Journal

of

Hospital

Infection

Clinically staphylococci:

(1992)

22,

19-31

significant identification K. Refsahl

Department

of Medical

coagulase-negative and resistance

patterns

and B. M. Andersen

Microbiology,

University

Accepted for publication

Hospital,

29 April

Tromso,

Norway

1992

Summary:

Coagulase-negative staphylococci (CNS) of clinical significance, isolated from 13 1 patients, were investigated during the period 1989-90 in northern Norway. The staphylococci were isolated from blood cultures (68; 51.9%), vascular catheters (6), osteomyelitis foci (13), postoperative and other wounds (15), and urine samples (29). The use of Gram-positive Identification Card (Vitek) and ‘Staph-zym’ (Rosco) both gave a primarily correct species identification in 95% of the cases. Staphylococcus epidermidis was the predominent species (72.3%). Methicillin-resistance was found in 40 of 131 (30.5%) of all CNS and in 34 of 96 (35.4%) of S. epidermidis. Methicillin-resistant (MR) S. epidermidis strains were usually resistant to gentamicin, tetracycline, chloramphenicol and trimethoprim. MR strains were, however, less resistant to sulphonamides than methicillin-sensitive strains (10 out of 34 vs. 55 out of 62). Methicillin-resistance implied resistance to all p-lactam antibiotics, including imipenem. Among S. epidermidis, MR isolates increased from 10% in 1987 to 35.4% in 1989-90. All strains were sensitive to vancomycin and rifampicin.

Keywords:

Staphylococcus

Micrococcaceae;

typing,

spp. bacterial;

identification; drug resistance.

Staphylococcus

epidermidis;

Introduction Coagulase-negative staphylococci, part of the normal skin flora, are of increasing clinical importance, especially as a cause of hospital infection.’ They are associated with the presence of foreign bodies such as prosthetic valves, cerebra-spinal fluid shunts, orthopaedic prostheses and intravascular, urinary and dialysis catheters. The genus Staphylococcus, of the family Micrococcaceae, is divided into more than 2.5 species.2 The most frequently isolated are Staphylococcus epidermidis and Staphylococcus saprophyticus.3 Increased methicillin resistance has been observed among isolates from hospital infections. 4 In the present study, 131 clinically significant isolates of coagulase-negative staphylococci were identified by two commercial test systems and their antibacterial susceptibility was investigated. Correspondence 0195-6701/92/090013+

to: K. Refsahl. 0 1992 The Hospml

19 EOS.OO/O

19

Infection

Society

20

K. Refsahl Materials

and B. M. Andersen and

methods

Bacterial

strains and sites of infection Consecutive isolates (13 1) of coagulase-negative staphylococci that appeared to be of clinical significance were collected during 1989 and 1990 at the Department of Microbiology, University Hospital, Tromso, Norway. The strains were isolated in pure culture and in heavy growth from the specimen sampled. The blood culture isolates were from 68 patients with two or more separate cultures positive and with signs of bacteraemia. The other isolates were from central vein catheters, osteomyelitis foci, postoperative and other wound infections and urine samples; 17 out of 29 urinary strains were from patients outside hospital; all other isolates were from hospitalized patients. IdentiJication The staphylococci were identified by Gram staining, catalase and coagulase (Staphylase Test, Oxoid Diagnostic Reagents, Oxoid Ltd, Hampshire, UK) reactions and sensitivity to furazolidone. Species identification was made with Gram-positive Identification Card (GPI) (Vitek Systems, 595 Anglum Drive, Hazelwood, Missouri 63042-2395, USA), and with ‘Staph-zym’ (Rosco Diagnostica, 2630 Taastrup, Denmark). GPI identifies a number of Gram-positive bacteria, among them 14 species of coagulase-negative staphylococci (Table I). The GPI card contains 30 wells with different biochemical substrates. The Vitek test is dependent on expensive technical equipment, but may identify most aerobic and anaerobic bacteria, including a number of Gram-positive species. The bacterial suspension is transferred to the wells and after a varying length of incubation the results are recorded automatically. The result of the catalase test has to be marked on the card. ‘Staph-zym’ may identify 18 species of coagulase-negative staphylococci and coagulase-positive staphylococci (Table I). A suspension of bacteria is transferred to 10 wells filled with different test substrates. In addition, sensitivity to polymyxin, novobiocin and furazolidone is tested. The results can be read as colour changes after incubation for 18 h, and after adding a few reagents to two of the wells. The ‘Staph-zym’ test is read visually and results are converted into a numerical code after values are given to the different tests and combinations of tests as follows: first digit from the combination of P-glucosidase (/3-GLU), P-galactosidase (ONPG) and P-glucuronidase (PGUA) with values of 1,2 and 4 respectively; second digit from the combination of trehalose (TRE), maltose (MAL) and urease (UR) with values of 1, 2 and 4 respectively; third digit from alkaline phosphatase (ALK.P) with value 1; fourth digit from the combination of novobiocin (NOVO) and polymyxin (POL) with values 1 and 3 respectively.

Other test systems API 20 Staph (BioMerieux,

la Balme les Grottes,

38390 Montalieu

Vercieu,

Coagulase-negative Table

I.

The repertoire

of

identification

21

staphylococci

systems for

coagulase-negative

staphylococci

System

Staphylococcus S. S. S. S. S. S.

species

GPI*

number

* GPI requires t x, The system

ATB 32 Staph x X

x X

cohnii epidermidis equorum gallinarum haemolyticus hominis hyicus intermedius kloosii lentus lugdunensis saprophyticus schleiferi sciuri simulans warneri xylosus

Total

API 20 Staph

xt

aureus arlettae auricularis capitis caprae carnosus

S. chromogenes

S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. s.

‘Staph-zym’

x x X x x X x X X x x

of species marking may

;4

of the coagulase identify

the

reaction

x x

X x

X X X

x x x

x X X X X X X X

X x X x x

x x X

x X x X X X

X x X x X X

X x

X

X X

19

;9

2x4

X X X X X X

on the card

species.

France) contains 20 wells filled with test substrates to which a suspension of bacteria is applied, and 18 h later the result can be read after adding some reagents to the wells. ATB 32 Staph (BioMerieux) is, in principle, the same method as API 20 Staph, but has 32 wells and should be read automatically. Interpretation Identical results in the GPI and the ‘Staph-zym’ were considered to be correct. When discrepancies occurred, the isolates were tested once again in both systems to exclude technical errors. If identification repeatedly gave different results, the isolates were tested in API 20 Staph and ATB 32 Staph. An isolate unidentified in either GPI or ‘Staph-zym’ was considered to be correctly species-identified if the two other systems showed an identical result with GPI or with Staph-zym. Resistance Methicillin resistance was evaluated by the oxacillin agar incorporation method’ and by the heavy inoculum agar diffusion method. The oxacillin agar method was performed on Mueller-Hinton agar with 4% NaCl and

K. Refsahl

22

and B. M. Andersen

6 mg 1-l oxacillin. Growth of the strain on the agar, after incubation at 35°C for 24 h, was interpreted as methicillin resistance. In the heavy inoculum method, a thick streak was made on Paper Disk Method (PDM) Medium (Biodisk); a tablet of oxacillin 5 pg was placed on the streak before incubation at 32°C. Oxacillin resistance was interpreted as no zone of inhibited growth around the oxacillin tablet or no narrowing of the streak against the tablet after 24 h of growth. The oxacillin-containing agar and the PDM medium were made freshly once per week. Resistance to other drugs was examined by the agar diffusion method using tablets (Rosco) on PDM medium. Sensitivity group 14 was determined from the zone sizes in the tablet test as recommended by the Norwegian Working Group on Antibacterial Resistance.6 Sensitivity group 1 was defined as having the following MIC values (mg 1-l): benzylpenicillin < 1, oxacillin < 1, erythromycin < 1, fusidic acid < 1, cephalothin < 2, cefotaxime < 2, ceftazidime < 2, gentamicin < 2, streptomycin ~4, vancomycin ~2, rifampicin < 0.01, clindamycin < 1, tetracycline < 1, chloramphenicol < 2, ciprofloxacin