Staphylococcus Speciesg - Journal of Clinical Microbiology

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Jul 29, 1974 - Bacterial strains. The 939 strains of ..... Subacute bacterial endocarditis due to coagulase-negative Staphylococcus albus. Acta Med. Scand.
Vol. 1, No. 1 Printed in U.S.A.

JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1975, p. 82-88

Copyright 0 1975 American Society for Microbiology

Simplified Scheme for Routine Identification of Human Staphylococcus Speciesg WESLEY E. KLOOS* AND KARL H. SCHLEIFER Department of Genetics, North Carolina State University, Raleigh, North Carolina 27607*. and Lehr.stuhl far Mikrobiologie, Universitdt Munchen, 8 Munich 19, Germany Received for publication 29 July 1974

From a total of 40 characters that were previously used to differentiate species of staphylococci, 13 key characters were selected to make a simplified scheme that could be easily used by the routine clinical laboratory for identifying human staphylococci. These key characters included coagulase activity, hemolysis, nitrate reduction, and aerobic acid production from fructose, xylose, arabinose, ribose, maltose, lactose, sucrose, trehalose, mannitol, and xylitol. In the simplified scheme, 924 strains of staphylococci were placed into 11 positions, each of which contained the major portion (>80%) of strains of one of the recognized species. Several positions contained a rare or few uncommon strains of one or more additional species and these could be resolved on the basis of other key characters.

Historically, the coagulase-positive staphylococci, known as Staphylococcus aureus, have been regarded as opportunistic pathogens, whereas the coagulase-negative staphylococci (including some strains erroneously identified as micrococci) have been generally regarded as nonpathogens. This distinction has been very useful for diagnosis in the clinical laboratory, but accumulating evidence is strongly suggesting that coagulase-negative staphylococci can cause a variety of pyogenic infections (3, 20, 21) and urinary-tract infections (12, 15, 19, 21, 23), and can colonize artificial internal prostheses in association with a bacteremia (7). Due to the growing recognition that coagulase-negative staphylococci are of medical importance, numerous studies were initiated in an attempt to classify these organisms (2, 13-15, 17, 22). Results of these preliminary studies have indicated that coagulase-negative staphylococci represent a heterogenous group. Recently we have reported on comprehensive systematic studies, aimed at determining natural relationships, that have resolved and characterized nine different coagulase-negative Staphylococcus species isolated from human skin (9, 24). In these studies, species were differentiated by an array of morphological, physiological, and biochemical characters and antibiotic susceptibility and cell wall composition. The purpose of this report is to condense and simplify the extensive findings into a working scheme form,

suitable for use in the routine clinical laboratories.

MATERIALS AND METHODS Bacterial strains. The 939 strains of staphylococci used in this study were isolated from the healthy skins of two groups of people (9, 10, 24). These included 778 strains isolated from 20 people living in Raleigh, N.C., and 161 strains isolated from 20 people living in Somerville and New Brunswick, N.J. All except seven of the above strains were identified as belonging to the species S. aureus, S. simulans, S. xylosus, S. cohnii, S. saprophyticus, S. haemolyticus, S. warneri, S. hominis, S. epidermidis, or S. capitis (9, 24). Five unusual coagulase-positive strains and two coagulase-negative strains were tentatively designated as unnamed Staphylococcus sp. until additional strains are found to provide better estimates of species character parameters. Character determinations. Procedures for determining the various key morphological, physiological, biochemical (including cell wall composition), and antibiotic characters have been previously described (10, 24, 25). To provide continuity between the earlier systematic studies and this report, we describe in the following paragraphs those procedures that can be easily adapted to the routine clinical laboratory. Inocula for tests and test cultures were prepared from 24- to 48-h-old agar slope cultures and all incubations of cultures were performed at 34 to 35 C. (i) Anaerobic growth in a thioglycolate medium. Tubes (150 by 13 mm) containing 8 ml of fluid and cooled (50 to 52 C) thioglycolate semisolid medium (Brewer's fluid thioglycolate medium plus 0.3% agar) (5) were inoculated with a 0.1-ml saline suspension of 1 Paper no. 4433 of the Journal Series of the North Caro- cells to a density of 10" to 107 colony-forming units lina Agricultural Experiment Station, Raleigh, N.C. 27607. (CFU)/ml. Cultures were incubated for 5 days at 82

VOL. 1, 1975

IDENTIFICATION OF HUMAN STAPHYLOCOCCUS SP.

which time growth characteristics were noted. Strains demonstrating dense uniform anaerobic growth usually show this property within 24 to 48 h and do not require further incubation for characterization. However, strains producing a gradient of dense to light growth down the medium or those failing to show evidence of anaerobic growth within 48 h should be incubated for the entire 5-day period to allow for the possibility of discrete colony development. (ii) Lysostaphin susceptibility. The minimal inhibitory concentration of lysostaphin for strains was previously determined (9, 24). In this report, for the purpose of simplicity and expediency, we recommend determining only the susceptibility of strains to 50 ,g of lysostaphin per ml of P agar. Species that were sensitive to this concentration usually had cell wall peptidoglycans of the L-Lys-Gly4.7_6.0 type, whereas species that were resistant to this concentration had the LdLys-Gly,.,_,.l, L-Sero.6-1.5 type. A loopful of a saline suspension (containing 107 to 10' CFU/ml) was inoculated on the surface of a 24-h dried, P agar plate containing lysostaphin and on a control P agar plate without lysostaphin. Up to 16 strains could be tested on a plate. Cultures were incubated for 24 h and then examined for growth. (iii) Coagulase. Clumping factor and coagulase activity were determined using the conventional slide and tube test, respectively, with rabbit plasma (Difco)

(1).

(iv) Hemolysis. Differences in the hemolytic activity of species were best demonstrated with bovine blood agar (5% blood in P agar) and secondly with human blood agar. The strong hemolysis of bovine and human blood agars by many strains of S. haemolyticus was very useful in distinguishing this species from other coagulase-negative staphylococci. These strains demonstrated weaker and somewhat less distinctive hemolysis on sheep or rabbit blood agars. In those situations where bovine blood is not available, laboratories may use citrated human blood with reasonable success. Many strains of S. simulans, S. epidermidis, and S. capitis produced stronger hemolysis with human blood than with bovine blood, but usually produced narrower hemolysis zones (trace to 2.5 mm) than most strains of S. haemolyticus (2.5 to 5.0 mm). A few strains of S. simulans produced hemolysis zones that were as wide ( n2.5 mm) as those found with some strains of S. haemolyticus, but could be distinguished by their clearer appearance. Culture streaks were made by lightly inoculating a 1-cm line on the surface of a blood agar plate with a loopful of cells. Up to eight streaks could be radially inoculated on each plate. Hemolysis was characterized after 24, 48, and 72 h of culture incubation and was interpreted according to the following scheme: +, strong, hemolysis extending > 1.5 mm (bovine blood) or >2.5 mm (human blood) out from the culture streak within 48 to 72 h; -, moderate to weak, hemolysis extending trace to < 1.5 mm (bovine blood) or trace to -H

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