genotyping of all MRSA isolates by macrorestriction analysis of chromosomal DNA revealed that 83% of clumping factor-negative MRSA are closely related to the “southern-German” epidemic strain. This is the first study demonstrating the Slidex Staph-Kit’s capability for identifying epidemic clumping factor-negative S. aureus strains as methicillin resistant even prior to antimicrobial susceptibility testing.     

Diversity of Staphylococcus aureus Strains Isolated from Two European Regions with Different Prevalences of Methicillin Resistance

The genodiversity of Staphylococcus aureus isolates from the Nottingham region of the United Kingdom was compared with isolates from the Freiburg region of Germany. The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) isolates was higher in Nottingham than in Freiburg. In patients from Nottingham hospitals, 80% of MRSA isolates were classical epidemic MRSA-15, but genotypic variants of epidemic MRSA-15 comprised 72% of isolates from Nottingham community-based patients. In contrast, MRSA isolates from Freiburg showed greater diversity, but 47% and 23% of isolates, respectively, belonged to two predominant MRSA genotypes found in isolates from both hospitalised and community-based patients. The results suggest that genodiversity becomes increasingly more confined in settings with a higher frequency and longer duration of MRSA prevalence. Electronic Publication     

Interactions between Methicillin and Vancomycin in Methicillin-Resistant Staphylococcus aureus Strains Displaying Different Phenotypes of Vancomycin Susceptibility

Vancomycin-sensitive, -intermediate, and -heterointermediate methicillin-resistant Staphylococcus aureus isolates were tested by using E-tests to explore the interaction of methicillin and vancomycin. For the vancomycin-intermediate and -heterointermediate strains both drugs showed antagonism at concentrations below their MICs but synergy at methicillin concentrations near the MIC. This property could be used to screen for heterointermediate S. aureus strains.     

Slime Production by Staphylococcus aureus and Staphylococcus epidermidis Strains Isolated from Patients with Diabetic Foot Ulcers

Slime production is a very important factor related to biofilm formation. The objective of the present study was to determine the frequency of slime production by Staphylococcus aureus and Staphylococcus epidermidis strains recovered from 50 patients with diabetic foot ulcers. Slime production was determined using the Congo red agar (CRA) method and compared with immunocytochemistry for the production of polysaccharide intercellular adhesin (PIA). Out of 55 S. aureus strains, 69% produced slime as shown by the CRA method. Of them, 84.2% also produced PIA. Of 17 CRA-negative strains, 70.6% produced PIA. Out of 20 S. epidermidis strains, 75% were CRA positive and 93.3% produced PIA. All CRA-negative S. epidermidis produced PIA. In conclusion, PIA production is a very common trait of S. aureus and S. epidermidis isolates obtained from diabetic foot ulcer patients.     

Antigenic Determinants of Staphylococcus aureus Type 5 and Type 8 Capsular Polysaccharide Vaccines

Bacterial capsular polysaccharides (CP) are carbohydrate polymers comprised of repeating saccharide units. Several of these CP have side chains attached to their backbone structures. The side chains may include O-acetyl, phosphate, sialic acid, and other moieties. Those moieties represent the immunodominant epitopes and the most functional ones. The clinically significant Staphylococcus aureus type 5 CP (CP 5) and type 8 CP (CP 8) are comprised of a trisaccharide repeat unit with one O-acetyl group attached to each repeat unit. The immunogenicity of these CP and the functionality of antibodies to the backbone and the O-acetyl moieties were investigated. Immunization with the native CP conjugates (CP with 75% O-acetylation) elicited a high proportion of antibodies directed against the O-acetyl moiety. Nonetheless, all of the vaccinees produced antibodies to the backbone moieties as well. Conjugate vaccines made of de-O-acetylated CP elicited backbone antibodies only. Antibodies to both backbone and O-acetyl groups were found to be opsonic against S. aureus strains which varied in their O-acetyl content. Absorption studies with O-acetylated and de-O-acetylated CP showed that (i) native CP conjugates generated antibodies to both backbone and O-acetyl groups and (ii) O-acetylated isolates were opsonized by both populations of antibodies while the non-O-acetylated strains were predominantly opsonized by the backbone antibodies. These results suggest that S. aureus CP conjugate vaccines elicit multiple populations of antibodies with diverse specificities. Moreover, the antibodies of different specificities (backbone or O-acetyl) are all functional and efficient against the variations in bacterial CP that may occur among clinically significant S. aureus pathogenic isolates.     

Rapid Detection of Staphylococcus aureus Strains with Reduced Susceptibility to Vancomycin by Isothermal Microcalorimetry

Methicillin-resistant Staphylococcus aureus (MRSA) usually harbors a vancomycin-susceptible phenotype (VSSA) but can exhibit reduced vancomycin susceptibility phenotypes that can be heterogeneous-intermediate (hVISA), intermediate (VISA), or fully resistant (VRSA). Current detection techniques (e.g., Etest and population analysis profiles [PAPs]) are slow and time-consuming. We investigated the potential of microcalorimetry to detect reduced susceptibilities to vancomycin in MRSA strains. Representative MSSA, VSSA, hVISA, VISA, and VRSA reference strains, as well as clinical isolates, were used. PAPs were performed by standard methods. Microcalorimetry was performed by inoculating 5 × 10⁷ CFU of overnight cultures into 3-ml vials of brain heart infusion broth supplemented with increasing concentrations of vancomycin, and growth-related heat production was measured at 37°C. For the reference strains, no heat production was detected in the VSSA isolates at vancomycin concentrations of >3 μg/ml during the 72 h of incubation. The hVISA and VISA strains showed heat production with concentration-proportional delays of up to 6 μg/ml in 48 h and up to 12 μg/ml in 72 h, respectively. The VRSA strain showed heat production at concentrations up to 16 μg/ml in 12 h. The testing of clinical strains indicated an excellent negative predictive value, allowing us to rule out a decreased vancomycin susceptibility phenotype in <8 h of incubation. Sequential isolates from a patient undergoing vancomycin therapy showed evolving microcalorimetric profiles up to a VISA phenotype. Microcalorimetry was able to detect strains with reduced susceptibilities to vancomycin in <8 h. The measurement of bacterial heat production might represent a simple and rapid method for the detection of reduced susceptibilities to vancomycin in MRSA strains.     

Comparison of Compact and Diffuse Variants of Strains of Staphylococcus aureus

Several biological characteristics of six diffuse-type variants and six compact-type variants of Staphylococcus aureus were compared on the basis of morphology, phage type, certain cellular products, growth rate, and mouse virulence. All compact variants gave a positive test for clumping-factor reaction, coagulase, deoxyribonuclease, mannitol fermentation, hemolysis, and pigmentation. Four of the six compact variants were phage typable and lacked capsules. None of the diffuse variants was phage-typable or possessed the clumping-factor reaction. Only one diffuse variant had hemolytic activity, and all of the diffuse strains were encapsulated. No differences between the compact and diffuse strains were noted with respect to mannitol fermentation or deoxyribonuclease activity. Coagulase and acid phosphatase activities of the culture supernatant fluid were diminished in most of the diffuse strains. Less β-carotene content was found in cells of diffuse variants. Virulence in mice was found to correlate with the capsule size, as the mortality rate was greatest for diffuse variants having the largest capsules. Growth rates of variants were generally not significantly different.