SEARCH

SEARCH BY CITATION

Coagulase-negative staphylococci differ phenotypically from Staphylococcus aureus by lacking the ability to form some extracellular products and cell wall-associated proteins [1]. In routine clinical microbiology the coagulase test-tube reaction has been used as the main species characteristic of S. aureus [1], as has DNase formation in food microbiology [2]. Although less sensitive and less specific than coagulase formation [3], the demonstration of clumping factor is often used in routine clinical microbiology. The clumping factor of S. aureus is a cell wall-associated protein which reacts with fibrinogen monomers, thus leading to aggregation of S. aureus cells [4]. The recent emergence of epidemic methicillin-resistant S. aureus (MRSA) lacking this capability [5] has rendered tests based only on the demonstration of protein agglutination unreliable [6]. Therefore, recently developed test kits have included the use of antibodies against cell wall-associated macro-molecules, and IgG specific for S. aureus protein A [7].

This communication reports on the sensitivity and specificity of the Dry Spot Staphytect Plus test (Oxoid, Basingstoke, UK) and three other agglutination tests for speciating staphylococcal species, especially S. aureus including methicillin-resistant isolates.

For methicillin-sensitive S. aureus (MSSA, n=120), strains belonging to the major clonal group of this species as deduced from genomic DNA fragment patterns [8] were tested. For MRSA (n=80), five isolates from each of six MRSA epidemics with an inter-regional dissemination in Germany [9] were included, and in addition 50 isolates which, according to their SmaI macrorestriction patterns, are different to both each other and these epidemic strains. All S. aureus strains included in the study were isolated from human sites of infection.

All S. aureus strains involved in this study produced coagulase and gave an expected 423-bp PCR product specific for the ribosomal RNA spacer region [10]. In addition, 100 coagulase-negative staphylococci (CNS) were included in the study: S. epidermidis (50), S. haemolyticus (25), S. hominis (6), S. simulans (1), S. auricularis. (1), S. sciuri (1), S. capitis (4), S. kloosii (2), S. warneri (2), S. schleiferi (2), S. cohnii (1), and S. xylosus (5). All strains were speciated by classical biochemical methods [3] and originated from human sites of infection or colonization.

The following agglutination tests have been compared: Staphyslide-Test (bioMérieux, Marcy l'Etoile, France), composed of fibrinogen bound to red blood cells; Pastorex Staph-Plus (Sanofi, Marne-LaCoguette, France), composed of latex coated with fibrinogen, IgG and antibodies against capsular polysaccharides; Staphaurex Plus (Murex Diagnostics Ltd, Dartford, UK), composed of fibrinogen as well as antibodies against serotype 5 capsular polysaccharides and somatic type 18 bound to latex; and Dry Spot Staphytect Plus (Oxoid Ltd, Basingstoke, UK), composed of latex particles coated with pig fibrinogen, IgG and antibodies to capsular polysaccharides dried on the surface of smooth pasteboard. All tests were performed as recommended by the manufacturers. S. aureus species type was confirmed by PCR of ribosomal RNA gene sequences specific for S. aureus. Whole cellular DNA was isolated as described previously [11]. Primers used were SA V (5′CATATTGTATTCAGTTTTGA-3′) and SA VI (5′-TCCACCATTTTTATAAGTC-3′) [10]. Replitherm-polymerase (Biozym, Markoldendorf, Germany) and reaction mixtures as previously described [11] were used with the following DNA amplification conditions: 30 cycles of 94°C for 1 min, 55°C for 2 min, and 72°C for 3 min.

For the demonstration of free coagulase using the test-tube method, five colonies were suspended in 0.5 mL of Oxoid nutrient broth. After incubation for 2 h at 37°C, 0.5 mL of human blood plasma was added. The tubes were inspected for formation of clots after 3 h at 37°C and again after 24 h at room temperature. For detection of clumping factor, one colony from a blood agar plate (incubated overnight) was picked with a sterile loop, suspended on a glass slide in approximately 20 μL of 0.9% NaCl solution and mixed with approximately 20 μL of human fibrinogen solution (2 mg/mL in 0.9% NaCl). Results of the comparison of different agglutination tests with the tube coagulase and clumping factor are shown in Table 1). For all four tests, the sensitivity for detecting MSSA was 100%.

Table 1. Comparison of different test kits for rapid diagnosis of Staphylococcus species
 Dry Spot Staphytect PlusPastorex Staph-PlusStaphaurex PlusStaphyslide-TestClumping reactionTube coagulase
  1. MSSA, methicillin-sensitive S. aureus; MRSA, methicillin resistant S. aureus; CNS, coagulase-negative staphylococci.

  2. aOne S. schleiferi.

  3. bOne S. schleiferi, one S. colnii.

MSSA, n=120
Negative
Intermediate
Positive120120120120120120
MRSA, n=80
Negative2121
Intermediate1155
Positive798079545480
CNS, n=100
Negative9798989999100
Intermediate211
Positive1a2b2b
Sensitivitiy (%)99.510099.58787100
Specificity (%)9798989999100

As is evident from the results obtained with the Staphyslide-Test, tests designed only for the detection of clumping factor are highly specific but not sensitive enough for correct detection of MRSA. in contrast to the increased sensitivity of test kits containing additional components such as IgG and antibodies against capsular polysaccharides. For Staphaurex Plus and for Pastorex Staph-Plus a high sensitivity for identification of MRSA has been previously reported [6]. The specificity of these test kits is only slightly reduced with regard to a positive reaction of S. schleiferi (Dry Spot Staphytect Plus) and S. schleiferi and S. cohnii (Pastorex Staph-Plus, Staphaurex Plus).

For S. schleiferi a positive clumping reaction has been described when suspended in human blood plasma [12]. The absence of clumping in fibrinogen solution (Table 1) suggests that S. schleiferi probably possesses another surface component as well as the clumping factor, reacting with plasma constituents other than fibrinogen.

Of the 80 MRSA strains, five gave intermediate results with tests based on demonstration of clumping factor alone, which may be due to weak expression of clumping factor. One strain was also intermediate when Dry Spot Staphytect Plus or Staphaurex Plus were used. These intermediate reactions were also found when isolates were retested following fresh subculture on blood agar. In clinical bacteriology the coagulase tube test should be performed for strains which are intermediate in agglutination tests. The few CNS exhibiting intermediate agglutination reactions were identified as S. epidermidis isolates and originated from blood cultures. The bases for this reaction are unknown.

In conclusion, Dry Spot Staphytect Plus has an excellent sensitivity for the identification of S. aureus in the clinical microbiology laboratory.

References

  1. Top of page
  2. References
  • 1
    Bergey's manual of systematic bacteriology, Vol. 20. Baltimore, Philadelphia: Williams & Wilkins, 1986: 101535.
  • 2
    Lachica RVF, Deibel RH. Detection of nuclease activity in semisolid and broth cultures. Appl Microbiol 1969; 18: 1746.
  • 3
    Kloos W, Schleifer K-H, Götz F. The genus Staphylococcus. In: BalowsA, TripesHG, DworkinM, HarderW, SchleiferK-H, eds. The procaryotes, 2nd edn. New York, Berlin, Heidelberg: Springer Verlag, 1991: 1369420.
  • 4
    Boden M, Flock J-I. Evidence for three different fibrinogen-binding proteins with unique properties from Staphylococcus aureus strain Newman. Microb Pathogen 1992; 12: 28998.
  • 5
    Schwarzkopf A, Karch H, Schmidt H, Lenz W, Heesemann J. Phenotypical and genotypical characterization of epidemic clumping factor for negative oxacillin resistant Staphylococcus aureus. J Clin Microbiol 1993; 31: 22815.
  • 6
    Luijendijk A, Van Belkum A, Verbrugh H. Klujtmons J. Comparison of five tests for identification of Straphylococcus aureus from clinical samples. J Clin Microbiol 1996; 34: 22679.
  • 7
    Grov A, Oeding P, Myklestad B, Aasen J. Reactions of staphylococcal antigens with normal sera, je-globulins and j-globulin fragments of various species origin. Acta Pathol Microbiol Scand 1970; 378: 10611.
  • 8
    Witte W, Cuny C, Claus H. Clonal relatedness of Staphylococcus aureus strains from infections in humans as deduced from genomic DNA fragment patterns. Med Microbiol Lett 1993; 2: 729.
  • 9
    Witte W, Kresken M, Braulke C. Cuny C. Increasing incidence and widespread dissemination of methicillin-resistant Straphylococcus aureus (MRSA) in hospitals in central Europe, with special reference to German hospitals. Clin Microbiol Infect 1997; 3: 41422.
  • 10
    Saruta K, Matsungana T, Kono M, et al. Rapid identification and typing of Staphylococcus aureus by nested PCR amplified ribosomal DNA spacer region. FEMS Microbiol Lett 1997: 146: 2718.
  • 11
    Cuny C, Witte W. Typing of Staphylococcus aureus by PCR for DNA sequences flanked by transposon Tn916 target region and ribosomal binding site. J Clin Microbiol 1996: 34: 15025.
  • 12
    Freenay J, Brun Y. Bes M, et al. Staphylococcus lugdunensis sp. nov. and Staphylococcus schleiferi sp. nov. two species from human clinical specimens. Int J Syst Bacteriol 1988: 38: 16872.